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The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion. [Display omitted] • Avian model of neural tube defects resembling human spina bifida. • Role of the tight junction protein Claudin-3 in fusion of the neural tube. • Effects of Claudin-3 in regulating apical protein localization patterns. [ABSTRACT FROM AUTHOR]

Cell division and cytoskeleton organization are fundamental processes participating in the development of Drosophila imaginal discs. In this manuscript we describe the phenotypes in the adult fly wing generated by knockdowns of 85% of Drosophila genes encoding proteins likely related to the regulation of cell division and cytoskeleton organization. We also compile a molecular classification of these proteins into classes that describe their expected or known main biochemical characteristics, as well as mRNA expression in the wing disc and likely protein subcellular localization for a subset of these genes. Finally, we analyze in more detail one protein family of cytoskeleton genes (Arp2/3 complex), and define the consequences of interfering with cell division for wing growth and patterning. [Display omitted] • We studied Drosophila wing phenotypes of cell division and the cytoskeleton genes. • Cell division and cytoskeletal genes affect mainly wing size. • The Arp2/3 complex influences cell division and survival in epithelial cells. • An identical venation pattern is accommodated into extremely different wing sizes. [ABSTRACT FROM AUTHOR]

Fibroblasts play an important role in maintaining tissue integrity by secreting components of the extracellular matrix and initiating response to injury. Although the function of fibroblasts has been extensively studied in adults, the embryonic origin and diversification of different fibroblast subtypes during development remain largely unexplored. Using zebrafish as a model, we show that the sclerotome, a sub-compartment of the somite, is the embryonic source of multiple fibroblast subtypes including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, fin mesenchymal cells, and interstitial fibroblasts. High-resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Long-term Cre-mediated lineage tracing reveals that the sclerotome also contributes to cells closely associated with the axial skeleton. Ablation of sclerotome progenitors results in extensive skeletal defects. Using photoconversion-based cell lineage analysis, we find that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single-cell clonal analysis combined with in vivo imaging suggests that the sclerotome mostly contains unipotent and bipotent progenitors prior to cell migration, and the fate of their daughter cells is biased by their migration paths and relative positions. Together, our work demonstrates that the sclerotome is the embryonic source of trunk fibroblasts as well as the axial skeleton, and local signals likely contribute to the diversification of distinct fibroblast subtypes. [Display omitted] • The sclerotome is the embryonic source of distinct fibroblasts in zebrafish. • The sclerotome contributes to the proper development of the axial skeleton. • The sclerotome mostly contains unipotent and bipotent progenitors. • Sclerotome differentiation is biased by the axial position and migratory path. [ABSTRACT FROM AUTHOR]

Charles Breder, a pioneering researcher of blind Mexican cavefish was the first to note extreme variation in the facial skeleton of this intriguing subterranean-dwelling organism. Using a system of polar coordinate plots, he identified substantial dysmorphic changes affecting bones of the orbital skeleton. A complication of his landmark publication from 1944 was an error in the number of orbital bones depicted for this species. Intriguingly, however, he proposed an unknown "organizing force" likely influences final bone position and associated dysmorphia. At the time this was merely hypothetical. Roughly eight decades since its publication, however, insights into sensory influences on facial bone development may explain dysmorphia and variation in bone numbers for Astyanax cavefish. A morphological association between mechano-sensory neuromasts of the lateral line and dermal bones of the facial skeleton had been appreciated in the classical literature, but the polarity of this interaction has long remained unclear. Here, we propose that sensory-skeletal integration between sensory neuromasts and bones explain the incomplete numbers of bones, and dysmorphic features such as fusion between neighboring elements. We propose that in closely-related surface fish (and most teleost fish) this developmental coupling enables the sensory and skeletal systems to become integrated into a functional unit over the course of life history. In this opinion article, we discuss the relevance of this (poorly understood) phenomenon as a potential evolutionary source of variation in the facial bone structures of taxa across deep geologic time. We provide three potential explanations for the error in Breder's drawings, that may be explained by natural developmental variation documented in other related species. Moreover, we argue that the natural variation in this "evolutionary" model system is useful for explaining diverse cranial features by uniting aberrations occurring during embryogenesis with long-term adult dysmorphia. [Display omitted] • Charles Breder represented Astyanax as having too few orbital bones. • Breder proposed an "unknown organizing force" governed development of these bones. • This "force" is likely the osteogenic capacity of sensory neuromasts. • Sensory neuromasts likely induce bone to prefigure their functional role. • Variations on sensory-skeletal integration explain aberrations in cavefish. [ABSTRACT FROM AUTHOR]

Ethanol is a known vertebrate teratogen that causes craniofacial defects as a component of fetal alcohol syndrome (FAS). Our results show that sea urchin embryos treated with ethanol similarly show broad skeletal patterning defects, potentially analogous to the defects associated with FAS. The sea urchin larval skeleton is a simple patterning system that involves only two cell types: the primary mesenchymal cells (PMCs) that secrete the calcium carbonate skeleton and the ectodermal cells that provide migratory, positional, and differentiation cues for the PMCs. Perturbations in RA biosynthesis and Hh signaling pathways are thought to be causal for the FAS phenotype in vertebrates. Surprisingly, our results indicate that these pathways are not functionally relevant for the teratogenic effects of ethanol in developing sea urchins. We found that developmental morphology as well as the expression of some ectodermal and PMC genes was delayed by ethanol exposure. Temporal transcriptome analysis revealed significant impacts of ethanol on signaling and metabolic gene expression, and a disruption in the timing of GRN gene expression that includes both delayed and precocious gene expression throughout the specification network. We conclude that the skeletal patterning perturbations in ethanol-treated embryos likely arise from a loss of temporal synchrony within and between the instructive and responsive tissues. [Display omitted] • Ethanol perturbs sea urchin skeletal patterning. • Ethanol-mediated phenotypes are not mediated by retinoic acid. • Ethanol desynchronizes specification network gene expression timing. [ABSTRACT FROM AUTHOR]

Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, we found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, we identified components of Mib2 complexes, which include E-cadherin and α- and β-catenins, as well as actin regulators. We also found that three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. We conclude that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms. [Display omitted] • Drosophila Mind bomb 2, an E3 ligase, is highly expressed in ovarian follicle cells. • mind bomb 2 is required for border cell migration and epithelial character. • Mind bomb 2 interactors, RhoGAP19D, Myosin heavy chain-like , and Supervillin , act in migration. • Loss of mind bomb 2 results in reduced adhesion complexes and F-actin. Summary statement: Mind bomb 2, an E3 ubiquitin ligase, enables collective cell migration in Drosophila and promotes epithelial character by stabilizing adhesion complexes and cytoskeletal proteins. [ABSTRACT FROM AUTHOR]

Sea urchin larvae spend weeks to months feeding on plankton prior to metamorphosis. When handled in the laboratory they are easily injured, suggesting that in the plankton they are injured with some frequency. Fortunately, larval wounds are repaired through an efficient wound response with mesenchymal pigment cells and blastocoelar cells assisting as the epithelium closes. An injury to the epithelium leads to an immediate calcium transient that rapidly spreads around the entire larva and is necessary for activating pigment cell migration toward the wound. If calcium transport is blocked, the pigment cells fail to activate and remain in place. When activated, pigment cells initiate directed migration to the wound site from distances of at least 85 ​μm. Upon arrival at the wound site they participate in an innate immune response. Blastocoelar cells are recruited to the injury site as well, though the calcium transient is unnecessary for activating these cells. At the wound site, blastocoelar cells participate in several functions including remodeling the skeleton if it protrudes through the epithelium. The graphic shows a skeletal rod protruding through the epithelium of the larval arm. Pigment cells and blastocoelar cells respond to provide an immune response plus skeletal remodeling functions. A Calcium transient, initiated by the wound, rapidly spreads around the entire larva to activate the pigment cell migration toward the wound. Upon arrival at the wound pigment cells (maroon) degranulate to participate in an innate immune response. Blastocoelar cells (green) are recruited to the injury site as well. These cells exit the larva, attach to, and excise the protruding skeleton thereby allowing the epithelium to close the wound. [Display omitted] • Sea urchin larval pigment and blastocoelar cells respond to assist in injury repair. • The pigment cell response is activated by a Calcium transient. • At the injury site pigment cells release echinochrome A, a bacteriocide. • Blastocoelar cells remodel the skeleton allowing the epithelium to close the wound. [ABSTRACT FROM AUTHOR]

We previously showed the importance of TGFβ signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFβ signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFβRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFβ signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFβRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFβ signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development. [Display omitted] • Injection of TGFBR1 inhibitor in chick somites resulted in defects in axial skeleton. • TGFβ signaling was required for normal resegmentation of sclerotome. • Inhibition of TGFβ signaling resulted in altered sclerotome polarity. • Lineage tracer dyes and drug inhibitors can be used to study somite development in chick. [ABSTRACT FROM AUTHOR]

The morphogenesis and plasticity of dendritic spines are associated with synaptic strength, learning, and memory. Dendritic spines are highly compartmentalized structures, which makes proteins involved in cellular polarization and membrane compartmentalization likely candidates regulating their formation and maintenance. Indeed, recent studies suggest polarity proteins help form and maintain dendritic spines by compartmentalizing the spine neck and head. Here, we review emerging evidence that polarity proteins regulate dendritic spine plasticity and stability through the cytoskeleton, scaffolding molecules, and signaling molecules. We specifically analyze various polarity complexes known to contribute to different forms of cell polarization processes and examine the essential conceptual context linking these groups of polarity proteins to dendritic spine morphogenesis, plasticity, and cognitive functions. The morphogenesis and plasticity of dendritic spines are associated with synaptic strength, learning, and memory. Dendritic spines are highly compartmentalized structures, which makes proteins involved in cellular polarization and membrane compartmentalization likely candidates regulating their formation and maintenance. Here, we review emerging evidence that polarity proteins regulate dendritic spine plasticity and stability through the cytoskeleton, scaffolding molecules, and signaling molecules. [Display omitted] • Polarity proteins regulate the compartmentalization of dendritic spines. • Many polarity proteins regulate synaptic and cognitive functions. • Neurodevelopmental disorders have been linked to polarity proteins. [ABSTRACT FROM AUTHOR]

The ability of stem cells to divide asymmetrically is crucial for cell-type diversity and tissue homeostasis. Drosophila neural stem cells, also knowns as neuroblasts, utilize asymmetric cell division to self-renew and give rise to differentiated daughter cells. Drosophila neuroblasts relies on the polarized protein complexes on the apical and basal cortex to govern cell polarity and asymmetry. Here, we review recent advances in our understanding of the neuroblast polarity focusing on how actin cytoskeleton, phosphoinositide lipids and liquid-liquid phase separation regulate the asymmetric cell division of Drosophila neuroblasts. [Display omitted] • Cortical flows mediated by actin cytoskeleton drive asymmetric division cycles of Drosophila neuroblasts. • Phosphatidylinositol lipids facilitate membrane targeting of polarity proteins and Myosin II in the asymmetric division. • Phase separation mediates the localization of polarity proteins in Drosophila neuroblasts. [ABSTRACT FROM AUTHOR]

