recent journal articles: developmental biology




Recent Articles in Annual Review of Cell and Developmental Biology

Zheng JQ, Poo MM
Calcium signaling in neuronal motility.
Annu Rev Cell Dev Biol. 2007;23375-404.
Neuronal motility is a fundamental feature that underlies the development, regeneration, and plasticity of the nervous system. Two major developmental events--directed migration of neuronal precursor cells to the proper positions and guided elongation of axons to their target cells--depend on large-scale neuronal motility. At a finer scale, motility is also manifested in many aspects of neuronal structures and functions, ranging from differentiation and refinement of axonal and dendritic morphology during development to synapse remodeling associated with learning and memory in the adult brain. As a primary second messenger that conveys the cytoplasmic actions of electrical activity and many neuroactive ligands, Ca(2+) plays a central role in the regulation of neuronal motility. Recent studies have revealed common Ca(2+)-dependent signaling pathways that are deployed for regulating cytoskeletal dynamics associated with neuronal migration, axon and dendrite development and regeneration, and synaptic plasticity. [Abstract]

Lobo NA, Shimono Y, Qian D, Clarke MF
The biology of cancer stem cells.
Annu Rev Cell Dev Biol. 2007;23675-99.
Cancers originally develop from normal cells that gain the ability to proliferate aberrantly and eventually turn malignant. These cancerous cells then grow clonally into tumors and eventually have the potential to metastasize. A central question in cancer biology is, which cells can be transformed to form tumors? Recent studies elucidated the presence of cancer stem cells that have the exclusive ability to regenerate tumors. These cancer stem cells share many characteristics with normal stem cells, including self-renewal and differentiation. With the growing evidence that cancer stem cells exist in a wide array of tumors, it is becoming increasingly important to understand the molecular mechanisms that regulate self-renewal and differentiation because corruption of genes involved in these pathways likely participates in tumor growth. This new paradigm of oncogenesis has been validated in a growing list of tumors. Studies of normal and cancer stem cells from the same tissue have shed light on the ontogeny of tumors. That signaling pathways such as Bmi1 and Wnt have similar effects in normal and cancer stem cell self-renewal suggests that common molecular pathways regulate both populations. Understanding the biology of cancer stem cells will contribute to the identification of molecular targets important for future therapies. [Abstract]

Jenik PD, Gillmor CS, Lukowitz W
Embryonic patterning in Arabidopsis thaliana.
Annu Rev Cell Dev Biol. 2007;23207-36.
Early embryonic development in the flowering plant Arabidopsis thaliana follows a predictable sequence of cell divisions. Anatomical hallmarks and the expression of marker genes in dynamic patterns indicate that new cell fates are established with virtually every round of mitosis. Although some of the factors regulating these early patterning events have been identified, the overall process remains relatively poorly understood. Starting at the globular stage, when the embryo has approximately 100 cells, the organization of development appears to be taken over by programs that regulate postembryonic patterning throughout the life cycle. [Abstract]

Tran TS, Kolodkin AL, Bharadwaj R
Semaphorin regulation of cellular morphology.
Annu Rev Cell Dev Biol. 2007;23263-92.
Semaphorin proteins, although initially characterized as repulsive neuronal guidance cues, are now appreciated as major contributors to morphogenesis and homeostasis for a wide range of tissue types. Semaphorin-mediated long- and short-range repulsive, and attractive, guidance has profound influences on cellular morphology. The diversity of semaphorin receptor complexes utilized by various semaphorin ligands, the ability of semaphorins themselves to serve as receptors, and the myriad of intracellular signaling components that comprise semaphorin signaling cascades all contribute to cell-type-specific responses to semaphorins. Analysis of the molecular and cellular mechanisms underlying semaphorin function in neural and vascular systems provides insight into principles governing how this large protein family contributes to organogenesis, function, and disease. [Abstract]

Pokutta S, Weis WI
Structure and mechanism of cadherins and catenins in cell-cell contacts.
Annu Rev Cell Dev Biol. 2007;23237-61.
Cadherins are Ca(2+)-dependent cell adhesion molecules found in several kinds of cell-cell contact, including adherens junctions and desmosomes. In the presence of Ca(2+), cells expressing the same type of cadherin form stable contacts with one another, a phenomenon designated homophilic, or homotypic, adhesion. Most cadherins are single-pass transmembrane proteins whose extracellular regions mediate specific cell-cell interactions. The intracellular faces of these contacts are associated with the actin cytoskeleton in adherens junctions or the intermediate-filament system in desmosomes. The close coordination of the transmembrane adhesion molecules with the cytoskeleton is believed to be essential in coordinating morphogenetic movements of tissues during development and in conferring the appropriate mechanical properties to cell-cell contacts. Structural, biochemical, and biophysical analysis of the molecules that comprise these contacts has provided unique mechanistic insights into the specificity of homophilic adhesion, the functional connection to the underlying cytoskeleton, and the dynamics of junction formation. [Abstract]

Gillingham AK, Munro S
The small G proteins of the Arf family and their regulators.
Annu Rev Cell Dev Biol. 2007;23579-611.
Small G proteins play a central role in the organization of the secretory and endocytic pathways. The majority of such small G proteins are members of the Rab family, which are anchored to the bilayer by C-terminal prenyl groups. However, the recruitment of some effectors, including vesicle coat proteins, is mediated by a second class of small G proteins that is unique in having an N-terminal amphipathic helix that becomes available for membrane insertion upon GTP binding. Sar1, Arf1, and Arf6 are the best-characterized members of this ADP-ribosylation factor (Arf) family. In addition, all eukaryotes contain additional distantly related G proteins, often called Arf like, or Arls. The complete Arf family in humans has 29 members. The roles of these related G proteins are poorly understood, but recent work has shown that some are involved in membrane traffic or organizing the cytoskeleton. Here we review what is known about all the members of the Arf family, along with the known regulatory molecules that convert them between GDP- and GTP-bound states. [Abstract]

Fagarasanu A, Fagarasanu M, Rachubinski RA
Maintaining peroxisome populations: a story of division and inheritance.
Annu Rev Cell Dev Biol. 2007;23321-44.
Eukaryotic cells divide their metabolic labor between functionally distinct, membrane-enveloped organelles, each precisely tailored for a specific set of biochemical reactions. Peroxisomes are ubiquitous, endoplasmic reticulum-derived organelles that perform requisite biochemical functions intimately connected to lipid metabolism. Upon cell division, cells have to strictly control peroxisome division and inheritance to maintain an appropriate number of peroxisomes in each cell. Peroxisome division follows a specific sequence of events that include peroxisome elongation, membrane constriction, and peroxisome fission. Pex11 proteins mediate the elongation step of peroxisome division, whereas dynamin-related proteins execute the final fission. The mechanisms responsible for peroxisome membrane constriction are poorly understood. Molecular players involved in peroxisome inheritance are just beginning to be elucidated. Inp1p and Inp2p are two recently identified peroxisomal proteins that perform antagonistic functions in regulating peroxisome inheritance in budding yeast. Inp1p promotes the retention of peroxisomes in mother cells and buds by attaching peroxisomes to as-yet-unidentified cortical structures. Inp2p is implicated in the motility of peroxisomes by linking them to the Myo2p motor, which then propels their movement along actin cables. The functions of Inp1p and Inp2p are cell cycle regulated and coordinated to ensure a fair distribution of peroxisomes at cytokinesis. [Abstract]

Stinchcombe JC, Griffiths GM
Secretory mechanisms in cell-mediated cytotoxicity.
Annu Rev Cell Dev Biol. 2007;23495-517.
Cytotoxic T lymphocytes (CTLs) play a critical role in the immune system; they are able to recognize and destroy virally infected and tumorigenic cells. Specific recognition of MHC class I-peptide complexes by the T cell receptor (TcR) results in precise delivery of lytic granules to the target cell, sparing neighboring cells and the CTL itself. Over the past 10 years various studies have eludicated the mechanisms that lead to the rapid polarization of the secretory apparatus in CTLs. These studies highlight similarities and differences between polarity and secretory mechanisms seen in other cell types and developmental systems. This review focuses on recent advances in our understanding of the molecular basis of polarized secretion from CTLs and the novel mechanism used by these cells to deliver their lethal hit. [Abstract]

Blair SS
Wing vein patterning in Drosophila and the analysis of intercellular signaling.
Annu Rev Cell Dev Biol. 2007;23293-319.
The positioning and elaboration of ectodermal veins in the wing of Drosophila melanogaster rely on widely utilized developmental signals, including those mediated by EGF, BMP, Hedgehog, Notch, and Wnt. Analysis of vein patterning mutants, using the molecular and genetic mosaic techniques available in Drosophila, has provided important insights into how a combination of short-range and long-range signaling can pattern a simple epidermal tissue. Moreover, venation has become a powerful system for isolating and analyzing novel components in these signaling pathways. I here review the basic events of vein patterning and give examples of how changes in venation have been used to identify important features of cell signaling pathways. [Abstract]

Shepherd JD, Huganir RL
The cell biology of synaptic plasticity: AMPA receptor trafficking.
Annu Rev Cell Dev Biol. 2007;23613-43.
The cellular processes that govern neuronal function are highly complex, with many basic cell biological pathways uniquely adapted to perform the elaborate information processing achieved by the brain. This is particularly evident in the trafficking and regulation of membrane proteins to and from synapses, which can be a long distance away from the cell body and number in the thousands. The regulation of neurotransmitter receptors, such as the AMPA-type glutamate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain, is a crucial mechanism for the modulation of synaptic transmission. The levels of AMPARs at synapses are very dynamic, and it is these plastic changes in synaptic function that are thought to underlie information storage in the brain. Thus, understanding the cellular machinery that controls AMPAR trafficking will be critical for understanding the cellular basis of behavior as well as many neurological diseases. Here we describe the life cycle of AMPARs, from their biogenesis, through their journey to the synapse, and ultimately through their demise, and discuss how the modulation of this process is essential for brain function. [Abstract]

Kimble J, Crittenden SL
Controls of germline stem cells, entry into meiosis, and the sperm/oocyte decision in Caenorhabditis elegans.
Annu Rev Cell Dev Biol. 2007;23405-33.
The Caenorhabditis elegans germ line provides an exceptional model for analysis of the molecular controls governing stem cell maintenance, the cell cycle transition from mitosis to meiosis, and the choice of sexual identity-sperm or oocyte. Germline stem cells are maintained in an undifferentiated state within a well-defined niche formed by a single somatic cell, the distal tip cell (DTC). In both sexes, the DTC employs GLP-1/Notch signaling and FBF/PUF RNA-binding proteins to maintain stem cells and promote mitotic divisions, three additional RNA regulators (GLD-1/quaking, GLD-2/poly(A) polymerase, and GLD-3/Bicaudal-C) control entry into meiosis, and FOG-1/CPEB and FOG-3/Tob proteins govern sperm specification. These key regulators are part of a robust regulatory network that controls germ cell proliferation, stem cell maintenance, and sex determination. Parallels with controls in other organisms include the use of PUF proteins for stem cell maintenance and the prominence of mRNA regulation for the control of germline development. [Abstract]

Piper RC, Katzmann DJ
Biogenesis and function of multivesicular bodies.
Annu Rev Cell Dev Biol. 2007;23519-47.
The two major cellular sites for membrane protein degradation are the proteasome and the lysosome. Ubiquitin attachment is a sorting signal for both degradation routes. For lysosomal degradation, ubiquitination triggers the sorting of cargo proteins into the lumen of late endosomal multivesicular bodies (MVBs)/endosomes. MVB formation occurs when a portion of the limiting membrane of an endosome invaginates and buds into its own lumen. Intralumenal vesicles are degraded when MVBs fuse to lysosomes. The proper delivery of proteins to the MVB interior relies on specific ubiquitination of cargo, recognition and sorting of ubiquitinated cargo to endosomal subdomains, and the formation and scission of cargo-filled intralumenal vesicles. Over the past five years, a number of proteins that may directly participate in these aspects of MVB function and biogenesis have been identified. However, major questions remain as to exactly what these proteins do at the molecular level and how they may accomplish these tasks. [Abstract]

Pardue ML
Following the chromosome path to the garden of the genome.
Annu Rev Cell Dev Biol. 2007;231-22.
I have been fascinated by chromosomes for longer than I care to mention; their beautiful structure, cell-type-specific changes in morphology, and elegant movements delight me. Shortly before I began graduate study, the development of nucleic acid hybridization made it possible to compare two nucleic acids whether or not their sequences were known. From this stemmed a progression of development in tools and techniques that continues to enhance our understanding of how chromosomes function. As my PhD project I contributed to this progression by developing in situ hybridization, a technique for hybridization to nucleic acids within their cellular context. Early studies with this technique initiated several lines of research, two of which I describe here, that I have pursued to this day. First, analysis of RNA populations by hybridization to polytene chromosomes (a proto-microarray-type experiment) led us to characterize levels of regulation during heat shock beyond those recognizable by puffing studies. We found also that one still-undeciphered major heat shock puff encodes a novel set of RNAs for which we propose a regulatory role. Second, localization of various multicopy DNA sequences has suggested roles for them in chromosome structure: Most recently we have found that Drosophila telomeres consist of and are maintained by special non-LTR (long terminal repeat) retrotransposons. [Abstract]

Bushati N, Cohen SM
microRNA functions.
Annu Rev Cell Dev Biol. 2007;23175-205.
microRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional gene regulation. In animal cells, miRNAs regulate their targets by translational inhibition and mRNA destabilization. Here, we review recent work in animal models that provide insight into the diverse roles of miRNAs in vivo. [Abstract]

Lipka V, Kwon C, Panstruga R
SNARE-ware: the role of SNARE-domain proteins in plant biology.
Annu Rev Cell Dev Biol. 2007;23147-74.
In yeast and animal cells, members of the superfamily of N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE)-domain-containing proteins are key players in vesicle-associated membrane fusion events during transport processes between individual compartments of the endomembrane system, including exocytosis and endocytosis. Compared with genomes of other eukaryotes, genomes of monocotyledonous and dicotyledonous plants encode a surprisingly high number of SNARE proteins, suggesting vital roles for this protein class in higher plant species. Although to date it remains elusive whether plant SNARE proteins function like their yeast and animal counterparts, genetic screens have recently begun to unravel the variety of biological tasks in which plant SNAREs are involved. These duties involve fundamental processes such as cytokinesis, shoot gravitropism, pathogen defense, symbiosis, and abiotic stress responses, suggesting that SNAREs contribute essentially to many facets of plant biology. [Abstract]

Ciofani M, Zúńiga-Pflücker JC
The thymus as an inductive site for T lymphopoiesis.
Annu Rev Cell Dev Biol. 2007;23463-93.
Like all hematopoietic cells, T lymphocytes are derived from bone-marrow-resident stem cells. However, whereas most blood lineages are generated within the marrow, the majority of T cell development occurs in a specialized organ, the thymus. This distinction underscores the unique capacity of the thymic microenvironment to support T lineage restriction and differentiation. Although the identity of many of the contributing thymus-derived signals is well established and rooted in highly conserved pathways involving Notch, morphogenetic, and protein tyrosine kinase signals, the manner in which the ensuing cascades are integrated to orchestrate the underlying processes of T cell development remains under investigation. This review focuses on the current definition of the early stages of T cell lymphopoiesis, with an emphasis on the nature of thymus-derived signals delivered to T cell progenitors that support the commitment and differentiation of T cells toward the alphabeta and gammadelta T cell lineages. [Abstract]

Poo MM, Zheng J
Calcium Signaling and Neuronal Motility.
Annu Rev Cell Dev Biol. 2006 Sep 8;
Neuronal motility is a fundamental feature of the nervous system that underlies its development, regeneration and plasticity. Two major developmental events-directed migration of neuronal precursor cells to the proper positions and guided elongation of axons to their target cells-depend on large-scale neuronal motility. At a finer scale, motility is also manifested in many aspects of neuronal structures and functions, ranging from differentiation and refinement of axonal and dendritic morphology during development to synapse remodeling associated with learning and memory in the adult brain. As a primary second messenger that conveys the cytoplasmic actions of electrical activity and many neuroactive ligands, Ca(2+) plays a central role in the regulation of neuronal motility. Recent studies have revealed common Ca(2+)-dependent signaling pathways that are deployed for regulating cytoskeletal dynamics associated with neuronal migration, axon and dendrite development and regeneration, and synaptic plasticity. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 23 is October 6, 2007. Please see for revised estimates. [Abstract]

Eggenschwiler JT, Anderson KV
Cilia and developmental signaling.
Annu Rev Cell Dev Biol. 2007;23345-73.
Recent studies have revealed unexpected connections between the mammalian Hedgehog (Hh) signal transduction pathway and the primary cilium, a microtubule-based organelle that protrudes from the surface of most vertebrate cells. Intraflagellar transport proteins, which are required for the construction of cilia, are essential for all responses to mammalian Hh proteins, and proteins required for Hh signal transduction are enriched in primary cilia. The phenotypes of different mouse mutants that affect ciliary proteins suggest that cilia may act as processive machines that organize sequential steps in the Hh signal transduction pathway. Cilia on vertebrate cells are likely to be important in additional developmental signaling pathways and are required for PDGF receptor alpha signaling in cultured fibroblasts. Cilia are not essential for either canonical or noncanonical Wnt signaling, although cell-type-specific modulation of cilia components may link cilia and Wnt signaling in some tissues. Because ciliogenesis in invertebrates is limited to a very small number of specialized cell types, the role of cilia in developmental signaling pathways is likely a uniquely vertebrate phenomenon. [Abstract]

Jiang D, Liang J, Noble PW
Hyaluronan in tissue injury and repair.
Annu Rev Cell Dev Biol. 2007;23435-61.
A hallmark of tissue injury and repair is the turnover of extracellular matrix components. This review focuses on the role of the glycosaminoglycan hyaluronan in tissue injury and repair. Both the synthesis and degradation of extracellular matrix are critical contributors to tissue repair and remodeling. Fragmented hyaluronan accumulates during tissue injury and functions in ways distinct from the native polymer. There is accumulating evidence that hyaluronan degradation products can stimulate the expression of inflammatory genes by a variety of immune cells at the injury site. CD44 is the major cell-surface hyaluronan receptor and is required to clear hyaluronan degradation products produced during lung injury; impaired clearance of hyaluronan results in persistent inflammation. However, hyaluronan fragment stimulation of inflammatory gene expression is not dependent on CD44 in inflammatory macrophages. Instead, hyaluronan fragments utilize both Toll-like receptor (TLR) 4 and TLR2 to stimulate inflammatory genes in macrophages. Hyaluronan also is present on the cell surface of lung alveolar epithelial cells and provides protection against tissue damage by interacting with TLR2 and TLR4 on these parenchymal cells. The simple repeating structure of hyaluronan appears to be involved in a number of important aspects of noninfectious tissue injury and repair that are dependent on the size and location of the polymer as well as the interacting cells. Thus, the interactions between the endogenous matrix component hyaluronan and its signaling receptors initiate inflammatory responses, maintain structural cell integrity, and promote recovery from tissue injury. [Abstract]

Buckingham M, Relaix F
The role of Pax genes in the development of tissues and organs: Pax3 and Pax7 regulate muscle progenitor cell functions.
Annu Rev Cell Dev Biol. 2007;23645-73.
Pax genes play key roles in the formation of tissues and organs during embryogenesis. Pax3 and Pax7 mark myogenic progenitor cells and regulate their behavior and their entry into the program of skeletal muscle differentiation. Recent results have underlined the importance of the Pax3/7 population of cells for skeletal muscle development and regeneration. We present our current understanding of different aspects of Pax3/7 function in myogenesis, focusing on the mouse model. This is compared with that of other Pax proteins in the emergence of tissue specific lineages and their differentiation as well as in cell survival, proliferation, and migration. Finally, we consider the molecular mechanisms that underlie the function of Pax transcription factors, including the cofactors and regulatory networks with which they interact. [Abstract]

Sillitoe RV, Joyner AL
Morphology, molecular codes, and circuitry produce the three-dimensional complexity of the cerebellum.
Annu Rev Cell Dev Biol. 2007;23549-77.
The most noticeable morphological feature of the cerebellum is its folded appearance, whereby fissures separate its anterior-posterior extent into lobules. Each lobule is molecularly coded along the medial-lateral axis by parasagittal stripes of gene expression in one cell type, the Purkinje cells (PCs). Additionally, within each lobule distinct combinations of afferents terminate and supply the cerebellum with synchronized sensory and motor information. Strikingly, afferent terminal fields are organized into parasagittal domains, and this pattern bears a close relationship to PC molecular coding. Thus, cerebellum three-dimensional complexity obeys a basic coordinate system that can be broken down into morphology and molecular coding. In this review, we summarize the sequential stages of cerebellum development that produce its laminar structure, foliation, and molecular organization. We also introduce genes that regulate morphology and molecular coding, and discuss the establishment of topographical circuits within the context of the two coordinate systems. Finally, we discuss how abnormal cerebellar organization may result in neurological disorders like autism. [Abstract]

Horwich AL, Fenton WA, Chapman E, Farr GW
Two families of chaperonin: physiology and mechanism.
Annu Rev Cell Dev Biol. 2007;23115-45.
Chaperonins are large ring assemblies that assist protein folding to the native state by binding nonnative proteins in their central cavities and then, upon binding ATP, release the substrate protein into a now-encapsulated cavity to fold productively. Two families of such components have been identified: type I in mitochondria, chloroplasts, and the bacterial cytosol, which rely on a detachable "lid" structure for encapsulation, and type II in archaea and the eukaryotic cytosol, which contain a built-in protrusion structure. We discuss here a number of issues under current study. What is the range of substrates acted on by the two classes of chaperonin, in particular by GroEL in the bacterial cytoplasm and CCT in the eukaryotic cytosol, and are all these substrates subject to encapsulation? What are the determinants for substrate binding by the type II chaperonins? And is the encapsulated chaperonin cavity a passive container that prevents aggregation, or could it be playing an active role in polypeptide folding? [Abstract]

Koster MI, Roop DR
Mechanisms regulating epithelial stratification.
Annu Rev Cell Dev Biol. 2007;2393-113.
The epidermis is a stratified epithelium that functions as a barrier protecting the organism from dehydration, mechanical trauma, and microbial insults. This barrier function is established during embryogenesis through a complex and tightly controlled stratification program. Whereas the morphological changes that occur during epidermal development have been extensively studied, the molecular mechanisms that govern this process remain poorly understood. In this review we summarize the current advances that have been made in understanding the molecular mechanisms that regulate epidermal morphogenesis. [Abstract]

Schäfer M, Werner S
Transcriptional control of wound repair.
Annu Rev Cell Dev Biol. 2007;2369-92.
Injury to the skin initiates a complex process of events involving inflammation as well as the formation and remodeling of new tissue. These processes result in at least partial reconstitution of the injured skin. However, wounds in adult mammals heal with a scar, which is accompanied by functional and aesthetic impairments. In addition to this problem, a large number of patients, in particular in the aged population, suffer from chronic, nonhealing ulcers. Therefore, there is a strong need to improve the wound healing process. This requires a thorough understanding of the underlying molecular and cellular mechanisms. During the past several years, important regulators of the wound healing process have been identified. In particular, the growth factors and matrix proteins, which orchestrate skin repair, have been characterized in detail. By contrast, much less is known about the transcription factors, which regulate gene expression at the wound site. This review summarizes recent data on the expression of transcription factors in skin wounds and their functions in the repair process. [Abstract]

Abu-Issa R, Kirby ML
Heart field: from mesoderm to heart tube.
Annu Rev Cell Dev Biol. 2007;2345-68.
In this review we discuss the major morphogenetic and regulative events that control myocardial progenitor cells from the time that they delaminate from the epiblast in the primitive streak to their differentiation into cardiomyocytes in the heart tube. During chick and mouse embryogenesis, myocardial progenitor cells go through four specific processes that are sequential but overlapping: specification of the cardiogenic mesoderm, determination of the bilaterally symmetric heart fields, patterning of the heart field, and finally cardiomyocyte differentiation and formation of the heart tube. We describe the morphological and molecular events that play a pivotal role in each of these four processes. [Abstract]

Tsai B
Penetration of nonenveloped viruses into the cytoplasm.
Annu Rev Cell Dev Biol. 2007;2323-43.
Although the precise mechanism by which nonenveloped viruses penetrate biological membranes is unclear, a more coherent understanding of this process is starting to emerge. To initiate membrane penetration, nonenveloped viruses engage host cell factors that impart conformational changes on the viral particles, resulting in the exposure of a hydrophobic moiety or the release of a lytic factor. The viruses' interactions with the limiting membrane subsequently compromise the bilayer integrity. This reaction presumably perforates the bilayer to enable the virus to cross the membrane and reach the cytosol. Valuable insights into this process can be gleaned from the membrane transport mechanisms of enveloped viruses and bacterial toxins. To identify systematically the cellular components that facilitate nonenveloped virus membrane penetration, sensitive assays that monitor the transport event directly must first be established. Moreover, higher-resolution structures of penetration intermediates, particularly those solved in complex with membranes, would provide important molecular details into this process. [Abstract]

Mombaerts P
Axonal wiring in the mouse olfactory system.
Annu Rev Cell Dev Biol. 2006;22713-37.
The main olfactory epithelium of the mouse is a mosaic of 2000 populations of olfactory sensory neurons (OSNs). Each population expresses one allele of one of the 1000 intact odorant receptor (OR) genes. An OSN projects a single unbranched axon to a single glomerulus, from an array of 1600-1800 glomeruli in the main olfactory bulb. Within a glomerulus the OSN axon synapses with the dendrites of second-order neurons and interneurons. Axons of OSNs that express the same OR project to the same glomeruli-typically one glomerulus per half-bulb and thus four glomeruli per mouse. These glomeruli are located at characteristic positions within the glomerular layer of the bulb. ORs determine both the odorant response profile of the OSN and the projection of its axon to a specific glomerulus. I focus on genetic approaches to the axonal wiring problem, particularly on how ORs may function in axonal wiring. [Abstract]

Dickson BJ, Gilestro GF
Regulation of commissural axon pathfinding by slit and its Robo receptors.
Annu Rev Cell Dev Biol. 2006;22651-75.
Commissural axons grow along complex pathways toward, across, and beyond the midline of the central nervous system. Taking commissural axons in the vertebrate spinal cord and the Drosophila ventral nerve cord as examples, we examine how commissural axon pathfinding is regulated by the Slit family of guidance cues and their Robo family receptors. We extract several principles that seem likely to apply to other axons and other contexts, such as the reiterative use of the same guidance molecules in distinct pathfinding decisions, the transcriptional specification of a pathway, the posttranscriptional regulation of growth along the pathway, and the possible role of feedback mechanisms to ensure the fidelity of pathfinding choices. Such mechanisms may help explain how a relatively small number of guidance molecules can generate complex and stereotyped wiring patterns. We also highlight the many gaps in our understanding of commissural axon pathfinding and question some widely accepted views. We hope that this review encourages further efforts to tackle these questions, in the expectation that this system will continue to reveal the general principles of axon pathfinding. [Abstract]

Hay BA, Guo M
Caspase-dependent cell death in Drosophila.
Annu Rev Cell Dev Biol. 2006;22623-50.
Cell death plays many roles during development, in the adult, and in the genesis of many pathological states. Much of this death is apoptotic in nature and requires the activity of members of the caspase family of proteases. It is now possible uniquely in Drosophila to carry out genetic screens for genes that determine the fate-life or death-of any population of cells during development and adulthood. This, in conjunction with the ability to obtain biochemical quantities of material, has made Drosophila a useful organism for exploring the mechanisms by which apoptosis is carried out and regulated. This review summarizes our knowledge of caspase-dependent cell death in Drosophila and compares that knowledge with what is known in worms and mammals. We also discuss the significance of recent work showing that a number of key cell death activators also play nonapoptotic roles. We highlight opportunities and outstanding questions along the way. [Abstract]

Stewart SA, Weinberg RA
Telomeres: cancer to human aging.
Annu Rev Cell Dev Biol. 2006;22531-57.
The cell phenotypes of senescence and crisis operate to circumscribe the proliferative potential of mammalian cells, suggesting that both are capable of operating in vivo to suppress the formation of tumors. The key regulators of these phenotypes are the telomeres, which are located at the ends of chromosomes and operate to protect the chromosomes from end-to-end fusions. Telomere erosion below a certain length can trigger crisis. The relationship between senescence and telomere function is more complex, however: Cell-physiological stresses as well as dysfunction of the complex molecular structures at the ends of telomeric DNA can trigger senescence. Cells can escape senescence by inactivating the Rb and p53 tumor suppressor proteins and can surmount crisis by activating a telomere maintenance mechanism. The resulting cell immortalization is an essential component of the tumorigenic phenotype of human cancer cells. Here we discuss how telomeres are monitored and maintained and how loss of a functional telomere influences biological functions as diverse as aging and carcinogenesis. [Abstract]

Recent Articles in Genes & Development

Peden E, Kimberly E, Gengyo-Ando K, Mitani S, Xue D
Control of sex-specific apoptosis in C. elegans by the BarH homeodomain protein CEH-30 and the transcriptional repressor UNC-37/Groucho.
Genes Dev. 2007 Dec 1;21(23):3195-207.
Apoptosis is essential for proper development and tissue homeostasis in metazoans. It plays a critical role in generating sexual dimorphism by eliminating structures that are not needed in a specific sex. The molecular mechanisms that regulate sexually dimorphic apoptosis are poorly understood. Here we report the identification of the ceh-30 gene as a key regulator of sex-specific apoptosis in Caenorhabditis elegans. Loss-of-function mutations in ceh-30 cause the ectopic death of male-specific CEM neurons. ceh-30 encodes a BarH homeodomain protein that acts downstream from the terminal sex determination gene tra-1, but upstream of, or in parallel to, the cell-death-initiating gene egl-1 to protect CEM neurons from undergoing apoptosis in males. The second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A and a POU-type homeodomain protein UNC-86 and acts as a sensor to regulate proper specification of the CEM cell fate. Surprisingly, the N terminus of CEH-30 but not its homeodomain is critical for CEH-30's cell death inhibitory activity in CEMs and contains a conserved eh1/FIL domain that is important for the recruitment of the general transcriptional repressor UNC-37/Groucho. Our study suggests that ceh-30 defines a critical checkpoint that integrates the sex determination signal TRA-1 and the cell fate determination and survival signal UNC-86 to control the sex-specific activation of the cell death program in CEMs through the general transcription repressor UNC-37. [Abstract]

Schwartz HT, Horvitz HR
The C. elegans protein CEH-30 protects male-specific neurons from apoptosis independently of the Bcl-2 homolog CED-9.
Genes Dev. 2007 Dec 1;21(23):3181-94.
The developmental control of apoptosis is fundamental and important. We report that the Caenorhabditis elegans Bar homeodomain transcription factor CEH-30 is required for the sexually dimorphic survival of the male-specific CEM (cephalic male) sensory neurons; the homologous cells of hermaphrodites undergo programmed cell death. We propose that the cell-type-specific anti-apoptotic gene ceh-30 is transcriptionally repressed by the TRA-1 transcription factor, the terminal regulator of sexual identity in C. elegans, to cause hermaphrodite-specific CEM death. The established mechanism for the regulation of specific programmed cell deaths in C. elegans is the transcriptional control of the BH3-only gene egl-1, which inhibits the Bcl-2 homolog ced-9; similarly, most regulation of vertebrate apoptosis involves the Bcl-2 superfamily. In contrast, ceh-30 acts within the CEM neurons to promote their survival independently of both egl-1 and ced-9. Mammalian ceh-30 homologs can substitute for ceh-30 in C. elegans. Mice lacking the ceh-30 homolog Barhl1 show a progressive loss of sensory neurons and increased sensory-neuron cell death. Based on these observations, we suggest that the function of Bar homeodomain proteins as cell-type-specific inhibitors of apoptosis is evolutionarily conserved. [Abstract]

Junion G, Bataillé L, Jagla T, Da Ponte JP, Tapin R, Jagla K
Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors.
Genes Dev. 2007 Dec 1;21(23):3163-80.
Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions we performed muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search for direct Ladybird targets. Our data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, we identified bric-a-brac 2 gene as a new component of identity code and inflated encoding alphaPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells. [Abstract]

Pöyry TA, Kaminski A, Connell EJ, Fraser CS, Jackson RJ
The mechanism of an exceptional case of reinitiation after translation of a long ORF reveals why such events do not generally occur in mammalian mRNA translation.
Genes Dev. 2007 Dec 1;21(23):3149-62.
The subgenomic mRNA of feline caliciviruses is bicistronic with the two cistrons overlapping by four nucleotides, ..AUGA. The upstream cistron encodes a 75-kDa major capsid protein precursor (pre-VP1), and the downstream cistron a 10-kDa minor capsid protein. The kinetics of translation in reticulocyte lysates show that the downstream cistron is translated by a termination-reinitiation process, which is unusual in not requiring eIF4G or the eIF4F complex. Reinitiation requires the 3'-terminal 87 nucleotides (nt) of the pre-VP1 ORF, but no other viral sequences. The reinitiation site is selected by virtue of its proximity to this 87-nt element, and not its proximity to the pre-VP1 ORF stop codon, although this must be located not more than approximately 30 nt downstream from the restart codon. This 87-nt element was shown to bind 40S ribosomal subunits and initiation factor eIF3, and addition of supplementary eIF3 enhanced reinitiation efficiency. Mutants defective in reinitiation showed reduced affinity for eIF3 or defective 40S subunit binding (or both). These results suggest a mechanism in which some of the eIF3/40S complexes formed during disassembly of post-termination ribosomes bind to this 87-nt element in a position appropriate for reinitiation following acquisition of an eIF2/GTP/Met-tRNA i ternary complex. [Abstract]

Funakoshi Y, Doi Y, Hosoda N, Uchida N, Osawa M, Shimada I, Tsujimoto M, Suzuki T, Katada T, Hoshino S
Mechanism of mRNA deadenylation: evidence for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases.
Genes Dev. 2007 Dec 1;21(23):3135-48.
In eukaryotes, shortening of the 3'-poly(A) tail is the rate-limiting step in the degradation of most mRNAs, and two major mRNA deadenylase complexes--Caf1-Ccr4 and Pan2-Pan3--play central roles in this process, referred to as deadenylation. However, the molecular mechanism triggering deadenylation remains elusive. Previously, we demonstrated that eukaryotic releasing factor eRF3 mediates deadenylation and decay of mRNA in a manner coupled to translation termination. Here, we report the mechanism of mRNA deadenylation. The eRF3-mediated deadenylation is catalyzed by both Caf1-Ccr4 and Pan2-Pan3. Interestingly, translation termination complexes eRF1-eRF3, Pan2-Pan3, and Caf1-Ccr4 competitively interact with polyadenylate-binding protein PABPC1. In each complex, eRF3, Pan3, and Tob, respectively, mediate PABPC1 binding, and a combination of a PAM2 motif and a PABC domain is commonly utilized for their contacts. A translation-dependent exchange of eRF1-eRF3 for the deadenylase occurs on PABPC1. Consequently, PABPC1 binding leads to the activation of Pan2-Pan3 and Caf1-Ccr4. From these results, we suggest a mechanism of mRNA deadenylation by Pan2-Pan3 and Caf1-Ccr4 in cooperation with eRF3 and PABPC1. [Abstract]

Linhart HG, Lin H, Yamada Y, Moran E, Steine EJ, Gokhale S, Lo G, Cantu E, Ehrich M, He T, Meissner A, Jaenisch R
Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing.
Genes Dev. 2007 Dec 1;21(23):3110-22.
Increased methylation of CpG islands and silencing of affected target genes is frequently found in human cancer; however, in vivo the question of causality has only been addressed by loss-of-function studies. To directly evaluate the role and mechanism of de novo methylation in tumor development, we overexpressed the de novo DNA methyltransferases Dnmt3a1 and Dnmt3b1 in Apc Min/+ mice. We found that Dnmt3b1 enhanced the number of colon tumors in Apc Min/+ mice approximately twofold and increased the average size of colonic microadenomas, whereas Dnmt3a1 had no effect. The overexpression of Dnmt3b1 caused loss of imprinting and increased expression of Igf2 as well as methylation and transcriptional silencing of the tumor suppressor genes Sfrp2, Sfrp4, and Sfrp5. Importantly, we found that Dnmt3b1 but not Dnmt3a1 efficiently methylates the same set of genes in tumors and in nontumor tissues, demonstrating that de novo methyltransferases can initiate methylation and silencing of specific genes in phenotypically normal cells. This suggests that DNA methylation patterns in cancer are the result of specific targeting of at least some tumor suppressor genes rather than of random, stochastic methylation followed by clonal selection due to a proliferative advantage caused by tumor suppressor gene silencing. [Abstract]

Leu JI, George DL
Hepatic IGFBP1 is a prosurvival factor that binds to BAK, protects the liver from apoptosis, and antagonizes the proapoptotic actions of p53 at mitochondria.
Genes Dev. 2007 Dec 1;21(23):3095-109.
Liver is generally refractory to apoptosis induced by the p53 tumor suppressor protein, but the molecular basis remains poorly understood. Here we show that p53 transcriptional activation leads to enhanced expression of hepatic IGFBP1 (insulin-like growth factor-binding protein-1). Exhibiting a previously unanticipated role, a portion of intracellular IGFBP1 protein localizes to mitochondria where it binds to the proapoptotic protein BAK and hinders BAK activation and apoptosis induction. Interestingly, in many cells and tissues p53 also has a direct apoptotic function at mitochondria that includes BAK binding and activation. When IGFBP1 is in a complex with BAK, formation of a proapoptotic p53/BAK complex and apoptosis induction are impaired, both in cultured cells and in liver. In contrast, livers of IGFBP1-deficient mice exhibit spontaneous apoptosis that is accompanied by p53 mitochondrial accumulation and evidence of BAK oligomerization. These data support the importance of BAK as a mediator of p53's mitochondrial function. The results also identify IGFBP1 as a negative regulator of the BAK-dependent pathway of apoptosis, whose expression integrates the transcriptional and mitochondrial functions of the p53 tumor suppressor protein. [Abstract]