Sea urchin larval skeletons are produced by skeletogenic primary mesenchyme cells (PMCs), which migrate to form two ventrolateral clusters (VLCs) at the sites where biomineralization is initiated. Both PMC migration and biomineralization are controlled by VEGF signals emitted from lateral ectodermal cells. In mammals, VEGF signaling can be activated by hypoxia-inducible factor alpha (HIFα), an oxygen-sensitive transcription factor. Our previous study showed that the sea urchin maternal HIFα is involved in regulating gene expression along the dorsoventral axis. In this study, we discovered that zygotic hifα is expressed in PMCs, and at the late gastrula stage, hifα transcripts display a graded pattern, with stronger signal in the ventral PMCs than in the dorsal PMCs. We further showed that PMCs are hypoxic, which is a condition typically required for HIFα function. In embryos injected with a splice-blocking morpholino against hifα , elongation of the skeleton was impaired, and expression of vegfr-10-Ig (encodes VEGF receptor; VEGFR) was significantly reduced. This morpholino-caused defect could be partially rescued by injection of vegfr-10-Ig mRNA. Expression patterns of transcription factor and biomineralization genes, such as alx1 , tbr , msp130 , and the sm30 family, were affected when HIFα was knocked down or when VEGF signaling was inhibited. These results suggest that zygotic HIFα acts upstream or in parallel with VEGF signaling to regulate skeletogenic gene expression and participate in spicule elongation. Our study therefore links HIFα with the known role of VEGF signaling in sea urchin biomineralization. [Display omitted] • The sea urchin hifα gene is activated zygotically in PMCs. • PMCs are hypoxic during normal sea urchin embryogenesis. • Zygotic HIFα is involved in spicule elongation in the sea urchin embryo. • Zygotic HIFα acts upstream or in parallel with VEGF signaling to regulate skeletogenic gene expression. [ABSTRACT FROM AUTHOR]

The microtubule cytoskeleton is critical for maintenance of long and long-lived neurons. The overlapping array of microtubules extends from the major site of synthesis in the cell body to the far reaches of axons and dendrites. New materials are transported from the cell body along these neuronal roads by motor proteins, and building blocks and information about the state of affairs in other parts of the cell are returned by motors moving in the opposite direction. As motor proteins walk only in one direction along microtubules, the combination of correct motor and correctly oriented microtubules is essential for moving cargoes in the right direction. In this review, we focus on how microtubule polarity is established and maintained in neurons. At first thought, it seems that figuring out how microtubules are organized in neurons should be simple. After all, microtubules are essentially sticks with a slow-growing minus end and faster-growing plus end, and arranging sticks within the constrained narrow tubes of axons and dendrites should be straightforward. It is therefore quite surprising how many mechanisms contribute to making sure they are arranged in the correct polarity. Some of these mechanisms operate to generate plus-end-out polarity of axons, and others control mixed or minus-end-out dendrites. [Display omitted] [ABSTRACT FROM AUTHOR]

Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans , and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans. [Display omitted] • In parthenogenetic D. pachys , the first embryonic cell division is asymmetric. • In D. pachys embryos, the nucleus inhabits principally the future posterior pole. • Embryo centrifugation revealed that the posterior pole is defined during meiosis. • Pre-meiotic D. pachys oocytes do not show signs of asymmetry. • The meiotic spindle appears to trigger the location of the future posterior pole. [ABSTRACT FROM AUTHOR]

Actin filament crosslinking, bundling and molecular motor proteins are necessary for the assembly of epithelial projections such as microvilli, stereocilia, hairs, and bristles. Mutations in such proteins cause defects in the shape, structure, and function of these actin - based protrusions. One protein necessary for stereocilia formation, Myosin VIIA, is an actin - based motor protein conserved throughout phylogeny. In Drosophila melanogaster, severe mutations in the MyoVIIA homolog crinkled (ck) are "semi - lethal" with only a very small percentage of flies surviving to adulthood. Such survivors show morphological defects related to actin bundling in hairs and bristles. To better understand ck /MyoVIIA's function in bundled - actin structures, we used dominant female sterile approaches to analyze the loss of maternal and zygotic (M/Z) ck/ MyoVIIA in the morphogenesis of denticles, small actin - based projections on the ventral epidermis of Drosophila embryos. M/Z ck mutants displayed severe defects in denticle morphology – actin filaments initiated in the correct location, but failed to elongate and bundle to form normal projections. Using deletion mutant constructs, we demonstrated that both of the C - terminal MyTH4 and FERM domains are necessary for proper denticle formation. Furthermore, we show that ck /MyoVIIA interacts genetically with dusky - like (dyl) , a member of the ZPD family of proteins that links the extracellular matrix to the plasma membrane, and when mutated also disrupts normal denticle formation. Loss of either protein alone does not alter the localization of the other; however, loss of the two proteins together dramatically enhances the defects in denticle shape observed when either protein alone was absent. Our data indicate that ck /MyoVIIA plays a key role in the formation and/or organization of actin filament bundles, which drive proper shape of cellular projections. • Loss of maternal and zygotic ck /MyoVIIA disrupts denticle morphology. • ck /MyoVIIA mutant denticles fail to elongate and have multiple apices. • MyTH4 - FERM domains are necessary for actin-based protrusion formation. • Loss of ck /MyoVIIA and d yl results in severe abnormalities in denticle morphology. [ABSTRACT FROM AUTHOR]

Mutations in non-muscle myosin 2A (NM2A) encompass a wide spectrum of anomalies collectively known as MYH9-Related Disease (MYH9-RD) in humans that can include macrothrombocytopenia, glomerulosclerosis, deafness, and cataracts. We previously created mouse models of the three mutations most frequently found in humans: R702C, D1424N, and E1841K. While homozygous R702C and D1424N mutations are embryonic lethal, we found homozygous mutant E1841K mice to be viable. However the homozygous male, but not female, mice were infertile. Here, we report that these mice have reduced testis size and defects in actin-associated junctions in Sertoli cells, resulting in inability to form the blood-testis barrier and premature germ cell loss. Moreover, compound double heterozygous (R702C/E1841K and D1424/E1841K) males show the same abnormalities in testes as E1841K homozygous males. Conditional ablation of either NM2A or NM2B alone in Sertoli cells has no effect on fertility and testis size, however deletion of both NM2A and NM2B in Sertoli cells results in infertility. Isolation of mutant E1841K Sertoli cells reveals decreased NM2A and F-actin colocalization and thicker NM2A filaments. Furthermore, AE1841K/AE1841K and double knockout Sertoli cells demonstrate microtubule disorganization and increased tubulin acetylation, suggesting defects in the microtubule cytoskeleton. Together, these results demonstrate that NM2A and 2B paralogs play redundant roles in Sertoli cells and are essential for testes development and normal fertility. •Human disease-causing mutations in Myh9 cause abnormalities in Sertoli cells, BTB defects, and infertility in male mice. •Mutant Sertoli cells are characterized by abnormal morphology and cytoskeletal structures. •Deletion of either NM2A or NM2B in Sertoli cells does not affect fertility, demonstrating redundant roles for NM2 paralogs. •Deletion of both NM2A and NM2B recapitulates these defects suggesting that mutant NM2A interferes with the function of NM2B. [ABSTRACT FROM AUTHOR]

Although somatic cells play an integral role in animal gametogenesis, their organization and function are usually poorly characterized, especially in non-model systems. One such example is a peculiar cell found in leech ovaries – the apical cell (AC). A single AC can be found at the apical tip of each ovary cord, the functional unit of leech ovaries, where it is surrounded by other somatic and germline cells. The AC is easily distinguished due to its enormous size and its numerous long cytoplasmic projections that penetrate the space between neighboring cells. It is also characterized by a prominent accumulation of mitochondria, Golgi complexes and electron-dense vesicles. ACs are also enriched in cytoskeleton, mainly in form of intermediate filaments. Additionally, the AC is connected to neighboring cells via junctions that structurally resemble hemidesmosomes. In spite of numerous descriptive data about the AC, its functions remain poorly understood. Its suggested functions include a role in forming skeleton for the germline cells, and a role in defining a niche for germline stem cells. The latter is more speculative, since germline stem cells have not been identified in leech ovaries. Somatic cells with similar morphological properties to those of the AC have been found in gonads of nematodes – the distal tip cell – and in insects – Verson's cell, hub cells and cap cells. In the present article we summarize information about the AC structure and its putative functions. AC is compared with other well-described somatic cells with potentially similar roles in gametogenesis. • At the tip of ovary cord of certain leeches, a huge and ramified apical cell occurs. • Apical cell is tightly adhered to neighboring germline and somatic cells. • Distal tip cell and Verson's cell are morphologically similar to leech apical cell. • Apical cell supposedly forms a niche for germline stem cells. [ABSTRACT FROM AUTHOR]

A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2 , required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells. • YAP1 release from TE nuclei occurs through actin cytoskeletal remodeling. • Contracted blastocysts release YAP1 from TE nuclei independent of Hippo signaling. • Actin cytoskeleton organization controls YAP1 subcellular localization. [ABSTRACT FROM AUTHOR]

We herein report that deletion of mTOR in dental epithelia caused defective development of multiple cell layers of the enamel organ, which culminated in tooth malformation and cystogenesis. Specifically, cells of the stellate reticulum and stratum intermedium were poorly formed, resulting in cystic changes. The pre-ameloblasts failed to elongate along the apical-basal axis and persisted vigorous expression of Sox2 and P63, which are normally downregulated during cytodifferentiation. Expression of amelogenic markers was also attenuated in mutants. Cell proliferation and cell sizes in mutants were significantly reduced over time. Importantly, we found reduced amounts and aberrant aggregations of cytoskeletal components in mutants, along with attenuated expression of cytoskeleton regulator Cdc42 , whose epithelial deletion causes a similar phenotype. Moreover, disruption of actin assembly in an organ culture system affected cell proliferation and cytodifferentiation of tooth germs, supporting a causative role of mTOR-regulated cytoskeleton dynamics for the observed phenotype of mTOR mutant mice. In further support of this view, we showed that mTOR overactivation caused increased cytoskeletal component synthesis and assembly, along with accelerated cytodifferentiation in the enamel organ. Finally, we demonstrated that mTOR regulated enamel organ development principally through the mTORC1 pathway. • mTOR deletion causes enamel organ defects and tooth malformation. • mTOR is required for cell adhesion, proliferation and differentiation in the enamel organ. • mTOR regulates cytoskeleton dynamics in the enamel organ. • mTOR regulates enamel organ development principally through mTORC1. [ABSTRACT FROM AUTHOR]

The proliferation and hypertrophy of chondrocytes play important roles in endochondral ossification, which is tightly regulated during skeleton development. However, the regulation mechanism remains largely unknown. Here we show that DDB1 (Damaged DNA Binding Protein 1) has a critical function in the development of growth plates. Using chondrocyte-specific DDB1 knockout mice, we found that DDB1 deletion in chondrocytes results in dwarfism due to the aberrant skeleton development. The structure of growth plate in tibia becomes disordered at P21, not in femur. But at P70, the changes are severer in femur than tibia. Chondrocyte proliferation and differentiation are attenuated and asynchronous in both tibia and femur at P7 and P21. Furthermore, DDB1 deficiency induces p27 upregulation and subsequent cell cycle arrest in primary chondrocytes. Therefore, our data reveal that DDB1 is essential for the skeleton development by controlling chondrocyte proliferation and differentiation. Image 1 • Chondrocyte-specific DDB1 knockout mice exhibit dwarfism and dyschondroplasia. • The mutant growth plates in tibia and femur are structurally disordered with abnormal proliferation and differentiation. • DDB1 controls cartilage development via p27-mediated cell cycle regulation. [ABSTRACT FROM AUTHOR]