Hogg JR, Collins K
Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control.
Genes Dev. 2007 Dec 1;21(23):3067-72.
Humans express four distinct non-protein-coding Y RNAs (ncRNAs). To investigate Y RNA functional diversification, we exploited an RNA-based affinity purification method to isolate ribonucleoproteins (RNPs) assembled on individual human Y RNAs. Silver staining and mass spectrometry revealed that the Ro and La proteins assemble with all Y RNAs, while additional proteins associate with specific Y RNAs. Unexpectedly, Y5 RNA uniquely copurified ribosomal protein L5 and its binding partner 5S RNA. These findings reveal a contribution of Y5 to 5S surveillance and suggest that interactions between Ro-Y5 and L5-5S RNPs establish 5S RNA as a target of quality control. [Abstract]

Juhász G, Erdi B, Sass M, Neufeld TP
Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila.
Genes Dev. 2007 Dec 1;21(23):3061-6.
Autophagy, a cellular process of cytoplasmic degradation and recycling, is induced in Drosophila larval tissues during metamorphosis, potentially contributing to their destruction or reorganization. Unexpectedly, we find that flies lacking the core autophagy regulator Atg7 are viable, despite severe defects in autophagy. Although metamorphic cell death is perturbed in Atg7 mutants, the larval-adult midgut transition proceeds normally, with extended pupal development compensating for reduced autophagy. Atg7-/- adults are short-lived, hypersensitive to nutrient and oxidative stress, and accumulate ubiquitin-positive aggregates in degenerating neurons. Thus, normal levels of autophagy are crucial for stress survival and continuous cellular renewal, but not metamorphosis. [Abstract]

Martinez-Agosto JA, Mikkola HK, Hartenstein V, Banerjee U
The hematopoietic stem cell and its niche: a comparative view.
Genes Dev. 2007 Dec 1;21(23):3044-60.
Stem cells have been identified as a source of virtually all highly differentiated cells that are replenished during the lifetime of an animal. The critical balance between stem and differentiated cell populations is crucial for the long-term maintenance of functional tissue types. Stem cells maintain this balance by choosing one of several alternate fates: self-renewal, commitment to differentiate, and senescence or cell death. These characteristics comprise the core criteria by which these cells are usually defined. The self-renewal property is important, as it allows for extended production of the corresponding differentiated cells throughout the life span of the animal. A microenvironment that is supportive of stem cells is commonly referred to as a stem cell niche. In this review, we first present some general concepts regarding stem cells and their niches, comparing stem cells of many different kinds from diverse organisms, and in the second part, we compare specific aspects of hematopoiesis and the niches that support hematopoiesis in Drosophila, zebrafish and mouse. [Abstract]

Schneider R, Grosschedl R
Dynamics and interplay of nuclear architecture, genome organization, and gene expression.
Genes Dev. 2007 Dec 1;21(23):3027-43.
The organization of the genome in the nucleus of a eukaryotic cell is fairly complex and dynamic. Various features of the nuclear architecture, including compartmentalization of molecular machines and the spatial arrangement of genomic sequences, help to carry out and regulate nuclear processes, such as DNA replication, DNA repair, gene transcription, RNA processing, and mRNA transport. Compartmentalized multiprotein complexes undergo extensive modifications or exchange of protein subunits, allowing for an exquisite dynamics of structural components and functional processes of the nucleus. The architecture of the interphase nucleus is linked to the spatial arrangement of genes and gene clusters, the structure of chromatin, and the accessibility of regulatory DNA elements. In this review, we discuss recent studies that have provided exciting insight into the interplay between nuclear architecture, genome organization, and gene expression. [Abstract]

Branzei D, Foiani M
RecQ helicases queuing with Srs2 to disrupt Rad51 filaments and suppress recombination.
Genes Dev. 2007 Dec 1;21(23):3019-26. [Abstract]

Adler AS, Sinha S, Kawahara TL, Zhang JY, Segal E, Chang HY
Motif module map reveals enforcement of aging by continual NF-{kappa}B activity.
Genes Dev. 2007 Nov 30;
Aging is characterized by specific alterations in gene expression, but their underlying mechanisms and functional consequences are not well understood. Here we develop a systematic approach to identify combinatorial cis-regulatory motifs that drive age-dependent gene expression across different tissues and organisms. Integrated analysis of 365 microarrays spanning nine tissue types predicted fourteen motifs as major regulators of age-dependent gene expression in human and mouse. The motif most strongly associated with aging was that of the transcription factor NF-kappaB. Inducible genetic blockade of NF-kappaB for 2 wk in the epidermis of chronologically aged mice reverted the tissue characteristics and global gene expression programs to those of young mice. Age-specific NF-kappaB blockade and orthogonal cell cycle interventions revealed that NF-kappaB controls cell cycle exit and gene expression signature of aging in parallel but not sequential pathways. These results identify a conserved network of regulatory pathways underlying mammalian aging and show that NF-kappaB is continually required to enforce many features of aging in a tissue-specific manner. [Abstract]

Wendel HG, Silva RL, Malina A, Mills JR, Zhu H, Ueda T, Watanabe-Fukunaga R, Fukunaga R, Teruya-Feldstein J, Pelletier J, Lowe SW
Dissecting eIF4E action in tumorigenesis.
Genes Dev. 2007 Nov 30;
Genetically engineered mouse models are powerful tools for studying cancer genes and validating targets for cancer therapy. We previously used a mouse lymphoma model to demonstrate that the translation initiation factor eIF4E is a potent oncogene in vivo. Using the same model, we now show that the oncogenic activity of eIF4E correlates with its ability to activate translation and become phosphorylated on Ser 209. Furthermore, constitutively activated MNK1, an eIF4E Ser 209 kinase, promotes tumorigenesis in a manner similar to eIF4E, and a dominant-negative MNK mutant inhibits the in vivo proliferation of tumor cells driven by mutations that deregulate translation. Phosphorylated eIF4E promotes tumorigenesis primarily by suppressing apoptosis and, accordingly, the anti-apoptotic protein Mcl-1 is one target of both phospho-eIF4E and MNK1 that contributes to tumor formation. Our results provide insight into how eIF4E contributes to tumorigenesis and pinpoint a level of translational control that may be suitable for therapeutic intervention. [Abstract]

Garcia SM, Casanueva MO, Silva MC, Amaral MD, Morimoto RI
Neuronal signaling modulates protein homeostasis in Caenorhabditis elegans post-synaptic muscle cells.
Genes Dev. 2007 Nov 15;21(22):3006-16.
Protein homeostasis maintains proper intracellular balance by promoting protein folding and clearance mechanisms while minimizing the stress caused by the accumulation of misfolded and damaged proteins. Chronic expression of aggregation-prone proteins is deleterious to the cell and has been linked to a wide range of conformational disorders. The molecular response to misfolded proteins is highly conserved and generally studied as a cell-autonomous process. Here, we provide evidence that neuronal signaling is an important modulator of protein homeostasis in post-synaptic muscle cells. In a forward genetic screen in Caenorhabditis elegans for enhancers of polyglutamine aggregation in muscle cells, we identified unc-30, a neuron-specific transcription factor that regulates the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We used additional sensors of protein conformational states to show that defective GABA signaling or increased acetylcholine (ACh) signaling causes a general imbalance in protein homeostasis in post-synaptic muscle cells. Moreover, exposure to GABA antagonists or ACh agonists has a similar effect, which reveals that toxins that act at the neuromuscular junction are potent modifiers of protein conformational disorders. These results demonstrate the importance of intercellular communication in intracellular homeostasis. [Abstract]

Sikder D, Kodadek T
The neurohormone orexin stimulates hypoxia-inducible factor-1 activity.
Genes Dev. 2007 Nov 15;21(22):2995-3005.
Orexin A and Orexin B (also known as hypocretins) are neuropeptides that bind two related G-coupled protein receptors (OXR1 and OXR2) and thus induce wakefulness, food consumption, and locomotion. Conversely, deletion of the orexin gene in mice produces a condition similar to canine and human narcolepsy. Despite the central importance of the orexin system in regulating wakefulness and feeding behavior, little is known about the downstream signaling mechanisms that achieve these effects. In this study, genomics techniques are used to probe this question and reveal that orexin activates the hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor whose pathogenic role in stimulating angiogenesis in hypoxic tumors has been the focus of intense investigation. Orexin-stimulated HIF-1 activity is due to both increased HIF-1alpha gene transcription and a down-regulation of von Hippel-Lindau (VHL), the E3 ubiquitin ligase that mediates the turnover of HIF-1 via the ubiquitin-proteasome pathway. Orexin-mediated activation of HIF-1 results in increased glucose uptake and higher glycolytic activity, as expected from studies of hypoxic cells. However, orexin receptor-expressing cells somehow override the HIF-1-mediated preference for funneling pyruvate into anaerobic glycolysis and instead favor ATP production through the tricarboxylic acid cycle and oxidative phosphorylation. These findings implicate HIF-1 as an important transcription factor in the hormone-mediated regulation of hunger and wakefulness. [Abstract]

Samuelson AV, Carr CE, Ruvkun G
Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants.
Genes Dev. 2007 Nov 15;21(22):2976-94.
Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. We conducted a genome-wide RNAi screen to identify genes necessary for the extended life span of daf-2 mutants and identified approximately 200 gene inactivations that shorten daf-2 life span. Some of these gene inactivations dramatically shorten daf-2 mutant life span but less dramatically shorten daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase sod-3 in a daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging. [Abstract]

Xu YX, Manley JL
Pin1 modulates RNA polymerase II activity during the transcription cycle.
Genes Dev. 2007 Nov 15;21(22):2950-62.
The C-terminal domain of the RNA polymerase (RNAP) II largest subunit (CTD) plays a critical role in coordinating multiple events in pre-mRNA transcription and processing. Previously we reported that the peptidyl prolyl isomerase Pin1 modulates RNAP II function during the cell cycle. Here we provide evidence that Pin1 affects multiple aspects of RNAP II function via its regulation of CTD phosphorylation. Using chromatin immunoprecipitation (ChIP) assays with CTD phospho-specific antibodies, we confirm that RNAP II displays a dynamic association with specific genes during the cell cycle, preferentially associating with transcribed genes in S phase, while disassociating in M phase in a matter that correlates with changes in CTD phosphorylation. Using inducible Pin1 cell lines, we show that Pin1 overexpression is sufficient to release RNAP II from chromatin, which then accumulates in a hyperphosphorylated form in nuclear speckle-associated structures. In vitro transcription assays show that Pin1 inhibits transcription in nuclear extract, while an inactive Pin1 mutant in fact stimulates it. Several assays indicate that the inhibition largely reflects Pin1 activity during transcription initiation and not elongation, suggesting that Pin1 modulates CTD phosphorylation, and RNAP II activity, during an early stage of the transcription cycle. [Abstract]

Delmas V, Beermann F, Martinozzi S, Carreira S, Ackermann J, Kumasaka M, Denat L, Goodall J, Luciani F, Viros A, Demirkan N, Bastian BC, Goding CR, Larue L
Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development.
Genes Dev. 2007 Nov 15;21(22):2923-35.
Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16(Ink4a) gene. Melanomas also frequently exhibit constitutive activation of the Wnt/beta-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. We show here that, contrary to expectations, stabilized beta-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16(Ink4a) promoter. Significantly, in a novel mouse model for melanoma, stabilized beta-catenin bypasses the requirement for p16(Ink4a) mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease. [Abstract]

Aggarwal P, Lessie MD, Lin DI, Pontano L, Gladden AB, Nuskey B, Goradia A, Wasik MA, Klein-Szanto AJ, Rustgi AK, Bassing CH, Diehl JA
Nuclear accumulation of cyclin D1 during S phase inhibits Cul4-dependent Cdt1 proteolysis and triggers p53-dependent DNA rereplication.
Genes Dev. 2007 Nov 15;21(22):2908-22.
Deregulation of cyclin D1 occurs in numerous human cancers through mutations, alternative splicing, and gene amplification. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms whereby they contribute to neoplasia are poorly understood. We now provide evidence derived from both mouse models and human cancer-derived cells revealing that nuclear accumulation of catalytically active mutant cyclin D1/CDK4 complexes triggers DNA rereplication, resulting from Cdt1 stabilization, which in turn triggers the DNA damage checkpoint and p53-dependent apoptosis. Loss of p53 through mutations or targeted deletion results in increased genomic instability and neoplastic growth. Collectively, the data presented reveal mechanistic insights into how uncoupling of critical cell cycle regulatory events will perturb DNA replication fidelity, thereby contributing to neoplastic transformation. [Abstract]

Chen S, de Vries MA, Bell SP
Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2 7 loading.
Genes Dev. 2007 Nov 15;21(22):2897-907.
The origin recognition complex (ORC) nucleates DNA replication initiation in eukaryotic cells. This six-protein complex binds replication origin DNA, recruits other initiation factors, and facilitates loading of the DNA helicase. Studying the function of individual ORC subunits during pre-RC formation has been hampered by the requirement of most subunits for DNA binding. In this study, we investigate the function of the Saccharomyces cerevisiae Orc6, the only ORC subunit not required for DNA binding. In vivo, depletion of Orc6 inhibits prereplicative complex (pre-RC) assembly and maintenance. In vitro, ORC lacking Orc6 fails to interact with Cdt1 and to load the Mcm2-7 helicase onto origin DNA. We demonstrate that two regions of Orc6 bind Cdt1 directly, and that the extreme C terminus of Orc6 (Orc6-CTD) interacts tightly with the remaining five ORC subunits. Replacing Orc6 with a fusion protein linking Cdt1 to the Orc6-CTD results in an ORC complex that loads Mcm2-7 onto DNA. Interestingly, this complex can only perform a single round of Mcm2-7 loading, suggesting that a dynamic association of Cdt1 with ORC is required for multiple rounds of Mcm2-7 loading. [Abstract]

Bocobza S, Adato A, Mandel T, Shapira M, Nudler E, Aharoni A
Riboswitch-dependent gene regulation and its evolution in the plant kingdom.
Genes Dev. 2007 Nov 15;21(22):2874-9.
Riboswitches are natural RNA sensors that affect gene control via their capacity to bind small molecules. Their prevalence in higher eukaryotes is unclear. We discovered a post-transcriptional mechanism in plants that uses a riboswitch to control a metabolic feedback loop through differential processing of the precursor RNA 3' terminus. When cellular thiamin pyrophosphate (TPP) levels rise, metabolite sensing by the riboswitch located in TPP biosynthesis genes directs formation of an unstable splicing product, and consequently TPP levels drop. When transformed in plants, engineered TPP riboswitches can act autonomously to modulate gene expression. In an evolutionary perspective, a TPP riboswitch is also present in ancient plant taxa, suggesting that this mechanism is active since vascular plants emerged 400 million years ago. [Abstract]

Mizushima N
Autophagy: process and function.
Genes Dev. 2007 Nov 15;21(22):2861-73.
Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. Despite its simplicity, recent progress has demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles, which are sometimes complex. Autophagy consists of several sequential steps--sequestration, transport to lysosomes, degradation, and utilization of degradation products--and each step may exert different function. In this review, the process of autophagy is summarized, and the role of autophagy is discussed in a process-based manner. [Abstract]

Reina JH, Hernandez N
On a roll for new TRF targets.
Genes Dev. 2007 Nov 15;21(22):2855-60. [Abstract]

Kerjan G, Gleeson JG
A missed exit: Reelin sets in motion Dab1 polyubiquitination to put the break on neuronal migration.
Genes Dev. 2007 Nov 15;21(22):2850-4. [Abstract]

Argiropoulos B, Yung E, Humphries RK
Unraveling the crucial roles of Meis1 in leukemogenesis and normal hematopoiesis.
Genes Dev. 2007 Nov 15;21(22):2845-9. [Abstract]

Katiyar-Agarwal S, Gao S, Vivian-Smith A, Jin H
A novel class of bacteria-induced small RNAs in Arabidopsis.
Genes Dev. 2007 Dec 1;21(23):3123-34.
Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential regulatory molecules of many cellular processes. Arabidopsis has at least three classes of endogenous siRNAs--chromatin-associated siRNAs, trans-acting siRNAs (tasiRNAs), and natural antisense transcript (NAT)-associated siRNAs (nat-siRNAs)--all 20-25 nucleotides (nt) in length. Here, we identified a novel class of small RNAs, long siRNAs (lsiRNAs), which are 30-40 nt and share many common features with known siRNAs. The lsiRNAs identified so far are induced by pathogen infection or under specific growth conditions. One of the lsiRNAs, AtlsiRNA-1, is generated from SRRLK/AtRAP NAT pair and specifically induced by the bacterium Pseudomonas syringae carrying effector avrRpt2. Recently, 25- to 31-nt PIWI-interacting RNAs (piRNAs) and repeat-associated siRNAs (rasiRNAs) were identified in animal germline cells. In contrast to the biogenesis of piRNAs/rasiRNAs, which is dicer independent and requires PIWI subfamily proteins, generation of AtlsiRNA-1 requires DCL1, DCL4, and the ARGONAUTE subfamily protein AGO7. It also depends on HYL1, HEN1, HST1, RDR6, and Pol IV. Induction of AtlsiRNA-1 silences AtRAP, which encodes a RAP-domain protein involved in disease resistance. Our further analysis implies that AtlsiRNA-1 may destabilize target mRNA through decapping and XRN4-mediated 5'-to-3' degradation. [Abstract]

Bugreev DV, Yu X, Egelman EH, Mazin AV
Novel pro- and anti-recombination activities of the Bloom's syndrome helicase.
Genes Dev. 2007 Dec 1;21(23):3085-94.
Bloom's syndrome (BS) is an autosomal recessive disorder characterized by a strong cancer predisposition. The defining feature of BS is extreme genome instability. The gene mutated in Bloom's syndrome, BLM, encodes a DNA helicase (BLM) of the RecQ family. BLM plays a role in homologous recombination; however, its exact function remains controversial. Mutations in the BLM cause hyperrecombination between sister chromatids and homologous chromosomes, indicating an anti-recombination role. Conversely, other data show that BLM is required for recombination. It was previously shown that in vitro BLM helicase promotes disruption of recombination intermediates, regression of stalled replication forks, and dissolution of double Holliday junctions. Here, we demonstrate two novel activities of BLM: disruption of the Rad51-ssDNA (single-stranded DNA) filament, an active species that promotes homologous recombination, and stimulation of DNA repair synthesis. Using in vitro reconstitution reactions, we analyzed how different biochemical activities of BLM contribute to its functions in homologous recombination. [Abstract]

Hu Y, Raynard S, Sehorn MG, Lu X, Bussen W, Zheng L, Stark JM, Barnes EL, Chi P, Janscak P, Jasin M, Vogel H, Sung P, Luo G
RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments.
Genes Dev. 2007 Dec 1;21(23):3073-84.
Members of the RecQ helicase family play critical roles in genome maintenance. There are five RecQ homologs in mammals, and defects in three of these (BLM, WRN, and RECQL4) give rise to cancer predisposition syndromes in humans. RECQL and RECQL5 have not been associated with a human disease. Here we show that deletion of Recql5 in mice results in cancer susceptibility. Recql5-deficient cells exhibit elevated frequencies of spontaneous DNA double-strand breaks and homologous recombination (HR) as scored using a reporter that harbors a direct repeat, and are prone to gross chromosomal rearrangements in response to replication stress. To understand how RECQL5 regulates HR, we use purified proteins to demonstrate that human RECQL5 binds the Rad51 recombinase and inhibits Rad51-mediated D-loop formation. By biochemical means and electron microscopy, we show that RECQL5 displaces Rad51 from single-stranded DNA (ssDNA) in a reaction that requires ATP hydrolysis and RPA. Together, our results identify RECQL5 as an important tumor suppressor that may act by preventing inappropriate HR events via Rad51 presynaptic filament disruption. [Abstract]

Georlette D, Ahn S, MacAlpine DM, Cheung E, Lewis PW, Beall EL, Bell SP, Speed T, Manak JR, Botchan MR
Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells.
Genes Dev. 2007 Nov 15;21(22):2880-96.
Myb-MuvB (MMB)/dREAM is a nine-subunit complex first described in Drosophila as a repressor of transcription, dependent on E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent on DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNA interference and microarray expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupied 3538 chromosomal sites and was promoter-proximal to 32% of Drosophila genes. MMB contains multiple DNA-binding factors, and the data highlighted the combinatorial way by which the complex was targeted and utilized for regulation. Interestingly, only a subset of chromatin-bound complexes repressed genes normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression, whereas at other nonoverlapping sites, Myb was critical for repression. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants. [Abstract]

Recent Articles in Developmental Cell

Mitiku N, Baker JC
Genomic analysis of gastrulation and organogenesis in the mouse.
Dev Cell. 2007 Dec;13(6):897-907.
We developed a comprehensive dataset that samples the mouse transcriptome every 6 hr, from gastrulation through organogenesis. We observe an abrupt increase in overall transcript diversity at the onset of organogenesis (e8.0); the genes that comprise these changes are preferentially clustered along chromosome 7 and contain a significant enrichment of Gli binding sites. Furthermore, we identify seven dominant patterns of gene expression during gastrulation and organogenesis. Genes clustered according to these seven patterns constitute distinct functional classes, including a cluster enriched for gastrulation and pluripotency genes, two clusters differentially regulating localization and ion metabolism, and three clusters involved in discrete aspects of organogenesis. The last cluster is defined by a dramatic transient decrease in the expression of genes that regulate RNA processing and the cell cycle. Drosophila homologs of these genes are also coordinately downregulated following gastrulation, suggesting that the combined function of these genes has been conserved during metazoan evolution. [Abstract]

Yamanaka Y, Tamplin OJ, Beckers A, Gossler A, Rossant J
Live imaging and genetic analysis of mouse notochord formation reveals regional morphogenetic mechanisms.
Dev Cell. 2007 Dec;13(6):884-96.
The node and notochord have been extensively studied as signaling centers in the vertebrate embryo. The morphogenesis of these tissues, particularly in mouse, is not well understood. Using time-lapse live imaging and cell lineage tracking, we show the notochord has distinct morphogenetic origins along the anterior-posterior axis. The anterior head process notochord arises independently of the node by condensation of dispersed cells. The trunk notochord is derived from the node and forms by convergent extension. The tail notochord forms by node-derived progenitors that actively migrate toward the posterior. We also reveal distinct genetic regulation within these different regions. We show that Foxa2 compensates for and genetically interacts with Noto in the trunk notochord, and that Noto has an evolutionarily conserved role in regulating axial versus paraxial cell fate. Therefore, we propose three distinct regions within the mouse notochord, each with unique morphogenetic origins. [Abstract]

de Frutos CA, Vega S, Manzanares M, Flores JM, Huertas H, Martínez-Frías ML, Nieto MA
Snail1 Is a Transcriptional Effector of FGFR3 Signaling during Chondrogenesis and Achondroplasias.
Dev Cell. 2007 Dec;13(6):872-83.
Achondroplasias are the most common genetic forms of dwarfism in humans. They are associated with activating mutations in FGFR3, which signal through the Stat and MAPK pathways in a ligand-independent manner to impair chondrocyte proliferation and differentiation. Snail1 has been implicated in chondrocyte differentiation as it represses Collagen II and aggrecan transcription in vitro. Here we demonstrate that Snail1 overexpression in the developing bone leads to achondroplasia in mice. Snail1 acts downstream of FGFR3 signaling in chondrocytes, regulating both Stat and MAPK pathways. Moreover, FGFR3 requires Snail1 during bone development and disease as the inhibition of Snail1 abolishes its signaling even through achondroplastic- and thanatophoric-activating FGFR3 forms. Significantly, Snail1 is aberrantly upregulated in thanatophoric versus normal cartilages from stillborns. Thus, Snail activity may likely be considered a target for achondroplasia therapies. [Abstract]

McBrayer Z, Ono H, Shimell M, Parvy JP, Beckstead RB, Warren JT, Thummel CS, Dauphin-Villemant C, Gilbert LI, O'Connor MB
Prothoracicotropic hormone regulates developmental timing and body size in Drosophila.
Dev Cell. 2007 Dec;13(6):857-71.
In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval. [Abstract]

Anastasiou E, Kenz S, Gerstung M, Maclean D, Timmer J, Fleck C, Lenhard M
Control of Plant Organ Size by KLUH/CYP78A5-Dependent Intercellular Signaling.
Dev Cell. 2007 Dec;13(6):843-56.
Plant organs grow to characteristic sizes that are genetically controlled. In animals, signaling by mobile growth factors is thought to be an effective mechanism for measuring primordium size, yet how plants gauge organ size is unclear. Here, we identify the Arabidopsis cytochrome P450 KLUH (KLU)/CYP78A5 as a stimulator of plant organ growth. While klu loss-of-function mutants form smaller organs because of a premature arrest of cell proliferation, KLU overexpression leads to larger organs with more cells. KLU promotes organ growth in a non-cell-autonomous manner, yet it does not appear to modulate the levels of known phytohormones. We therefore propose that KLU is involved in generating a mobile growth signal distinct from the classical phytohormones. The expression dynamics of KLU suggest a model of how the arrest of cell proliferation is coupled to the attainment of a certain primordium size, implying a common principle of size measurement in plants and animals. [Abstract]

Bai J, Hartwig JH, Perrimon N
SALS, a WH2-Domain-Containing Protein, Promotes Sarcomeric Actin Filament Elongation from Pointed Ends during Drosophila Muscle Growth.
Dev Cell. 2007 Dec;13(6):828-42.
Organization of actin filaments into a well-organized sarcomere structure is critical for muscle development and function. However, it is not completely understood how sarcomeric actin/thin filaments attain their stereotyped lengths. In an RNAi screen in Drosophila primary muscle cells, we identified a gene, sarcomere length short (sals), which encodes an actin-binding, WH2 domain-containing protein, required for proper sarcomere size. When sals is knocked down by RNAi, primary muscles display thin myofibrils with shortened sarcomeres and increased sarcomere number. Both loss- and gain-of-function analyses indicate that SALS may influence sarcomere lengths by promoting thin-filament lengthening from pointed ends. Furthermore, the complex localization of SALS and other sarcomeric proteins in myofibrils reveals that the full length of thin filaments is achieved in a two-step process, and that SALS is required for the second elongation phase, most likely because it antagonizes the pointed-end capping protein Tropomodulin. [Abstract]

Bratman SV, Chang F
Stabilization of Overlapping Microtubules by Fission Yeast CLASP.
Dev Cell. 2007 Dec;13(6):812-27.
Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components. [Abstract]

Toyoshima F, Matsumura S, Morimoto H, Mitsushima M, Nishida E
PtdIns(3,4,5)P3 Regulates Spindle Orientation in Adherent Cells.
Dev Cell. 2007 Dec;13(6):796-811.
Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum. [Abstract]

Skwarek LC, Garroni MK, Commisso C, Boulianne GL
Neuralized contains a phosphoinositide-binding motif required downstream of ubiquitination for delta endocytosis and notch signaling.
Dev Cell. 2007 Dec;13(6):783-95.
The Notch signaling pathway, which plays a critical role in cell-fate decisions throughout development, is regulated by endocytosis of both the ligand and receptor. Endocytosis of the Drosophila ligands, Delta and Serrate, is required in the signaling cell for signal initiation and requires one of two ubiquitin ligases, Neuralized or Mind bomb. Through in vitro binding assays we have identified an interaction between Neuralized and phosphoinositides, modified membrane lipids that mediate membrane trafficking and signaling. We show that interactions between phosphoinositides and Neuralized contribute to the membrane localization of Neuralized in the absence of Delta, and that the phosphoinositide-binding motif is required for Neuralized to endocytose Delta downstream of Delta ubiquitination. Lastly, we provide evidence that this interaction may also be important for vertebrate Neuralized function. These results demonstrate that, through interactions with Neuralized, phosphoinositides may regulate Delta endocytosis and, by extension, Notch signal transduction. [Abstract]

Langton PF, Colombani J, Aerne BL, Tapon N
Drosophila ASPP Regulates C-Terminal Src Kinase Activity.
Dev Cell. 2007 Dec;13(6):773-82.
Src-family kinases (SFKs) control a variety of biological processes, from cell proliferation and differentiation to cytoskeletal rearrangements. Abnormal activation of SFKs has been implicated in a wide variety of cancers and is associated with metastatic behavior (Yeatman, 2004). SFKs are maintained in an inactive state by inhibitory phosphorylation of their C-terminal region by C-terminal Src kinase (Csk). We have identified Drosophila Ankyrin-repeat, SH3-domain, and Proline-rich-region containing Protein (dASPP) as a regulator of Drosophila Csk (dCsk) activity. dASPP is the homolog of the mammalian ASPP proteins, which are known to bind to and stimulate the proapoptotic function of p53. We show that dASPP is a positive regulator of dCsk. First, dASPP loss-of-function strongly enhances the specific phenotypes of dCsk mutants in wing epithelial cells. Second, dASPP interacts physically with dCsk to potentiate the inhibitory phosphorylation of Drosophila Src (dSrc). Our results suggest a role for dASPP in maintaining epithelial integrity through dCsk regulation. [Abstract]

Hogan B
A shared vision.
Dev Cell. 2007 Dec;13(6):769-71.
One of today's most powerful technologies in biomedical research-the creation of mutant mice by gene targeting in embryonic stem (ES) cells-was finally celebrated in this year's Nobel Prize in Medicine. The history of how ES cells were first discovered and genetically manipulated highlights the importance of collaboration among scientists from different backgrounds with a shared vision. [Abstract]

Hersh B
Hox en provence.
Dev Cell. 2007 Dec;13(6):763-8.
In the nearly 25 years since the cloning of the first Hox genes, the broad brushstrokes of their functions in axial patterning have become familiar motifs in developmental biology. The October 2007 Fondation des Treilles workshop on "Hox Genes in Development and Evolution" in Les Treilles, France, highlighted some of the finer details regarding the function of these genes in shaping animal morphology. [Abstract]

Fossat N, Pfister S, Tam PP
A transcriptome landscape of mouse embryogenesis.
Dev Cell. 2007 Dec;13(6):761-2.
Coordinated regulation of genetic activity underpins formation of the body plan and morphogenesis of embryonic structures. In this issue of Developmental Cell, Mitiku and Baker describe a chronological series of transcriptomes of postimplantation mouse embryos at gastrulation and early organogenesis, providing a valuable resource for studying the dynamics of both genome-wide and gene-specific transcriptional activities that accompany mouse embryogenesis. [Abstract]

Zhou Z
New phosphatidylserine receptors: clearance of apoptotic cells and more.
Dev Cell. 2007 Dec;13(6):759-60.
Phagocytes recognize apoptotic cells using cell surface receptors, and subsequently engulf these cells. In a recent issue of Nature, two papers reported the identification of novel phagocytic receptors that directly interact with apoptotic cell surface phosphatidylserine (PS). The studies provide new insights into the apoptotic cell clearance process and implicate PS receptors in additional signaling events. [Abstract]

Vanderzalm P, Garriga G
Losing Their Minds: Mena/VASP/EVL Triple Knockout Mice.
Dev Cell. 2007 Dec;13(6):757-8.
A recent Neuron paper by Kwiatkowski et al. that analyzes the phenotypes of mouse embryos lacking all members of the Ena/VASP family reveals new roles for these molecules in cortical development. [Abstract]

Verheyen EM
Opposing Effects of Wnt and MAPK on BMP/Smad Signal Duration.
Dev Cell. 2007 Dec;13(6):755-6.
Signaling downstream of BMP receptors relies on activated nuclear Smad proteins. Recent studies, one of which is published in the November 30 issue of Cell, shed light on a mechanism that balances inputs from both activated MAPK and Wnt pathways to regulate the proteasomal degradation of Smad1 and thus modulate the extent of BMP signaling. [Abstract]

Gachet Y, Tournier S
Lost Your Orientation? Find Your Way with PtdIns(3,4,5)P3!
Dev Cell. 2007 Dec;13(6):753-4.
Faithful chromosome segregation requires correct positioning of the spindle during mitosis. In this issue of Developmental Cell, Toyoshima et al. describe a new mechanism for spindle orientation involving phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3]. They found that in metaphase cells, dynactin was associated with the cortex through the actin cytoskeleton, and accumulated in the midsections in a PtdIns(3,4,5)P3-dependent manner. Thus, PtdIns(3,4,5)P3 regulates spindle orientation through dynein-dynactin motor complexes. [Abstract]

Saito K, Fujimura-Kamada K, Hanamatsu H, Kato U, Umeda M, Kozminski KG, Tanaka K
Transbilayer Phospholipid Flipping Regulates Cdc42p Signaling during Polarized Cell Growth via Rga GTPase-Activating Proteins.
Dev Cell. 2007 Nov;13(5):743-51.
An important problem in polarized morphogenesis is how polarized transport of membrane vesicles is spatiotemporally regulated. Here, we report that a local change in the transbilayer phospholipid distribution of the plasma membrane regulates the axis of polarized growth. Type 4 P-type ATPases Lem3p-Dnf1p and -Dnf2p are putative heteromeric phospholipid flippases in budding yeast that are localized to polarized sites on the plasma membrane. The lem3Delta mutant exhibits prolonged apical growth due to a defect in the switch to isotropic bud growth. In lem3Delta cells, the small GTPase Cdc42p remains polarized at the bud tip where phosphatidylethanolamine remains exposed on the outer leaflet. Intriguingly, phosphatidylethanolamine and phosphatidylserine stimulate GTPase-activating protein (GAP) activity of Rga1p and Rga2p toward Cdc42p, whereas PI(4,5)P(2) inhibits it. We propose that a redistribution of phospholipids to the inner leaflet of the plasma membrane triggers the dispersal of Cdc42p from the apical growth site, through activation of GAPs. [Abstract]

Corrigall D, Walther RF, Rodriguez L, Fichelson P, Pichaud F
Hedgehog signaling is a principal inducer of Myosin-II-driven cell ingression in Drosophila epithelia.
Dev Cell. 2007 Nov;13(5):730-42.
Cell constriction promotes epithelial sheet invagination during embryogenesis across phyla. However, how this cell response is linked to global patterning information during organogenesis remains unclear. To address this issue, we have used the Drosophila eye and studied the formation of the morphogenetic furrow (MF), which is characterized by cells undergoing a synchronous apical constriction and apicobasal contraction. We show that this cell response relies on microtubules and F-actin enrichment within the apical domain of the constricting cell as well as on the activation of nonmuscle myosin. In the MF, Hedgehog (Hh) signaling is required to promote cell constriction downstream of cubitus interruptus (ci), and, in this context, Ci155 functions redundantly with mad, the main effector of dpp/BMP signaling. Furthermore, ectopically activating Hh signaling in fly epithelia reveals a direct relationship between the duration of exposure to this signaling pathway, the accumulation of activated Myosin II, and the degree of tissue invagination. [Abstract]

Escudero LM, Bischoff M, Freeman M
Myosin II regulates complex cellular arrangement and epithelial architecture in Drosophila.
Dev Cell. 2007 Nov;13(5):717-29.
Remodeling epithelia is a primary driver of morphogenesis. Here, we report a central role of myosin II in regulating several aspects of complex epithelial architecture in the Drosophila eye imaginal disc. The epithelial indentation of the morphogenetic furrow is established from a pattern of myosin II activation defined by the developmental signals Hedgehog and Decapentaplegic. More generally, patterned myosin activation can control diverse three-dimensional epithelial sculpting. We have developed a technique to image eye disc development in real time, and we show that myosin II also regulates higher-order organization of cells in the plane of the epithelium. This includes the clustering of cells into ommatidial units and their subsequent coordinated rotation. This later clustering function of myosin II depends on EGF receptor signaling. Our work implies that regulation of the actomyosin cytoskeleton can control morphogenesis by regulating both individual cell shapes and their complex two-dimensional arrangement within epithelia. [Abstract]

Wright A, Reiley WW, Chang M, Jin W, Lee AJ, Zhang M, Sun SC
Regulation of early wave of germ cell apoptosis and spermatogenesis by deubiquitinating enzyme CYLD.
Dev Cell. 2007 Nov;13(5):705-16.
Spermatogenesis involves an early wave of germ cell apoptosis, which is required for maintaining the balance between germ cells and supporting Sertoli cells. However, the signaling mechanism regulating this apoptotic event is poorly defined. Here we show that genetic deficiency of Cyld, a recently identified deubiquitinating enzyme, attenuates the early wave of germ cell apoptosis and causes impaired spermatogenesis in mice. Interestingly, the loss of CYLD in testicular cells leads to activation of the transcription factor NF-kappaB and aberrant expression of antiapoptotic genes. We further show that CYLD negatively regulates a ubiquitin-dependent NF-kappaB activator, RIP1. CYLD binds to RIP1 and inhibits its ubiquitination and signaling function. These findings establish CYLD as a pivotal deubiquitinating enzyme (DUB) that regulates germ cell apoptosis and spermatogenesis and suggest an essential role for CYLD in controlling the RIP1/NF-kappaB signaling axis in testis. [Abstract]