Dorsal closure during Drosophila embryogenesis provides a robust genetic platform to study the basic cellular mechanisms that govern epithelial wound healing and morphogenesis. As dorsal closure proceeds, the lateral epithelial tissue (LE) adjacent to the dorsal opening advance contra-laterally, with a simultaneous retraction of the amnioserosa. The process involves a fair degree of coordinated cell shape changes in the dorsal most epithelial (DME) cells as well as a few penultimate rows of lateral epithelial (LE) cells (collectively referred here as Dorsolateral Epithelial (DLE) cells), lining the periphery of the amnioserosa, which in due course of time extend contra-laterally and ultimately fuse over the dorsal hole, giving rise to a dorsal epithelial continuum. The JNK-Dpp signaling in the dorsolateral epidermis, plays an instrumental role in guiding their fate during this process. A large array of genes have been reported to be involved in the regulation of this core signaling pathway, yet the mechanisms by which they do so is hitherto unclear, which forms the objective of our present study. Here we show a probable mechanism via which lgl , a conserved tumour suppressor gene, regulates the JNK–Dpp pathway during dorsal closure and epithelial morphogenesis. A conditional/targeted knock-down of lgl in the dorsolateral epithelium of embryos results in failure of dorsal closure. Interestingly, we also observed a similar phenotype in a Rab11 knockdown condition. Our experiment suggests Rab11 to be interacting with lgl as they seem to synergize in order to regulate the core JNK-Dpp signaling pathway during dorsal closure and also during adult thorax closure process. • Rab11 or lgl knockdown in the dorso-lateral epidermis results in the failure of dorsal closure in Drosophila embryos. • A targeted knockdown of lgl or Rab11 in the dorsolateral epidermal cells results in an improper morphology. • Rab11 expression increases significantly in the dorso-lateral embryonic epidermis where lgl is knocked down. • A simultaneous knockdown of Rab11 in an lgl depleted background in the dorsolateral epidermis results in a rescue. • Rab11 and lgl interact to regulate the JNK-Dpp pathway by maintenance of cell junctions and cortical cytoskeleton. [ABSTRACT FROM AUTHOR]

Congenital heart disease (CHD) is the most common birth defect, and the leading cause of death due to birth defects, yet causative molecular mechanisms remain mostly unknown. We previously implicated a novel CHD candidate gene, SHROOM3 , in a patient with CHD. Using a Shroom3 gene trap knockout mouse (Shroom3 gt/gt ) we demonstrate that SHROOM3 is downstream of the noncanonical Wnt planar cell polarity signaling pathway (PCP) and loss-of-function causes cardiac defects. We demonstrate Shroom3 expression within cardiomyocytes of the ventricles and interventricular septum from E10.5 onward, as well as within cardiac neural crest cells and second heart field cells that populate the cardiac outflow tract. We demonstrate that Shroom3 gt/gt mice exhibit variable penetrance of a spectrum of CHDs that include ventricular septal defects, double outlet right ventricle, and thin left ventricular myocardium. This CHD spectrum phenocopies what is observed with disrupted PCP. We show that during cardiac development SHROOM3 interacts physically and genetically with, and is downstream of, key PCP signaling component Dishevelled 2. Within Shroom3 gt/gt hearts we demonstrate disrupted terminal PCP components, actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation and morphology. Together, these data demonstrate SHROOM3 functions during cardiac development as an actomyosin cytoskeleton effector downstream of PCP signaling, revealing SHROOM3's novel role in cardiac development and CHD. • Shroom3 is expressed within cardiomyocytes and cardiac neural crest cells of the embryonic heart during important stages of cardiac development. • SHROOM3 loss-of-function leads to previously unreported cardiac defects in addition to neural tube, gut, kidney and lens defects. • SHROOM3 has a role during cardiac development as an actomyosin effector downstream of PCP & is a novel contributor to cardiac development and CHD. [ABSTRACT FROM AUTHOR]

We show for the first time endoskeletal regeneration in the developing pectoral fin of zebrafish. The developing pectoral fin contains an aggregation plate of differentiated chondrocytes (endochondral disc; primordium for endoskeletal components, proximal radials). The endochondral disc can be regenerated after amputation in the middle of the disc. The regenerated disc sufficiently forms endoskeletal patterns. Early in the process of regenerating the endochondral disc, epithelium with apical ectodermal ridge (AER) marker expression rapidly covers the amputation plane, and mesenchymal cells start to actively proliferate. Taken together with re-expression of a blastema marker gene, msxb , and other developmental genes, it is likely that regeneration of the endochondral disc recaptures fin development as epimorphic limb regeneration does. The ability of endoskeletal regeneration declines during larval growth, and adult zebrafish eventually lose the ability to regenerate endoskeletal components such that amputated endoskeletons become enlarged. Endoskeletal regeneration in the zebrafish pectoral fin will serve as a new model system for successful appendage regeneration in mammals. • Endoskeletal regeneration in the developing pectoral fin of zebrafish is shown for the first time. • The endochondral disc can be regenerated after amputation in the middle of the disc. • Adult zebrafish eventually loses the ability to regenerate endoskeletal components. • msxb is re-expressed in the process of regeneration of the endochondral disc. [ABSTRACT FROM AUTHOR]

The notochord is an embryonic tissue that acts as a hydrostatic skeleton until ossification begins in vertebrates. It is composed of outer sheath cells and inner vacuolated cells, which are generated from a common pool of disc-shaped precursors. Notochord extension during early embryogenesis is driven by the growth of vacuolated cells, reflecting in turn the expansion of their inner vacuole. Here we use desmogon , a novel desmosomal cadherin, to follow notochord development and regeneration in medaka (Oryzias latipes). We trace desmogon ​+ disc-shaped precursors at the single cell level to demonstrate that they operate as unipotent progenitors, giving rise to either sheath or vacuolated cells. We reveal that once specified, vacuolated cells grow asynchronously and drive notochord expansion bi-directionally. Additionally, we uncover distinct regenerative responses in the notochord, which depend on the nature of the injury sustained. By generating a desmogon CRISPR mutant we demonstrate that this cadherin is essential for proper vacuolated cell shape and therefore correct notochord and spine morphology. Our work expands the repertoire of model systems to study dynamic aspects of the notochord in vivo , and provides new insights in its development and regeneration properties. • Desmogon is a novel desmoglein that sustains notochord integrity in medaka. • Notochord progenitors are unipotent, and generate either VCs or ShCs. • The notochord displays a local regeneration response when few VCs collapse. • The notochord displays a global regeneration response upon cell leakage. [ABSTRACT FROM AUTHOR]