Chicoine J, Benoit P, Gamberi C, Paliouras M, Simonelig M, Lasko P
Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression.
Dev Cell. 2007 Nov;13(5):691-704.
Bicaudal-C (Bic-C) encodes an RNA-binding protein required maternally for patterning the Drosophila embryo. We identified a set of mRNAs that associate with Bic-C in ovarian ribonucleoprotein complexes. These mRNAs are enriched for mRNAs that function in oogenesis and in cytoskeletal regulation, and include Bic-C RNA itself. Bic-C binds specific segments of the Bic-C 5' untranslated region and negatively regulates its own expression by binding directly to NOT3/5, a component of the CCR4 core deadenylase complex, thereby promoting deadenylation. Bic-C overexpression induces premature cytoplasmic-streaming, a posterior-group phenotype, defects in Oskar and Kinesin heavy chain:betaGal localization as well as dorsal-appendage defects. These phenotypes are largely reciprocal to those of Bic-C mutants, and they affect cellular processes that Bic-C-associated mRNAs are known, or predicted, to regulate. We conclude that Bic-C regulates expression of specific germline mRNAs by controlling their poly(A)-tail length. [Abstract]

Joo E, Surka MC, Trimble WS
Mammalian SEPT2 is required for scaffolding nonmuscle myosin II and its kinases.
Dev Cell. 2007 Nov;13(5):677-90.
Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis. [Abstract]

Kiyomitsu T, Obuse C, Yanagida M
Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1.
Dev Cell. 2007 Nov;13(5):663-76.
The spindle checkpoint controls mitotic progression. Checkpoint proteins are temporally recruited to kinetochores, but their docking site is unknown. We show that a human kinetochore oncoprotein, AF15q14/blinkin, a member of the Spc105/Spc7/KNL-1 family, directly links spindle checkpoint proteins BubR1 and Bub1 to kinetochores and is required for spindle checkpoint and chromosome alignment. Blinkin RNAi causes accelerated mitosis due to a checkpoint failure and chromosome misalignment resulting from the lack of kinetochore and microtubule attachment. Blinkin RNAi phenotypes resemble the double RNAi phenotypes of Bub1 and BubR1 in living cells. While the carboxy domain associates with the c20orf172/hMis13 and DC8/hMis14 subunits of the hMis12 complex in the inner kinetochore, association of the amino and middle domain of blinkin with the TPR domains in the amino termini of BubR1 and Bub1 is essential for BubR1 and Bub1 to execute their distinct mitotic functions. Blinkin may be the center of the network for generating kinetochore-based checkpoint signaling. [Abstract]

Delorme V, Machacek M, DerMardirossian C, Anderson KL, Wittmann T, Hanein D, Waterman-Storer C, Danuser G, Bokoch GM
Cofilin activity downstream of pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks.
Dev Cell. 2007 Nov;13(5):646-62.
Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals. [Abstract]

Bi X, Mancias JD, Goldberg J
Insights into COPII coat nucleation from the structure of Sec23.Sar1 complexed with the active fragment of Sec31.
Dev Cell. 2007 Nov;13(5):635-45.
The COPII vesicular coat forms on the endoplasmic reticulum from Sar1-GTP, Sec23/24 and Sec13/31 protein subunits. Here, we define the interaction between Sec23/24.Sar1 and Sec13/31, involving a 40 residue Sec31 fragment. In the crystal structure of the ternary complex, Sec31 binds as an extended polypeptide across a composite surface of the Sec23 and Sar1-GTP molecules, explaining the stepwise character of Sec23/24.Sar1 and Sec13/31 recruitment to the membrane. The Sec31 fragment stimulates GAP activity of Sec23/24, and a convergence of Sec31 and Sec23 residues at the Sar1 GTPase active site explains how GTP hydrolysis is triggered leading to COPII coat disassembly. The Sec31 active fragment is accommodated in a binding groove supported in part by Sec23 residue Phe380. Substitution of the corresponding residue F382L in human Sec23A causes cranio-lenticulo-sutural dysplasia, and we suggest that this mutation disrupts the nucleation of COPII coat proteins at endoplasmic reticulum exit sites. [Abstract]

Fromme JC, Ravazzola M, Hamamoto S, Al-Balwi M, Eyaid W, Boyadjiev SA, Cosson P, Schekman R, Orci L
The genetic basis of a craniofacial disease provides insight into COPII coat assembly.
Dev Cell. 2007 Nov;13(5):623-34.
Proteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in SEC23A, a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission. [Abstract]

Goldstein B, Macara IG
The PAR proteins: fundamental players in animal cell polarization.
Dev Cell. 2007 Nov;13(5):609-22.
The par genes were discovered in genetic screens for regulators of cytoplasmic partitioning in the early embryo of C. elegans, and encode six different proteins required for asymmetric cell division by the worm zygote. Some of the PAR proteins are localized asymmetrically and form physical complexes with one another. Strikingly, the PAR proteins have been found to regulate cell polarization in many different contexts in diverse animals, suggesting they form part of an ancient and fundamental mechanism for cell polarization. Although the picture of how the PAR proteins function remains incomplete, cell biology and biochemistry are beginning to explain how PAR proteins polarize cells. [Abstract]

Menon AK
A flip-flop switch in polarity signaling.
Dev Cell. 2007 Nov;13(5):607-8.
The Rho GTPase Cdc42 is essential for polarized growth of budding yeast. Temporal control of Cdc42 depends partly on the activity of its GTPase-activating proteins (GAPs). In this issue of Developmental Cell, Saito et al. report that Cdc42 GAP activity is regulated by the phospholipid composition of the bud-tip membrane, under control of the phospholipid flippases Lem3-Dnf1 and Lem3-Dnf2. [Abstract]

Chan SP, Slack FJ
And now introducing mammalian mirtrons.
Dev Cell. 2007 Nov;13(5):605-7.
Mirtrons are short hairpin introns recently found in flies and nematodes that provide an alternative source for animal microRNA biogenesis and use the splicing machinery to bypass Drosha cleavage in initial maturation. The presence of mirtrons outside of invertebrates was not previously known. In the October 26 issue of Molecular Cell, Berezikov et al. expose a number of short mammalian introns as mirtrons. [Abstract]

Recent Articles in Current Topics in Developmental Biology

Bellomo N, Forni G
17 complex multicellular systems and immune competition: new paradigms looking for a mathematical theory.
Curr Top Dev Biol. 2007;81485-502.
This chapter deals with the modeling and simulation of large systems of interacting entities whose microscopic state includes not only geometrical and mechanical variables (typically position and velocity), but also biological functions or specific activities. The main issue looks at the development of a biological mathematical theory for multicellular systems. The first part is devoted to the derivation of mathematical structures to be properly used to model a variety of biological phenomena with special focus on immune competition. Then, some specific applications are proposed referring to the competition between neoplastic and immune cells. Finally, the last part is devoted to research perspectives towards the objective of developing a mathematical-biological theory. A critique is presented of what has already been achieved towards the above target and what is still missing with special focus on multiscale systems. [Abstract]

Flenner E, Marga F, Neagu A, Kosztin I, Forgacs G
16 relating biophysical properties across scales.
Curr Top Dev Biol. 2007;81461-83.
A distinguishing feature of a multicellular living system is that it operates at various scales, from the intracellular to organismal. Genes and molecules set up the conditions for the physical processes to act, in particular to shape the embryo. As development continues the changes brought about by the physical processes lead to changes in gene expression. It is this coordinated interplay between genetic and generic (i.e., physical and chemical) processes that constitutes the modern understanding of early morphogenesis. It is natural to assume that in this multiscale process the smaller defines the larger. In case of biophysical properties, in particular, those at the subcellular level are expected to give rise to those at the tissue level and beyond. Indeed, the physical properties of tissues vary greatly from the liquid to solid. Very little is known at present on how tissue level properties are related to cell and subcellular properties. Modern measurement techniques provide quantitative results at both the intracellular and tissue level, but not on the connection between these. In the present work we outline a framework to address this connection. We specifically concentrate on the morphogenetic process of tissue fusion, by following the coalescence of two contiguous multicellular aggregates. The time evolution of this process can accurately be described by the theory of viscous liquids. We also study fusion by Monte Carlo simulations and a novel Cellular Particle Dynamics (CPD) model, which is similar to the earlier introduced Subcellular Element Model (SEM; Newman, 2005). Using the combination of experiments, theory and modeling we are able to relate the measured tissue level biophysical quantities to subcellular parameters. Our approach has validity beyond the particular morphogenetic process considered here and provides a general way to relate biophysical properties across scales. [Abstract]

Grima R
15 multiscale modeling of biological pattern formation.
Curr Top Dev Biol. 2007;81435-60.
In the past few decades, it has become increasingly popular and important to utilize mathematical models to understand how microscopic intercellular interactions lead to the macroscopic pattern formation ubiquitous in the biological world. Modeling methodologies come in a large variety and presently it is unclear what is their interrelationship and the assumptions implicit in their use. They can be broadly divided into three categories according to the spatial scale they purport to describe: the molecular, the cellular and the tissue scales. Most models address dynamics at the tissue-scale, few address the cellular scale and very few address the molecular scale. Of course there would be no dissent between models or at least the underlying assumptions would be known if they were all rigorously derived from a molecular level model, in which case the laws of physics and chemistry are very well known. However in practice this is not possible due to the immense complexity of the problem. A simpler approach is to derive models at a coarse scale from an intermediate scale model which has the special property of being based on biology and physics which are experimentally well studied. In this article we use such an approach to understand the assumptions inherent in the use of the most popular models, the tissue-level ones. Such models are found to invariably rely on the hidden assumption that statistical correlations between cells can be neglected. This often means that the predictions of these models are qualitatively correct but may fail in spatial regions where cell concentration is small, particularly if there are strong long-range correlations in cell movement. Such behavior can only be properly taken into account by cellular models. However such models unlike the tissue-level models are frequently not easily amenable to analysis, except when the number of interacting cells is small or when the interactions are weak, and thus are rather more suited for simulation. Hence it is our conclusion that the simultaneous theoretical and numerical analysis of models of the same biological system at different spatial scales provides a more robust method of understanding biological systems than the utilization of a single scale model. In particular this enables one to clearly separate nonphysical predictions stemming from model artifacts from those due to genuine physiological behavior. [Abstract]

Hatzikirou H, Deutsch A
14 cellular automata as microscopic models of cell migration in heterogeneous environments.
Curr Top Dev Biol. 2007;81401-34.
Understanding the precise interplay of moving cells with their typically heterogeneous environment is crucial for central biological processes as embryonic morphogenesis, wound healing, immune reactions or tumor growth. Mathematical models allow for the analysis of cell migration strategies involving complex feedback mechanisms between the cells and their microenvironment. Here, we introduce a cellular automaton (especially lattice-gas cellular automaton-LGCA) as a microscopic model of cell migration together with a (mathematical) tensor characterization of different biological environments. Furthermore, we show how mathematical analysis of the LGCA model can yield an estimate for the cell dispersion speed within a given environment. Novel imaging techniques like diffusion tensor imaging (DTI) may provide tensor data of biological microenvironments. As an application, we present LGCA simulations of a proliferating cell population moving in an external field defined by clinical DTI data. This system can serve as a model of in vivo glioma cell invasion. [Abstract]

Meyer-Hermann M
13 Delaunay-Object-Dynamics: Cell Mechanics with a 3D Kinetic and Dynamic Weighted Delaunay-Triangulation.
Curr Top Dev Biol. 2007;81373-99.
Mathematical methods in Biology are of increasing relevance for understanding the control and the dynamics of biological systems with medical relevance. In particular, agent-based methods turn more and more important because of fast increasing computational power which makes even large systems accessible. An overview of different mathematical methods used in Theoretical Biology is provided and a novel agent-based method for cell mechanics based on Delaunay-triangulations and Voronoi-tessellations is explained in more detail: The Delaunay-Object-Dynamics method. It is claimed that the model combines physically realistic cell mechanics with a reasonable computational load. The power of the approach is illustrated with two examples, avascular tumor growth and genesis of lymphoid tissue in a cell-flow equilibrium. [Abstract]

Salazar-Ciudad I
12 Tooth Morphogenesis in vivo, in vitro, and in silico.
Curr Top Dev Biol. 2007;81341-71.
One of the aims of developmental biology is to understand how a single egg cell gives rise to the complex spatial distributions of cell types and extracellular components of the adult phenotype. This review discusses the main genetic and epigenetic interactions known to play a role in tooth development and how they can be integrated into coherent models. Along the same lines, several hypotheses about aspects of tooth development that are currently not well understood are evaluated. This is done from their morphological consequences from the model and how these fit known morphological variation and change during tooth development. Thus the aim of this review is two-fold. On one hand the model and its comparison with experimental evidence will be used to outline our current understanding about tooth morphogenesis. On the other hand these same comparisons will be used to introduce a computational model that makes accurate predictions on three-dimensional morphology and patterns of gene expression by implementing cell signaling, proliferation and mechanical interactions between cells. In comparison with many other models of development this model includes reaction-diffusion-like dynamics confined to a diffusion chamber (the developing tooth) that changes in shape in three-dimensions over time. These changes are due to mechanical interactions between cells triggered by the proliferation enhancing effect of the reactants (growth factors). In general, tooth morphogenesis can be understood from the indirect cross-regulation between extracellular signals, the local regulation of proliferation and differentiation rates by these signals and the effect of intermediate developing morphology on the diffusion, dilution, and spatial distribution of these signals. Overall, this review should be interesting to either readers interested in the mechanistic bases of tooth morphogenesis, without necessarily being interested in modeling per se, and readers interested in development modeling in general. [Abstract]

Newman SA, Christley S, Glimm T, Hentschel HG, Kazmierczak B, Zhang YT, Zhu J, Alber M
11 multiscale models for vertebrate limb development.
Curr Top Dev Biol. 2007;81311-40.
Dynamical systems in which geometrically extended model cells produce and interact with diffusible (morphogen) and nondiffusible (extracellular matrix) chemical fields have proved very useful as models for developmental processes. The embryonic vertebrate limb is an apt system for such mathematical and computational modeling since it has been the subject of hundreds of experimental studies, and its normal and variant morphologies and spatiotemporal organization of expressed genes are well known. Because of its stereotypical proximodistally generated increase in the number of parallel skeletal elements, the limb lends itself to being modeled by Turing-type systems which are capable of producing periodic, or quasiperiodic, arrangements of spot- and stripe-like elements. This chapter describes several such models, including, (i) a system of partial differential equations in which changing cell density enters into the dynamics explicitly, (ii) a model for morphogen dynamics alone, derived from the latter system in the "morphostatic limit" where cell movement relaxes on a much slower time-scale than cell differentiation, (iii) a discrete stochastic model for the simplified pattern formation that occurs when limb cells are placed in planar culture, and (iv) several hybrid models in which continuum morphogen systems interact with cells represented as energy-minimizing mesoscopic entities. Progress in devising computational methods for handling 3D, multiscale, multimodel simulations of organogenesis is discussed, as well as for simulating reaction-diffusion dynamics in domains of irregular shape. [Abstract]

Miura T
10 modeling lung branching morphogenesis.
Curr Top Dev Biol. 2007;81291-310.
Vertebrate lung has tree-like structure which facilitates gas exchange. After discovery of the involvement of several key toolkit genes-FGF10, BMP4, and Shh, huge amount of molecular information on lung development is now available. However, how their interactions result in a branched structure has not been elucidated. Recently, some studies have utilized mathematical models to understand the mechanism of branching morphogenesis, and we now have some models which are reliable enough to make experimental predictions in the in vitro system. In addition, a different type of modeling, which generates tree-like branching pattern by repeatedly applying a set of simple rules iteratively, is also utilized to model lung function. In this review, I focus on how these models can contribute to understand pattern formation phenomena from experimental biologist's point of view. [Abstract]

Czirok A, Zamir EA, Szabo A, Little CD
9 Multicellular Sprouting during Vasculogenesis.
Curr Top Dev Biol. 2007;81269-89.
Living organisms, from bacteria to vertebrates, are well known to generate sophisticated multicellular patterns. Numerous recent interdisciplinary studies have focused on the formation and regulation of these structures. Advances in automatized microscopy allow the time-resolved tracking of embryonic development at cellular resolution over an extended area covering most of the embryo. The resulting images yield simultaneous information on the motion of multiple tissue components-both cells and extracellular matrix (ECM) fibers. Recent studies on ECM displacements in bird embryos resulted in a method to distinguish tissue deformation and cell-autonomous motion. Patterning of the primary vascular plexus results from a collective action of primordial endothelial cells. The emerging "polygonal" vascular structure is shown to be formed by cell-cell and cell-ECM interactions: adhesion and protrusive activity (sprouting). Utilizing avb3 integrins, multicellular sprouts invade rapidly into avascular areas. Sprout elongation, in turn, depends on a continuous supply of endothelial cells. Endothelial cells migrate along the sprout, towards its tip, in a vascular endothelial (VE) cadherin-dependent process. The observed abundance of multicellular sprout formation in various in vitro and in vivo systems can be explained by a general mechanism based on preferential attraction to elongated structures. Our interacting particle model exhibits robust sprouting dynamics and results in patterns with morphometry similar to native primordial vascular plexuses-without ancillary assumptions involving chemotaxis or mechanochemical signaling. [Abstract]

Lubkin SR
8 branched organs: mechanics of morphogenesis by multiple mechanisms.
Curr Top Dev Biol. 2007;81249-68.
Branching morphogenesis is ubiquitous and important in creating bulk transport systems. Branched ducts can be generated by several different mechanisms including growth, cell rearrangements, contractility, adhesion changes, and other mechanisms. We have developed several models of the mechanics of cleft formation, which we review. We discuss the implications of several candidate mechanisms and review what has been found in models and in experiments. [Abstract]

Glazier JA, Zhang Y, Swat M, Zaitlen B, Schnell S
7 Coordinated Action of N-CAM, N-cadherin, EphA4, and ephrinB2 Translates Genetic Prepatterns into Structure during Somitogenesis in Chick.
Curr Top Dev Biol. 2007;81205-47.
During gastrulation in vertebrates, mesenchymal cells at the anterior end of the presomitic mesoderm (PSM) periodically compact, transiently epithelialize and detach from the posterior PSM to form somites. In the prevailing clock-and-wavefront model of somitogenesis, periodic gene expression, particularly of Notch and Wnt, interacts with an FGF8-based thresholding mechanism to determine cell fates. However, this model does not explain how cell determination and subsequent differentiation translates into somite morphology. In this paper, we use computer simulations of chick somitogenesis to show that experimentally-observed temporal and spatial patterns of adhesive N-CAM and N-cadherin and repulsive EphA4-ephrinB2 pairs suffice to reproduce the complex dynamic morphological changes of somitogenesis in wild-type and N-cadherin (-/-) chick, including intersomitic separation, boundary-shape evolution and sorting of misdifferentiated cells across compartment boundaries. Since different models of determination yield the same, experimentally-observed, distribution of adhesion and repulsion molecules, the patterning is independent of the details of this mechanism. [Abstract]

Baker RE, Schnell S, Maini PK
6 mathematical models for somite formation.
Curr Top Dev Biol. 2007;81183-203.
Somitogenesis is the process of division of the anterior-posterior vertebrate embryonic axis into similar morphological units known as somites. These segments generate the prepattern which guides formation of the vertebrae, ribs and other associated features of the body trunk. In this work, we review and discuss a series of mathematical models which account for different stages of somite formation. We begin by presenting current experimental information and mechanisms explaining somite formation, highlighting features which will be included in the models. For each model we outline the mathematical basis, show results of numerical simulations, discuss their successes and shortcomings and avenues for future exploration. We conclude with a brief discussion of the state of modeling in the field and current challenges which need to be overcome in order to further our understanding in this area. [Abstract]

Newman TJ
5 grid-free models of multicellular systems, with an application to large-scale vortices accompanying primitive streak formation.
Curr Top Dev Biol. 2007;81157-82.
This paper is comprised of two parts. In the first we provide a brief overview of grid-free methods for modeling multicellular systems. We focus on an approach based on Langevin equations, in which inertia is ignored, and stochastic effects on cell motion are included. The discussion starts with simpler models, in which cells are modeled as adhesive spheres. We then turn to more sophisticated approaches in which nontrivial cell shape is accommodated, including the recently introduced Subcellular Element Model, in which each cell is described as a cluster of adhesively coupled over-damped subcellular elements, representing patches of cytoskeleton. In the second part of the paper we illustrate the use of a standard grid-free cell-based model to computationally probe interesting new features associated with primitive streak formation in the chick embryo. Streak formation is a key developmental step in amniotes (i.e., birds, reptiles, and mammals), and can be observed in detail in the chick embryo, where the streak extends across a tightly-packed two-dimensional sheet (the epiblast) comprised of about 50,000 cells. The Weijer group [Cui, Yang, Chuai, Glazier, and Weijer, Dev. Biol. 284 (2005) 37-47] recently observed that streak formation is accompanied by coordinated cell movement lateral to the streak, resulting in two large counter-rotating vortices. We study a mechanism based on cell polarity (in the plane of the epiblast) that provides an explanation for these vortices, and test it successfully using computer simulations. This mechanism is robust, since the emergent vortex formation depends only on the gross features of the initial spatial distribution of planar polarity in the epiblast. [Abstract]

Chuai M, Weijer CJ
4 the mechanisms underlying primitive streak formation in the chick embryo.
Curr Top Dev Biol. 2007;81135-56.
Formation of the primitive streak is one of the key events in the early development of amniote embryos. The streak is the site where during gastrulation the mesendoderm cells ingress to take up their correct topographical positions in the embryo. The process of streak formation can be conveniently observed in the chick embryo, where the streak forms as an accumulation of cells in the epiblast in the posterior pole of the embryo and extends subsequently in anterior direction until it covers 80% of the epiblast. A prerequisite for streak formation is the differentiation of mesoderm, which is induced in the epiblast at the interface between the posterior Area Opaca and Area Pellucida in a sickle shaped domain overlying Koller's sickle. Current views on the molecular mechanisms of mesoderm induction by inducing signals from the Area Opaca and inhibitory signals from the hypoblast are briefly discussed. During streak formation the sickle of mesoderm cells transforms into an elongated structure in the central midline of the embryo. We discuss possible cellular mechanisms underlying this process, such as oriented cell division, cell-cell intercalation, chemotactic cell movement in response to attractive and repulsive signals and a combination of chemotaxis and contact following. We review current experimental evidence in favor and against these different hypotheses and outline some the outstanding questions. Since many of the interactions between cells signaling and moving are dynamic and nonlinear in nature they will require detailed modeling and computer simulations to be understood in detail. [Abstract]

Davidson LA
3 integrating morphogenesis with underlying mechanics and cell biology.
Curr Top Dev Biol. 2007;81113-33.
Morphogenesis integrates a wide range of cellular processes into a self-organizing, self-deforming tissue. No single molecular "magic bullet" controls morphogenesis. Wide ranging cellular processes, often without parallels in conventional cell culture systems, work together to generate the architecture and modulate forces that produce and guide shape changes in the embryo. In this review we summarize the early development of the frog Xenopus laevis from a biomechanical perspective. We describe processes operating in the embryo from whole embryo scale, the tissue-scale, to the cellular and extracellular matrix scale. We focus on describing cells, their behaviors and the unique microenvironments they traverse during gastrulation and discuss the role of tissue mechanics in these processes. [Abstract]

Umulis D, O'Connor MB, Othmer HG
2 Robustness of Embryonic Spatial Patterning in Drosophila melanogaster.
Curr Top Dev Biol. 2007;8165-111. [Abstract]

Meinhardt H
1 models of biological pattern formation: from elementary steps to the organization of embryonic axes.
Curr Top Dev Biol. 2007;811-63.
An inroad into an understanding of the complex molecular interactions on which development is based can be achieved by uncovering the minimum requirements that describe elementary steps and their linkage. Organizing regions and other signaling centers can be generated by reactions that involve local self-enhancement coupled to antagonistic reactions of longer range. More complex patterns result from a chaining of such reactions in which one pattern generates the prerequisites for the next. Patterning along the single axis of radial symmetric animals including the small freshwater polyp hydra can be explained in this way. The body pattern of such ancestral organisms evolved into the brain of higher organisms, while trunk and midline formation are later evolutionary additions. The equivalent of the hydra organizer is the blastopore, for instance, the marginal zone in amphibians. It organizes the anteroposterior axis. The Spemann organizer, located on this primary organizer, initiates and elongates the midline, which is responsible for the dorsoventral pattern. In contrast, midline formation in insects is achieved by an inhibitory signal from a dorsal organizer that restricts the midline to the ventral side. Thus, different modes of midline formation are proposed to be the points of no return in the separation of phyla. The conversion of the transient patterns of morphogenetic signaling into patterns of stable gene activation can be achieved by genes whose gene products have a positive feedback on the activity of their own gene. If several such autoregulatory genes mutually exclude each other, a cell has to make an unequivocal decision to take a particular pathway. Under the influence of a gradient, sharply confined regions with particular determinations can emerge. Borders between regions of different gene activities, and the areas of intersection of two such borders, become the new signaling centers that initiate secondary embryonic fields. As required for leg and wing formation, these new fields emerge in pairs at defined positions, with defined orientation and left-right handedness. Recent molecular-genetic results provide strong support for theoretically predicted interactions. By computer simulations it is shown that the regulatory properties of these models correspond closely to experimental observations (animated simulations are available at [Abstract]

Schnell S, Maini PK, Newman SA, Newman TJ
Curr Top Dev Biol. 2007;81xvii-xxv. [Abstract]

Filippova GN
Genetics and epigenetics of the multifunctional protein CTCF.
Curr Top Dev Biol. 2008;80337-60.
Recent advances in studying long-range chromatin interactions have shifted focus from the transcriptional regulation by nearby regulatory elements to recognition of the role of higher-order chromatin organization within the nucleus. These advances have also suggested that CCCTC-binding factor (CTCF), a known chromatin insulator protein, may play a central role in mediating long-range chromatin interactions, directing DNA segments into transcription factories and/or facilitating interactions with other DNA regions. Several models that describe possible mechanisms for multiple functions of CTCF in establishment and maintenance of epigenetic programs are now emerging. Epigenetics plays an important role in normal development and disease including cancer. CTCF involvement in multiple aspects of epigenetic regulation, including regulation of genomic imprinting and X-chromosome inactivation, has been well established. More recently, CTCF was found to play a role in regulation of noncoding transcription and establishing local chromatin structure at the repetitive elements in mammalian genomes, suggesting a new epigenetic basis for several repeat-associated genetic disorders. Emerging evidence also points to the role of CTCF deregulation in the epigenetic imbalance in cancer. These studies provide some of the important missing links in our understanding of epigenetic control of both development and cancer. [Abstract]

Liu J, DeNofrio J, Yuan W, Wang Z, McFadden AW, Parise LV
Genetic manipulation of megakaryocytes to study platelet function.
Curr Top Dev Biol. 2008;80311-35.
Proper platelet function is essential for hemostasis. However, understanding platelet function is complicated by the fact that platelets are anucleate and therefore not amenable to direct genetic manipulations. To study platelet function, several laboratories have developed CHO cell lines expressing platelet proteins or used megakaryocyte-like cell lines. However, these cell culture models are unable to mimic critical platelet functions, most notably agonist-induced activation of integrin alphaIIbbeta3. Mature megakaryocytes, which are platelet precursors, express platelet-specific proteins, and the function of such proteins and signaling pathways appears conserved between the two cell types. Murine megakaryocytes have been successfully differentiated in cultures from bone marrow, fetal liver, and embryonic stem (ES) cells, while human megakaryocytes have been cultured from human cord blood, peripheral blood, and ES cells. The various sources of megakaryocyte progenitors provide choices to researchers, allowing them to access the most applicable systems. As examples, both bone marrow-derived and ES cell-derived murine megakaryocytes have been used to study proteins involved in integrin alphaIIbbeta3 regulation such as CIB1 and H-Ras. Therefore, megakaryocytes have provided an invaluable resource for better understanding the biology of platelets. In this chapter, we will describe: (1) approaches to obtain, generate, and characterize megakaryocytes, (2) molecular manipulation of these cells to elevate or decrease expression levels of specific proteins, and (3) current uses and future applications of megakaryocytes. [Abstract]

Hawkins BT, Egleton RD
Pathophysiology of the blood-brain barrier: animal models and methods.
Curr Top Dev Biol. 2008;80277-309.
The specialized cerebral microvascular endothelium interacts with the cellular milieu of the brain and extracellular matrix to form a neurovascular unit, one aspect of which is a regulated interface between the blood and central nervous system (CNS). The concept of this blood-brain barrier (BBB) as a dynamically regulated system rather than a static barrier has wide-ranging implications for pathophysiology of the CNS. While in vitro models of the BBB are useful for screening drugs targeted to the CNS and indispensable for studies of cerebral endothelial cell biology, the complex interactions of the neurovascular unit make animal-based models and methods essential tools for understanding the pathophysiology of the BBB. BBB dysfunction is a complication of neurodegenerative disease and brain injury. Studies on animal models have shown that diseases of the periphery, such as diabetes and inflammatory pain, have deleterious effects on the BBB which may contribute to neurological complications associated with these conditions. Furthermore, genetic and/or epigenetic abnormalities in constituents of the BBB may be significant contributing factors in disease etiology. Research that approaches the BBB as a dynamic system integrated with both the CNS and the periphery is therefore critical to understanding and treating diseases of the CNS. Herein, we review various methodological approaches used to study BBB function in the context of disease. These include measurement of transport between blood and brain, imaging-based technologies, and genomic/proteomic approaches. [Abstract]

Marin F, Luquet G, Marie B, Medakovic D
Molluscan shell proteins: primary structure, origin, and evolution.
Curr Top Dev Biol. 2008;80209-76.
In the last few years, the field of molluscan biomineralization has known a tremendous mutation, regarding fundamental concepts on biomineralization regulation as well as regarding the methods of investigation. The most recent advances deal more particularly with the structure of shell biominerals at nanoscale and the identification of an increasing number of shell matrix protein components. Although the matrix is quantitatively a minor constituent in the shell of mollusks (less than 5% w/w), it is, however, the major component that controls different aspects of the shell formation processes: synthesis of transient amorphous minerals and evolution to crystalline phases, choice of the calcium carbonate polymorph (calcite vs aragonite), organization of crystallites in complex shell textures (microstructures). Until recently, the classical paradigm in molluscan shell biomineralization was to consider that the control of shell synthesis was performed primarily by two antagonistic mechanisms: crystal nucleation and growth inhibition. New concepts and emerging models try now to translate a more complex reality, which is remarkably illustrated by the wide variety of shell proteins, characterized since the mid-1990s, and described in this chapter. These proteins cover a broad spectrum of pI, from very acidic to very basic. The primary structure of a number of them is composed of different modules, suggesting that these proteins are multifunctional. Some of them exhibit enzymatic activities. Others may be involved in cell signaling. The oldness of shell proteins is discussed, in relation with the Cambrian appearance of the mollusks as a mineralizing phylum and with the Phanerozoic evolution of this group. Nowadays, the extracellular calcifying shell matrix appears as a whole integrated system, which regulates protein-mineral and protein-protein interactions as well as feedback interactions between the biominerals and the calcifying epithelium that synthesized them. Consequently, the molluscan shell matrix may be a source of bioactive molecules that would offer interesting perspectives in biomaterials and biomedical fields. [Abstract]

Braendle C, Milloz J, Félix MA
Mechanisms and evolution of environmental responses in Caenorhabditis elegans.
Curr Top Dev Biol. 2008;80171-207.
We review mechanistic and evolutionary aspects of interactions between the model organism Caenorhabditis elegans and its environment. In particular, we focus on environmental effects affecting developmental mechanisms. We describe natural and laboratory environments of C. elegans and provide an overview of the different environmental responses of this organism. We then show how two developmental processes respond to changes in the environment. First, we discuss the development of alternative juvenile stages, the dauer and non-dauer larva. This example illustrates how development responds to variation in the environment to generate complex phenotypic variation. Second, we discuss the development of the C. elegans vulva. This example illustrates how development responds to variation in the environment while generating an invariant final phenotype. [Abstract]

Westholm DE, Rumbley JN, Salo DR, Rich TP, Anderson GW
Organic anion-transporting polypeptides at the blood-brain and blood-cerebrospinal fluid barriers.
Curr Top Dev Biol. 2008;80135-70.
Organic anion-transporting polypeptides (Oatps) are solute carrier family members that exhibit marked evolutionary conservation. Mammalian Oatps exhibit wide tissue expression with an emphasis on expression in barrier cells. In the brain, Oatps are expressed in the blood-brain barrier endothelial cells and blood-cerebrospinal fluid barrier epithelial cells. This expression profile serves to illustrate a central role for Oatps in transporting endo- and xenobiotics across brain barrier cells. This chapter will detail the expression patterns and substrate specificities of Oatps expressed in the brain, and will place special emphases on the role of Oatps in prostaglandin synthesis and in the transport of conjugated endobiotics. [Abstract]

Oien DB, Moskovitz J
Substrates of the methionine sulfoxide reductase system and their physiological relevance.
Curr Top Dev Biol. 2008;8093-133.
Posttranslational modifications can change a protein's structure, function, and solubility. One specific modification caused by reactive oxygen species is the oxidation of the sulfur atom in the methionine (Met) side chain. This modified amino acid is denoted as methionine sulfoxide (MetO). MetOs in proteins are of considerable interest as they are involved in early posttranslational modification events. Thus, various organisms produce specific enzymes that can reverse these modifications. MetO reductases, known collectively as the methionine sulfoxide reductase (Msr) system, are the only known enzymes that can reduce MetOs. The current research field of Met redox cycles is consumed with elucidating its role in regulation, redox homeostasis, prevention of irreversible modifications, pathogenesis, and the aging process. Substrates of the Msr system can be loosely classified by the overall effect of the MetO on the protein. Regulated substrates utilize Met as a molecular switch to modulate activation; scavenging substrates use Mets to detoxify oxidants and protect important regions of the protein; and modified substrates are altered by Met oxidation resulting in various changes in their properties, including function, activity, structure, and degradation resistance. [Abstract]

Yan HH, Mruk DD, Cheng CY
Junction restructuring and spermatogenesis: the biology, regulation, and implication in male contraceptive development.
Curr Top Dev Biol. 2008;8057-92.
Spermatogenesis that occurs in the seminiferous epithelium of adult mammalian testes is associated with extensive junction restructuring at the Sertoli-Sertoli cell, Sertoli-germ cell, and Sertoli-basement membrane interface. While this morphological phenomenon is known and has been described in great details for decades, the biochemical and molecular changes as well as the mechanisms/signaling pathways that define changes at the cell-cell and cell-matrix interface remain largely unknown until recently. In this chapter, we summarize and discuss findings in the field regarding the coordinated efforts of the anchoring [e.g., adherens junction (AJ), such as basal ectoplasmic specialization (basal ES)] and tight junctions (TJs) that are present in the same microenvironment, such as at the blood-testis barrier (BTB), or at distinctly opposite ends of the Sertoli cell epithelium, such as between apical ectoplasmic specialization (apical ES) in the apical compartment, and the BTB adjacent to the basal compartment of the epithelium. These efforts, in turn, regulate and coordinate different cellular events that occur during the seminiferous epithelial cycle. For instance, the events of spermiation and of preleptotene spermatocyte migration across the BTB both take place concurrently at stage VIII of the epithelial cycle of spermatogenesis. Recent findings suggest that these events are coordinated by protein complexes found at the apical and basal ES and TJ, which are located at different ends of the Sertoli cell epithelium. Besides, we highlight important areas of research that can now be undertaken, and functional studies that can be designed to tackle different issues pertinent to junction restructuring during spermatogenesis. [Abstract]

Zacchigna S, Ruiz de Almodovar C, Carmeliet P
Similarities between angiogenesis and neural development: what small animal models can tell us.
Curr Top Dev Biol. 2008;801-55.
During evolution vertebrates had to evolve in order to perform more and more complex tasks. To achieve this goal, they developed specialized tissues: a highly branched vascular system to ensure that all tissues receive adequate blood supply, and an intricate nervous system in which nerves branch to transmit electrical signals to peripheral organs. The development of both systems is tightly controlled by a series of developmental cues, which ensure the accomplishment of a complex and highly stereotyped mature network. Vessels and nerves use similar signals and principles to grow, differentiate, and navigate toward their final targets. Both systems share several molecular pathways, highlighting an important link between vascular biology and neuroscience. Moreover, the vascular and the nervous system crosstalk and, when deregulated, contribute to medically relevant diseases. This new phenomenon, named the neurovascular link, promises to accelerate the discovery of new pathogenetic insights and therapeutic strategies for the treatment of both vascular and neurological diseases. To study the development of both systems, scientists are taking advantage of the use of several vertebrate and invertebrate animal models. In the first part of this chapter, we will discuss the more commonly used animal models; in the second part, the striking similarities occurring during the development of the vascular and the neural systems will be revised. [Abstract]

Salipante SJ, Horwitz MS
A phylogenetic approach to mapping cell fate.
Curr Top Dev Biol. 2007;79157-84.
Recent, surprising, and controversial discoveries have challenged conventional concepts regarding the origins and plasticity of stem cells, and their contributions to tissue regeneration, and highlight just how little is known about mammalian development in comparison to simpler model organisms. In the case of the transparent worm, Caenorhabditis elegans, Sulston and colleagues used a microscope to record the birth and death of every cell during its life, and the compilation of this "fate map" represents a milestone achievement of developmental biology. Determining a fate map for mammals or other higher organisms is more complicated because they are opaque, take a long time to mature, and have a tremendous number of cells. Consequently, fate mapping experiments have relied on tagging a progenitor cell with a dye or genetic marker in order to later identify its descendants. This approach, however, extracts little information because it demonstrates that a population of cells, all having inherited the same label, shares a common ancestor, but it does not reveal how cells in that population are related to one another. To avoid that problem, as well as technical limitations of current methods for mapping cell fate, we, and others, have developed a new strategy for retrospectively deriving cell fate maps by using phylogenetics to infer the order in which somatic mutations have arisen in the genomes of individual cells during development in multicellular organisms. DNA replication inevitably introduces mutations, particularly at repetitive sequences, every time a cell divides. It is thus possible to deduce the history of cell divisions by cataloging somatic mutations and phylogenetically reconstructing cell lineage. This approach has the potential to produce a complete mammalian cell fate map that, in principle, could describe the developmental lineage of any cell and help resolve outstanding questions of stem cell biology, tissue repair and maintenance, and aging. [Abstract]

Antonicelli F, Bellon G, Debelle L, Hornebeck W
Elastin-elastases and inflamm-aging.
Curr Top Dev Biol. 2007;7999-155.
Degradation of elastin, the main amorphous component of elastic fibers, by elastases belonging to the serine, metallo, or cysteine families leads to the generation of elastin fragments, designated as elastokines in keeping with their cytokine-like properties. Generation of elastokines from one of the longest lived protein in human might represent a strong tissue repair signal. Indeed, they (1) exhibit potent chemotactic activity for leukocytes, (2) stimulate fibroblast and smooth muscle cell proliferation, and (3) display proangiogenic activity as potent as VEGF. However, continuous exposure of cells to these matrikines, through increased elastase(s) expression with age, can contribute to the formation of a chronic inflammatory state, that is, inflamm-aging. Importantly, binding of elastokines to S-Gal, their cognate receptor, proved to stimulate matrix metalloproteinase expression in normal and cancer cells. Besides, these elastin fragments can polarize lymphocytes toward a Th-1 response or induce an osteogenic response in smooth muscle cells, and arterial wall calcification. In this chapter, emphasis will be made on the contribution of elastokines on the genesis of age-related arterial wall diseases, particularly abdominal aortic aneurysms (AAAs). An elastokine theory of AAAs progression will be proposed. Age is one main risk factor of cancer incidence and development. The myriad of biological effects exerted by elastokines on stromal and inflammatory cells led us to hypothesize that they might be main actors in elaborating a favorable cancerization field in melanoma; for instance these peptides could catalyze the vertical growth phase transition in melanoma through increased expression of gelatinase A and membrane-type 1 matrix metalloproteinase. [Abstract]

Jakovcevic D, Harder DR
Role of astrocytes in matching blood flow to neuronal activity.
Curr Top Dev Biol. 2007;7975-97.
The brain is critically dependent on oxygen and glucose supply for normal function. Various neurovascular control mechanisms assure that the blood supply of the brain is adequate to meet the energy needs of its components. Emerging evidence shows that neuronal activity can control microcirculation using astrocytes as a mediator. Astrocytes can sense neuronal activity and are involved in signal transmission. Synaptic activity triggers an increase in the intracellular calcium concentration [Ca(2+)]i of adjacent astrocytes, stimulating the release of adenosine triphosphate (ATP) and glutamate. The released ATP mediates the propagation of Ca(2+) waves between neighboring astrocytes, thereby recruiting them to mediate adequate cerebrovascular response to neuronal activation. Simultaneously, sodium-dependent glutamate uptake in astrocytes generates Na(+) waves and subsequently increases glucose uptake and metabolism that leads to the formation of lactate, which is then delivered to neurons as an energy substrate. Further, astrocytic Ca(2+) elevations can lead to secretion of vasodilatory substances from perivascular endfeet, such as epoxyeicosatrienoic acid (EETs), adenosine, nitric oxide (NO), and cyclooxygenase-2 (COX-2) metabolites, resulting in increased local blood flow. Thus, astrocytes by releasing vasoactive molecules mediate the neuron-astrocyte-endothelial signaling pathway and play a profound role in coupling blood flow to neuronal activity. [Abstract]

Recent Articles in Development

Wu Y, Wang G, Scott SA, Capecchi MR
Hoxc10 and Hoxd10 regulate mouse columnar, divisional and motor pool identity of lumbar motoneurons.
Development. 2008 Jan;135(1):171-82.
A central question in neural development is how the broad diversity of neurons is generated in the vertebrate CNS. We have investigated the function of Hoxc10 and Hoxd10 in mouse lumbar motoneuron development. We show that Hoxc10 and Hoxd10 are initially expressed in most newly generated lumbar motoneurons, but subsequently become restricted to the lateral division of the lateral motor column (lLMC). Disruption of Hoxc10 and Hoxd10 caused severe hindlimb locomotor defects. Motoneurons in rostral lumbar segments were found to adopt the phenotype of thoracic motoneurons. More caudally the lLMC and dorsal-projecting axons were missing, yet most hindlimb muscles were innervated. The loss of the lLMC was not due to decreased production of motoneuron precursors or increased apoptosis. Instead, presumptive lLMC neurons failed to migrate to their normal position, and did not differentiate into other motoneurons or interneurons. Together, these results show that Hoxc10 and Hoxd10 play key roles in establishing lumbar motoneuron columnar, divisional and motor pool identity. [Abstract]

Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa K, Urasaki A, Horikawa K, Ikeo K, Takeda H, Kawakami K
Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like.
Development. 2008 Jan;135(1):159-69.
Gene trap and enhancer trap methods using transposon or retrovirus have been recently described in zebrafish. However, insertional mutants using these methods have not been reported. We report here development of an enhancer trap method by using the Tol2 transposable element and identification and characterization of insertional mutants. We created 73 fish lines that carried single copy insertions of an enhancer trap construct, which contained the zebrafish hsp70 promoter and the GFP gene, in their genome and expressed GFP in specific cells, tissues and organs, indicating that the hsp70 promoter is highly capable of responding to chromosomal enhancers. First, we analyzed genomic DNA surrounding these insertions. Fifty-one of them were mapped onto the current version of the genomic sequence and 43% (22/51) were located within transcribed regions, either exons or introns. Then, we crossed heterozygous fish carrying the same insertions and identified two insertions that caused recessive mutant phenotypes. One disrupted the tcf7 gene, which encodes a transcription factor of the Tcf/Lef family mediating Wnt signaling, and caused shorter and wavy median fin folds and pectoral fins. We knocked down Lef1, another member of the Tcf/Lef family also expressed in the fin bud, in the tcf7 mutant, and revealed functional redundancy of these factors and their essential role in establishment of the apical ectodermal ridge (AER). The other disrupted the synembryn-like gene (synbl), a homolog of the C. elegans synembryn gene, and caused embryonic lethality and small pigment spots. The pigment phenotype was rescued by application of forskolin, an activator of adenylyl cyclase, suggesting that the synbl gene activates the Galpha(S) pathway leading to activation of adenylyl cyclase. We thus demonstrated that the transposon-mediated enhancer trap approach can indeed create insertional mutations in developmental genes. Our present study provides a basis for the development of efficient transposon-mediated insertional mutagenesis in a vertebrate. [Abstract]

Smith J, Alfred J
Whither Development and developmental biology?
Development. 2008 Jan;135(1):1. [Abstract]

Ohhata T, Hoki Y, Sasaki H, Sado T
Crucial role of antisense transcription across the Xist promoter in Tsix-mediated Xist chromatin modification.
Development. 2007 Dec 5;
Expression of Xist, which triggers X inactivation, is negatively regulated in cis by an antisense gene, Tsix, transcribed along the entire Xist gene. We recently demonstrated that Tsix silences Xist through modification of the chromatin structure in the Xist promoter region. This finding prompted us to investigate the role of antisense transcription across the Xist promoter in Tsix-mediated silencing. Here, we prematurely terminated Tsix transcription before the Xist promoter and addressed its effect on Xist silencing in mouse embryos. We found that although 93% of the region encoding Tsix was transcribed, truncation of Tsix abolished the antisense regulation of Xist. This resulted in a failure to establish the repressive chromatin configuration at the Xist promoter on the mutated X, including DNA methylation and repressive histone modifications, especially in extraembryonic tissues. These results suggest a crucial role for antisense transcription across the Xist promoter in Xist silencing. [Abstract]

Ogino H, Fisher M, Grainger RM
Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification.
Development. 2007 Dec 5;
Xenopus is ideal for systematic decoding of cis-regulatory networks because its evolutionary position among vertebrates allows one to combine comparative genomics with efficient transgenic technology in one system. Here, we have identified and analyzed the major enhancer of FoxE3 (Lens1), a gene essential for lens formation that is activated in the presumptive lens ectoderm (PLE) when commitment to the lens fate occurs. Deletion and mutation analyses of the enhancer based on comparison of Xenopus and mammalian sequences and in vitro and in vivo binding assays identified two essential transcriptional regulators: Otx2, a homeodomain protein expressed broadly in head ectoderm including the PLE, and Su(H), a nuclear signal transducer of Notch signaling. A Notch ligand, Delta2, is expressed in the optic vesicle adjacent to the PLE, and inhibition of its activity led to loss, or severe reduction, of FoxE3 expression followed by failure of placode formation. Ectopic activation of Notch signaling induced FoxE3 expression within head ectoderm expressing Otx2, and additional misexpression of Otx2 in trunk ectoderm extended the Notch-induced FoxE3 expression posteriorly. These data provide the first direct evidence of the involvement of Notch signaling in lens induction. The obligate integration of inputs of a field-selector (Otx2) and localized signaling (Notch) within target cis-regulatory elements might be a general mechanism of organ-field specification in vertebrates (as it is in Drosophila). This concept is also consistent with classical embryological studies of many organ systems involving a 'multiple-step induction'. [Abstract]

Yamada Y, Davis KD, Coffman CR
Programmed cell death of primordial germ cells in Drosophila is regulated by p53 and the Outsiders monocarboxylate transporter.
Development. 2007 Dec 5;
Primordial germ cell development uses programmed cell death to remove abnormal, misplaced or excess cells. Precise control of this process is essential to maintain the continuity and integrity of the germline, and to prevent germ cells from colonizing locations other than the gonads. Through careful analyses of primordial germ cell distribution in developing Drosophila melanogaster embryos, we show that normal germ cell development involves extensive programmed cell death during stages 10-12 of embryogenesis. This germ cell death is mediated by Drosophila p53 (p53). Mutations in p53 result in excess primordial germ cells that are ectopic to the gonads. Initial movements of the germ cells appear normal, and wild-type numbers of germ cells populate the gonads, indicating that p53 is required for germ cell death, but not migration. To our knowledge, this is the first report of a loss-of-function phenotype for Drosophila p53 in a non-sensitized background. The p53 phenotype is remarkably similar to that of outsiders (out) mutants. Here, we show that the out gene encodes a putative monocarboxylate transporter. Mutations in p53 and out show nonallelic noncomplementation. Interestingly, overexpression of p53 in primordial germ cells of out mutant embryos partially suppresses the out germ cell death phenotype, suggesting that p53 functions in germ cells either downstream of out or in a closely linked pathway. These findings inform models in which signaling between p53 and cellular metabolism are integrated to regulate programmed cell death decisions. [Abstract]

Enshell-Seijffers D, Lindon C, Morgan BA
The serine protease Corin is a novel modifier of the agouti pathway.
Development. 2007 Dec 5;
The hair follicle is a model system for studying epithelial-mesenchymal interactions during organogenesis. Although analysis of the epithelial contribution to these interactions has progressed rapidly, the lack of tools to manipulate gene expression in the mesenchymal component, the dermal papilla, has hampered progress towards understanding the contribution of these cells. In this work, Corin was identified in a screen to detect genes specifically expressed in the dermal papilla. It is expressed in the dermal papilla of all pelage hair follicle types from the earliest stages of their formation, but is not expressed elsewhere in the skin. Mutation of the Corin gene reveals that it is not required for morphogenesis of the hair follicle. However, analysis of the 'dirty blonde' phenotype of these mice reveals that the transmembrane protease encoded by Corin plays a critical role in specifying coat color and acts downstream of agouti gene expression as a suppressor of the agouti pathway. [Abstract]

Ohsugi M, Zheng P, Baibakov B, Li L, Dean J
Maternally derived FILIA-MATER complex localizes asymmetrically in cleavage-stage mouse embryos.
Development. 2007 Dec 5;
Initial cell lineages that presage the inner cell mass and extra-embryonic trophectoderm are established when eight blastomeres compact to form polarized morulae in preimplantation mouse development. FILIA has been identified as a binding partner to MATER (maternal antigen that embryos require; also known as NLRP5), which is encoded by a maternal effect gene. Products of each gene are detected in growing oocytes and, although transcripts are degraded before fertilization, the cognate proteins persist in early blastocysts. The two proteins co-localize to the cytocortex of ovulated eggs, where the stability of FILIA is dependent on the presence of MATER. After fertilization, FILIA-MATER complexes become asymmetrically restricted in the apical cytocortex of two-cell embryos due to their absence in regions of cell-cell contact. This asymmetry is reversible upon disaggregation of blastomeres of the two- and four-cell embryo. Each protein persists in cells of the preimplantation embryo, but the continuous cell-cell contact of 'inner' cells of the morulae seemingly precludes formation of the subcortical FILIA-MATER complex and results in cell populations that are marked by its presence ('outer') or absence ('inner'). Thus, the FILIA-MATER complex provides a molecular marker of embryonic cell lineages, but it remains to be determined if the molecular asymmetry established after the first cell division plays a role in cell fate determinations in the early mouse embryo. If so, the plasticity of the FILIA-MATER complex localization may reflect the regulative nature of preimplantation mouse development. [Abstract]

Alvarez-Medina R, Cayuso J, Okubo T, Takada S, Martí E
Wnt canonical pathway restricts graded Shh/Gli patterning activity through the regulation of Gli3 expression.
Development. 2007 Dec 5;
Dorsoventral patterning of the vertebrate nervous system is achieved by the combined activity of morphogenetic signals secreted from dorsal and ventral signalling centres. The Shh/Gli pathway plays a major role in patterning the ventral neural tube; however, the molecular mechanisms that limit target gene responses to specific progenitor domains remain unclear. Here, we show that Wnt1/Wnt3a, by signalling through the canonical beta-catenin/Tcf pathway, control expression of dorsal genes and suppression of the ventral programme, and that this role in DV patterning depends on Gli activity. Additionally, we show that Gli3 expression is controlled by Wnt activity. Identification and characterization of highly conserved non-coding DNA regions around the human Gli3 gene revealed the presence of transcriptionally active Tcf-binding sequences. These indicated that dorsal Gli3 expression might be directly regulated by canonical Wnt activity. In turn, Gli3, by acting as a transcriptional repressor, restricted graded Shh/Gli ventral activity to properly pattern the spinal cord. [Abstract]

Schneider I, Houston DW, Rebagliati MR, Slusarski DC
Calcium fluxes in dorsal forerunner cells antagonize -catenin and alter left-right patterning.
Development. 2008 Jan;135(1):75-84.
Establishment of the left-right axis is essential for normal organ morphogenesis and function. Ca(2+) signaling and cilia function in the zebrafish Kuppfer's Vesicle (KV) have been implicated in laterality. Here we describe an endogenous Ca(2+) release event in the region of the KV precursors (dorsal forerunner cells, DFCs), prior to KV and cilia formation. Manipulation of Ca(2+) release to disrupt this early flux does not impact early DFC specification, but results in altered DFC migration or cohesion in the tailbud at somite stages. This leads to disruption of KV formation followed by bilateral expression of asymmetrical genes, and randomized organ laterality. We identify beta-catenin inhibition as a Ca(2+)-signaling target and demonstrate that localized loss of Ca(2+) within the DFC region or DFC-specific activation of beta-catenin is sufficient to alter laterality in zebrafish. We identify a previously unknown DFC-like cell population in Xenopus and demonstrate a similar Ca(2+)-sensitive stage. As in zebrafish, manipulation of Ca(2+) release results in ectopic nuclear beta-catenin and altered laterality. Overall, our data support a conserved early Ca(2+) requirement in DFC-like cell function in zebrafish and Xenopus. [Abstract]

Zhu X, Bergles DE, Nishiyama A
NG2 cells generate both oligodendrocytes and gray matter astrocytes.
Development. 2008 Jan;135(1):145-57.
NG2 glia constitute a fourth major glial cell type in the mammalian central nervous system (CNS) that is distinct from other cell types. Although circumstantial evidence suggests that some NG2 glia differentiate into oligodendrocytes, their in vivo fate has not been directly examined. We have used the bacterial artificial chromosome (BAC) modification technique to generate transgenic mice that express DsRed or Cre specifically in NG2-expressing (NG2+) cells. In NG2DsRedBAC transgenic mice, DsRed was expressed specifically in NG2+ cells throughout the postnatal CNS. When the differentiation potential of NG2+ cells in vitro was examined using DsRed+NG2+ cells purified from perinatal transgenic brains, the majority of the cells either remained as NG2+ cells or differentiated into oligodendrocytes. In addition, DsRed+NG2+ cells also differentiated into astrocytes. The in vivo fate of NG2 glia was examined in mice that were double transgenic for NG2creBAC and the Cre reporter Z/EG. In the double transgenic mice, the Cre reporter EGFP was detected in myelinating oligodendrocytes and in a subpopulation of protoplasmic astrocytes in the gray matter of ventrolateral forebrain but not in fibrous astrocytes of white matter. These observations suggest that NG2+ cells are precursors of oligodendrocytes and some protoplasmic astrocytes in gray matter. [Abstract]

Su YQ, Sugiura K, Wigglesworth K, O'Brien MJ, Affourtit JP, Pangas SA, Matzuk MM, Eppig JJ
Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells.
Development. 2008 Jan;135(1):111-21.
Oocyte-derived bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control cumulus cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15(-/-) and Bmp15(-/-) Gdf9(+/-) double mutant cumulus cells, and in wild-type cumulus cells after removal of oocytes from cumulus-cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these cumulus cells. This indicates that oocytes regulate cumulus cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in cumulus cells compared with oocytes; and oocytes, in the absence of the surrounding cumulus cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant cumulus cells, lower levels were also detected in cumulus-cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant cumulus cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in cumulus cells, probably as compensation for oocyte deficiencies in cholesterol production. [Abstract]

Dunty WC, Biris KK, Chalamalasetty RB, Taketo MM, Lewandoski M, Yamaguchi TP
Wnt3a/ -catenin signaling controls posterior body development by coordinating mesoderm formation and segmentation.
Development. 2008 Jan;135(1):85-94.
Somitogenesis is thought to be controlled by a segmentation clock, which consists of molecular oscillators in the Wnt3a, Fgf8 and Notch pathways. Using conditional alleles of Ctnnb1 (beta-catenin), we show that the canonical Wnt3a/beta-catenin pathway is necessary for molecular oscillations in all three signaling pathways but does not function as an integral component of the oscillator. Small, irregular somites persist in abnormally posterior locations in the absence of beta-catenin and cycling clock gene expression. Conversely, Notch pathway genes continue to oscillate in the presence of stabilized beta-catenin but boundary formation is delayed and anteriorized. Together, these results suggest that the Wnt3a/beta-catenin pathway is permissive but not instructive for oscillating clock genes and that it controls the anterior-posterior positioning of boundary formation in the presomitic mesoderm (PSM). The Wnt3a/beta-catenin pathway does so by regulating the activation of the segment boundary determination genes Mesp2 and Ripply2 in the PSM through the activation of the Notch ligand Dll1 and the mesodermal transcription factors T and Tbx6. Spatial restriction of Ripply2 to the anterior PSM is ensured by the Wnt3a/beta-catenin-mediated repression of Ripply2 in posterior PSM. Thus, Wnt3a regulates somitogenesis by activating a network of interacting target genes that promote mesodermal fates, activate the segmentation clock, and position boundary determination genes in the anterior PSM. [Abstract]

Chen Z, Tan JL, Ingouff M, Sundaresan V, Berger F
Chromatin assembly factor 1 regulates the cell cycle but not cell fate during male gametogenesis in Arabidopsis thaliana.
Development. 2008 Jan;135(1):65-73.
The interdependence of cell cycle control, chromatin remodeling and cell fate determination remains unclear in flowering plants. Pollen development provides an interesting model, as it comprises only two cell types produced by two sequential cell divisions. The first division separates the vegetative cell from the generative cell. The generative cell divides and produces the two sperm cells, transported to the female gametes by the pollen tube produced by the vegetative cell. We show in Arabidopsis thaliana that loss of activity of the Chromatin assembly factor 1 (CAF1) pathway causes delay and arrest of the cell cycle during pollen development. Prevention of the second pollen mitosis generates a fraction of CAF1-deficient pollen grains comprising a vegetative cell and a single sperm cell, which both express correctly cell fate markers. The single sperm is functional and fertilizes indiscriminately either female gamete. Our results thus suggest that pollen cell fate is independent from cell cycle regulation. [Abstract]

Wills AA, Holdway JE, Major RJ, Poss KD
Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish.
Development. 2008 Jan;135(1):183-92.
The heart maintains structural and functional integrity during years of continual contraction, but the extent to which new cell creation participates in cardiac homeostasis is unclear. Here, we assessed cellular and molecular mechanisms of cardiac homeostasis in zebrafish, which display indeterminate growth and possess an unusual capacity to regenerate after acute cardiac injury. Lowering fish density in the aquarium triggered rapid animal growth and robust cardiomyocyte proliferation throughout the adult ventricle, greater than that observed during slow animal growth or size maintenance. Rapid animal growth also induced strong expression of the embryonic epicardial markers raldh2 (aldh1a2) and tbx18 in adult epicardial tissue. Pulse-chase dye labeling experiments revealed that the epicardium recurrently contributes cells to the ventricular wall, indicating an active homeostatic process. Inhibition of signaling by Fibroblast growth factors (Fgfs) decreased this epicardial supplementation of the ventricular wall in growing zebrafish, and led to spontaneous ventricular scarring in animals maintaining cardiac size. Our results demonstrate that the adult zebrafish ventricle grows and is maintained by cardiomyocyte hyperplasia, and that epicardial cells are added to the ventricle in an Fgf-dependent fashion to support homeostasis. [Abstract]

Akbari OS, Bae E, Johnsen H, Villaluz A, Wong D, Drewell RA
A novel promoter-tethering element regulates enhancer-driven gene expression at the bithorax complex in the Drosophila embryo.
Development. 2008 Jan;135(1):123-31.
A key question in our understanding of the cis-regulation of gene expression during embryonic development has been the molecular mechanism that directs enhancers to specific promoters within a gene complex. Promoter competition and insulators are thought to play a role in regulating these interactions. In the bithorax complex of Drosophila, the IAB5 enhancer is located 55 kb 3' of the Abdominal-B (Abd-B) promoter and 48 kb 5' of the abdominal-A (abd-A) promoter. Although roughly equidistant from the two promoters, IAB5 specifically interacts only with the Abdominal-B promoter, even though the enhancer and promoter are separated by at least two insulators. Here we demonstrate that a 255 bp element, located 40 bp 5' of the Abd-B transcriptional start site, has a novel cis-regulatory activity as it is able to tether IAB5 to the Abd-B promoter in transgenic embryos. The tethering element is sufficient to direct IAB5 to an ectopic promoter in competition assays. Deletion of the promoter-tethering element results in the redirection of enhancer-driven gene expression on transgenes. Taken together, these results provide evidence that specific long-range enhancer-promoter interactions in the bithorax complex are regulated by a tethering element 5' of the Abd-B promoter. We discuss a bioinformatic analysis of the tethering element across different Drosophila species and a possible molecular mechanism by which this element functions. We also examine existing evidence that this novel class of cis-regulatory elements might regulate enhancer-promoter specificity at other gene complexes. [Abstract]

Miller CM, Page-McCaw A, Broihier HT
Matrix metalloproteinases promote motor axon fasciculation in the Drosophila embryo.
Development. 2008 Jan;135(1):95-109.
Matrix metalloproteinases (MMPs) are a large conserved family of extracellular proteases, a number of which are expressed during neuronal development and upregulated in nervous system diseases. Primarily on the basis of studies using pharmaceutical inhibitors, MMPs have been proposed to degrade the extracellular matrix to allow growth cone advance during development and hence play largely permissive roles in axon extension. Here we show that MMPs are not required for axon extension in the Drosophila embryo, but rather are specifically required for the execution of several stereotyped motor axon pathfinding decisions. The Drosophila genome contains only two MMP homologs, Mmp1 and Mmp2. We isolated Mmp1 in a misexpression screen to identify molecules required for motoneuron development. Misexpression of either MMP inhibits the regulated separation/defasciculation of motor axons at defined choice points. Conversely, motor nerves in Mmp1 and Mmp2 single mutants and Mmp1 Mmp2 double mutant embryos are loosely bundled/fasciculated, with ectopic axonal projections. Quantification of these phenotypes reveals that the genetic requirement for Mmp1 and Mmp2 is distinct in different nerve branches, although generally Mmp2 plays the predominant role in pathfinding. Using both an endogenous MMP inhibitor and MMP dominant-negative constructs, we demonstrate that MMP catalytic activity is required for motor axon fasciculation. In support of the model that MMPs promote fasciculation, we find that the defasciculation observed when MMP activity is compromised is suppressed by otherwise elevating interaxonal adhesion - either by overexpressing Fas2 or by reducing Sema-1a dosage. These data demonstrate that MMP activity is essential for embryonic motor axon fasciculation. [Abstract]

Lee TV, Ding T, Chen Z, Rajendran V, Scherr H, Lackey M, Bolduc C, Bergmann A
The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously.
Development. 2008 Jan;135(1):43-52.
Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene. [Abstract]

Liu R, Woolner S, Johndrow JE, Metzger D, Flores A, Parkhurst SM
Sisyphus, the Drosophila myosin XV homolog, traffics within filopodia transporting key sensory and adhesion cargos.
Development. 2008 Jan;135(1):53-63.
Unconventional myosin proteins of the MyTH-FERM superclass are involved in intrafilopodial trafficking, are thought to be mediators of membrane-cytoskeleton interactions, and are linked to several forms of deafness in mammals. Here we show that the Drosophila myosin XV homolog, Sisyphus, is expressed at high levels in leading edge cells and their cellular protrusions during the morphogenetic process of dorsal closure. Sisyphus is required for the correct alignment of cells on opposing sides of the fusing epithelial sheets, as well as for adhesion of the cells during the final zippering/fusion phase. We have identified several putative Sisyphus cargos, including DE-cadherin (also known as Shotgun) and the microtubule-linked proteins Katanin-60, EB1, Milton and aPKC. These cargos bind to the Sisyphus FERM domain, and their binding is in some cases mutually exclusive. Our data suggest a mechanism for Sisyphus in which it maintains a balance between actin and microtubule cytoskeleton components, thereby contributing to cytoskeletal cross-talk necessary for regulating filopodial dynamics during dorsal closure. [Abstract]

Dos Santos G, Simmonds AJ, Krause HM
A stem-loop structure in the wingless transcript defines a consensus motif for apical RNA transport.
Development. 2008 Jan;135(1):133-43.
Although the subcellular localization of mRNA transcripts is a well-established mechanism for controlling protein localization, the basis for the recognition of mRNA localization elements is only now emerging. For example, although localization elements have been defined for many mRNAs that localize to apical cytoplasm in Drosophila embryos, no unifying properties have been identified within these elements. In this study, we identify and characterize an apical localization element in the 3'UTR of the Drosophila wingless mRNA. We show that this element, referred to as WLE3, is both necessary and sufficient for apical RNA transport. Full, unrestricted activity, however, requires the presence of one of several downstream potentiating elements. Comparison of WLE3 sequences within the Drosophila genus, and their predicted secondary structures, defines a highly conserved stem-loop structure. Despite these high levels of sequence and predicted structure conservation, however, mutagenesis shows significant leeway for both sequence and structure variation in the predicted stem-loop. Importantly, the features that emerge as crucial include an accessible distal helix sequence motif, which is also found in the predicted structures of other apical localization elements. [Abstract]

Moret F, Renaudot C, Bozon M, Castellani V
Semaphorin and neuropilin co-expression in motoneurons sets axon sensitivity to environmental semaphorin sources during motor axon pathfinding.
Development. 2007 Dec;134(24):4491-501.
Class III semaphorins (SemaIIIs) are intercellular cues secreted by surrounding tissues to guide migrating cells and axons in the developing organism. This chemotropic activity is crucial for the formation of nerves and vasculature. Intriguingly, SemaIIIs are also synthesized by neurons during axon pathfinding, but their function as intrinsic cues remains unknown. We have explored the role of Sema3A expression in motoneurons during spinal nerve development. Loss- and gain-of-function in the neural tube of the chick embryo were undertaken to target Sema3A expression in motoneurons while preserving Sema3A sources localized in peripheral tissues, known to provide important repulsive information for delineating the routes of motor axons towards their ventral or dorsal targets. Strikingly, Sema3A overexpression induced defasciculation and exuberant growth of motor axon projections into these normally non-permissive territories. Moreover, knockdown studies showed that motoneuronal Sema3A is required for correct spinal nerve compaction and dorsal motor axon extension. Further analysis of Sema3A gain- and loss-of-function in ex vivo models revealed that Sema3A in motoneurons sets the level of sensitivity of their growth cones to exogenous Sema3A exposure. This regulation is associated with post-transcriptional and local control of the availability of the Sema3A receptor neuropilin 1 at the growth cone surface. Thus, by modulating the strength of Sema3A-mediated environmental repulsive constraints, Sema3A in motoneurons enables axons to extend more or less far away from these repulsive sources. Such interplay between intrinsic and extrinsic Sema3A may represent a fundamental mechanism in the accurate specification of axon pathways. [Abstract]

Hui K, Fei GH, Saab BJ, Su J, Roder JC, Feng ZP
Neuronal calcium sensor-1 modulation of optimal calcium level for neurite outgrowth.
Development. 2007 Dec;134(24):4479-89.
Neurite extension and branching are affected by activity-dependent modulation of intracellular Ca(2+), such that an optimal window of [Ca(2+)] is required for outgrowth. Our understanding of the molecular mechanisms regulating this optimal [Ca(2+)](i) remains unclear. Taking advantage of the large growth cone size of cultured primary neurons from pond snail Lymnaea stagnalis combined with dsRNA knockdown, we show that neuronal calcium sensor-1 (NCS-1) regulates neurite extension and branching, and activity-dependent Ca(2+) signals in growth cones. An NCS-1 C-terminal peptide enhances only neurite branching and moderately reduces the Ca(2+) signal in growth cones compared with dsRNA knockdown. Our findings suggest that at least two separate structural domains in NCS-1 independently regulate Ca(2+) influx and neurite outgrowth, with the C-terminus specifically affecting branching. We describe a model in which NCS-1 regulates cytosolic Ca(2+) around the optimal window level to differentially control neurite extension and branching. [Abstract]

Estrada B, Gisselbrecht SS, Michelson AM
The transmembrane protein Perdido interacts with Grip and integrins to mediate myotube projection and attachment in the Drosophila embryo.
Development. 2007 Dec;134(24):4469-78.
The molecular mechanisms underlying muscle guidance and formation of myotendinous junctions are poorly understood both in vertebrates and in Drosophila. We have identified a novel gene that is essential for Drosophila embryonic muscles to form proper projections and stable attachments to epidermal tendon cells. Loss-of-function of this gene - which we named perdido (perd)-results in rounded, unattached muscles. perd is expressed prior to myoblast fusion in a subset of muscle founder cells, and it encodes a conserved single-pass transmembrane cell adhesion protein that contains laminin globular extracellular domains and a small intracellular domain with a C-terminal PDZ-binding consensus sequence. Biochemical experiments revealed that the Perd intracellular domain interacts directly with one of the PDZ domains of the Glutamate receptor interacting protein (Grip), another factor required for formation of proper muscle projections. In addition, Perd is necessary to localize Grip to the plasma membrane of developing myofibers. Using a newly developed, whole-embryo RNA interference assay to analyze genetic interactions, perd was shown to interact not only with Grip but also with multiple edematous wings, which encodes one subunit of the alphaPS1-betaPS integrin expressed in tendon cells. These experiments uncovered a previously unrecognized role for the alphaPS1-betaPS integrin in the formation of muscle projections during early stages of myotendinous junction development. We propose that Perd regulates projection of myotube processes toward and subsequent differentiation of the myotendinous junction by priming formation of a protein complex through its intracellular interaction with Grip and its transient engagement with the tendon cell-expressed laminin-binding alphaPS1-betaPS integrin. [Abstract]

Rao S, Lobov IB, Vallance JE, Tsujikawa K, Shiojima I, Akunuru S, Walsh K, Benjamin LE, Lang RA
Obligatory participation of macrophages in an angiopoietin 2-mediated cell death switch.
Development. 2007 Dec;134(24):4449-58.
Macrophages have a critical function in the recognition and engulfment of dead cells. In some settings, macrophages also actively signal programmed cell death. Here we show that during developmentally scheduled vascular regression, resident macrophages are an obligatory participant in a signaling switch that favors death over survival. This switch occurs when the signaling ligand angiopoietin 2 has the dual effect of suppressing survival signaling in vascular endothelial cells (VECs) and stimulating Wnt ligand production by macrophages. In response to the Wnt ligand, VECs enter the cell cycle and in the absence of survival signals, die from G1 phase of the cell cycle. We propose that this mechanism represents an adaptation to ensure that the macrophage and its disposal capability are on hand when cell death occurs. [Abstract]

Dyer N, Rebollo E, Domínguez P, Elkhatib N, Chavrier P, Daviet L, González C, González-Gaitán M
Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes.
Development. 2007 Dec;134(24):4437-47.
The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression. [Abstract]

Raft S, Koundakjian EJ, Quinones H, Jayasena CS, Goodrich LV, Johnson JE, Segil N, Groves AK
Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development.
Development. 2007 Dec;134(24):4405-15.
Temporal and spatial coordination of multiple cell fate decisions is essential for proper organogenesis. Here, we define gene interactions that transform the neurogenic epithelium of the developing inner ear into specialized mechanosensory receptors. By Cre-loxP fate mapping, we show that vestibular sensory hair cells derive from a previously neurogenic region of the inner ear. The related bHLH genes Ngn1 (Neurog1) and Math1 (Atoh1) are required, respectively, for neural and sensory epithelial development in this system. Our analysis of mouse mutants indicates that a mutual antagonism between Ngn1 and Math1 regulates the transition from neurogenesis to sensory cell production during ear development. Furthermore, we provide evidence that the transition to sensory cell production involves distinct autoregulatory behaviors of Ngn1 (negative) and Math1 (positive). We propose that Ngn1, as well as promoting neurogenesis, maintains an uncommitted progenitor cell population through Notch-mediated lateral inhibition, and Math1 irreversibly commits these progenitors to a hair-cell fate. [Abstract]

Esguerra CV, Nelles L, Vermeire L, Ibrahimi A, Crawford AD, Derua R, Janssens E, Waelkens E, Carmeliet P, Collen D, Huylebroeck D
Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination.
Development. 2007 Dec;134(24):4381-93.
During vertebrate development, signaling by the TGFbeta ligand Nodal is critical for mesoderm formation, correct positioning of the anterior-posterior axis, normal anterior and midline patterning, and left-right asymmetric development of the heart and viscera. Stimulation of Alk4/EGF-CFC receptor complexes by Nodal activates Smad2/3, leading to left-sided expression of target genes that promote asymmetric placement of certain internal organs. We identified Ttrap as a novel Alk4- and Smad3-interacting protein that controls gastrulation movements and left-right axis determination in zebrafish. Morpholino-mediated Ttrap knockdown increases Smad3 activity, leading to ectopic expression of snail1a and apparent repression of e-cadherin, thereby perturbing cell movements during convergent extension, epiboly and node formation. Thus, although the role of Smad proteins in mediating Nodal signaling is well-documented, the functional characterization of Ttrap provides insight into a novel Smad partner that plays an essential role in the fine-tuning of this signal transduction cascade. [Abstract]

Robertson EJ, Charatsi I, Joyner CJ, Koonce CH, Morgan M, Islam A, Paterson C, Lejsek E, Arnold SJ, Kallies A, Nutt SL, Bikoff EK
Blimp1 regulates development of the posterior forelimb, caudal pharyngeal arches, heart and sensory vibrissae in mice.
Development. 2007 Dec;134(24):4335-45.
The zinc-finger transcriptional repressor Blimp1 (Prdm1) controls gene expression patterns during differentiation of B lymphocytes and regulates epigenetic changes required for specification of primordial germ cells. Blimp1 is dynamically expressed at diverse tissue sites in the developing mouse embryo, but its functional role remains unknown because Blimp1 mutant embryos arrest at E10.5 due to placental insufficiency. To explore Blimp1 activities at later stages in the embryo proper, here we used a conditional inactivation strategy. A Blimp1-Cre transgenic strain was also exploited to generate a fate map of Blimp1-expressing cells. Blimp1 plays essential roles in multipotent progenitor cell populations in the posterior forelimb, caudal pharyngeal arches, secondary heart field and sensory vibrissae and maintains key signalling centres at these diverse tissues sites. Interestingly, embryos carrying a hypomorphic Blimp1(gfp) reporter allele survive to late gestation and exhibit similar, but less severe developmental abnormalities, whereas transheterozygous Blimp1(gfp/-) embryos with further reduced expression levels, display exacerbated defects. Collectively, the present experiments demonstrate that Blimp1 requirements in diverse cell types are exquisitely dose dependent. [Abstract]