Plant growth and development are driven by extended phases of irreversible cell expansion generating cells that increase in volume from 10- to 100-fold. Some specialized cell types define cortical sites that reinitiate polarized growth and generate branched cell morphology. This structural specialization of individual cells has a major importance for plant adaptation to diverse environments and practical importance in agricultural contexts. The patterns of cell shape are defined by highly integrated cytoskeletal and cell wall systems. Microtubules and actin filaments locally define the material properties of a tough outer cell wall to generate complex shapes. Forward genetics, powerful live cell imaging experiments, and computational modeling have provided insights into understanding of mechanisms of cell shape control. In particular, finite element modeling of the cell wall provides a new way to discover which cell wall heterogeneities generate complex cell shapes, and how cell shape and cell wall stress can feedback on the cytoskeleton to maintain growth patterns. This review focuses on cytoskeleton-dependent cell wall patterning during cell branching, and how combinations of multi-scale imaging experiments and computational modeling are being used to unravel systems-level control of morphogenesis. • Genetic pathways for branch morphogenesis are being defined. • Multivariate live cell imaging enables systems level analysis of gene activities in vivo. • Feedback control among actin filaments, microtubules, and cell wall stress patterns cell morphogenesis. • Finite element computational modeling reveals linkages between the cytoskeleton, cell wall, and cell shape change. • Future multi-scale models that include cytoplasm-cell wall interactions are needed. [ABSTRACT FROM AUTHOR]

A neuron's contribution to the information flow within a neural circuit is governed by the structure of its dendritic arbor. The geometry of the dendritic arbor directly determines synaptic density and the size of the receptive field, both of which influence the firing pattern of the neuron. Importantly, the position of individual dendritic branches determines the identity of the neuron's presynaptic partner and thus the nature of the incoming sensory information. To generate the unique stereotypic architecture of a given neuronal subtype, nascent branches must emerge from the dendritic shaft at preprogramed branch points. Subsequently, a complex array of extrinsic factors regulates the degree and orientation of branch expansion to ensure maximum coverage of the receptive field whilst constraining growth within predetermined territories. In this review we focus on studies that best illustrate how environmental cues such as the Wnts and Netrins and their receptors sculpt the dendritic arbor. We emphasize the pivotal role played by the actin cytoskeleton and its upstream regulators in branch initiation, outgrowth and navigation. Finally, we discuss how protocadherin and DSCAM contact-mediated repulsion prevents inappropriate synapse formation between sister dendrites or dendrites and the axon from the same neuron. Together these studies highlight the clever ways evolution has solved the problem of constructing complex branch geometries. • The architecture of the dendritic tree is unique to each neuronal subtype. • Development of functional neural circuitry is determined by dendrite shape. • Dendrite branch geometry is tightly regulated by adhesion and guidance receptors. • Branch initiation and growth is driven by dynamic actin remodelling. [ABSTRACT FROM AUTHOR]

The terminal cells of the tracheal epithelium in Drosophila melanogaster are one of the few known cell types that undergo subcellular morphogenesis to achieve a stable, branched shape. During the animal's larval stages, the cells repeatedly sprout new cytoplasmic processes. These grow very long, wrapping around target tissues to which the terminal cells adhere, and are hollowed by a gas-filled subcellular tube for oxygen delivery. Our understanding of this ramification process remains rudimentary. This review aims to provide a comprehensive summary of studies on terminal cells to date, and attempts to extrapolate how terminal branches might be formed based on the known genetic and molecular components. Next to this cell-intrinsic branching mechanism, we examine the extrinsic regulation of terminal branching by the target tissue and the animal's environment. Finally, we assess the degree of similarity between the patterns established by the branching programs of terminal cells and other branched cells and tissues from a mathematical and conceptual point of view. • Branch growth in terminal cells depends on concomittant growth of a subcellular tube. • Existing evidence allows synthesising a working model of terminal branch formation. • Terminal cells sense hypoxia like most cells but respond uniquely. • The classic model of terminal branch guidance is inconsistent with newer evidence. [ABSTRACT FROM AUTHOR]

In the first ascidian cell cycle, cytoplasmic and cortical reorganization is required for distributing maternal factors to their appropriate positions, resulting in the formation of the embryonic axis. This cytoplasmic reorganization is considered to depend on the cortical microfilament network in the first phase and on the sperm astral microtubule (MT) in the second phase. Recently, we described three novel MT structures: a deeply extended MT meshwork (DEM) in the entire subcortical region of the unfertilized egg, transiently accumulated MT fragments (TAF) in the vegetal pole, and a cortical MT array in the posterior vegetal cortex (CAMP). Particularly, our previous study showed CAMP to contribute to the movement of myoplasm. In addition, it is very similar to the parallel MT array, which appears during cortical rotation in Xenopus eggs. However, how these MT structures are organized is still unclear. Here, we investigated the relationship between the egg activation pathway and MT structures during the first ascidian cell cycle. First, we carefully analyzed cell cycle progression through meiosis I and II and the first mitosis, and successfully established a standard time table of cell cycle events. Using this time table as a reference, we precisely described the behavior of novel MT structures and revealed that it was closely correlated with cell cycle events. Moreover, pharmacological experiments supported the relationship between these MT structures and the signal transduction mechanisms that begin after fertilization, including Ca2+ signaling, MPF signaling, and MEK/MAPK signaling. Especially, CAMP formation was directed by activities of cyclin-dependent kinases. As these MT structures are conserved, at least, within chordate group, we emphasize the importance of understanding the controlling mechanisms of MT dynamics, which is important for embryonic axis determination in the ascidian egg. • Microtubules in the egg were regulated in accordance with cell-cycle progression. • Ca2+ influx triggered breakdown of deeply-extended subcortical microtubule meshwork. • Actin contraction was required for transient accumulation of microtubule fragments. • Cyclin dependent kinases regulated the cortical microtubule array formation. [ABSTRACT FROM AUTHOR]