Other recent books of interest.
Development. 2007 Dec;134(24):4314. [Abstract]

Huylebroeck D
An ;all in' account of Smad biology and TGF signaling.
Development. 2007 Dec;134(24):4313-4. [Abstract]

Recent Articles in Developmental Biology

Ignatius MS, Moose HE, El-Hodiri HM, Henion PD
colgate/hdac1 repression of foxd3 expression is required to permit mitfa-dependent melanogenesis.
Dev Biol. 2007 Nov 9; .
Neural crest-derived pigment cell development has been used extensively to study cell fate specification, migration, proliferation, survival and differentiation. Many of the genes and regulatory mechanisms required for pigment cell development are conserved across vertebrates. The zebrafish mutant colgate (col)/histone deacetylase1 (hdac1) has reduced numbers, delayed differentiation and decreased migration of neural crest-derived melanophores and their precursors. In hdac1(col) mutants normal numbers of premigratory neural crest cells are induced. Later, while there is only a slight reduction in the number of neural crest cells in hdac1(col) mutants, there is a severe reduction in the number of mitfa-positive melanoblasts suggesting that hdac1 is required for melanoblast specification. Concomitantly, there is a significant increase in and prolonged expression of foxd3 in neural crest cells in hdac1(col) mutants. We found that partially reducing Foxd3 expression in hdac1(col) mutants rescues mitfa expression and the melanophore defects in hdac1(col) mutants. Furthermore, we demonstrate the ability of Foxd3 to physically interact at the mitfa promoter. Because mitfa is required for melanoblast specification and development, our results suggest that hdac1 is normally required to suppress neural crest foxd3 expression thus de-repressing mitfa resulting in melanogenesis by a subset of neural crest-derived cells. [Abstract]

Matus DQ, Magie CR, Pang K, Martindale MQ, Thomsen GH
The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution.
Dev Biol. 2007 Sep 26;
Hedgehog signaling is an important component of cell-cell communication during bilaterian development, and abnormal Hedgehog signaling contributes to disease and birth defects. Hedgehog genes are composed of a ligand ("hedge") domain and an autocatalytic intein ("hog") domain. Hedgehog (hh) ligands bind to a conserved set of receptors and activate downstream signal transduction pathways terminating with Gli/Ci transcription factors. We have identified five intein-containing genes in the anthozoan cnidarian Nematostella vectensis, two of which (NvHh1 and NvHh2) contain definitive hedgehog ligand domains, suggesting that to date, cnidarians are the earliest branching metazoan phylum to possess definitive Hh orthologs. Expression analysis of NvHh1 and NvHh2, the receptor NvPatched, and a downstream transcription factor NvGli (a Gli3/Ci ortholog) indicate that these genes may have conserved roles in planar and trans-epithelial signaling during gut and germline development, while the three remaining intein-containing genes (NvHint1,2,3) are expressed in a cell-type-specific manner in putative neural precursors. Metazoan intein-containing genes that lack a hh ligand domain have previously only been identified within nematodes. However, we have identified intein-containing genes from both Nematostella and in two newly annotated lophotrochozoan genomes. Phylogenetic analyses suggest that while nematode inteins may be derived from an ancestral true hedgehog gene, the newly identified cnidarian and lophotrochozoan inteins may be orthologous, suggesting that both true hedgehog and hint genes may have been present in the cnidarian-bilaterian ancestor. Genomic surveys of N. vectensis suggest that most of the components of both protostome and deuterostome Hh signaling pathways are present in anthozoans and that some appear to have been lost in ecdysozoan lineages. Cnidarians possess many bilaterian cell-cell signaling pathways (Wnt, TGFbeta, FGF, and Hh) that appear to act in concert to pattern tissues along the oral-aboral axis of the polyp. Cnidarians represent a diverse group of animals with a predominantly epithelial body plan, and perhaps selective pressures to pattern epithelia resulted in the ontogeny of the hedgehog pathway in the common ancestor of the Cnidaria and Bilateria. [Abstract]

Takaesu NT, Bulanin DS, Johnson AN, Orenic TV, Newfeld SJ
A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles of Drosophila.
Dev Biol. 2007 Oct 24;
A previous genetic analysis of a reporter gene carrying a 375-bp region from a dpp intron (dppMX-lacZ) revealed that the Wingless and Dpp pathways are required to activate dpp expression in posterior spiracle formation. Here we report that within the dppMX region there is an enhancer with binding sites for TCF and Mad that are essential for activating dppMX expression in posterior spiracles. There is also a binding site for Brinker likely employed to repress dppMX expression. This combinatorial enhancer may be the first identified with the ability to integrate temporally distinct positive (TCF and Mad) and negative (Brinker) inputs in the same cells. Cuticle studies on a unique dpp mutant lacking this enhancer showed that it is required for viability and that the Filzkorper are U-shaped rather than straight. Together with gene expression data from these mutants and from brk mutants, our results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is associated with dorsal-ventral patterning and is necessary for designating the posterior spiracle field. The second is governed by the combinatorial enhancer and begins during germ band retraction. The second round appears necessary for proper spiracle internal morphology and fusion with the remainder of the tracheal system. Intriguingly, several aspects of dpp posterior spiracle expression and function are similar to demonstrated roles for Wnt and BMP signaling in proximal-distal outgrowth of the mammalian embryonic lung. [Abstract]

Hsu HJ, Lafever L, Drummond-Barbosa D
Diet controls normal and tumorous germline stem cells via insulin-dependent and -independent mechanisms in Drosophila.
Dev Biol. 2007 Nov 17;
The external environment influences stem cells, but this process is poorly understood. Our previous work showed that germline stem cells (GSCs) respond to diet via neural insulin-like peptides (DILPs) that act directly on the germ line to upregulate stem cell division and cyst growth under a protein-rich diet in Drosophila. Here, we report that DILPs specifically control the G2 phase of the GSC cell cycle via phosphoinositide-3 kinase (PI3K) and dFOXO, and that a separate diet mediator regulates the G1 phase. Furthermore, GSC tumors, which escape the normal stem cell regulatory microenvironment, or niche, still respond to diet via both mechanisms, indicating that niche signals are not required for GSCs to sense or respond to diet. Our results document the effects of diet and insulin-like signals on the cell cycle of stem cells within an intact organism and demonstrate that the response to diet requires multiple signals. Moreover, the retained ability of GSC tumors to respond to diet parallels the long known connections between diet, insulin signaling, and cancer risk in humans. [Abstract]

Fiehler RW, Wolff T
Nemo is required in a subset of photoreceptors to regulate the speed of ommatidial rotation.
Dev Biol. 2007 Oct 30;
Both dramatic and subtle morphogenetic movements are of paramount importance in molding cells and tissues into functional form. Cells move either independently or as populations and the distance traversed by cells varies greatly, but in all cases, the output is common: to organize cells into or within organs and epithelia. In the developing Drosophila eye, a highly specialized, 90 degrees rotational movement of subsets of cells imposes order by polarizing the retinal epithelium across its dorsoventral axis. This process was proposed to take place in two 45 degrees steps, with the second under control of the gene nemo (nmo), a serine/threonine kinase. While our analysis confirms that these subsets of cells, the ommatidial precursors, do stall at 45 degrees , we demonstrate that nmo is also required through most of the first 45 degrees of rotation to regulate the speed at which the ommatidial precursors move. In addition, although the precursors reach only the halfway point by the end of larval life, this work demonstrates that patterning events that occur during pupal life move the ommatidial units an additional 15 degrees . A re-analysis of nmo mosaic clones indicates that nmo is required in photoreceptors R1, R6 and R7 for normal orientation. This work also demonstrates that two major isoforms of nmo rescue the nmo(P1) phenotype. Finally, a dominant modifier screen of a nmo misexpression background identified genomic regions that potentially regulate rotation. The results presented here suggest a model in which a motor for rotation is established in a nemo-dependent fashion in a subset of cells. [Abstract]

Ralston A, Rossant J
Cdx2 acts downstream of cell polarization to cell-autonomously promote trophectoderm fate in the early mouse embryo.
Dev Biol. 2007 Nov 13;
The first lineage decision during mouse development is the establishment of trophectoderm and inner cell mass lineages, morphologically distinguishable at the blastocyst stage. The Caudal-like transcription factor Cdx2 is required for repression of inner cell mass genes Oct4 and Nanog in the trophectoderm. Expression of Cdx2 in the trophectoderm is thus one of the earliest known events in lineage determination. However, it is not clear whether the Cdx2 expression pattern is the cause or the consequence of this first lineage decision. Here, we show that Cdx2 is initially ubiquitously expressed, and becomes progressively upregulated in outside, future trophectoderm cells prior to blastocyst formation. Ubiquitous Cdx2 expression begins around the time of cell polarization, but we show that cell polarization is independent of zygotic Cdx2. Finally, we show functionally that Cdx2 is downstream of lineage allocation since Cdx2 mutant cells, which show cell-autonomous defects in expression of Oct4, Nanog, and the trophectoderm marker Eomesodermin, do not preferentially contribute to inner cell mass in chimeric blastocysts. Cdx2 therefore appears to act downstream of the first lineage decision, suggesting that processes influencing lineage allocation or morphogenesis may regulate Cdx2 expression along the inside/outside axis of the embryo. [Abstract]

Surkova S, Kosman D, Kozlov K, Manu E, Samsonova AA, Spirov A, Vanario-Alonso CE, Samsonova M, Reinitz J
Characterization of the Drosophila segment determination morphome.
Dev Biol. 2007 Nov 4;
Here we characterize the expression of the full system of genes which control the segmentation morphogenetic field of Drosophila at the protein level in one dimension. The data used for this characterization are quantitative with cellular resolution in space and about 6 min in time. We present the full quantitative profiles of all 14 segmentation genes which act before the onset of gastrulation. The expression patterns of these genes are first characterized in terms of their average or typical behavior. At this level, the expression of all of the genes has been integrated into a single atlas of gene expression in which the expression levels of all genes in each cell are specified. We show that expression domains do not arise synchronously, but rather each domain has its own specific dynamics of formation. Moreover, we show that the expression domains shift position in the direction of the cephalic furrow, such that domains in the anlage of the segmented germ band shift anteriorly while those in the presumptive head shift posteriorly. The expression atlas of integrated data is very close to the expression profiles of individual embryos during the latter part of the blastoderm stage. At earlier times gap gene domains show considerable variation in amplitude, and significant positional variability. Nevertheless, an average early gap domain is close to that of a median individual. In contrast, we show that there is a diversity of developmental trajectories among pair-rule genes at a variety of levels, including the order of domain formation and positional accuracy. We further show that this variation is dynamically reduced, or canalized, over time. As the first quantitatively characterized morphogenetic field, this system and its behavior constitute an extraordinarily rich set of materials for the study of canalization and embryonic regulation at the molecular level. [Abstract]

Liu J, Qian L, Han Z, Wu X, Bodmer R
Spatial specificity of mesodermal even-skipped expression relies on multiple repressor sites.
Dev Biol. 2007 Oct 25;
Individual cardiac progenitors emerge at defined positions within each segment in the trunk mesoderm. Their specification depends on segmental information from the pre-patterned ectoderm, which provides positional information to the underlying cardiac mesoderm via inductive signals. This pattern is further reinforced by repressive interactions between transcription factors that are expressed in neighboring sets of cardiac progenitors. For example, even-skipped (eve) and ladybird early (lbe) gene products mark adjacent cardiac cell clusters within a segment, and their antagonistic interaction results in mutually exclusive expression domains. Lbe acts directly on the eve mesodermal enhancer (eme) to participate in restricting its expression anteriorly. We hypothesized that additional repressive activities must regulate the precise pattern of eve expression in the cardiac mesoderm via this enhancer. In this study, we identified two additional repressor motifs: 4 copies of an "AT"-rich motif (M1a-d) and 2 copies of an "GC"-rich motif (M2a,b), which when mutated cause expansion of eme-dependent reporter gene expression. We have also examined potential negative regulators of eve and found that their overexpression is sufficient to repress eve as well as the eme enhancer via these sites. Our data suggest that a combination of factors is likely to interact with multiple essential repressor sites to confer precise spatial specificity of eve expression in the cardiac mesoderm. [Abstract]

Nimmo R, Woollard A
Worming out the biology of Runx.
Dev Biol. 2007 Nov 17;
Runx family transcription factors have risen to prominence over the last few years because of the increasing evidence implicating them as key regulators of the choice between cell proliferation and differentiation during development and carcinogenesis. Runx factors have been found to be involved in diverse developmental processes, ranging from hematopoiesis to neurogenesis, and are increasingly being linked with various human cancers. In this review, we examine the case for Runx factors as key regulators of cell proliferation in various developmental situations, a role that predisposes Runx mutations as causative agents in oncogenesis. We discuss the evidence that Runx factors regulate, and are regulated by, core components of the cell cycle machinery, and focus our attention on the solo Runx gene, rnt-1, in Caenorhabditis elegans, an organism that we feel has much to offer the Runx field. [Abstract]

Zammit PS, Cohen A, Buckingham ME, Kelly RG
Integration of embryonic and fetal skeletal myogenic programs at the myosin light chain 1f/3f locus.
Dev Biol. 2007 Nov 9;
The genetic control of skeletal muscle differentiation at the onset of myogenesis in the embryo is relatively well understood compared to the formation of muscle during the fetal period giving rise to the bulk of skeletal muscle fibers at birth. The Mlc1f/3f (Myl1) locus encodes two alkali myosin light chains, Mlc1f and Mlc3f, from two promoters that are differentially regulated during development. The Mlc1f promoter is active in embryonic, fetal and adult fast skeletal muscle whereas the Mlc3f promoter is upregulated during fetal development and remains on in adult fast skeletal muscle. Two enhancer elements have been identified at the mammalian Mlc1f/3f locus, a 3' element active at all developmental stages and an intronic enhancer activated during fetal development. Here, using transgenesis, we demonstrate that these enhancers act combinatorially to confer the spatial, temporal and quantitative expression profile of the endogenous Mlc3f promoter. Using double reporter transgenes we demonstrate that each enhancer can activate both Mlc1f and Mlc3f promoters in vivo, revealing enhancer sharing rather than exclusive enhancer-promoter interactions. Finally, we demonstrate that the fetal activated enhancer contains critical E-box myogenic regulatory factor binding sites and that enhancer activation is impaired in vivo in the absence of myogenin but not in the absence of innervation. Together our observations provide insights into the regulation of fetal myogenesis and the mechanisms by which temporally distinct genetic programs are integrated at a single locus. [Abstract]

Bonner J, Gribble SL, Veien ES, Nikolaus OB, Weidinger G, Dorsky RI
Proliferation and patterning are mediated independently in the dorsal spinal cord downstream of canonical Wnt signaling.
Dev Biol. 2007 Nov 9;
Canonical Wnt signaling can regulate proliferation and patterning in the developing spinal cord, but the relationship between these functions has remained elusive. It has been difficult to separate the distinct activities of Wnts because localized changes in proliferation could conceivably alter patterning, and gain and loss of function experiments have resulted in both types of defects. To resolve this issue we have investigated canonical Wnt signaling in the zebrafish spinal cord using multiple approaches. We demonstrate that Wnt signaling is required initially for proliferation throughout the entire spinal cord, and later for patterning dorsal progenitor domains. Furthermore, we find that spinal cord patterning is normal in embryos after cell division has been pharmacologically blocked. Finally, we determine the transcriptional mediators of Wnt signaling that are responsible for patterning and proliferation. We show that tcf7 gene knockdown results in dorsal patterning defects without decreasing the mitotic index in dorsal domains. In contrast, tcf3 gene knockdown results in a reduced mitotic index without affecting dorsal patterning. Together, our work demonstrates that proliferation and patterning in the developing spinal cord are separable events that are regulated independently by Wnt signaling. [Abstract]

Prodon F, Sardet C, Nishida H
Cortical and cytoplasmic flows driven by actin microfilaments polarize the cortical ER-mRNA domain along the a-v axis in ascidian oocytes.
Dev Biol. 2007 Nov 17;
Cellular mechanisms generating the polarized redistribution of maternal Type I postplasmic/PEM mRNAs in ascidian oocytes remain unknown. We have previously shown that PEM-1 mRNA is associated with a network of rough cortical Endoplasmic Reticulum (cER) polarized along the animal-vegetal (a-v) axis forming a cER-mRNA domain in mature oocytes. We now investigate the a-v polarization of this cER-mRNA domain during meiotic maturation using H. roretzi and C. intestinalis. We show that the cER and Hr-PEM-1 aggregate as interconnected cortical patches at the cell periphery before maturation, which uniformly spread out during maturation and form a reticulated organization enriched in the vegetal hemisphere at the end of maturation. Time-lapse video recordings coupled with micromanipulations reveal that stereotyped surface, cortical and cytoplasmic flows accompany the vegetal shift of the cER-mRNA domain and mitochondria-rich myoplasm. Treatments with cytochalasin B and nocodazole indicate that both polarization of the cER-mRNA domain and mitochondria-rich myoplasm and cortical and cytoplasmic flows depend on actin cytoskeleton, but not microtubules. Using cortical fragments prepared from maturing oocytes coupled with high resolution immuno/in situ localization, we have further analyzed the effects of these inhibitors on the reorganizations the cER network and Hr-PEM-1 mRNA. We show that before maturation starts, Hr-PEM-1 mRNAs are already associated with the cER, and actin cytoskeleton inhibitors disturb their association. Finally, we hypothesize that Germinal Vesicle Break Down (GVBD) triggers an actomyosin-dependant cortical flow which directs the a-v polarization of ascidian oocytes. [Abstract]

Perryn ED, Czirók A, Little CD
Vascular sprout formation entails tissue deformations and VE-cadherin-dependent cell-autonomous motility.
Dev Biol. 2007 Nov 4;
Embryonic and fetal vascular sprouts form within constantly expanding tissues. Nevertheless, most biological assays of vascular spouting are conducted in a static mechanical milieu. Here we study embryonic mouse allantoides, which normally give raise to an umbilical artery and vein. However, when placed in culture, allantoides assemble a primary vascular network. Unlike other in vitro assays, allantoic primordial vascular cells are situated on the upper surface of a cellular layer that is engaged in robust spreading motion. Time-lapse imaging allows quantification of primordial vascular cell motility as well as the underlying mesothelial tissue motion. Specifically, we calculate endothelial cell-autonomous motion by subtracting the tissue-level mesothelial motion from the total endothelial cell displacements. Formation of new vascular polygons is hindered by administration of function-blocking VE-cadherin antibodies. Time-lapse recordings reveal that (1) cells at the base of sprouts normally move distally "over" existing sprout cells to form new tip-cells; and (2) loss of VE-cadherin activity prevents this motile behavior. Thus, endothelial cell-cell-adhesion-based motility is required for the advancement of vascular sprouts within a moving tissue environment. To the best of our knowledge, this is the first study that couples endogenous tissue dynamics to assembly of vascular networks in a mammalian system. [Abstract]

Raschperger E, Neve EP, Wernerson A, Hultenby K, Pettersson RF, Majumdar A
The coxsackie and adenovirus receptor (CAR) is required for renal epithelial differentiation within the zebrafish pronephros.
Dev Biol. 2007 Nov 12;
The coxsackie and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily and a component of vertebrate tight junctions. CAR protein is widely expressed in fish and mammals in organs of epithelial origin suggesting possible functions in epithelial biology. In order to gain insight into its function, we knocked the CAR gene down in zebrafish using antisense morpholinos. We identified a requirement for CAR in the terminal differentiation of glomerular podocytes and pronephric tubular epithelia. Podocytes differentiate in CAR morphants but are not able to elaborate a regularly patterned architecture of foot processes. In the tubules, CAR was required for the apposition of plasma membranes from adjacent epithelial cells but did not appear to be necessary for the formation of tight junctions. Additionally, tubular epithelia lacking CAR were not able to elaborate apical brush border microvilli. These results establish a requirement for CAR in the terminal differentiation of renal glomerular and tubular cell types. [Abstract]

Le Gall M, De Mattei C, Giniger E
Molecular separation of two signaling pathways for the receptor, Notch.
Dev Biol. 2007 Dec 4;
Notch is required for many aspects of cell fate specification and morphogenesis during development, including neurogenesis and axon guidance. We here provide genetic and biochemical evidence that Notch directs axon growth and guidance in Drosophila via a "non-canonical", i.e. non-Su(H)-mediated, signaling pathway, characterized by association with the adaptor protein, Disabled, and Trio, an accessory factor of the Abl tyrosine kinase. We find that forms of Notch lacking the binding sites for its canonical effector, Su(H), are nearly inactive for the cell fate function of the receptor, but largely or fully active in axon patterning. Conversely, deletion from Notch of the binding site for Disabled impairs its action in axon patterning without disturbing cell fate control. Finally, we show by co-immunoprecipitation that Notch protein is physically associated in vivo with both Disabled and Trio. Together, these data provide evidence for an alternate Notch signaling pathway that mediates a postmitotic, morphogenetic function of the receptor. [Abstract]

Lundin VF, Srayko M, Hyman AA, Leroux MR
Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans.
Dev Biol. 2007 Oct 24;
The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in alpha-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development. [Abstract]

Kim J, Lauderdale JD
Overexpression of pairedless Pax6 in the retina disrupts corneal development and affects lens cell survival.
Dev Biol. 2007 Nov 9;
The Pax6 transcription factor is required for multiple aspects of vertebrate eye development. The Pax6 gene encodes isoforms that either contain (Pax6+PD) or lack (Pax6DeltaPD) the N-terminal paired-box DNA-binding domain, in addition to the homeodomain. Alternative promoters control the expression of Pax6+PD and Pax6DeltaPD in the eye. Using a modified bacterial artificial chromosome (BAC) transgene that specifically expresses Pax6DeltaPD, but not paired-containing Pax6, in the normal endogenous pattern, we show that overexpression of Pax6DeltaPD causes a severe microphthalmic phenotype in both wild-type and Pax6-deficient (Sey(/+)) mice in a dosage-dependent manner. The microphthalmic phenotype is due to lens degeneration during embryonic development. Lens development initiates correctly, but cells in the lens undergo apoptotic cell death between E12 and E13. Concomitantly, in these mice, changes in Bmp4, Msx1, and Wnt2b expression were observed in the mesenchymal cells of the developing cornea. To visualize Pax6DeltaPD expression, we developed a dual-reporter Pax6 BAC transgene in which EGFP and DsRed demonstrate paired-containing and pairedless transcripts, respectively. In BAC transgenic mice, DsRed is predominantly expressed in the peripheral neural retina during early eye development, but not in the developing lens or cornea. Later DsRed is strongly expressed in the developing ciliary body, but not in the iris. We suggest that the ratio of Pax6+PD and Pax6DeltaPD isoforms in the distal retina is important for both cornea and lens development, either directly by controlling transcription of necessary growth factors or indirectly by controlling development of the distal neural retina. [Abstract]

Shovlin TC, Durcova-Hills G, Surani A, McLaren A
Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells.
Dev Biol. 2007 Nov 17;
Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, "early", in comparison with EG cells derived after PGC colonisation of the genital ridge, "late" and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification. [Abstract]

Smith J, Kraemer E, Liu H, Theodoris C, Davidson E
A spatially dynamic cohort of regulatory genes in the endomesodermal gene network of the sea urchin embryo.
Dev Biol. 2007 Nov 9;
A gene regulatory network subcircuit comprising the otx, wnt8, and blimp1 genes accounts for a moving torus of gene expression that sweeps concentrically across the vegetal domain of the sea urchin embryo. Here we confirm by mutation the inputs into the blimp1cis-regulatory module predicted by network analysis. Its essential design feature is that it includes both activation and autorepression sites. The wnt8 gene is functionally linked into the subcircuit in that cells receiving this ligand generate a beta-catenin/Tcf input required for blimp1 expression, while the wnt8 gene in turn requires a Blimp1 input. Their torus-like spatial expression patterns and gene regulatory analysis indicate that the genes even-skipped and hox11/13b are also entrained by this subcircuit. We verify the cis-regulatory inputs of even-skipped predicted by network analysis. These include activation by beta-catenin/Tcf and Blimp1, repression within the torus by Hox11/13b, and repression outside the torus by Tcf in the absence of Wnt8 signal input. Thus even-skipped and hox11/13b, along with blimp1 and wnt8, are members of a cohort of torus genes with similar regulatory inputs and similar, though slightly out-of-phase, expression patterns, which reflect differences in cis-regulatory design. [Abstract]

Devine CA, Key B
Robo-Slit interactions regulate longitudinal axon pathfinding in the embryonic vertebrate brain.
Dev Biol. 2007 Nov 7;
The early network of axons in the embryonic brain provides connectivity between functionally distinct regions of the nervous system. While many of the molecular interactions driving commissural pathway formation have been deciphered, the mechanisms underlying the development of longitudinal tracts remain unclear. We have identified here a role for the Roundabout (Robo) family of axon guidance receptors in the positioning of longitudinally projecting axons along the dorsoventral axis in the embryonic zebrafish forebrain. Using a loss-of-function approach, we established that Robo family members exhibit complementary functions in the tract of the postoptic commissure (TPOC), the major longitudinal tract in the forebrain. Robo2 acted initially to split the TPOC into discrete fascicles upon entering a broad domain of Slit1a expression in the ventrocaudal diencephalon. In contrast, Robo1 and Robo3 restricted the extent of defasciculation of the TPOC. In this way, the complementary roles of Robo family members balance levels of fasciculation and defasciculation along this trajectory. These results demonstrate a key role for Robo-Slit signaling in vertebrate longitudinal axon guidance and highlight the importance of context-specific guidance cues during navigation within complex pathways. [Abstract]

Ronco M, Uda T, Mito T, Minelli A, Noji S, Klingler M
Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus.
Dev Biol. 2007 Oct 10;
Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb'hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb'wingless, Gb'dachshund, Gb'aristaless and Gb'Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb'hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb'hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb'hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments. [Abstract]

Boyle S, Misfeldt A, Chandler KJ, Deal KK, Southard-Smith EM, Mortlock DP, Baldwin HS, de Caestecker M
Fate mapping using Cited1-CreER(T2) mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia.
Dev Biol. 2007 Oct 24;
Classic tissue recombination and in vitro lineage tracing studies suggest that condensed metanephric mesenchyme (MM) gives rise to nephronic epithelium of the adult kidney. However, these studies do not distinguish between cap mesenchyme and pre-tubular aggregates comprising the condensed MM, nor do they establish whether these cells have self-renewing capacity. To address these questions, we generated Cited1-CreER(T2) BAC transgenic mice, which express tamoxifen-regulated Cre recombinase exclusively in the cap mesenchyme. Fate mapping was performed by crossing these mice with the Rosa26R(LacZ) reporter line and evaluating the location and cellular characteristics of LacZ positive cells at different time points following tamoxifen injection. These studies confirmed expected results from previous in vitro analysis of MM cell fate, and provide in vivo evidence that the cap mesenchyme does not contribute to collecting duct epithelium in the adult. Furthermore, by exploiting the temporally regulated Cre recombinase, these studies show that nephronic epithelium arising at different stages of nephrogenesis has distinct spatial distribution in the adult kidney, and demonstrate for the first time that the cap mesenchyme includes a population of self-renewing epithelial progenitor cells. [Abstract]

Stollewerk A, Seyfarth EA
Evolutionary changes in sensory precursor formation in arthropods: Embryonic development of leg sensilla in the spider Cupiennius salei.
Dev Biol. 2007 Nov 17;
We describe here for the first time the development of mechanosensory organs in a chelicerate, the spider Cupiennius salei. It has been shown previously that the number of external sense organs increases with each moult. While stage 1 larvae do not have any external sensory structures, stage 2 larvae show a stereotyped pattern of touch sensitive 'tactile hairs' on their legs. We show that these mechanosensory organs develop during embryogenesis. In contrast to insects, groups of sensory precursors are recruited from the leg epithelium, rather than single sensory organ progenitors. The groups increase by proliferation, and neural cells delaminate from the cluster, which migrate away to occupy a position proximal to the accessory cells of the sense organ. In addition, we describe the development of putative internal sense organs, which do not differentiate until larval stage 2. We show by RNA interference that, similar to Drosophila, proneural genes are responsible for the formation and subtype identity of sensory organs. Furthermore, we demonstrate an additional function for proneural genes in the coordinated invagination and migration of neural cells during sensory organ formation in the spider. [Abstract]

Akiyama T, Kamimura K, Firkus C, Takeo S, Shimmi O, Nakato H
Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface.
Dev Biol. 2007 Nov 1;
Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp(DeltaN)), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp(DeltaN) shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex. [Abstract]

Andrews W, Barber M, Hernadez-Miranda LR, Xian J, Rakic S, Sundaresan V, Rabbitts TH, Pannell R, Rabbitts P, Thompson H, Erskine L, Murakami F, Parnavelas JG
The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons.
Dev Biol. 2007 Nov 13;
Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1(-/-), but not Robo2(-/-), animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1(-/-) mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1(-/-)/Slit2(-/-) mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1(-/-), Robo2(-/-) and Slit1(-/-)/Slit2(-/-), but not in Slit1(-/-) mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons. [Abstract]

Wyngaarden L, Hopyan S
Plasticity of proximal-distal cell fate in the mammalian limb bud.
Dev Biol. 2007 Nov 7;
Normal morphogenesis depends on location-specific behaviours of cells. There are circumstances in the embryo where it would be advantageous for cells to be flexible in establishing their fate, such as in the limb bud where some cells cross between Homeobox (Hox) expression domains. It is not known how flexible cell fate determination is in the embryonic limb bud, nor is the sequence and timing of this process clear. By transposing small groups of dye and genetically labelled limb bud cells in cultured mouse embryos, we show that mesenchymal cells are capable of altering their expression of Hox genes to match that of their local environment in response to instructive mesenchymal cues. Plasticity of Hox expression wanes during embryogenesis, and is lost earlier in the proximal limb bud than distally. These findings are consistent with a model of progressive fate determination in a zone that encompasses the entirety of the limb bud mesenchyme, and suggest a mechanism for the maintenance of sharp Hox boundaries despite cell transit across them. [Abstract]

Wang L, Wang ZB, Zhang X, Fitzharris G, Baltz JM, Sun QY, Liu XJ
Brefeldin A disrupts asymmetric spindle positioning in mouse oocytes.
Dev Biol. 2007 Oct 13;
Polar body formation in oocytes is an extreme form of asymmetric cell division, but what regulates the asymmetric spindle positioning and cytokinesis is poorly understood. During mouse oocyte maturation, the metaphase I spindle forms at the center but then moves to the cortex prior to anaphase I and first polar body emission. We show here that treating denuded mouse oocytes with brefeldin A, an inhibitor of Golgi-based membrane fusion, abolished the asymmetric positioning of the metaphase I spindle and resulted in the formation of two half-size metaphase II eggs, instead of a full-sized egg and a polar body. The normal metaphase II spindle is similarly asymmetrically positioned in the mature egg, where the spindle lies with its axis parallel to the cortex but becomes perpendicular before anaphase II and emission of the second polar body. When ovulated eggs were activated with strontium in the presence of brefeldin A, the metaphase II spindle failed to assume perpendicular position, and the chromosomes separated without the extrusion of the second polar body. Remarkably, symmetric cytokinesis began following a 3 h delay, forming two half-size eggs each containing a pronucleus. BFA-sensitive intracellular vesicular transport is therefore required for spindle positioning in both MI and MII. [Abstract]

Chen J, Downs SM
AMP-activated protein kinase is involved in hormone-induced mouse oocyte meiotic maturation in vitro.
Dev Biol. 2007 Sep 29;
We have previously shown that AMP-activated protein kinase (AMPK) can induce the resumption of meiosis in mouse oocytes maintained in meiotic arrest in vitro. The present study was carried out to determine whether AMPK activation is involved in hormone-induced maturation. Follicle-stimulating hormone (FSH) and the EGF-like peptide, amphiregulin (AR), are potent inducers of maturation in cumulus cell-enclosed oocytes (CEO). Within 3 h of FSH treatment, phospho-acetyl CoA carboxylase (ACC) levels were increased in germinal vesicle (GV)-stage oocytes when compared to non-stimulated controls and remained elevated throughout 9 h of culture, indicating AMPK activation. A similar response to AR was observed after 6 h of culture. Using anti-PT172 antibody (binds only to activated AMPK), Western analysis demonstrated active AMPK in both FSH- or AR-treated GV-stage oocytes within 6 h. The AMPK inhibitors, compound C and adenine 9-beta-d-arabinofuranoside (araA), blocked FSH- or AR-induced meiotic resumption and ACC phosphorylation, further supporting a causal role for AMPK in hormone-induced meiotic resumption. Immunocytochemistry using anti-PT172-AMPK antibody showed an increased diffuse cytoplasmic staining and more intense punctate staining in the germinal vesicles of oocytes following treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or with FSH or AR, and this staining was eliminated by compound C or a blocking peptide for the anti-PT172 antibody. Staining of oocytes from hCG-stimulated mice with the anti-PT172 antibody also showed pronounced label in the germinal vesicles within 1-2 h. Furthermore, in oocytes from all groups, active AMPK was always observed in association with the condensed chromosomes of maturing oocytes. Taken together, these results support a role for AMPK in FSH and AR-induced maturation in vitro and hCG-induced maturation in vivo. [Abstract]

Kurihara Y, Kawamura Y, Uchijima Y, Amamo T, Kobayashi H, Asano T, Kurihara H
Maintenance of genomic methylation patterns during preimplantation development requires the somatic form of DNA methyltransferase 1.
Dev Biol. 2007 Oct 30;
DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development. [Abstract]

Kurzhals RL, Tie F, Stratton CA, Harte PJ
Drosophila ESC-like can substitute for ESC and becomes required for Polycomb silencing if ESC is absent.
Dev Biol. 2007 Oct 25;
The Drosophila esc-like gene (escl) encodes a protein very similar to ESC. Like ESC, ESCL binds directly to the E(Z) histone methyltransferase via its WD region. In contrast to ESC, which is present at highest levels during embryogenesis and low levels thereafter, ESCL is continuously present throughout development and in adults. ESC/E(Z) complexes are present at high levels mainly during embryogenesis but ESCL/E(Z) complexes are found throughout development. While depletion of either ESCL or ESC by RNAi in S2 and Kc cells has little effect on E(Z)-mediated methylation of histone H3 lysine 27 (H3K27), simultaneous depletion of ESCL and ESC results in loss of di- and trimethyl-H3K27, indicating that either ESC or ESCL is necessary and sufficient for di- and trimethylation of H3K27 in vivo. While E(Z) complexes in S2 cells contain predominantly ESC, in ESC-depleted S2 cells, ESCL levels rise dramatically and ESCL replaces ESC in E(Z) complexes. A mutation in escl that produces very little protein is viable and exhibits no phenotypes but strongly enhances esc mutant phenotypes, suggesting they have similar functions. esc escl double homozygotes die at the end of the larval period, indicating that the well-known "maternal rescue" of esc homozygotes requires ESCL. Furthermore, maternal and zygotic over-expression of escl fully rescues the lethality of esc null mutant embryos that contain no ESC protein, indicating that ESCL can substitute fully for ESC in vivo. These data thus indicate that ESC and ESCL play similar if not identical functions in E(Z) complexes in vivo. Despite this, when esc is expressed normally, escl appears to be entirely dispensable, at least for development into morphologically normal fertile adults. Furthermore, the larval lethality of esc escl double mutants, together with the lack of phenotypes in the escl mutant, further suggests that in wild-type (esc(+)) animals it is the post-embryonic expression of esc, not escl, that is important for development of normal adults. Thus escl appears to function in a backup capacity during development that becomes important only when normal esc expression is compromised. [Abstract]

Recent Articles in Seminars in Cell & Developmental Biology

Greiling TM, Clark JI
The transparent lens and cornea in the mouse and zebra fish eye.
Semin Cell Dev Biol. 2007 Oct 30; .
The lens and cornea combine to form a single optical element in which transparency and refraction are the fundamental biophysical characteristics required for a functional visual system. Although lens and cornea have different cellular and extracellular specializations that contribute to transparency and refraction, their development is closely related. In the embryonic mouse, the developing cornea and lens separate early. In contrast, zebra fish lens and cornea remain connected during early development and the optical properties of the cornea and lens observed by slit lamp and quasielastic laser light scattering spectroscopy (QLS) are more similar in the zebra fish eye than in the mouse eye. Optical similarities between cornea and lens of zebra fish may be the result of similarities in the cellular development of the cornea and lens. [Abstract]

Yoshinaga K
Review of factors essential for blastocyst implantation for their modulating effects on the maternal immune system.
Semin Cell Dev Biol. 2007 Oct 16;
Pituitary and ovarian hormones prepare the endometrium for successful blastocyst implantation and support its process directly or indirectly through the action of growth factors, cytokines and other molecules. Many of the blastocyst implantation essential factors (BIEFs) are modulators of the maternal immune system. Since little is known as to the action of these molecules on the uterine lymphocytes, its clarification is imperative to the understanding of the process of blastocyst implantation. [Abstract]

Piatigorsky J
Lens and cornea: The "refracton hypothesis"
Semin Cell Dev Biol. 2007 Oct 30; [Abstract]

Hejtmancik JF
Congenital cataracts and their molecular genetics.
Semin Cell Dev Biol. 2007 Oct 10;
Cataract can be defined as any opacity of the crystalline lens. Congenital cataract is particularly serious because it has the potential for inhibiting visual development, resulting in permanent blindness. Inherited cataracts represent a major contribution to congenital cataracts, especially in developed countries. While cataract represents a common end stage of mutations in a potentially large number of genes acting through varied mechanisms in practice most inherited cataracts have been associated with a subgroup of genes encoding proteins of particular importance for the maintenance of lens transparency and homeostasis. The increasing availability of more detailed information about these proteins and their functions and is making it possible to understand the pathophysiology of cataracts and the biology of the lens in general. [Abstract]

Aghajanova L, Hamilton AE, Giudice LC
Uterine receptivity to human embryonic implantation: Histology, biomarkers, and transcriptomics.
Semin Cell Dev Biol. 2007 Oct 22;
Embryonic implantation is a dynamic process of paracrine interactions between the maternal compartment and the conceptus and involves a receptive endometrium and a developmentally competent blastocyst. Herein, we review histology, clinical approaches, and the promise of transcriptomics in elucidating mechanisms underlying implantation and development of biomarkers of uterine receptivity-with an eye to diagnose and treat implantation-based disorders of miscarriage, fetal growth restriction, pre-eclampsia, and infertility. [Abstract]

Jonasova K, Kozmik Z
Eye evolution: Lens and cornea as an upgrade of animal visual system.
Semin Cell Dev Biol. 2007 Oct 13;
Lens-containing eyes are a feature of surprisingly broad spectrum of organisms across the animal kingdom that represent a significant improvement of simple eye composed of just photoreceptor cells and pigment cells. It is apparent that such an upgrade of animal visual system has originated numerous times during evolution since many distinct strategies to enhance light refraction through the use of lens and cornea have been utilized. In addition to having an ancient role in prototypical eye formation Pax transcription factors were convergently recruited for regulation of structurally diverse crystallins and genes affecting morphogenesis of various lens-containing eyes. [Abstract]

Zelenka PS, Arpitha P
Coordinating cell proliferation and migration in the lens and cornea.
Semin Cell Dev Biol. 2007 Oct 18;
Migration is a complex process for epithelial tissues, because the epithelium must move as an intact sheet to preserve its barrier function. The requirement for structural integrity is met by coupling cell-to-matrix and cell-to-cell adhesion at the cellular level, and by coordinating cell proliferation and cell migration in the tissue as a whole. Proliferation is suppressed at the migrating cell front, allowing cells in this region to remain tightly packed while advancing rapidly. At the same time, proliferation is enhanced in a region behind the advancing cell front to expand the epithelial cell sheet. This review considers the extracellular signals and intracellular signaling pathways that regulate these processes in the lens and corneal epithelium, with emphasis on the commonalities that link these tissues. [Abstract]

Roberts RM, Chen Y, Ezashi T, Walker AM
Interferons and the maternal-conceptus dialog in mammals.
Semin Cell Dev Biol. 2007 Oct 16;
Two-way communication between the conceptus and the mother during early pregnancy is essential if the pregnancy is to survive. In this review, our primary focus is on biochemical communication between the conceptus and mother in the ruminant ungulate species. We emphasize, in particular, the role played by interferon-tau (IFNT) in triggering maternal responses in cattle and sheep and how maternal factors intervene to up-regulate IFNT gene (IFNT) expression in trophoblast. However, we also consider the possibility that different signaling cytokines or the physical presence of trophoblast may induce a partial IFN response in endometrium of those species where there is no evidence for large scale trophoblast IFN production. Conceivably, disparate signaling mechanisms trigger common downstream events necessary to secure a successful pregnancy. [Abstract]

McNiece I
Delivering cellular therapies: Lessons learned from ex vivo culture and clinical applications of hematopoietic cells.
Semin Cell Dev Biol. 2007 Dec;18(6):
Advances in stem cell biology and cellular therapy have led to promising treatments in a range of incurable diseases. However, it is unclear whether primitive stem cells can be delivered to damage tissue for regeneration of functional mature cells or stem cells must be stimulated to differentiate into mature cells in vitro and these cells delivered to patients. A range of other questions remains to be determined including how to formulate cellular products for in vivo delivery and how to undertake pharmacological testing of cellular products. Insights into these questions can be obtained from hematopoietic stem cells (HSC) which have been used for the past 50 years in bone marrow transplantation for regeneration of blood cells in patients undergoing high dose chemotherapy to treat cancer. The differentiation of HSC into mature blood cells is controlled by proteins called hematopoietic growth factors and these factors have been used to generate cellular products in vitro for clinical applications. This chapter will review some of the results of cellular therapies performed with HSC and the lessons that can be learned from these studies. [Abstract]

Brooke G, Cook M, Blair C, Han R, Heazlewood C, Jones B, Kambouris M, Kollar K, McTaggart S, Pelekanos R, Rice A, Rossetti T, Atkinson K
Therapeutic applications of mesenchymal stromal cells.
Semin Cell Dev Biol. 2007 Dec;18(6):
Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. They have been demonstrated to play a role in tissue repair and regeneration in both preclinical and clinical studies. They also have remarkable immunosuppressive properties. We describe their application in settings that include the cardiovascular, central nervous, gastrointestinal, renal, orthopaedic and haematopoietic systems. Manufacturing of MSC for clinical trials is also discussed. Since tissue matching between MSC donor and recipient does not appear to be required, MSC may be the first cell type able to be used as an "off-the-shelf" therapeutic product. [Abstract]

Colman A
Semin Cell Dev Biol. 2007 Dec;18(6): [Abstract]

Malhotra JD, Kaufman RJ
The endoplasmic reticulum and the unfolded protein response.
Semin Cell Dev Biol. 2007 Dec;18(6):
The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases. [Abstract]

Sambasivan R, Tajbakhsh S
Skeletal muscle stem cell birth and properties.
Semin Cell Dev Biol. 2007 Dec;18(6):
Development and maintenance of an abundant tissue such as skeletal muscle poses several challenges. Curiously, not all skeletal muscle stem cells are born alike, since diverse genetic pathways can specify their birth. Stem and progenitor cells that establish the tissue during development, those that maintain its homeostasis, as well as participate in its regeneration have generated considerable interest. The ability to distinguish stem cells from more committed progenitors throughout prenatal and postnatal life has guided researchers to identify stem cell properties and characterise their niche. These properties include markers that influence cell behaviour and mode of division during normal development, after trauma and cell transplantations. This review addresses these issues from a developmental perspective. [Abstract]

Zweigerdt R
The art of cobbling a running pump-Will human embryonic stem cells mend broken hearts?
Semin Cell Dev Biol. 2007 Dec;18(6):
The heart is one of the least regenerative organs in the body, and highly vulnerable to the increasing incidence of cardiovascular diseases in an aging world population. Cell-based approaches aimed at cardiac repair have recently caused great public excitement. But clinical trials of patients' own skeletal myoblasts or bone marrow cells for transplantation have been disappointing. Human embryonic stem cells (hESCs) form bona fide cardiomyocytes in vitro which are readily generated in mass culture and are being tested in animal models of heart damage. The early results, while encouraging, underscore that much remains to be done. This review focuses on the many challenges that remain before hESCs-mediated repair of the human heart becomes a reality. [Abstract]

Jester JV
Corneal crystallins and the development of cellular transparency.
Semin Cell Dev Biol. 2007 Oct 2;
Past studies have established that the cornea like the lens abundantly expresses a few water-soluble enzyme/proteins in a taxon specific fashion. Based on these similarities it has been proposed that the lens and the cornea form a structural unit, the 'refracton', that has co-evolved through gene sharing to maximize light transmission and refraction to the retina. Thus far, the analogy between corneal crystallins and lens crystallins has been limited to similarities in the abundant expression, with few reports concerning their structural function. This review covers recent studies that establish a clear relationship between expression of corneal crystallins and light scattering from corneal stromal cells, i.e. keratocytes, that support a structural role for corneal crystallins in the development of transparency similar to that of lens crystallins that would be consistent with the 'refracton' hypothesis. [Abstract]

Alison MR, Choong C, Lim S
Application of liver stem cells for cell therapy.
Semin Cell Dev Biol. 2007 Dec;18(6):
The worldwide shortage of donor livers to transplant end stage liver disease patients has prompted the search for alternative cell therapies for intractable liver disease. Embryonic stem cells can be readily differentiated into hepatocytes, and their transplantation into animals has improved liver function in the absence of teratoma formation: their use in bioartificial liver support is an obvious application. In animal models of liver disease, adopting strategies to provide a selective advantage for transplanted foetal or adult hepatocytes have proved highly effective in repopulating recipient livers, but the poor success of today's hepatocyte transplants can be attributed to the lack of a clinically applicable procedure to force a similar repopulation of the human liver. The activation of bipotential hepatic progenitor cells is clearly vital for survival in many cases of acute liver failure, but surprisingly little progress has been made with these cells in terms of transplantation. Finally there is the controversial subject of autologous bone marrow, and while the contribution of these indigenous cells to liver turnover seems at best, trivial, results from a small number of phase 1 studies of transplantation of bone marrow to cirrhotic patients have been moderately encouraging. [Abstract]

Caramelo JJ, Parodi AJ
How sugars convey information on protein conformation in the endoplasmic reticulum.
Semin Cell Dev Biol. 2007 Dec;18(6):
The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic reticulum to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and not completely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase and a glucosidase that creates monoglucosylated epitopes in glycans transferred in protein N-glycosylation or removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The purpose of the review is to describe the most significant recent findings on the mechanism of glycoprotein folding and assembly quality control and to discuss the main still unanswered questions. [Abstract]

Buck TM, Wright CM, Brodsky JL
The activities and function of molecular chaperones in the endoplasmic reticulum.
Semin Cell Dev Biol. 2007 Dec;18(6):
Most proteins in the secretory pathway are translated, folded, and subjected to quality control at the endoplasmic reticulum (ER). These processes must be flexible enough to process diverse protein conformations, yet specific enough to recognize when a protein should be degraded. Molecular chaperones are responsible for this decision making process. ER associated chaperones assist in polypeptide translocation, protein folding, and ER associated degradation (ERAD). Nevertheless, we are only beginning to understand how chaperones function, how they are recruited to specific substrates and assist in folding/degradation, and how unique chaperone classes make quality control "decisions". [Abstract]

van Driel R
Chromatin is wonderful stuff.
Semin Cell Dev Biol. 2007 Oct;18(5): [Abstract]

Hall VJ, Li JY, Brundin P
Restorative cell therapy for Parkinson's disease: A quest for the perfect cell.
Semin Cell Dev Biol. 2007 Dec;18(6):
The development of a cell therapy for the neurodegenerative disorder Parkinson's disease is a realistic ambition. It is pursued by researchers and companies alike, and spans different donor tissue types of embryonic, fetal and adult origins. In this review, we briefly outline the past and current status of research and clinical trials with cell transplantation in Parkinson's disease. We discuss studies on donor tissue derived from embryonic ventral mesencephalon and assess the current research on various forms of stem cells of both embryonic and adult origins in the quest to develop a cell-based therapy for this debilitating movement disorder. [Abstract]

Pellegrini G, De Luca M, Arsenijevic Y
Towards therapeutic application of ocular stem cells.
Semin Cell Dev Biol. 2007 Dec;18(6):
The first example of cell therapy using cultured stem cells dates back to 1981, when it was demonstrated that human epidermis could be grown in the laboratory and transplanted onto burnt patients to reconstitute a functional epidermis [Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979;76(11):5665-8: [1]; Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;1:75-8: [2]; Gallico 3rd GG, O'Connor NEMJ, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311(7):448-51: [3]]. This was the onset of regenerative medicine, which is now being developed also in many other fields including ophthalmology. Emerging cell therapies for the restoration of sight have focused on two areas of the eye that are critical for visual function, the cornea and the retina. The relatively easy access of the cornea, the homogeneity of the cells forming the different layers of the corneal epithelium and the improvement of cell culture protocols are leading to considerable success in corneal epithelium restoration. Rebuilding the entire cornea is however still far from reality. The restoration of the retina has recently been achieved in different animal models of retinal degeneration using immature photoreceptors, and two other promising strategies have been demonstrated: transplantation of endothelial precursors to rescue retinal vessels and neurons, and transplantation of retinal pigmented epithelial cells to preserve vision over the long term. The relevance of these approaches will be discussed in function of the disease targeted. [Abstract]

Docherty K, Bernardo AS, Vallier L
Embryonic stem cell therapy for diabetes mellitus.
Semin Cell Dev Biol. 2007 Dec;18(6):
There is a compelling need to develop novel therapies for diabetes mellitus. Recent successes in the transplantation of islets of Langerhans are seen as a major breakthrough. However, there is huge disparity between potential recipients and the availability of donor tissue. Human embryonic stem cells induced to form pancreatic beta cells could provide a replenishable supply of tissue. Early studies on the spontaneous differentiation of mouse embryonic stem cells have laid the foundation for a more directed approach based on recapitulating the events that occur during the development of the pancreas in the mouse. A high yield of definitive endoderm has been achieved, and although beta-like cells can be generated in a step-wise manner, the efficiency is still low and the final product is not fully differentiated. Future challenges include generating fully functional islet cells under Xeno-free and chemically defined conditions and circumventing the need for immunosuppression. [Abstract]

Sexton T, Umlauf D, Kurukuti S, Fraser P
The role of transcription factories in large-scale structure and dynamics of interphase chromatin.
Semin Cell Dev Biol. 2007 Oct;18(5):
The genome is spatially organized inside nuclei, with chromosomes and genes occupying preferential positions relative to each other and to various nuclear landmarks. What drives this organization is unclear, but recent findings suggest there are extensive intra- and inter-chromosomal communications between various genomic regions that appear to play important roles in genome function. Here we review transcription factories, distinct sub-nuclear foci where nascent transcription occurs. We argue that the spatially restricted, limited number of transcription sites compels transcribed regions of the genome to dynamically self-organize into tissue-specific conformations, thus playing a major role in the three-dimensional interphase organization of the genome. [Abstract]

Kostova Z, Tsai YC, Weissman AM
Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation.
Semin Cell Dev Biol. 2007 Dec;18(6):
Endoplasmic reticulum-associated degradation (ERAD) represents the primary means of quality control within the secretory pathway. Critical to this process are ubiquitin protein ligases (E3s) which, together with ubiquitin conjugating enzymes (E2s), mediate the ubiquitylation of proteins targeted for degradation from the ER. In this chapter we review our knowledge of both Saccharomyces cerevisiae and mammalian ERAD ubiquitin ligases. We focus on recent insights into these E3s, their associated proteins and potential mechanisms of action. [Abstract]

Suzuki T
Cytoplasmic peptide:N-glycanase and catabolic pathway for free N-glycans in the cytosol.
Semin Cell Dev Biol. 2007 Dec;18(6):
Peptide:N-glycanase (PNGase) releases N-glycans from glycoproteins/glycopeptides. Cytoplasmic PNGase is widely recognized as a component of machinery for ER-associated degradation (ERAD), i.e. proteasomal degradation of misfolded, newly synthesized (glyco)proteins that have been exported from the ER. The enzyme belongs to the "transglutaminase superfamily" that contains a putative catalytic triad of cysteine, histidine, and aspartic acid. The mammalian orthologues of PNGase contain the N-terminal PUB domain that serves as the protein-protein interaction domain. The C-terminus of PNGase was recently found to be a novel carbohydrate-binding domain. Taken together, these observations indicate that C-terminus of mammalian PNGase is important for recognition of the substrates while N-terminus of this enzyme is involved in assembly of a degradation complex. [Abstract]

Kanehara K, Kawaguchi S, Ng DT
The EDEM and Yos9p families of lectin-like ERAD factors.
Semin Cell Dev Biol. 2007 Dec;18(6):
Protein quality control pathways monitor the folding of newly synthesized proteins throughout the cell. Irreversibly misfolded proteins are sorted and degraded to neutralize their potential toxicity. In the secretory pathway, multiple strategies have evolved to test the wide diversity of molecules that traffic through the endoplasmic reticulum. The organelle has adapted the use of N-linked glycans to signal protein folding states. The signals are read by the EDEM and Yos9 protein families that take substrates out of folding cycles for degradation. [Abstract]

Veatch SL
From small fluctuations to large-scale phase separation: Lateral organization in model membranes containing cholesterol.
Semin Cell Dev Biol. 2007 Oct;18(5):
Macroscopic coexisting liquid phases are readily observed in certain model membranes containing cholesterol and at least two other lipid components. Recent fluorescence microscopy and deuterium NMR work indicates that submicron fluctuations are also found, in vesicles with near-critical lipid compositions. In principle, the magnitude of critical fluctuations can be controlled by changing temperature, or through other means of shifting the phase boundary such as including impurities or cross-linking components. Critical fluctuations are dynamic submicron domains in model membranes, and provide a plausible physical mechanism to produce putative 'raft' domains in cholesterol-rich biomembranes. [Abstract]

Raasi S, Wolf DH
Ubiquitin receptors and ERAD: A network of pathways to the proteasome.
Semin Cell Dev Biol. 2007 Dec;18(6):
The elimination of misfolded proteins, known as protein quality control, is an essential cellular process. Removal of misfolded proteins from the secretory pathway depends on their recognition in the endoplasmic reticulum (ER) followed by their retrograde transport into the cytosol for degradation. The AAA-ATPase Cdc48/p97 facilitates the translocation of misfolded ER-proteins into the cytosol. Cdc48/p97 can dock onto the ER-membrane via direct interaction with ER-membrane proteins and/or indirectly via its substrate-recruiting cofactors, which interact with the ubiquitylated substrates at the membrane. This tight interaction in conjunction with the conformational changes induced upon ATP hydrolysis within Cdc48/p97 is thought to provide the driving force for the translocation reaction. Subsequently, a series of protein-protein interactions between the Cdc48/p97 complex, its cofactors, and the ubiquitylated substrates is instrumental for the proper delivery of the ER substrates to the proteasome. These protein-protein interactions are governed mainly by ubiquitin-fold and ubiquitin-binding domains. [Abstract]

Langowski J, Heermann DW
Computational modeling of the chromatin fiber.
Semin Cell Dev Biol. 2007 Oct;18(5):
The packing of the genomic DNA in the living cell is essential for its biological function. While individual aspects of the genome architecture, such as DNA and nucleosome structure or the arrangement of chromosome territories are well studied, much information is missing for a unified description of cellular DNA at all its structural levels. Computer modeling can contribute to such a description. We present here some typical approaches to models of the chromatin fiber, including different amounts of detail in the description of the local nucleosome structure. The main results from our simulations are that the physical properties of the chromatin fiber can be well described by a simplified model consisting of cylinder-like nucleosomes connected by flexible DNA segments, with a geometry determined by the bending and twisting angles between nucleosomes. Randomness in the local geometry - such as random absence of linker histone H1 - leads to a dramatic increase in the chromatin fiber flexibility. Furthermore, we show that chromatin is much more flexible to bending than to stretching, and that the structure of the chromatin fiber favors the formation of sharp bends. [Abstract]

Bantseev V, McCanna DJ, Driot JY, Sivak JG
The effects of toxicological agents on the optics and mitochondria of the lens and the mitochondria of the corneal epithelium.
Semin Cell Dev Biol. 2007 Aug 19;
This review describes how the morphology and distribution of the mitochondria of the epithelium and the superficial fibre layers of the lens were studied using confocal scanning laser microscopy. This research was correlated with an effort to use the optical properties of the intact lens in culture as a proxy for the cornea in measuring ocular toxicity. In turn, this work led to the confocal study of the in vitro and then the in vivo cornea and their possible use in using confocal microscopy to evaluate the effect of various treatments on the integrity of the surface of the eye. Finally, confocal examination of the mitochondria of the lens has provided an avenue to the study of mitochondrial dynamics. [Abstract]

Recent Articles in Differentiation

Rauch J, Knoch TA, Solovei I, Teller K, Stein S, Buiting K, Horsthemke B, Langowski J, Cremer T, Hausmann M, Cremer C
Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei.
Differentiation. 2007 Nov 26; .
Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to achieve this goal are discussed. [Abstract]

Nogai H, Rosowski M, Grün J, Rietz A, Debus N, Schmidt G, Lauster C, Janitz M, Vortkamp A, Lauster R
Follistatin antagonizes transforming growth factor-beta3-induced epithelial-mesenchymal transition in vitro: implications for murine palatal development supported by microarray analysis.
Differentiation. 2007 Nov 20;
Epithelial-mesenchymal transition (EMT) is involved in normal embryonic development as well as in tumor progression and invasiveness. This process is also known to be a crucial step in palatogenesis during fusion of the bi-lateral palatal processes. Disruption of this step results in a cleft palate, which is among the most frequent birth defects in humans. A number of genes and encoded proteins have been shown to play a role in this developmental stage. The central role is attributed to the cytokine transforming growth factor-beta3 (TGF-beta3), which is expressed in the medial edge epithelium (MEE) already before the fusion process. The MEE covers the tips of the growing palatal shelves and eventually undergoes EMT or programmed cell death (apoptosis). TGF-beta3 is described to induce EMT in embryonic palates. With regard to the early expression of this molecule before the fusion process, it is not well understood which mechanisms prevent the TGF-beta3 producing epithelial cells from undergoing differentiation precociously. We used the murine palatal fusion to study the regulation of EMT. Specifically, we analyzed the MEE for the expression of known antagonists of TGF-beta molecules using in situ hybridization and detected the gene coding for Follistatin to be co-expressed with TGF-beta3. Further, we could show that Follistatin directly binds to TGF-beta3 and that it completely blocks TGF-beta3-induced EMT of the normal murine mammary gland (NMuMG) epithelial cell line in vitro. In addition, we analyzed the gene expression profile of NMuMG cells during TGF-beta3-induced EMT by microarray hybridization, detecting strong changes in the expression of apoptosis-regulating genes. [Abstract]

Alvisi M, De Arcangelis V, Ciccone L, Palombi V, Alessandrini M, Nemoz G, Molinaro M, Adamo S, Naro F
V1a vasopressin receptor expression is modulated during myogenic differentiation.
Differentiation. 2007 Nov 16;
Neurohypophyseal peptides potently stimulate myogenic differentiation by acting through different receptors of the same family. Here, we show that L6C5 myogenic cells express, at a high density, a single class of V1a Arg8-vasopressin (AVP) receptor. The expression of the vasopressin receptor of type 1a (V1aR) is significantly higher in proliferating myoblasts than in differentiated myotubes. The differentiation-related decrease of V1aR expression was evident both at the mRNA and at the protein level as shown by the reduction of [(3)H]-AVP binding. However, in L6C5 cells transfected with a synthetic construct containing the luciferase gene driven by the 2 kb upstream region of V1aR, we observed a stimulation of the activity of the promoter when the cells were cultured in differentiative medium. The down-regulation of the V1aR correlated with a decreased half-life of its mRNA (half-life 5.86+/-0.74 hr in 10% fetal bovine serum [FBS] versus 3.53+/-0.72 hr in 1% FBS). Cyclosporine A and dexamethasone, but not 5'-azacytidine, treatments of cells in differentiation medium restored the V1aR level to that measured in proliferating L6C5 cells, thus confirming the role of post-transcriptional mechanisms in the modulation of V1aR expression. Taken together, these data show that mRNA stability plays a role in modulating protein expression during the myogenic differentiation process. [Abstract]

Gilon M, Sher N, Cohen S, Gat U
Transcriptional activation of a subset of hair keratin genes by the NF-kappaB effector p65/RelA.
Differentiation. 2007 Nov 16;
The hair follicle is an intricate miniature organ dedicated to the production of the structural hair fiber, which is largely composed of hair keratin (HK) proteins. Many developmental pathways contribute to hair follicle development; however, the molecular control of HK genes is still far from being resolved. Because the nuclear factor (NF)-kappaB pathway is known to be involved in the morphogenesis of the hair follicle, we explored the possibility that it may also regulate HK expression. To this end, we analyzed the effect of p65/RelA, an NF-kappaB effector, on HK regulatory regions using transient transfections into tissue culture cells. Reporter assays on cells transfected with HK promoter constructs and real-time polymerase chain reaction analysis of endogenous HK gene activity demonstrated that p65 induces transcriptional activation of several HK genes of human and mouse origin, primarily that of acidic hair keratin 5 (Ha5). Focusing on the highly responsive human Ha5 gene, we defined the major NF-kappaB/RelA binding sites in its regulatory region and showed the direct binding of p65 to these sites using gel shift assays. We further show, using immunohistochemistry on human hair follicle sections, that p65 is co-expressed with HKs in the hair shaft compartment and may thus be the effector that mediates the NF-kappaB pathway's activity, which recently was genetically demonstrated to be active in the same region. Thus, we provide evidence for a previously unknown function of NF-kappaB in hair formation-direct activation of HK target genes-a function that may shed light on some of the symptoms of ectodermal dysplasias. [Abstract]

Humphrey GW, Wang YH, Hirai T, Padmanabhan R, Panchision DM, Newell LF, McKay RD, Howard BH
Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells.
Differentiation. 2007 Nov 15;
In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His-->Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons. [Abstract]

Tomokiyo A, Maeda H, Fujii S, Wada N, Shima K, Akamine A
Development of a multipotent clonal human periodontal ligament cell line.
Differentiation. 2007 Nov 15;
The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1-17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog, as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1-17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 (FGF-R1), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1-17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1-17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1-17 based on its differentiation stage. [Abstract]

Wiesmeijer K, Krouwels IM, Tanke HJ, Dirks RW
Chromatin movement visualized with photoactivable GFP-labeled histone H4.
Differentiation. 2007 Nov 15;
The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles. [Abstract]

Graichen R, Xu X, Braam SR, Balakrishnan T, Norfiza S, Sieh S, Soo SY, Tham SC, Mummery C, Colman A, Zweigerdt R, Davidson BP
Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.
Differentiation. 2007 Nov 15;
Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations <10 muM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations >/=15 muM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process. [Abstract]

Jean S, Moss T
A ubiquitin-conjugating enzyme, ube2d3.2, regulates xMLK2 and pronephros formation in Xenopus.
Differentiation. 2007 Nov 15;
The Jun N-terminal kinase kinase kinase MLK2 is required for the formation of the pronephros during early Xenopus development. Here, we have used a yeast 2-hybrid screen to identify proteins that interact with and regulate xMLK2. Using an N-terminal polypeptide encompassing the SH3 and kinase domains of xMLK2 as bait, five independent cDNAs were identified, all of which encoded a Xenopus ubiquitin conjugating enzyme, ube2d3.2. Ube2d3.2 is a functional E2 enzyme expressed maternally and in a tissue-restricted fashion during development. Ectopic expression of ube2d3.2 inhibits formation of the pronephric tubules, resulting in a phenotype very similar to the loss of xMLK2 function. Because ube2d3.2 is also shown to limit accumulation of xMLK2, it is likely that this effect is direct, although other explanations are possible. Ube2d3.2 is thus probably an endogenous regulator of xMLK2, and hence of JNK activity. [Abstract]

Ning H, Lin G, Fandel T, Banie L, Lue TF, Lin CS
Insulin growth factor signaling mediates neuron-like differentiation of adipose tissue-derived stem cells.
Differentiation. 2007 Nov 15;
Our previous study showed that adipose tissue-derived stem cells (ADSC) could be induced by isobutylmethylxanthine (IBMX) to differentiate into neuron-like cells. In the present study, ADSC were treated with IBMX in the presence or in the absence of each of eight specific inhibitors of different signaling pathways (JAK/STAT, PKA, PI3K, MEK, Wnt/Frizzled, ERK/MAPK, TGF-beta, and insulin growth factor [IGF]-I). PPP, a specific inhibitor of IGF-I signaling, was the only inhibitor that showed significant inhibition of IBMX-induced ADSC neuronal differentiation, as determined by changes in cell morphology in the initial screening. Further examination by immunofluorescence staining showed that the neuronal marker, beta-III-tubulin, was highly induced in IBMX-treated ADSC, and the induction was significantly suppressed by PPP. Western blotting, followed by densitometry showed that PPP suppressed IBMX-induced beta-III-tubulin expression by 43%, 88%, and 84% when used to treat the cells for 1, 3, and 24 hr, respectively. Treatment of ADSC with IBMX also led to the phosphorylation of IGF-I receptor at tyrosine 1136 (Y1136), as determined by immunofluorescence staining with an antibody that reacts specifically with Y1136. This effect was also abrogated by PPP. Thus, the IBMX-induced neuron-like differentiation of ADSC is mediated by IGF signaling through the phosphorylation of IGF-IR at Y1136. [Abstract]

Differentiation. 2007 Nov;75(9): [Abstract]

Campbell JS, Johnson MM, Bauer RL, Hudkins KL, Gilbertson DG, Riehle KJ, Yeh MM, Alpers CE, Fausto N
Targeting stromal cells for the treatment of platelet-derived growth factor C-induced hepatocellular carcinogenesis.
Differentiation. 2007 Nov;75(9):
Non-invasive therapies for the treatment of hepatocellular carcinoma (HCC) would be of great benefit to public health. To this end, we have developed a platelet-derived growth factor-C (PDGF-C) transgenic (Tg) mouse model, which mimics many aspects of human liver carcinogenesis. Specifically, overexpression of PDGF-C results in liver fibrosis, which is preceded by activation and proliferation of hepatic stellate cells, and is followed by the development of dysplastic lesions and angiogenesis, and progression to HCCs by 8 months of age. Here, we show that PDGF-C overexpression induces the proliferation of endothelial-like cells that are present in tumors and adjacent non-neoplastic parenchyma. The protein tyrosine kinase inhibitor, imatinib (Gleevec), decreases the proliferation of non-parenchymal cells (NPC) in vitro and in vivo, with concomitant inhibition of Akt. In vivo treatment with imatinib also blocks the expression of CD34 in PDGF-C Tg mice. Decreased NPC proliferation and CD34 expression correlated with lower levels of active ERK1/2 and total levels of PDGF receptor alpha (PDGFRalpha). In summary, the small molecule inhibitor imatinib attenuates stromal cell proliferation in PDGF-C-induced HCC, which coincides with decreased expression of both CD34 and PDGFRalpha, and activated Akt. Our findings suggest that imatinib may be efficacious in the treatment of hepatocarcinogenesis, particularly when neovascularization is present. [Abstract]

Vellon L, Menendez JA, Liu H, Lupu R
Up-regulation of alpha(v)beta(3) integrin expression is a novel molecular response to chemotherapy-induced cell damage in a heregulin-dependent manner.
Differentiation. 2007 Nov;75(9):
alpha(v)beta(3) integrin has a dual role in apoptosis. Whereas ligated alpha(v)beta(3) activates cell survival pathways and suppresses pro-apoptotic signals, unligated alpha(v)beta(3) or integrins bound to soluble ligands promote apoptosis. In this study, we assessed the role of alpha(v)beta(3) in chemosensitivity of breast cancer cells expressing different levels of heregulin (HRG). Expression levels of the RGD-binding integrins alpha(v)beta(3) were measured in MDA-MB-231 human breast cancer cells and its low HRG-expressing derivative (MDA-MB-231/AS31) treated with the microtubule-interfering agents (MIAs) paclitaxel and vincristine. Following treatment, only alpha(v)beta(3) levels were significantly increased in MDA-MB-231 cells. Interestingly, alpha(v)beta(3) expression was more significantly up-regulated in the MDA-MB-231/AS31 cells than in the parental cells. This MIA-induced increase of alpha(v)beta(3) expression was correlated with a decrease in cell viability and an increase in apoptosis in MDA-MB-231/AS31 cells, indicating that overexpression of alpha(v)beta(3) is linked to chemotherapy-induced cell death in low HRG-expressing breast cancer models. Moreover, a paclitaxel-induced increase of alpha(v)beta(3) was also observed in MCF-7 cells but not in an doxorubicin-resistant derivative that shows cross-resistance to paclitaxel, further providing evidence that the extent of alpha(v)beta(3) up-regulation is related to cell damage. These results indicate that alpha(v)beta(3) integrin is dramatically up-regulated in low HRG-expressing breast cancer models that are highly responsive to MIAs, thus providing a novel molecular marker of chemosensitivity influenced by HRG levels in breast cancer cells. [Abstract]

Sibilia M, Kroismayr R, Lichtenberger BM, Natarajan A, Hecking M, Holcmann M
The epidermal growth factor receptor: from development to tumorigenesis.
Differentiation. 2007 Nov;75(9):
The epidermal growth factor receptor (EGFR) is activated by many ligands and belongs to a family of tyrosine kinase receptors, including ErbB2, ErbB3, and ErbB4. These receptors are de-regulated in many human tumors, and EGFR amplification, overexpression, and mutations are detected at a high frequency in carcinomas and glioblastomas, which are tumors of epithelial and glial origin, respectively. From the analysis of EGFR-deficient mice, it seems that the cell types mostly affected by the absence of EGFR are epithelial and glial cells, the same cell types where the EGFR is found to be overexpressed in human tumors. Therefore, it is important to define molecularly the function of EGFR signaling in the development of these cell types, because this knowledge will be of fundamental importance to understand how aberrant EGFR signaling can lead to tumor formation and progression. A molecular understanding of the pathways that control the development of a given tissue or cell type will also provide the basis for developing better combination therapies targeting different key components of the EGFR signaling network in the respective cancerous cells. Here, we will review the current knowledge, mostly derived from the analysis of genetically modified mice and cells, about the function of the EGFR in specific organs and tissues and in sites where the EGFR is found to be overexpressed in human tumors. [Abstract]

Grunt TW
Receptor signaling in cancer.
Differentiation. 2007 Nov;75(9): [Abstract]

Traweger A, Lehner C, Farkas A, Krizbai IA, Tempfer H, Klement E, Guenther B, Bauer HC, Bauer H
Nuclear Zonula occludens-2 alters gene expression and junctional stability in epithelial and endothelial cells.
Differentiation. 2007 Oct 31;
Zonula occludens proteins (ZOPs) are essential scaffold proteins involved in the organization of epithelial and endothelial intercellular junctions. Based on their molecular domain architecture, they are members of the large family of membrane-associated guanylate kinase-like (MAGUK) proteins. As all other MAGUKs, ZOPs contain a core of several PDZ, an src homology-3, and a guanylate kinase-like domain, indicating that these proteins may serve both structural and signaling functions. In addition, ZOPs exhibit some unique motifs not shared by other MAGUKs, i.e., several nuclear localization (NLS) and nuclear export signals (NES), allowing these proteins to shuttle between the cytoplasm and the nucleus. However, the stimuli leading to the nuclear accumulation of ZOPs and the resulting physiological consequences remain poorly defined. We have previously reported the direct binding of nuclear ZO-2 to scaffold attachment factor B, a heterogeneous nuclear ribonucleoprotein involved in chromatin organization and the transcriptional control of eukaryotic genes. We now report that the nuclear accumulation of ZO-2 leads to an increase in the expression of the M2 type of pyruvate kinase (M2-PK) in epithelial and endothelial cells. Further, the proliferative activity was increased, while the intercellular junctional stability of Madin-Darby canine kidney cells was reduced. Our data provide evidence to suggest that ZO-2 exerts a junction-unrelated function that further supports the notion of a general "dual" role of junctional MAGUKs, being an indispensable structural component at cell-cell junctions and a nuclear factor influencing gene expression and cell behavior. [Abstract]

Zucchetti I, Marino R, Pinto MR, Lambris JD, Du Pasquier L, De Santis R
ciCD94-1, an ascidian multipurpose C-type lectin-like receptor expressed in Ciona intestinalis hemocytes and larval neural structures.
Differentiation. 2007 Oct 9;
C-type lectins play an important role in the immune system and are part of a large superfamily that includes C-type lectin-like domain (CTLD)-containing proteins. Divergent evolution, acting on the CTLD fold, has generated the Ca(2+)-dependent carbohydrate-binding lectins and molecules, as the lectin-like natural killer (NK) receptors that bind proteins, rather than sugars, in a Ca(2+)-independent manner. We have studied ciCD94-1, a CTLD-containing protein from the tunicate Ciona intestinalis, which is a homolog of the CD94 vertebrate receptor that is expressed on NK cells and modulates their cytotoxic activity by interacting with MHC class I molecules. ciCD94-1 shares structural features with the CTLD-containing molecules that recognize proteins, suggesting that it could be located along the evolutionary pathway leading to the NK receptors. ciCD94-1 was up-regulated in response to inflammation induced by lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating blood. Furthermore, an anti-ciCD94-1 antibody specifically inhibited the phagocytic activity of these cells. ciCD94-1 was also expressed during development in the larva and in the early stages of metamorphosis in structures related to the nervous system, and loss of its function affected the correct differentiation of these territories. These findings suggest that ciCD94-1 has different roles in immunity and in development, thus strengthening the concept of gene co-option during evolution and of an evolutionary relationship between the nervous and the immune systems. [Abstract]