Abstract Epithelial cell polarity, adhesion, proliferation, differentiation and survival are essential for morphogenesis of various organs and tissues including the ocular lens. The molecular mechanisms regulating the lens epithelial phenotype however, are not well understood. Here we investigated the role of scaffolding protein ankyrin-G (AnkG) in mouse lens development by conditional suppression of AnkG expression using the Cre-LoxP recombination approach. AnkG, which serves to link integral membrane proteins to the spectrin/actin cytoskeleton, was found to distribute predominantly to the lateral membranes of lens epithelium with several isoforms of the protein being detected in the mouse lens. Conditional deficiency of AnkG impaired mouse lens morphogenesis starting from embryonic stage E15.5, with neonatal (P1) AnkG cKO lenses exhibiting overt abnormalities in shape, size, epithelial cell height, sheet length and lateral membrane assembly together with defective fiber cell orientation relative to lenses from littermate AnkG floxed or Cre expressing mice. Severe disruptions in E-cadherin/β-catenin-based adherens junctions, and the membrane organization of spectrin-actin cytoskeleton, ZO-1, connexin-50 and Na+-K+-ATPase were noted in AnkG deficient lenses, along with detection in lens epithelium of α-smooth muscle actin, a marker of epithelial to mesenchymal transition. Moreover, lens epithelial cell proliferation and survival were severely compromised while differentiation appears to be normal in AnkG deficient mouse lenses. Collectively, these results indicate that AnkG regulates establishment of the epithelial phenotype via lateral membrane assembly, stabilization of E-cadherin-based cell-cell junctions, polarity and membrane organization of transport and adhesion proteins and the spectrin-actin skeleton, and provide evidence for an obligatory role for AnkG in lens morphogenesis and growth. Highlights • Ankyrin-G exhibits preferential distribution to the epithelium within mouse lens. • Ankyrin-G conditional deficiency impairs lens morphogenesis, growth and shape. • Ankyrin-G deficiency disrupts lens epithelial phenotype. • Ankyrin-G deficiency impairs lens epithelial lateral membrane assembly. • Ankyrin-G deficiency impairs lens epithelial cell proliferation and survival. [ABSTRACT FROM AUTHOR]

Intercellular bridges are conserved structures that allow neighboring cells to exchange cytoplasmic material; defects in intercellular bridges can lead to infertility in many organisms. Here, we use the Drosophila egg chamber to study the mechanisms that regulate intercellular bridges. Within the developing egg chamber, the germ cells (15 nurse cells and 1 oocyte) are connected to each other through intercellular bridges called ring canals, which expand over the course of oogenesis to support the transfer of materials from the nurse cells to the oocyte. The ring canals are enriched in actin and actin binding proteins, and many proteins have been identified that localize to the germline ring canals and control their expansion and stability. Here, we demonstrate a novel role for the Ste20 family kinase, Misshapen (Msn), in regulation of the size of the germline ring canals. Msn localizes to ring canals throughout most of oogenesis, and depletion of Msn led to the formation of larger ring canals. Over-expression of Msn decreased ring canal diameter, and expression of a membrane tethered form of Msn caused ring canal detachment and nurse cell fusion. Altering the levels or localization of Msn also led to changes in the actin cytoskeleton and altered the localization of E-cadherin, which suggests that Msn could be indirectly limiting ring canal size by altering the structure or dynamics of the actin cytoskeleton and/or adherens junctions. [ABSTRACT FROM AUTHOR]

GSK3 plays a central role in orchestrating key biological signaling pathways, including cell migration. Here, we identify GlkA as a GSK3 family kinase with functions that overlap with and are distinct from those of GskA. We show that GlkA, as previously shown for GskA, regulates the cell's cytoskeleton through MyoII assembly and control of Ras and Rap1 function, leading to aberrant cell migration. However, there are both qualitative and quantitative differences in the regulation of Ras and Rap1 and their downstream effectors, including PKB, PKBR1, and PI3K, with glkA - cells exhibiting a more severe chemotaxis phenotype than gskA - cells. Unexpectedly, the severe glkA - phenotypes, but not those of gskA - , are only exhibited when cells are grown attached to a substratum but not in suspension, suggesting that GlkA functions as a key kinase of cell attachment signaling. Using proteomic iTRAQ analysis we show that there are quantitative differences in the pattern of protein expression depending on the growth conditions in wild-type cells. We find that GlkA expression affects the cell's proteome during vegetative growth and development, with many of these changes depending on whether the cells are grown attached to a substratum or in suspension. These changes include key cytoskeletal and signaling proteins known to be essential for proper chemotaxis and signal relay during the aggregation stage of Dictyostelium development. [ABSTRACT FROM AUTHOR]

Control of microtubule dynamics is crucial for cell migration. We analyzed regulation of microtubule network dynamics in the zebrafish yolk cell during epiboly, the earliest coordinated gastrulation movement. We labeled microtubules with EMTB-3GFP and EB3-mCherry to visualize and measure microtubule dynamics by TIRF microscopy live imaging. Yolk cell microtubules dynamics is temporally modulated during epiboly progression. We used maternal zygotic Pou5f3 mutant (MZ spg ) embryos, which develop strong distortions of microtubule network organization and epiboly retardation, to investigate genetic control of microtubule dynamics. In MZ spg embryos, microtubule plus-end growth tracks move slower and are less straight compared to wild-type. MZ spg embryos have altered steroidogenic enzyme expression, resulting in increased pregnenolone and reduced progesterone levels. We show that progesterone positively affects microtubule plus-end growth and track straightness. Progesterone may thus act as a non-cell-autonomous regulator of microtubule dynamics across the large yolk cell, and may adjust differing demands on microtubule dynamics and stability during initiation and progression phases of epiboly. [ABSTRACT FROM AUTHOR]