Dinnes DL, Santerre JP, Labow RS
Influence of biodegradable and non-biodegradable material surfaces on the differentiation of human monocyte-derived macrophages.
Differentiation. 2007 Oct 9;
Monocyte-derived macrophages (MDM) and multinucleated foreign body giant cells (FBGC) are the primary cell types that remain at the cell-material interface of polyurethane (PU)-based medical devices as a result of chronic inflammatory responses. In vitro studies have demonstrated that MDM possess degradative potential toward PU, which can result in device failure. Because most studies have followed the degradation potential, morphology, and function of these cells only once fully differentiated, the current study investigated the influence of a non-degradable control tissue culture-grade polystyrene (TCPS) surface relative to two degradable model polycarbonate-urethanes (PCNU), of different chemistry, on various parameters of MDM morphology and function during a 14-day differentiation time course. The differentiation of human monocytes isolated from whole blood on PCNU materials resulted in increased cell attachment, decreased multinucleation, and significant decreases in cell spreading when compared with cells differentiated on TCPS. Actin-stained podosome-like cell adhesion structures were increased in PCNU-adherent cells, accompanied by an alteration in beta-actin and vinculin protein expression. The expression of the CD68 macrophage marker was reduced when cells were adherent to the PCNU materials and compared with TCPS, suggesting altered cell activation by the degradable relative to non-degradable materials. The degradative potential of these cells was altered by the material surface they were exposed to as measured by esterase activity and protein expression of monocyte-specific esterase. This was also supported by physical material breakdown evident in scanning electron microscopy images that illustrated holes in the PCNU films generated by the presence of differentiating MDM. It was concluded from these studies that PCNU materials significantly alter the function and morphology of differentiating MDM. This must be taken into consideration when studying cell-material interactions because these cells will receive cues from their immediate environment (including the biomaterial) upon differentiation, thereby affecting their resulting phenotype. [Abstract]

Ka?par P, Dvo?ák M
Involvement of phosphatidylserine externalization in the down-regulation of c-myb expression in differentiating C2C12 cells.
Differentiation. 2007 Oct 9;
Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression. [Abstract]

Ricke WA, Wang Y, Cunha GR
Steroid hormones and carcinogenesis of the prostate: the role of estrogens.
Differentiation. 2007 Nov;75(9):
Androgens have long been known to be the major sex hormones that target the prostate during development, maturation, and carcinogenesis. It is now apparent that estrogens, both those synthesized by the body as well as those from our environment, also target the prostate during all stages of development. Little is known about the mechanisms involved in estrogen stimulation of carcinogenesis and less is known about how to prevent or treat prostate cancer through estrogenic pathways. To better understand how estrogens mediate their carcinogenic effects, the respective roles of estrogen receptor (ER)-alpha and ER-beta must be elucidated in the epithelial and stromal cells that constitute the prostate. Lastly, the significance of ER signaling during various ontogenic periods must be determined. Answers to these questions will further our understanding of the mechanisms of estrogen/ER signaling and will serve as a basis for chemopreventive and/or chemotherapeutic strategies for prostate cancer. [Abstract]

Battula VL, Treml S, Abele H, Bühring HJ
Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody.
Differentiation. 2007 Oct 9;
We have recently shown that frizzled-9 (FZD9, CD349) is expressed on the cell surface of cultured mesenchymal stromal cells (MSC) derived from the human bone marrow (BM) and chorionic placenta (PL). To study whether FZD9 is also a marker for naive mesenchymal stem cells (MSC), we analyzed the expression pattern of FZD9 on freshly isolated PL cells and determined the clonogenic potential of isolated FZD9(+) cells using the colony-forming units-fibroblastic (CFU-F) assay. About 0.2% of isolated PL cells were positive for FZD9. Two-color analysis revealed that FZD9(+) PL cells uniformly express CD9, CD63, and CD90, but are heterogeneous for CD10, CD13, and CD26 expression. In contrast to BM-derived MSC, PL-derived MSC expressed only low levels of CD271. Colony assays of sorted cells showed that clonogenic CFU-F reside exclusively in the FZD9(+) but not in the FZD9(-) fraction. Further analysis revealed that CFU-F were enriched by 60-fold in the FZD9(+)CD10(+)CD29(+) fraction but were absent in the FZD9(+)CD10(-)CD29(-) population. Cultured FZD9(+) cells expressed the embryonic stem cell makers Oct-4 and nanog as well as SSEA-4 and TRA1-2-49/6E. In addition, they could be differentiated into functional adipocytes and osteoblasts. This report describes for the first time that FZD9 is a novel and specific marker for the prospective isolation of MSC from human term PL. [Abstract]

Gasa R, Mrejen C, Lynn FC, Skewes-Cox P, Sanchez L, Yang KY, Lin CH, Gomis R, German MS
Induction of pancreatic islet cell differentiation by the neurogenin-neuroD cascade.
Differentiation. 2007 Oct 9;
The related basic helix-loop-helix transcription factors neurogenin3 (Neurog3) and neurogenic differentiation 1 (NeuroD1) regulate pancreatic islet cell formation. The transient expression of Neurog3 initiates endocrine differentiation and activates its target, NeuroD1, which continues the endocrine differentiation process. Despite their distinct developmental roles, the expression of either factor can drive islet differentiation in progenitor cells. To determine whether Neurog3 and NeuroD1 function by targeting a common set of genes, we compared gene expression patterns in cells ectopically expressing these two factors using cDNA microarrays. The array data demonstrated that both factors regulated largely overlapping sets of genes, providing the molecular basis for their functional equivalence in gain-of-functions approaches. Distinct differences in the timing and level of expression of a subset of target genes, however, show that the functions of these two factors are not completely redundant. Interestingly, in addition to NeuroD1, Neurog3 also induced both NeuroD2 and NeuroD4 gene expression. NeuroD2 mRNA peaked in the embryonic pancreas during endocrine differentiation and induced endocrine differentiation in vitro. These data suggest possible redundant roles for the NeuroD1 paralogs NeuroD2 and NeuroD4 in pancreatic endocrine differentiation and their potential utility in cell-based therapies for diabetes mellitus. [Abstract]

Kirilenko P, Weierud FK, Zorn AM, Woodland HR
The efficiency of Xenopus primordial germ cell migration depends on the germplasm mRNA encoding the PDZ domain protein Grip2.
Differentiation. 2007 Oct 9;
A microarray analysis of vegetal pole sequences in the egg and early Xenopus laevis embryo identified Unigene Xl.14891 as a vegetally localized RNA. Analysis of the Xenopus tropicalis genome showed this Unigene to be localized near the 3' end of the Grip2 (glutamate receptor interacting protein 2) transcription unit. RACE showed that the Unigene represented the 3' UTR of Grip2 mRNA. Grip2 mRNA is present in the mitochondrial cloud of late pre-vitellogenic oocytes and then in the germplasm through oogenesis and early development until tailbud tadpole stages. Interference with Grip2 mRNA translation using two antisense morpholino oligos (MOs) impairs primordial germ cell (PGC) migration to the germinal ridges. Both MOs also inhibit swimming movements of the tailbud tadpole, known to involve glutamate receptors. We conclude that Grip2 has several functions in the embryo, including enabling efficient PGC migration. [Abstract]

Du Y, Deng H
Human embryonic stem cells: the practical handbook Edited by Stephen Sullivan, Chad A. Cowan, and Kevin Eggan Reviewed by Yuanyuan Du and Hongkui Deng.
Differentiation. 2007 Oct 9; [Abstract]

Schöfer C, Weipoltshammer K
Gene dynamics and nuclear architecture during differentiation.
Differentiation. 2007 Oct 1;
Recent advances have demonstrated that placing genes in a specific nuclear context plays an important role in the regulation of coordinated gene expression, thus adding an additional level of complexity to the mechanisms of gene regulation. Differentiation processes are characterized by dynamic changes in gene activation and silencing. These alterations are often accompanied by gene relocations in relation to other genomic regions or to nuclear compartments. Unraveling of mechanisms and dynamics of chromatin positioning will thus expand our knowledge about cellular differentiation. [Abstract]

Hirayama E, Kim J
Identification and characterization of a novel neural cell adhesion molecule (NCAM)-associated protein from quail myoblasts: relationship to myotube formation and induction of neurite-like protrusions.
Differentiation. 2007 Sep 6;
We identified a novel neural cell adhesion molecule (NCAM)-associated protein, myogenesis-related and NCAM-associated protein (MYONAP), the expression of which increases during the formation of myotubes in quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells). MYONAP shares homology with PL48 in human cytotrophoblasts and KIAA0386 in human brain. Excess expression of MYONAP in presumptive QM-RSV myoblasts induced long protrusions like neurites in cooperation with microtubules. Suppression of MYONAP by antisense cDNA prevented myotubes from forming in spite of the expression of myogenin, creatine kinase, and myosin, and rendered myoblast membranes resistant to fusion. Yeast two-hybrid screening showed that MYONAP interacted with NCAM specifically. Deletion of the NCAM-associated domain resulted in a loss of the function that induces neurite-like protrusions to form and disturbed the elongation of microtubules. The results suggested that MYONAP influenced the functions of microtubules and was involved in the formation of myotubes via its interaction with NCAM. [Abstract]

Nadri S, Soleimani M, Kiani J, Atashi A, Izadpanah R
Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells.
Differentiation. 2007 Sep 6;
Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2(+), SH3(+), CD29(+), CD44(+), CD166(+), CD13(+)) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers. [Abstract]

Hirata K, Amagai A, Chae SC, Hirose S, Maeda Y
Involvements of a novel protein, DIA2, in cAMP signaling and spore differentiation during Dictyostelium development.
Differentiation. 2007 Sep 6;
The novel gene dia2 (differentiation-associated gene 2) was originally isolated as a gene expressed specifically in response to initial differentiation of Dictyostelium discoideum Ax-2 cells. Using dia2(AS) cells in which the dia2 expression was inactivated by the antisense RNA method, DIA2 protein was found to be required for cAMP signaling during cell aggregation. During late development, the DIA2 protein changed its location from the endoplasmic reticulum (ER) to prespore-specific vacuoles (PSVs) that are specifically present in prespore cells of the slug. In differentiating prestalk cells, however, DIA2 was found to be nearly lost from the cells. Importantly, exocytosis of PSVs from prespore cells and the subsequent spore differentiation were almost completely impaired in dia2(AS) cells. In addition, spore induction by externally applied 8-bromo cAMP was significantly suppressed in dia2(AS) cells. Taken together, these results strongly suggested that DIA2 might be closely involved in cAMP signaling and spore differentiation as well as in the initiation of differentiation during Dictyostelium development. [Abstract]

Neildez-Nguyen TM, Parisot A, Vignal C, Rameau P, Stockholm D, Picot J, Allo V, Le Bec C, Laplace C, Paldi A
Epigenetic gene expression noise and phenotypic diversification of clonal cell populations.
Differentiation. 2007 Sep 6;
Spontaneous emergence of phenotypic heterogeneity in cultures of genetically identical cells is a frequently observed phenomenon that provides a simple in vitro experimental system to model the problems of in vivo differentiation. In the present study, we have investigated whether stochastic variation of gene expression levels could contribute to phenotypic change in human cells. We have applied the two fluorescence-coding gene method and the expression variability of the two reporter genes to human cells in culture. We have quantified the portion of gene expression variation determined by global, promoter-specific, or by epigenetic sources. These two types of variation appear to contribute, in different ways, to the phenotypic diversification of clonal cell populations. Global, or promoter-specific, gene expression noise increases with cellular stress and contributes to the emergence of cellular diversity by diversifying the gene-expression levels. Epigenetic mechanisms act to increase the robustness of the cellular state by stabilizing gene transcription levels or by reinforcing the silenced state. [Abstract]

Probst AV, Almouzni G
Pericentric heterochromatin: dynamic organization during early development in mammals.
Differentiation. 2007 Aug 28;
Constitutive heterochromatin in mammals is essentially found at centromeres, which are key chromosomal elements that ensure proper chromosome segregation. These regions are considered to be epigenetically defined, given that it is not sequence composition but chromatin organization that defines centromere function. How such an epigenetically defined domain, like the centromere, can be established during development and maintained during somatic cell life are fundamental questions. This review discusses the most recent insights into centromeric heterochromatin organization and replication. We further highlight the plasticity of this domain by describing the large-scale re-organization that occurs during development. [Abstract]

Recent Articles in Mechanisms of Development

Ochiai H, Sakamoto N, Momiyama A, Akasaka K, Yamamoto T
Analysis of cis-regulatory elements controlling spatio-temporal expression of T-brain gene in sea urchin, Hemicentrotus pulcherrimus.
Mech Dev. 2007 Oct 25;
In sea urchin development, micromere descendants play important roles in skeletogenesis and induction of gastrulation. We previously reported that the T-brain homolog of sea urchin Hemicentrotus pulcherrimus, HpTb expresses specifically in micromere descendants and is required for induction of gastrulation and skeletogenesis. Thus, HpTb is thought to play important roles in the function of micromere-lineage cells. To identify cis-regulatory regions responsible for spatio-temporal gene expression of HpTb, we isolated approximately 7kb genomic region of HpTb gene and showed that GFP expression driven by this region exhibits the spatio-temporal pattern corresponding substantially to that of endogenous HpTb expression. Deletion of interspecifically conserved C2 and C4 regions resulted in an increase of ectopic expression. Mutations in Hairy family and Snail family consensus sequences in C1 and C2 regions also increased ectopic expression. Furthermore, we demonstrated that C4 region functions as enhancer, and that three Ets family consensus sequences are involved in this activity but not in spatial regulation. Therefore, we concluded that expression of HpTb gene is regulated by multiple cis-regulatory elements. [Abstract]

Wu J, Jenny A, Mirkovic I, Mlodzik M
Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila.
Mech Dev. 2007 Oct 17;
Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/beta-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Our mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. We examined the role of a Dsh-binding PCP molecule, Diego (Dgo), in both Wnt-Fz/beta-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/beta-catenin signaling. Our data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell. [Abstract]

Cavaleri FM, Balbach ST, Gentile L, Jauch A, Böhm-Steuer B, Han YM, Schöler HR, Boiani M
Subsets of cloned mouse embryos and their non-random relationship to development and nuclear reprogramming.
Mech Dev. 2007 Sep 29;
An important question in oocyte-mediated nuclear reprogramming is whether gene expression of the donor nucleus changes randomly or follows a pattern. Since cloned embryos are very heterogenous and arrest frequently during preimplantation development, a random scenario is generally accepted. In the present study, we resolve the heterogeneity of cumulus cell-derived mouse clones by recognizing structured subsets, and we analyze their relationship to reprogramming of donor nuclei. We utilize live cell imaging of the Oct4 promoter-driven GFP transgene to resolve the populations of cloned and ICSI-fertilized morulae, and we sort them both into three subsets based on different GFP expression. Functionally, subsets of cloned but not ICSI morulae form blastocysts and ES cells proportional to Oct4-GFP expression. Regulatively, the subsets of cloned morulae are characterized by small differences of transcript level for the pluripotency-associated genes Oct4, Nanog and Sox2. Small differences of the level of select mRNAs across subsets suggest a uniform rather than random course of reprogramming from the morula stage on. Since these small differences correspond with substantial differences in developmental competence, we propose that developmental potential of clones relates to levels of gene expression in a different way than fertilized embryos. [Abstract]

Zheng L, Carthew RW
Lola regulates cell fate by antagonizing Notch induction in the Drosophila eye.
Mech Dev. 2007 Oct 22;
Lola is a transcription repressor that regulates axon guidance in the developing embryonic nervous system of Drosophila. Here, we show that Lola regulates two binary cell fate decisions guided by Notch inductive signaling in the developing eye: the R3-R4 and the R7-cone cell fate choices. Lola is required cell-autonomously in R3 for its specification, and Lola transforms R4 into R3 if overexpressed. Lola also promotes R7 fate at the expense of cone cell fate. Lola antagonizes Notch-dependent gene expression and Notch-dependent fate transformation. Expression analysis shows that Lola is constitutively present in all photoreceptors and cone cells. We propose that when a precursor cell receives a weak Notch inductive signal, it is not sufficiently strong to overcome the constitutive repression of target gene transcription by Lola. A precursor that receives a strong Notch inductive signal expresses target genes despite a constitutive repression by Lola. The predicted consequence of this mechanism is to sharpen a cell's responsiveness to Notch signaling by creating a threshold. [Abstract]

Wang Y, Riechmann V
Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm.
Mech Dev. 2007 Sep 29;
The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent. [Abstract]

Sakami S, Etter P, Reh TA
Activin signaling limits the competence for retinal regeneration from the pigmented epithelium.
Mech Dev. 2007 Oct 10;
Regeneration of the retina in amphibians is initiated by the transdifferentiation of the retinal pigmented epithelium (RPE) into neural progenitors. A similar process occurs in the early embryonic chick, but the RPE soon loses this ability. The factors that limit the competence of RPE cells to regenerate neural retina are not understood; however, factors normally involved in the development of the eye (i.e. FGF and Pax6) have also been implicated in transdifferentiation. Therefore, we tested whether activin, a TGFbeta family signaling protein shown to be important in RPE development, contributes to the loss in competence of the RPE to regenerate retina. We have found that addition of activin blocks regeneration from the RPE, even during stages when the cells are competent. Conversely, a small molecule inhibitor of the activin/TGFbeta/nodal receptors can delay, and even reverse, the developmental restriction in FGF-stimulated neural retinal regeneration. [Abstract]

Fromental-Ramain C, Vanolst L, Delaporte C, Ramain P
pannier encodes two structurally related isoforms that are differentially expressed during Drosophila development and display distinct functions during thorax patterning.
Mech Dev. 2007 Oct 24;
Previous studies have shown that the pannier (pnr) gene of Drosophila encodes a GATA transcription factor which is involved in various biological processes, including heart development, dorsal closure during embryogenesis as well as neurogenesis and regulation of wingless (wg) expression during imaginal development. We demonstrate here that pnr encodes two highly related isoforms that share functional domains but are differentially expressed during development. Moreover, we describe two genomic regions of the pnr locus that drive expression of a reporter in transgenic flies in patterns that recapitulate essential features of the expression of the isoforms, suggesting that these regions encompass crucial regulatory elements. These elements contain, in particular, sequences mediating regulation of expression by Decapentaplegic (Dpp) signaling, during both embryogenesis and imaginal development. Analysis of pnr alleles reveals that the isoforms differentially regulate expression of both wg and proneural achaete/scute (as/sc) targets during imaginal development. Pnr function has been demonstrated to be necessary both for activation of wg and, together with U-shaped (Ush), for its repression in the dorsal-most region of the presumptive notum. Expression of the isoforms define distinct longitudinal domains and, in this regard, we importantly show that the dual function of pnr during regulation of wg is achieved by one isoform repressing expression of the morphogen in the dorsal-most region of the disc while the other laterally promotes activation of the notal wg expression. Our study provides novel insights into pnr function during Drosophila development and extends our knowledge of the roles of prepattern factors during thorax patterning. [Abstract]

Duong HA, Wang CW, Sun YH, Courey AJ
Transformation of eye to antenna by misexpression of a single gene.
Mech Dev. 2007 Oct 4;
In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance our understanding of the mechanisms that restrict stem cell fate. We show here that Dip3, a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and have taken advantage of this observation to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, our results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae. [Abstract]

Lee JA, Anholt RR, Cole GJ
Olfactomedin-2 mediates development of the anterior central nervous system and head structures in zebrafish.
Mech Dev. 2007 Sep 29;
Olfactomedins comprise a diverse family of secreted glycoproteins, which includes noelin, tiarin, pancortin and gliomedin, implicated in development of the nervous system, and the glaucoma-associated protein myocilin. Here we show in zebrafish that olfactomedin-2 (OM2) is a developmentally regulated gene, and that knockdown of protein expression by morpholino antisense oligonucleotides leads to perturbations of nervous system development. Interference with OM2 expression results in impaired development of branchiomotor neurons, specific disruption of the late phase branchiomotor axon guidance, and affects development of the caudal pharyngeal arches, olfactory pits, eyes and optic tectum. Effects of OM2 knockdown on eye development are likely associated with Pax6 signaling in developing eyes, as Pax6.1 and Pax6.2 mRNA expression patterns are altered in the eyes of OM2 morphants. The specific absence of most cartilaginous structures in the pharyngeal arches indicates that the observed craniofacial phenotypes may be due to perturbed differentiation of cranial neural crest cells. Our studies show that this member of the olfactomedin protein family is an important regulator of development of the anterior nervous system. [Abstract]

Ishibashi H, Matsumura N, Hanafusa H, Matsumoto K, Robertis EM, Kuroda H
Expression of Siamois and Twin in the blastula Chordin/Noggin signaling center is required for brain formation in Xenopus laevis embryos.
Mech Dev. 2007 Oct 12;
The blastula Chordin- and Noggin-expressing (BCNE) center located in the dorsal animal region of the Xenopus blastula embryo contains both prospective anterior neuroectoderm and Spemann organizer precursor cells. Here we show that, contrary to previous reports, the canonical Wnt target homeobox genes, Double knockdown of these genes using antisense morpholinos in Xenopus laevis blocked head formation, reduced the expression of the other BCNE center genes, upregulated Bmp4 expression, and nullified hyperdorsalization by lithium chloride. Moreover, gain- and loss-of-function experiments showed that Siamois and Twin expression is repressed by the vegetal transcription factor VegT. We propose that VegT expression causes maternal beta-Catenin signals to restrict Siamois and Twin expression to the BCNE region. A two-step inhibition of BMP signals by Siamois and Twin - first by transcriptional repression of Bmp4 and then by activation of the expression of the BMP inhibitors Chordin and Noggin - in the BCNE center is required for head formation. [Abstract]

Gavis ER, Chatterjee S, Ford NR, Wolff LJ
Dispensability of nanos mRNA localization for abdominal patterning but not for germ cell development.
Mech Dev. 2007 Oct 12;
The development of a functional germline is essential for species propagation. The nanos (nos) gene plays an evolutionarily conserved role in germline development and is also essential for abdominal patterning in Drosophila. A small fraction of nos mRNA is localized to the germ plasm at the posterior pole of the Drosophila embryo, where it becomes incorporated into the germ cells. Germ plasm associated nos mRNA is translated to produce a gradient of Nos protein that patterns the abdomen, whereas the remaining unlocalized RNA is translationally repressed to allow anterior development. Using transgenes that compromise nos mRNA localization and translational regulation, we show that wild-type body patterning can ensue without nos mRNA localization provided that nos translation is properly modulated. In contrast, localization of nos to the germ plasm, but not translational regulation, is essential for nos function in the developing germ cells. We propose that an imperative for nos localization in producing a functional germline has preserved an inefficient localization mechanism. [Abstract]

Minakuchi C, Zhou X, Riddiford LM
Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster.
Mech Dev. 2007 Oct 11;
Juvenile hormone (JH) given at pupariation inhibits bristle formation and causes pupal cuticle formation in the abdomen of Drosophila melanogaster due to its prolongation of expression of the transcription factor Broad (BR). In a microarray analysis of JH-induced gene expression in abdominal integument, we found that Krüppel homolog 1 (Kr-h1) was up-regulated during most of adult development. Quantitative real-time PCR analyses showed that Kr-h1 up-regulation began at 10h after puparium formation (APF), and Kr-h1 up-regulation occurred in imaginal epidermal cells, persisting larval muscles, and larval oenocytes. Ectopic expression of Kr-h1 in abdominal epidermis using T155-Gal4 to drive UAS-Kr-h1 resulted in missing or short bristles in the dorsal midline. This phenotype was similar to that seen after a low dose of JH or after misexpression of br between 21 and 30h APF. Ectopic expression of Kr-h1 prolonged the expression of BR protein in the pleura and the dorsal tergite. No Kr-h1 was seen after misexpression of br. Thus, Kr-h1 mediates some of the JH signaling in the adult abdominal epidermis and is upstream of br in this pathway. We also show for the first time that the JH-mediated maintenance of br expression in this epidermis is patterned and that JH delays the fusion of the imaginal cells and the disappearance of Dpp in the dorsal midline. [Abstract]

McClure KD, Schubiger G
A screen for genes that function in leg disc regeneration in Drosophila melanogaster.
Mech Dev. 2007 Oct 12;
Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported here aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. We screened a collection of 97 recessive lethal P-lacZ enhancer trap lines for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, we identified six genes (Krüppel-homolog-1, rpd3, jing, combgap, Aly and S6 kinase) that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration. [Abstract]

Murakami Y, Hirata H, Miyamoto Y, Nagahashi A, Sawa Y, Jakt M, Asahara T, Kawamata S
Isolation of cardiac cells from E8.5 yolk sac by ALCAM (CD166) expression.
Mech Dev. 2007 Nov-Dec;124(11-12):830-9.
It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM- populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1- cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/- fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1- cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system. [Abstract]

International Society of Developmental Biologists (ISDB) 2007 Interim Report.
Mech Dev. 2007 Nov-Dec;124(11-12):935-6. [Abstract]

Kettunen P, Spencer-Dene B, Furmanek T, Kvinnsland IH, Dickson C, Thesleff I, Luukko K
Fgfr2b mediated epithelial-mesenchymal interactions coordinate tooth morphogenesis and dental trigeminal axon patterning.
Mech Dev. 2007 Nov-Dec;124(11-12):868-83.
Dental trigeminal nerve fiber growth and patterning are strictly integrated with tooth morphogenesis, but it is still unknown, how these two developmental processes are coordinated. Here we show that targeted inactivation of the dental epithelium expressed Fgfr2b results in cessation of the mouse mandibular first molar development at the degenerated cap stage and the failure of the trigeminal molar nerve to establish the lingual branch at E13.5 stage while the buccal branch develops properly. This axon patterning defect correlates to the histological absence of the mesenchymal dental follicle and adjacent Semaphorin3A-free dental follicle target field as well as appearance of ectopic Sema3A expression domain in the lingual side of the epithelial bud. Although the mesenchymal ligands for Fgfr2b, Fgf3 and -10 were present in the Fgfr2b(-/)(-) dental mesenchyme, mutant dental epithelium showed dramatically reduced proliferation and the lack of Fgf3. Tgfbeta1, which controls Sema3A was absent from the Fgfr2b(-/-) tooth germ, and Sema3A was specifically downregulated in the dental mesenchyme at the bud and cap stage. In addition, the epithelial primary enamel knot signaling center although being molecularly present neither was histologically detectable nor expressed Bmp4 and Fgf3 as well as Fgf4, which is essential for tooth morphogenesis and stimulates mesenchymal Fgf3 and Tgfbeta1. Fgf4 beads rescued Tgfbeta1 in the Fgfr2b(-/-) dental mesenchyme explants and Tgfbeta1 induced de novo Sema3A expression in the dental mesenchyme. Collectively these results demonstrate that epithelial Fgfr2b controls tooth morphogenesis and dental axon patterning, and suggests that Fgfr2b, by mediating local epithelial-mesenchymal interactions, integrates these two distinct developmental processes during odontogenesis. [Abstract]

Qiu Z, Tsoi SC, Macrae TH
Gene expression in diapause-destined embryos of the crustacean, Artemia franciscana.
Mech Dev. 2007 Nov-Dec;124(11-12):856-67.
Diapause-destined embryos of the crustacean Artemia franciscana cease development as gastrulae, encyst, and enter a resting stage characterized by greatly reduced metabolic activity and extreme stress resistance. To better understand diapause induction and maintenance in Artemia embryos gene expression was analyzed by subtractive hybridization at two days post-fertilization, a time early in this developmental process. Eighty-five of 264 cDNA clones sequenced matched GenBank entries and they fell into categories designated as environmental information processing, cellular processes, genetic information processing and metabolism. Semi-quantitative RT-PCR of cDNAs populating the subtractive library identified seventeen up-regulated and four down-regulated transcripts, the former including those encoding a human transcription cofactor homologue, three small heat shock proteins, putative cell growth suppressor proteins and several enzymes. As examples, p8 may modulate gene expression during diapause in Artemia embryos. BRCA1 associated protein-1 (BAP1) and other functionally related proteins may influence cell growth and division during transition into diapause, a time when these processes are inhibited, whereas small heat shock proteins protect embryos from stress. This study represents the first systematic molecular characterization of diapause in crustaceans. Several differentially expressed genes were identified, expanding the repertoire of proteins potentially modified during diapause and suggesting mechanistic pathways indigenous to the initiation and maintenance of this physiological state. [Abstract]

Fernández BG, Arias AM, Jacinto A
Dpp signalling orchestrates dorsal closure by regulating cell shape changes both in the amnioserosa and in the epidermis.
Mech Dev. 2007 Nov-Dec;124(11-12):884-97.
During the final stages of embryogenesis, the Drosophila embryo exhibits a dorsal hole covered by a simple epithelium of large cells termed the amnioserosa (AS). Dorsal closure is the process whereby this hole is closed through the coordination of cellular activities within both the AS and the epidermis. Genetic analysis has shown that signalling through Jun N-terminal Kinase (JNK) and Decapentaplegic (Dpp), a Drosophila member of the BMP/TGF-beta family of secreted factors, controls these activities. JNK activates the expression of dpp in the dorsal-most epidermal cells, and subsequently Dpp acts as a secreted signal to control the elongation of lateral epidermis. Our analysis shows that Dpp function not only affects the epidermal cells, but also the AS. Embryos defective in Dpp signalling display defects in AS cell shape changes, specifically in the reduction of their apical surface areas, leading to defective AS contraction. Our data also demonstrate that Dpp regulates adhesion between epidermis and AS, and mediates expression of the transcription factor U-shaped in a gradient across both the AS and the epidermis. In summary, we show that Dpp plays a crucial role in coordinating the activity of the AS and its interactions with the LE cells during dorsal closure. [Abstract]

Takebayashi-Suzuki K, Arita N, Murasaki E, Suzuki A
The Xenopus POU class V transcription factor XOct-25 inhibits ectodermal competence to respond to bone morphogenetic protein-mediated embryonic induction.
Mech Dev. 2007 Nov-Dec;124(11-12):840-55.
Bone morphogenetic proteins (BMPs) have been shown to play a key role in controlling ectodermal cell fates by inducing epidermis at the expense of neural tissue during gastrulation. Here, we present evidence that the Xenopus POU class V transcription factor XOct-25 regulates ectodermal cell fate decisions by inhibiting the competence of ectodermal cells to respond to BMP during Xenopus embryogenesis. When overexpressed in the ectoderm after the blastula stage, XOct-25 suppressed early BMP responses of ectodermal cells downstream of BMP receptor activation and promoted neural induction while suppressing epidermal differentiation. In contrast, inhibition of XOct-25 function in the prospective neuroectoderm resulted in expansion of epidermal ectoderm at the expense of neuroectoderm. The reduction of neural tissue by inhibition of XOct-25 function could be rescued by decreasing endogenous BMP signaling, suggesting that XOct-25 plays a role in the formation of neural tissue at least in part by inhibiting BMP-mediated epidermal induction (neural inhibition). This hypothesis is supported by the observation that ectodermal cells from XOct-25 morphants were more sensitive to BMP signaling than cells from controls in inducing both immediate early BMP target genes and epidermis at the expense of neural tissue, while cells overexpressing XOct-25 are less competent to respond to BMP-mediated induction. These results document an essential role for XOct-25 in commitment to neural or epidermal cell fates in the ectoderm and highlight the importance of a regulatory mechanism that limits competence to respond to BMP-mediated embryonic induction. [Abstract]

Wang S, Zhang J, Zhao A, Hipkens S, Magnuson MA, Gu G
Loss of Myt1 function partially compromises endocrine islet cell differentiation and pancreatic physiological function in the mouse.
Mech Dev. 2007 Nov-Dec;124(11-12):898-910.
Myelin transcription factor 1 (Myt1) is one of the three vertebrate C2HC-type zinc finger transcription factors that include Myt1 (Nzf1), Myt1L (Png1), and Myt3 (Nzf3, St18). All three paralogs are widely expressed in developing neuronal cells. Yet their function for mammalian development has not been investigated directly. Here we report that only Myt1 is expressed in the embryonic pancreas, in both endocrine progenitors and differentiated islet cells. Myt1(-/-) animals die postnatally, likely due to confounding effects in multiple tissues. The endocrine tissues in the embryonic Myt1(-/-) pancreas contained abnormal islet cells that expressed multiple hormones; although hormone levels were normal. We also created pancreas-specific Myt1 knockout mice. These mutant animals had no obvious physical defects from their wild-type littermates. Male mutant animals had reduced glucose-clearing abilities and abnormal multi-hormone-expressing cells present in their endocrine islets. In addition, they also had reduced Glut2 expression, and attenuated glucose-induced insulin secretion in the adult islets. Surprisingly, the expression of the Myt1 paralogs, Myt1l and Myt3, was induced in the embryonic Myt1(-/-) pancreas. The consequences of Myt1 inactivation in the developing pancreas could be masked by activation of its paralogs, Myt1l and Myt3. These findings suggest Myt1 is involved in proper endocrine differentiation and function. [Abstract]

Von Ohlen T, Syu LJ, Mellerick DM
Conserved properties of the Drosophila homeodomain protein, Ind.
Mech Dev. 2007 Nov-Dec;124(11-12):925-34.
Ind-Gsh-type homeodomain proteins are critical to patterning of intermediate domains in the developing CNS; yet, the molecular basis for the activities of these homeodomain proteins is not well understood. Here we identify domains within the Ind protein that are responsible for transcriptional repression, as well as those required for its interaction with the co-repressor, Groucho. To do this, we utilized a combination of chimeric transient transfection assays, co-immunoprecipitation and in vivo expression assays. We show that Ind's candidate Eh1 domain is essential to the embryonic repression activity of this protein, and that Groucho interacts with Ind via this domain. However, when activity is assayed in transient transfection assays using Ind-Gal4 DNA binding domain chimeras to determine domain activity, the repression activity of the Eh1 domain is minimal. This result is similar to previous results on the transcription factors, Vnd and Engrailed. Furthermore, the Eh1 domain is necessary, but not sufficient, for binding to Groucho; the C terminus of Ind, including the homeodomain also affects the interaction with this co-repressor in co-immunoprecipitations. Finally, we show that aspects of the cross-repressive activities of Ind/Gsh2-Ey/Pax6 are evolutionarily conserved. Taken together, these results point to conserved mechanisms used by Gsh/Ind-type homeodomain protein in regulating the expression of target genes. [Abstract]

Rakovitsky N, Buganim Y, Swissa T, Kinel-Tahan Y, Brenner S, Cohen MA, Levine A, Wides R
Drosophila Ten-a is a maternal pair-rule and patterning gene.
Mech Dev. 2007 Nov-Dec;124(11-12):911-24.
The Ten-a gene of Drosophila melanogaster encodes several alternative variants of a full length member of the Odz/Tenm protein family. A number of Ten-a mutants created by inexact excisions of a resident P-element insertion are embryonic lethal, but show no pair-rule phenotype. In contrast, these mutants, and deficiencies removing Ten-a, do enhance the segmentation phenotype of a weak allele of the paralog gene odz (or Ten-m) to the odz amorphic phenotype. Germ line clone derived Ten-a(-) embryos display a pair-rule phenotype which phenocopies that of odz. Post segmentation eye patterning phenotypes of Ten-a mutants establish it as a pleiotropic patterning co-partner of odz. [Abstract]

Vugler A, Lawrence J, Walsh J, Carr A, Gias C, Semo M, Ahmado A, da Cruz L, Andrews P, Coffey P
Embryonic stem cells and retinal repair.
Mech Dev. 2007 Nov-Dec;124(11-12):807-29.
In this review we examine the potential of embryonic stem cells (ESCs) for use in the treatment of retinal diseases involving photoreceptors and retinal pigment epithelium (RPE). We outline the ontogenesis of target retinal cell types (RPE, rods and cones) and discuss how an understanding of developmental processes can inform our manipulation of ESCs in vitro. Due to their potential for cellular therapy, special emphasis is placed upon the derivation and culture of human embryonic stem cells (HESCs) and their differentiation towards a retinal phenotype. In terms of achieving this goal, we suggest that much of the success to date reflects permissive in vitro environments provided by established protocols for HESC derivation, propagation and neural differentiation. In addition, we summarise key factors that may be important for enhancing efficiency of retinal cell-type derivation from HESCs. The retina is an amenable component of the central nervous system (CNS) and as such, diseases of this structure provide a realistic target for the application of HESC-derived cellular therapy to the CNS. In order to further this goal, the second component of our review focuses on the cellular and molecular cues within retinal environments that may influence the survival and behaviour of transplanted cells. Our analysis considers both the potential barriers to transplant integration in the retina itself together with the remodelling in host visual centres that is known to accompany retinal dystrophy. [Abstract]

Nie S, Chang C
PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation.
Mech Dev. 2007 Sep-Oct;124(9-10):657-67.
ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis. [Abstract]

Nambiar RM, Ignatius MS, Henion PD
Zebrafish colgate/hdac1 functions in the non-canonical Wnt pathway during axial extension and in Wnt-independent branchiomotor neuron migration.
Mech Dev. 2007 Sep-Oct;124(9-10):682-98.
Vertebrate gastrulation involves the coordinated movements of populations of cells. These movements include cellular rearrangements in which cells polarize along their medio-lateral axes leading to cell intercalations that result in elongation of the body axis. Molecular analysis of this process has implicated the non-canonical Wnt/Frizzled signaling pathway that is similar to the planar cell polarity pathway (PCP) in Drosophila. Here we describe a zebrafish mutant, colgate (col), which displays defects in the extension of the body axis and the migration of branchiomotor neurons. Activation of the non-canonical Wnt/PCP pathway in these mutant embryos by overexpressing DeltaNdishevelled, rho kinase2 and van gogh-like protein 2 (vangl2) rescues the extension defects suggesting that col acts as a positive regulator of the non-canonical Wnt/PCP pathway. Further, we show that col normally regulates the caudal migration of nVII facial hindbrain branchiomotor neurons and that the mutant phenotype can be rescued by misexpression of vangl2 independent of the Wnt/PCP pathway. We cloned the col locus and found that it encodes histone deacetylase1 (hdac1). Our previous results and studies by others have implicated hdac1 in repressing the canonical Wnt pathway. Here, we demonstrate novel roles for zebrafish hdac1 in activating non-canonical Wnt/PCP signaling underlying axial extension and in promoting Wnt-independent caudal migration of a subset of hindbrain branchiomotor neurons. [Abstract]

Gardiner MR, Gongora MM, Grimmond SM, Perkins AC
A global role for zebrafish klf4 in embryonic erythropoiesis.
Mech Dev. 2007 Sep-Oct;124(9-10):762-74.
There are two waves of erythropoiesis, known as primitive and definitive waves in mammals and lower vertebrates including zebrafish. The founding member of the Kruppel-like factor (KLF) family of CACCC-box binding proteins, EKLF/Klf1, is essential for definitive erythropoiesis in mammals but only plays a minor role in primitive erythropoiesis. Morpholino knockdown experiments have shown a role for zebrafish klf4 in primitive erythropoiesis and hatching gland formation. In order to generate a global understanding of how klf4 might influence gene expression and differentiation, we have performed expression profiling of klf4 morphants, and then performed validation of many putative target genes by qRT-PCR and whole mount in situ hybridization. We found a critical role for klf4 in embryonic globin, heme synthesis and hatching gland gene expression. In contrast, there was an increase in expression of definitive hematopoietic specific genes such as larval globin genes, runx1 and c-myb from 24 hpf, suggesting a selective role for klf4 in primitive rather than definitive erythropoiesis. In addition, we show klf4 preferentially binds CACCC box elements in the primitive zebrafish beta-like globin gene promoters. These results have global implications for primitive erythroid gene regulation by KLF-CACCC box interactions. [Abstract]

Maung SM, Jarman AP
Functional distinctness of closely related transcription factors: a comparison of the Atonal and Amos proneural factors.
Mech Dev. 2007 Sep-Oct;124(9-10):647-56.
Using the well-characterised paradigm of Drosophila sensory nervous system development, we examine the functional distinctness of the Amos and Atonal (Ato) proneural transcription factors, which have different mutant phenotypes but share very high similarity in their signature bHLH domains. Using misexpression and mutant rescue assays, we show that Ato and Amos proteins have abundantly distinct intrinsic proneural capabilities in much of the ectoderm. The eye, however, is an exception: here both proteins share the capability to direct the R8 photoreceptor fate choice. Therefore, functional distinctness between these closely related transcription factors vary with developmental context, indicating different molecular mechanisms of specificity in different contexts. Consistent with this, the structural basis for their distinctness also varies depending upon the function in question. In previous studies of neural bHLH factors, specificity invariably mapped to the bHLH domain sequence. Similarly, and despite their high similarity, much of the Amos' specificity relative to Ato maps to Amos-specific residues in its bHLH domain. For Ato-specific functions, however, the Amos bHLH domain can substitute for that of Ato. Consequently, Ato's specificity relative to Amos requires the non-bHLH portion of the Ato protein. Ato provides a powerful precedence for a role of non-bHLH sequences in modulating bHLH functional specificity. This has implications for structural and functional comparisons of other closely related transcription factors, and for understanding the molecular basis of specificity. [Abstract]

Jansen HJ, Wacker SA, Bardine N, Durston AJ
The role of the Spemann organizer in anterior-posterior patterning of the trunk.
Mech Dev. 2007 Sep-Oct;124(9-10):668-81.
The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk. [Abstract]

Yamamoto M, Watt CD, Schmidt RJ, Kuscuoglu U, Miesfeld RL, Goldhamer DJ
Cloning and characterization of a novel MyoD enhancer-binding factor.
Mech Dev. 2007 Sep-Oct;124(9-10):715-28.
Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1(nlacZ) heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1(nlacZ) embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors. [Abstract]

Welsh IC, Hagge-Greenberg A, O'Brien TP
A dosage-dependent role for Spry2 in growth and patterning during palate development.
Mech Dev. 2007 Sep-Oct;124(9-10):746-61.
The formation of the palate involves the coordinated outgrowth, elevation and midline fusion of bilateral shelves leading to the separation of the oral and nasal cavities. Reciprocal signaling between adjacent fields of epithelial and mesenchymal cells directs palatal shelf growth and morphogenesis. Loss of function mutations in genes encoding FGF ligands and receptors have demonstrated a critical role for FGF signaling in mediating these epithelial-mesenchymal interactions. The Sprouty family of genes encode modulators of FGF signaling. We have established that mice carrying a deletion that removes the FGF signaling antagonist Spry2 have cleft palate. We show that excessive cell proliferation in the Spry2-deficient palate is accompanied by the abnormal progression of shape changes and movements required for medially directed shelf outgrowth and midline contact. Expression of the FGF responsive transcription factors Etv5, Msx1, and Barx1, as well as the morphogen Shh, is restricted to specific regions of the developing palate. We detected elevated and ectopic expression of these transcription factors and disorganized Shh expression in the Spry2-deficient palate. Mice carrying a targeted disruption of Spry2 fail to complement the craniofacial phenotype characterized in Spry2 deletion mice. Furthermore, a Spry2-BAC transgene rescues the palate defect. However, the BAC transgenic mouse lines express reduced levels of Spry2. The resulting hypomorphic phenotype demonstrates that palate development is Spry2 dosage sensitive. Our results demonstrate the importance of proper FGF signaling thresholds in regulation of epithelial-mesenchymal interactions and cellular responses necessary for coordinated morphogenesis of the face and palate. [Abstract]

Recent Articles in Evolution & Development

Allen JD, Pernet B
Intermediate modes of larval development: bridging the gap between planktotrophy and lecithotrophy.
Evol Dev. 2007 Nov-Dec;9(6):643-53.
The extraordinary diversity of larval form and function in marine invertebrates has motivated many studies of development, ecology, and evolution. Among organisms with pelagic development via a larval stage, this diversity is often reduced to a dichotomy between two broad nutritional categories: planktotrophy and lecithotrophy. Despite the clear utility of the planktotrophy-lecithotrophy dichotomy to those interested in the history or consequences of life history patterns, it is also clear that a number of larval forms do not fit neatly into either of these general categories. Here we review studies of these intermediate larval forms, focusing on descriptions of larvae known as facultative feeders. Recent descriptions of larval development suggest that facultative feeders and other intermediate larval forms are not as rare as commonly assumed. We assess the importance of these forms for models of life-history evolution and call for a more-detailed and nuanced view of larval biology to account for their existence. Clearer knowledge of the phylogenetic distribution and frequency of occurrence of larvae that exhibit intermediate nutritional requirements is also essential for evaluating current ideas on evolutionary transitions between planktotrophy and lecithotrophy. Finally, intermediate larval types provide valuable and underutilized opportunities for testing hypotheses in the fields of larval ecology and the evolution of development. [Abstract]

Nevo U, Hauben E
Ecoimmunity: immune tolerance by symmetric co-evolution.
Evol Dev. 2007 Nov-Dec;9(6):632-42.
It is widely accepted that immune tolerance toward "self" is established by central and peripheral adaptations of the immune system. Mechanisms that have been demonstrated to play a role in the induction and maintenance of tolerance include thymic deletion of self-reactive T cells, peripheral T cell anergy and apoptosis, as well as thymic and peripheral induction of regulatory T cells. However, a large body of experimental findings cannot be rationalized solely based on adaptations of the immune system to its environment. Here we propose a new model termed Ecoimmunity, where the immune system and the tissue are viewed as two sides of a continuously active and co-evolving predator-prey system. Ecoimmunity views self-tolerance, not as an equilibrium in which autoimmunity is chronically suppressed, but as a symmetrical balanced conflict between the ability of immune cells to destroy tissue cells by numerous mechanisms, and the capacity of adapted tissue cells to avoid predation. This balance evolves during ontogeny, in parallel to immune adaptations, embryonic tissue cells adapt their phenotype to the corresponding immune activity by developing the ability to escape or modulate damaging local immune responses. This phenotypic plasticity of tissue cells is directed by epigenetic selection of gene expression pattern and cellular phenotype amidst an ongoing immune pressure. Thus, whereas some immune cells prey predominantly on pathogens and infected cells, self-reactive cells continuously prey on incompetent tissue cells that fail to express the adapted phenotype and resist predation. This model uses ecological generalization to reconcile current contradictory observations as well as classical enigmas related to both autoimmunity and to tolerance toward foreign tissues. Finally, it provides empirical predictions and alternative strategies toward clinical challenges. [Abstract]

Riesgo A, Taylor C, Leys SP
Reproduction in a carnivorous sponge: the significance of the absence of an aquiferous system to the sponge body plan.
Evol Dev. 2007 Nov-Dec;9(6):618-31.
Sponges usually produce, release, and capture gametes via the aquiferous system, and so the absence of both choanocytes and an aquiferous system in the carnivorous sponge Asbestopluma occidentalis has led to unusual characteristics of development for this Phylum. Sperm are highly specialized elongate cells tightly packed into spermatic cysts in the peripheral tissue of the sponge. Mature spermatozoa have proacrosomal vesicles at the anterior end and a ciliary pit surrounding the flagellum. Clusters of four to five oocytes are in synchronous stages of cleavage, suggesting that fertilization is synchronous. All stages of embryos occur in the same individual. Early cleavage was holoblastic and equal; blastomeres in two-, four- and eight-cell embryos were compact and 16-cell stage embryos were bi-layered. Late-stage embryos show three cellular regions along the anterior-posterior axis: the anterior hemisphere with heterogeneous cells, a mid-region with cells lying perpendicular to the A-P axis in a collagenous matrix, and small cells at the posterior pole. Unusually for Porifera, multiciliated cells cover all but the posterior pole. It is inferred that fertilization occurs by capture of intact spermatic cysts whose surrounding forceps spicules become trapped in the anisochelae of neighboring sponges. The elongate shape of sperm may be designed to penetrate the loose collagenous mesohyl, such that the arrival of a packet of sperm would lead to simultaneous fertilization of oocytes in a cluster. Loss of the water canal system in carnivorous sponges has allowed the evolution of features that are highly specialized for the habitat of this animal, but such modifications were not necessarily a prerequisite for the subsequent evolution of metazoans. Given the extremely versatile mechanisms of gametogenesis, embryogenesis, and tissue/body structure in sponges, generalizations regarding basal metazoan reproduction, development, and structure must be approached with caution. [Abstract]

Bergter A, Hunnekuhl VS, Schniederjans M, Paululat A
Evolutionary aspects of pattern formation during clitellate muscle development.
Evol Dev. 2007 Nov-Dec;9(6):602-17.
As a taxon of the lophotrochozoans, annelids have re-entered scientific investigations focusing on plesiomorphic bilaterian features and the evolutionary changes therein. The view of a clitellate-like plesiomorphic muscle arrangement in annelids has been challenged by recent investigations of polychaete muscle organization. However, there are few investigations of muscle formation in clitellate species that address this problem. Direct comparison of potential homologous muscles between these annelid groups is thus hampered. Somatic muscle formation during embryogenesis of two clitellates-the oligochaete Limnodrilus sp. and the hirudinean Erpobdella octoculata-occurs by distinct processes in each species, even though they share a closed outer layer of circular and an inner layer of longitudinal muscles characteristic of clitellates. In E. octoculata, the first emerging longitudinal muscles are distributed irregularly on the body surface of the embryo whereas the circular muscles appear in an orderly repetitive pattern along the anterioposterior axis. Both primary muscle types consist of fiber-bundles that branch at both their ends. This way the circular muscle bundles divide into a fine muscle-grid. The primary longitudinal muscles are incorporated into a second type of longitudinal muscles, the latter starting to differentiate adjacent to the ventral nerve cord. Those secondary muscles emerge in a ventral to dorsal manner, enclosing the embryo of E. octoculata. In Limnodrilus sp., one dorsal and one ventral bilateral pair of primary longitudinal muscles are established initially, elongating toward posterior. Initial circular muscles are emerging in a segmental pattern. Both muscle layers are completed later in development by the addition of secondary longitudinal and circular muscles. Some features of embryonic longitudinal muscle patterns in Limnodrilus sp. are comparable to structures found in adult polychaete muscle systems. Our findings show that comparative studies of body-wall muscle formation during clitellate embryogenesis are a promising approach to gain further information on annelid muscle arrangements. [Abstract]

Tokita M, Kiyoshi T, Armstrong KN
Evolution of craniofacial novelty in parrots through developmental modularity and heterochrony.
Evol Dev. 2007 Nov-Dec;9(6):590-601.
Parrots (order Psittaciformes) have developed novel cranial morphology. At the same time, they show considerable morphological diversity in the cranial musculoskeletal system, which includes two novel structures: the suborbital arch and the musculus (M.) pseudomasseter. To understand comprehensively the evolutionary pattern and process of novel cranial morphology in parrots, phylogenetic and developmental studies were conducted. Firstly, we undertook phylogenetic analyses based on mitochondrial ribosomal RNA gene sequences to obtain a robust phylogeny among parrots, and secondly we surveyed the cranial morphology of parrots extensively to add new information on the character states. Character mapping onto molecular phylogenies indicated strongly the repeated evolution of both the suborbital arch and the well-developed M. pseudomasseter within parrots. These results also suggested that the direction of evolutionary change is not always identical in the two characters, implying that these characters are relatively independent or decoupled structures behaving as separate modules. Finally, we compared the developmental pattern of jaw muscles among bird species and found a difference in the timing of M. pseudomasseter differentiation between the cockatiel Nymphicus hollandicus (representative of a well-developed condition) and the peach-faced lovebird Agapornis roseicollis (representative of an underdeveloped condition). On the basis of this study, we suggest that in the development of novel traits, modularity and heterochrony facilitate the diversification of parrot cranial morphology. [Abstract]

O'Sullivan FM, Murphy SK, Simel LR, McCann A, Callanan JJ, Nolan CM
Imprinted expression of the canine IGF2R, in the absence of an anti-sense transcript or promoter methylation.
Evol Dev. 2007 Nov-Dec;9(6):579-89.
Imprinted genes are epigenetically modified in a parent of origin-dependent manner, and as a consequence, are differentially expressed. Although the evolution of genomic imprinting is a subject of intense debate, imprinted genes have been studied primarily in mice and humans and in a small number of marsupial mammals. Comparative studies involving rodents and primates are of limited value, as they belong to the same superordinal group of eutherian mammals (Euarchontoglires). On the other hand, comparisons involving marsupials may not be informative, due to phylogenetic distance. Canis familiaris belongs to Laurasiatheria, a sister-group of Euarchontoglires, and should prove useful in comparative studies of imprinted genes. Using RT-PCR we demonstrate monoallelic expression of the canine IGF2R in several tissues, including uterus and umbilical cord. In the case of umbilical cord, we identify the expressed allele as maternally derived. The canine IGF2R is thus an imprinted gene. Using bisulfite sequencing, we show that the canine IGF2R resembles the imprinted mouse Igf2r in having a CpG island in intron 2 that is hemi-methylated. However, it differs from the mouse gene in that maintenance of the monoallelic expression of canine IGF2R does not require expression of an anti-sense transcript from the paternally derived allele, or methylation of the repressed IGF2R promoter. In these two important features, the imprinted canine gene resembles the imprinted opossum IGF2R. Our data suggest that these features were properties of the ancestral imprinted IGF2R and that the anti-sense transcript (Air) and promoter methylation observed in mouse are derived features of the mouse Igf2r locus. [Abstract]

Piekarski N, Olsson L
Muscular derivatives of the cranialmost somites revealed by long-term fate mapping in the Mexican axolotl (Ambystoma mexicanum).
Evol Dev. 2007 Nov-Dec;9(6):566-78.
The fate of single somites has not been analyzed from a comparative perspective with modern cell-marking methods. Most of what we know is based on work using quail-chick chimeras. Consequently, to what degree cell fate has been conserved despite the anatomical differences among vertebrates is unknown. We have analyzed the cell fate of the cranialmost somites, with the focus on somite two, in the Mexican axolotl (Ambystoma mexicanum). Somite cells were marked by injection of dextran-fluorescein and detected using immunofluorescence after 2 months of development in paraffin sections. Our data confirm and extend earlier studies based on classical histology in salamanders. We show that somite two contributes to different muscles, skeletal elements, and connective tissues of the head and cranial trunk region. Cells from somites two and three migrate latero-ventrally and contribute to the hypobranchial muscles mm. geniohyoideus and rectus cervicis. We provide evidence that the specific formation of the hypobranchial musculature from ventral processes of the somites might be variable in different classes of vertebrates. We further demonstrate that mm. cucullaris and dilatator laryngis, which were earlier thought to have a branchial origin, arise from somitic material in a manner very similar to the findings in quail-chick chimeras. Our findings indicate that the pattern of somitic derivatives is highly conserved within tetrapods. [Abstract]

Sears KE, Goswami A, Flynn JJ, Niswander LA
The correlated evolution of Runx2 tandem repeats, transcriptional activity, and facial length in carnivora.
Evol Dev. 2007 Nov-Dec;9(6):555-65.
To assess the ability of protein-coding mutations to contribute to subtle, inter-specific morphologic evolution, here, we test the hypothesis that mutations within the protein-coding region of runt-related transcription factor 2 (Runx2) have played a role in facial evolution in 30 species from a naturally evolving group, the mammalian order Carnivora. Consistent with this hypothesis, we find significant correlations between changes in Runx2 glutamine-alanine tandem-repeat ratio, and both Runx2 transcriptional activity and carnivoran facial length. Furthermore, we identify a potential evolutionary mechanism for the correlation between Runx2 tandem repeat ratio and facial length. Specifically, our results are consistent with the Runx2 tandem repeat system providing a flexible genetic mechanism to rapidly change the timing of ossification. These heterochronic changes, in turn, potentially act on existing allometric variation in carnivoran facial length to generate the disparity in adult facial lengths observed among carnivoran species. Our results suggest that despite potentially great pleiotropic effects, changes to the protein-coding regions of genes such as Runx2 do occur and have the potential to affect subtle morphologic evolution across a diverse array of species in naturally evolving lineages. [Abstract]

Hurley IA, Scemama JL, Prince VE
Consequences of hoxb1 duplication in teleost fish.
Evol Dev. 2007 Nov-Dec;9(6):540-54.
Vertebrate evolution is characterized by gene and genome duplication events. There is strong evidence that a whole-genome duplication occurred in the lineage leading to the teleost fishes. We have focused on the teleost hoxb1 duplicate genes as a paradigm to investigate the consequences of gene duplication. Previous analysis of the duplicated zebrafish hoxb1 genes suggested they have subfunctionalized. The combined expression pattern of the two zebrafish hoxb1 genes recapitulates the expression pattern of the single Hoxb1 gene of tetrapods, possibly due to degenerative changes in complementary cis-regulatory elements of the duplicates. Here we have tested the hypothesis that all teleost duplicates had a similar fate post duplication, by examining hoxb1 genes in medaka and striped bass. Consistent with this theory, we found that the ancestral Hoxb1 expression pattern is subdivided between duplicate genes in a largely similar fashion in zebrafish, medaka, and striped bass. Further, our analysis of hoxb1 genes reveals that sequence changes in cis-regulatory regions may underlie subfunctionalization in all teleosts, although the specific changes vary between species. It was previously shown that zebrafish hoxb1 duplicates have also evolved different functional capacities. We used misexpression to compare the functions of hoxb1 duplicates from zebrafish, medaka and striped bass. Unexpectedly, we found that some biochemical properties, which were paralog specific in zebrafish, are conserved in both duplicates of other species. This work suggests that the fate of duplicate genes varies across the teleost group. [Abstract]

Kingsley EP, Chan XY, Duan Y, Lambert JD
Widespread RNA segregation in a spiralian embryo.
Evol Dev. 2007 Nov-Dec;9(6):527-39.
Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3-4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four-cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24-cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups. [Abstract]

Patterson JS, Klingenberg CP
Developmental buffering: how many genes?
Evol Dev. 2007 Nov-Dec;9(6):525-6. [Abstract]

Oakley TH
Today's multiple choice exam: (a) gene duplication; (b) structural mutation; (c) co-option; (d) regulatory mutation; (e) all of the above.
Evol Dev. 2007 Nov-Dec;9(6):523-4. [Abstract]

Frank U
The evolution of a malignant dog.
Evol Dev. 2007 Nov-Dec;9(6):521-2. [Abstract]

Evol Dev. 2007 Sep-Oct;9(5):520. [Abstract]

Balmer O
A review of Evolutionary Ecology of Parasites, by Robert Poulin.
Evol Dev. 2007 Sep-Oct;9(5):517-9. [Abstract]

Oakley TH
A review of Gene Sharing and Evolution: The Diversity of Protein Functions, by Joram Piatigorsky.
Evol Dev. 2007 Sep-Oct;9(5):514-6. [Abstract]

Zera AJ
Endocrine analysis in evolutionary-developmental studies of insect polymorphism: hormone manipulation versus direct measurement of hormonal regulators.
Evol Dev. 2007 Sep-Oct;9(5):499-513.
"Hormone manipulation" is being used increasingly in evo-devo studies as the sole or primary technique to investigate the regulation of insect polymorphism by hormones, most notably juvenile hormone (JH). This manuscript critically evaluates the limitations and strengths of this indirect method for inferring aspects of endocrine regulation, and conclusions derived from recent endocrine studies of evolution and development in which data have been obtained primarily or exclusively by this method. The main conclusions of this critique are as follows: first, when used alone, or as the primary empirical technique, hormone manipulation is a superficial method that is fraught with problems with respect to identifying a hormone that regulates developmental-morphological variation, let alone identifying its mode of action. Second, conclusions reported in studies using this technique as the exclusive, or nearly exclusive experimental approach, most notably recent studies of JH regulation of horn polymorphism in dung beetles, and some studies of wing polymorphism should be considered, at best, weakly supported until substantiated by well-validated, direct methods. Finally, there are many reliable and well-validated techniques that can be used to directly and accurately quantify JH levels, and activities of JH regulators, in many insects, even in small, nonmodel species. Some of the most important of these assays will be briefly described and their strengths and weaknesses will be discussed. [Abstract]

Hunda BR, Hughes NC
Evaluating paedomorphic heterochrony in trilobites: the case of the diminutive trilobite Flexicalymene retrorsa minuens from the Cincinnatian Series (Upper Ordovician), Cincinnati region.
Evol Dev. 2007 Sep-Oct;9(5):483-98.
Flexicalymene retrorsa minuens from the uppermost 3 m of the Waynesville Formation of the Cincinnatian Series (Upper Ordovician) of North America lived approximately 445 Ma and exhibited marked reduction in maximum size relative to its stratigraphically subjacent sister subspecies, Flexicalymene retrorsa retrorsa. Phylogenetic analysis is consistent with the notion that F. retrorsa retrorsa was the ancestor of F. retrorsa minuens. F. retrorsa minuens has been claimed to differ from F. retrorsa retrorsa"in size alone," and thus presents a plausible example of global paedomorphic evolution in trilobites. Despite strong similarity in the overall form of the two subspecies, F. retrorsa minuens is neither a dwarf nor a simple progenetic descendant of F. retrorsa retrorsa. More complex patterns of global heterochronic paedomorphosis, such as a neotonic decrease in the rate of progress along a common ontogenetic trajectory with respect to size, coupled with growth cessation at a small size, "sequential" progenesis, or non-uniform changes in the rate of progress along a shared ontogenetic trajectory with respect to size, can also be rejected. Rather, differences between these subspecies are more consistent with localized changes in rates of character development than with a global heterochronic modification of the ancestral ontogeny. The evolution of F. retrorsa minuens from F. retrorsa retrorsa was largely dominated by modifications of the development of characters already evident in the ancestral ontogeny, not by the origin of novel structures. Factors promoting size reduction in F. retrorsa minuens appear to have been specific to this subspecies, because other co-occurring taxa, including other trilobite species, do not show marked differences in mean size. [Abstract]

Gerber S, Neige P, Eble GJ
Combining ontogenetic and evolutionary scales of morphological disparity: a study of early Jurassic ammonites.
Evol Dev. 2007 Sep-Oct;9(5):472-82.
Two major research themes in Evolutionary Developmental Biology and in Paleobiology, respectively, have each become central for the analysis and interpretation of morphological changes in evolution: the study of ontogeny/phylogeny connections, mainly within the widespread and controversial framework of heterochrony; and the study of morphological disparity, the morphological signal of biodiversity, describing secular changes in morphospace occupation during the history of any given clade. Although enriching in their respective fields, these two themes have remained rather isolated to date, despite the potential value of integrating them as some recent studies begin to suggest. Here, we explore the recent notion of developmental morphospace-morphospace carrying ontogenetic information-as a potential tool for bridging the gap between disparity dynamics and developmental dynamics. We elaborate this approach with a case study of Early Jurassic ammonite family Hildoceratidae (Mollusca, Cephalopoda). Morphometric analyses of the shell shape of 20 species spanning the morphological spectrum of the family are used to quantify and contrast juvenile and adult disparity levels. Adult disparity is significantly greater than juvenile disparity at the family level; yet, some subclades also display different patterns. In addition, comparisons of ontogenetic trajectories underline the prevalence of heterochrony-based evolutionary modifications within subfamilies (via ontogenetic scaling); they also point to the probable existence of pervasive developmental constraints structuring inhomogeneous morphospace occupation. [Abstract]

Penet L, Laurin M, Gouyon PH, Nadot S
Constraints and selection: insights from microsporogenesis in Asparagales.
Evol Dev. 2007 Sep-Oct;9(5):460-71.
Developmental constraints have been proposed to interfere with natural selection in limiting the available set of potential adaptations. Whereas this concept has long been debated on theoretical grounds, it has been investigated empirically only in a few studies. In this article, we evaluate the importance of developmental constraints during microsporogenesis (male meiosis in plants), with an emphasis on phylogenetic patterns in Asparagales. Different developmental constraints were tested by character reshuffling or by simulated distributions. Among the different characteristics of microsporogenesis, only cell wall formation appeared as constrained. We show that constraints may also result from biases in the correlated occurrence of developmental steps (e.g., lack of successive cytokinesis when wall formation is centripetal). We document such biases and their potential outcomes, notably the establishment of intermediate stages, which allow development to bypass such constraints. These insights are discussed with regard to potential selection on pollen morphology. [Abstract]

Vázquez-Lobo A, Carlsbecker A, Vergara-Silva F, Alvarez-Buylla ER, Pińero D, Engström P
Characterization of the expression patterns of LEAFY/FLORICAULA and NEEDLY orthologs in female and male cones of the conifer genera Picea, Podocarpus, and Taxus: implications for current evo-devo hypotheses for gymnosperms.
Evol Dev. 2007 Sep-Oct;9(5):446-59.
The identity of genes causally implicated in the development and evolutionary origin of reproductive characters in gymnosperms is largely unknown. Working within the framework of plant evolutionary developmental biology, here we have cloned, sequenced, performed phylogenetic analyses upon and tested the expression patterns of LEAFY/FLORICAULA and NEEDLY orthologs in reproductive structures from selected species of the conifer genera Picea, Podocarpus, and Taxus. Contrary to expectations based on previous assessments, expression of LFY/FLO and NLY in cones of these taxa was found to occur simultaneously in a single reproductive axis, initially overlapping but later in mutually exclusive primordia and/or groups of developing cells in both female and male structures. These observations directly affect the status of the "mostly male theory" for the origin of the angiosperm flower. On the other hand, comparative spatiotemporal patterns of the expression of these genes suggest a complex genetic regulatory network of cone development, as well as a scheme of functional divergence for LFY/FLO with respect to NLY homologs in gymnosperms, both with clear heterochronic aspects. Results presented in this study contribute to the understanding of the molecular-genetic basis of morphological evolution in conifer cones, and may aid in establishing a foundation for gymnosperm-specific, testable evo-devo hypotheses. [Abstract]

Byrne M, Nakajima Y, Chee FC, Burke RD
Apical organs in echinoderm larvae: insights into larval evolution in the Ambulacraria.
Evol Dev. 2007 Sep-Oct;9(5):432-45.
The anatomy and cellular organization of serotonergic neurons in the echinoderm apical organ exhibits class-specific features in dipleurula-type (auricularia, bipinnaria) and pluteus-type (ophiopluteus, echinopluteus) larvae. The apical organ forms in association with anterior ciliary structures. Apical organs in dipleurula-type larvae are more similar to each other than to those in either of the pluteus forms. In asteroid bipinnaria and holothuroid auricularia the apical organ spans ciliary band sectors that traverse the anterior-most end of the larvae. The asteroid apical organ also has prominent bilateral ganglia that connect with an apical network of neurites. The simple apical organ of the auricularia is similar to that in the hemichordate tornaria larva. Apical organs in pluteus forms differ markedly. The echinopluteus apical organ is a single structure on the oral hood between the larval arms comprised of two groups of cells joined by a commissure and its cell bodies do not reside in the ciliary band. Ophioplutei have a pair of lateral ganglia associated with the ciliary band of larval arms that may be the ophiuroid apical organ. Comparative anatomy of the serotonergic nervous systems in the dipleurula-type larvae of the Ambulacraria (Echinodermata+Hemichordata) suggests that the apical organ of this deuterostome clade originated as a simple bilaterally symmetric nerve plexus spanning ciliary band sectors at the anterior end of the larva. From this structure, the apical organ has been independently modified in association with the evolution of class-specific larval forms. [Abstract]

Gonzales EE, van der Zee M, Dictus WJ, van den Biggelaar J
Brefeldin A and monensin inhibit the D quadrant organizer in the polychaete annelids Arctonoe vittata and Serpula columbiana.
Evol Dev. 2007 Sep-Oct;9(5):416-31.
The D quadrant organizer is a developmental signaling center that is localized to the vegetal D quadrant in different spiral-cleaving lophotrochozoan embryos and may be homologous to axial organizing regions in other metazoans. Patterning by this organizing center creates a secondary developmental axis and is required for the transition from spiral to bilateral cleavage and later establishment of the adult body plan. Organizer specification in equal-cleaving embryos is thought to involve inductive interactions between opposing animal and vegetal blastomeres. To date, experimental demonstration of this interaction has been limited to molluscs and nemerteans. Here, we examine three families of equal-cleaving polychaete annelids for evidence of animal-vegetal contact. We find that contact is present in the polynoid, Arctonoe vittata, but is absent in the serpulid, Serpula columbiana, and in the oweniid, Oweniia fusiformis. To interfere with cell signaling during the period predicted for organizer specification and patterning in A. vittata and S. columbiana, we use two general inhibitors of protein processing and secretion: Brefeldin A (BFA) and monensin. In A. vittata, we detail subsequent embryonic and larval adult development and show that treatment with either chemical results in radialization of the embryo and subsequent body plan. Radialized larvae differentiate many larval and adult structures despite the loss of bilateral symmetry but do so in either a radially symmetric or four-fold radially symmetric fashion. Our results suggest that the D quadrant organizer is functionally conserved in equal-cleaving polychaetes, but that details of its specification, induction, and patterning have diverged relative to other spiral-cleaving phyla. [Abstract]

Sempere LF, Martinez P, Cole C, Baguńŕ J, Peterson KJ
Phylogenetic distribution of microRNAs supports the basal position of acoel flatworms and the polyphyly of Platyhelminthes.
Evol Dev. 2007 Sep-Oct;9(5):409-15.
Phylogenetic analyses based on gene sequences suggest that acoel flatworms are not members of the phylum Platyhelminthes, but instead are the most basal branch of triploblastic bilaterians. Nonetheless, this result has been called into question. An alternative test is to use qualitative molecular markers that should, in principle, exclude the possibility of convergent (homoplastic) evolution in unrelated groups. microRNAs (miRNAs), noncoding regulatory RNA molecules that are under intense stabilizing selection, are a newly discovered set of phylogenetic markers that can resolve such taxonomic disputes. The acoel Childia sp. has recently been shown to possess a subset of the conserved core of miRNAs found across deuterostomes and protostomes, whereas a polyclad flatworm-in addition to this core subset-possesses miRNAs restricted to just protostomes. Here, we examine another acoel, Symsagittifera roscoffensis, and three other platyhelminths. Our results show that the distribution of miRNAs in S. roscoffensis parallels that of Childia. In addition, two of 13 new miRNAs cloned from a triclad flatworm are also found in other lophotrochozoan protostomes, but not in ecdysozoans, deuterostomes, or in basal metazoans including acoels. The limited set of miRNAs found in acoels, intermediate between the even more reduced set in cnidarians and the larger and expanding set in the rest of bilaterians, is compelling evidence for the basal position of acoel flatworms and the polyphyly of Platyhelminthes. [Abstract]

Peterson KJ, Sperling EA
Poriferan ANTP genes: primitively simple or secondarily reduced?
Evol Dev. 2007 Sep-Oct;9(5):405-8. [Abstract]

Hendrikse JL, Parsons TE, Hallgrímsson B
Evolvability as the proper focus of evolutionary developmental biology.
Evol Dev. 2007 Jul-Aug;9(4):393-401.
Research conducted under the label of evolutionary developmental biology has tended to revolve around a few central issues such as modularity, integration, and canalization. Yet, as the field has grown, it has become increasingly difficult to define in terms of its central question and relation to broader evolutionary concerns. We argue that these central issues of evo-devo gain their currency from connections to a central question that defines the field, and we propose that this central question is about the nature of evolvability. However, not all research currently carried out under the label of "evo-devo" speaks to this focal concern. The aim of this article is therefore to argue for a precise formulation of evolutionary developmental biology's core question. [Abstract]

Sharp JA, Lefčvre C, Nicholas KR
Molecular evolution of monotreme and marsupial whey acidic protein genes.
Evol Dev. 2007 Jul-Aug;9(4):378-92.
Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation. [Abstract]

Orgogozo V, Muro NM, Stern DL
Variation in fiber number of a male-specific muscle between Drosophila species: a genetic and developmental analysis.
Evol Dev. 2007 Jul-Aug;9(4):368-77.
We characterize a newly discovered morphological difference between species of the Drosophila melanogaster subgroup. The muscle of Lawrence (MOL) contains about four to five fibers in D. melanogaster and Drosophila simulans and six to seven fibers in Drosophila mauritiana and Drosophila sechellia. The same number of nuclei per fiber is present in these species but their total number of MOL nuclei differs. This suggests that the number of muscle precursor cells has changed during evolution. Our comparison of MOL development indicates that the species difference appears during metamorphosis. We mapped the quantitative trait loci responsible for the change in muscle fiber number between D. sechellia and D. simulans to two genomic regions on chromosome 2. Our data eliminate the possibility of evolving mutations in the fruitless gene and suggest that a change in the twist might be partly responsible for this evolutionary change. [Abstract]

Marlow HQ, Martindale MQ
Embryonic development in two species of scleractinian coral embryos: Symbiodinium localization and mode of gastrulation.
Evol Dev. 2007 Jul-Aug;9(4):355-67.
Reef-building scleractinian corals widely engage in symbiotic relationships with Symbiodinium dinoflagellates (zooxanthellae), which reside inside cells of the gastrodermis. In most cases, sexually produced larvae acquire their symbionts from the environment in the early developmental stages preceding settlement; however, some scleractinian corals maternally "seed" their oocytes with symbionts, and these symbionts are reported to be restricted to the gastrodermis at the time of its formation (gastrulation). A precise mechanism for how Symbiodinium are translocated to endoderm in these seeded species was previously unknown. In order to examine the process of endoderm formation and Symbiodinium localization during gastrulation, we have examined two species of "robust" clade scleractinians: Fungia scutaria (nonseeded) and Pocillopora meandrina (maternally seeded). We determined that both species, independent of whether or not they are seeded, undergo a "nutritive" stage before gastrulation, wherein lipid-rich cells (F. scutaria) or membrane-bound cellular fragments (P. meandrina) are passed to the blastocoel where they are subsequently taken up by the definitive endoderm. This emergent property of anthozoan development has been co-opted to facilitate the movement of Symbiodinium to the blastocoel (future site of endoderm), in the seeded species, where they are later phagocytosed by the newly formed definitive endoderm. Additionally, both species of robust clade scleractinians examined gastrulate by way of invagination, as do the majority of anthozoans. This invagination differs from the prawn chip-type gastrulation seen in the complex clade corals and provides evidence for a possible linkage between gastrulation type and phylogenetic history. [Abstract]

Bowsher JH, Nijhout HF
Evolution of novel abdominal appendages in a sepsid fly from histoblasts, not imaginal discs.
Evol Dev. 2007 Jul-Aug;9(4):347-54.
Abdominal appendages in male sepsid flies are a complex novel structure of unknown developmental and evolutionary origin. Although these abdominal appendages superficially resemble serially homologous insect appendages, they do not develop from imaginal discs like other dipteran appendages. Cauterization of the genital disc and ventral abdominal histoblasts in Themira biloba (Sepsidae, Diptera) revealed that these abdominal appendages develop from the ventral histoblast nests of the fourth abdominal segment. Cell counts of the histoblasts in males and females revealed that the ventral histoblast nests on the fourth abdominal segment in males were significantly larger than other histoblast nests, indicating that the specification of that segment as the location of the abdominal appendages occurs before the last larval instar. The recruitment of histoblasts to produce appendages has not been documented before, and implies a developmental and evolutionary potential for histoblasts that was previously unknown. [Abstract]