The Caenorhabditis elegans germline is widely used as a model to study stem cell development, chromosome dynamics and apoptosis. Major readouts of germline phenotypes such as cell counting and protein expression profiling are routinely analyzed manually and in a two-dimensional manner. The major disadvantages of the existing approaches are 1) they are time-consuming and laborious and 2) there is an inability to study the effects of genetic mutations in three dimensions. Here, we demonstrate a rapid, automated method for analyzing the three-dimensional distribution of proteins, germline nuclei and cytoskeletal structures in the C. elegans germline. Using this method, we have revealed previously unappreciated germline organization and cytoskeletal structures that will have a major impact on the characterization of germline phenotypes. To conclude, our new method dramatically enhances the efficiency and resolution of C. elegans germline analysis and may be applied to other cellular structures. [ABSTRACT FROM AUTHOR]

Arginylation is an emerging protein modification mediated by arginyltransferase ATE1, shown to regulate embryogenesis and actin cytoskeleton, however its functions in different physiological systems are not well understood. Here we analyzed the role of ATE1 in brain development and neuronal growth by producing a conditional mouse knockout with Ate1 deletion in the nervous system driven by Nestin promoter (Nes- Ate1 mice). These mice were weaker than wild type, resulting in low postnatal survival rates, and had abnormalities in the brain that suggested defects in neuronal migration. Cultured Ate1 knockout neurons showed a reduction in the neurite outgrowth and the levels of doublecortin and F-actin in the growth cones. In wild type, ATE1 prominently localized to the growth cones, in addition to the cell bodies. Examination of the Ate1 mRNA sequence reveals the existence of putative zipcode-binding sequences involved in mRNA targeting to the cell periphery and local translation at the growth cones. Fluorescence in situ hybridization showed that Ate1 mRNA localized to the tips of the growth cones, likely due to zipcode-mediated targeting, and this localization coincided with spots of localization of arginylated β-actin, which disappeared in the presence of protein synthesis inhibitors. We propose that zipcode-mediated co-targeting of Ate1 and β-actin mRNA leads to localized co-translational arginylation of β-actin that drives the growth cone migration and neurite outgrowth. [ABSTRACT FROM AUTHOR]

Functional movement and stability of the limb depends on an organized and fully integrated musculoskeletal system composed of skeleton, muscle, and tendon. Much of our current understanding of musculoskeletal development is based on studies that focused on the development and differentiation of individual tissues. Likewise, research on patterning events have been largely limited to the primary skeletal elements and the mechanisms that regulate soft tissue patterning, the development of the connections between tissues, and their interdependent development are only beginning to be elucidated. This review will therefore highlight recent exciting discoveries in this field, with an emphasis on tendon and muscle patterning and their integrated development with the skeleton and skeletal attachments. [ABSTRACT FROM AUTHOR]

More than sixty years ago, while studying feather tracks on the shoulder of the chick embryo, Dr. John Saunders used Nile Blue dye to stain the tissue. There, he noticed a darkly stained line of cells that neatly rims the tip of the growing limb bud. Rather than ignoring this observation, he followed it up by removing this tissue and found that it led to a striking truncation of the limb skeletons. This landmark experiment marks the serendipitous discovery of the apical ectodermal ridge (AER), the quintessential embryonic structure that drives the outgrowth of the limb. Dr. Saunders continued to lead the limb field for the next fifty years, not just through his own work, but also by inspiring the next generation of researchers through his infectious love of science. Together, he and those who followed ushered in the discovery of fibroblast growth factor (FGF) as the AER molecule. The seamless marriage of embryology and molecular biology that led to the decoding of the AER serves as a shining example of how discoveries are made for the rest of the developmental biology field. [ABSTRACT FROM AUTHOR]

Collective cell migration is involved in numerous processes both physiological, such as embryonic development, and pathological such as metastasis. Compared to single cell migration, collective motion requires cell behaviour coordination through an as-yet poorly understood but critical cell-cell communication mechanism. Using Drosophila border cell migration, we show here that the small Rho GTPase Cdc42 regulates cell-cell communication. Indeed, we demonstrate that Cdc42 controls protrusion formation in a cell non-autonomous manner. Moreover, we found that the endocytic small GTPase Rab11, controls Cdc42 localisation to the periphery of migrating border cell clusters. Accordingly, over-expression of Cdc42 in border cells rescues the loss of Rab11 function. In addition, we showed that Cdc42 acts upstream of Moesin, a cytoskeletal regulator known to function downstream of rab11. Thus, our study positions Cdc42 as a new key player in cell-cell communication, acting downstream of Rab11. [ABSTRACT FROM AUTHOR]

Cell-cell signaling plays a prominent role in the formation of the embryonic skeleton of sea urchins, but the mechanisms are poorly understood. In the present study, we uncover an essential role for TGF-β sensu stricto signaling in this process. We show that TgfbrtII, a type II receptor dedicated to signaling through TGF-β sensu stricto , is expressed selectively in skeletogenic primary mesenchyme cells (PMCs) during skeleton formation. Morpholino (MO) knockdowns and studies with a specific TgfbrtII inhibitor (ITD-1) in both S. purpuratus and Lytechinus variegatus embryos show that this receptor is required for biomineral deposition. We provide pharmacological evidence that Alk4/5/7 is the cognate TGF-β type I receptor that pairs with TgfbrtII and show by inhibitor treatments of isolated micromeres cultured in vitro that both Alk4/5/7 and TgfbrtII function cell-autonomously in PMCs. Gene expression and gene knockdown studies suggest that TGF-β sensu stricto may be the ligand that interacts with TgfbrtII and support the view that this TGF-β superfamily ligand provides an essential, permissive cue for skeletogenesis, although it is unlikely to provide spatial patterning information. Taken together, our findings reveal that this model morphogenetic process involves an even more diverse suite of cell signaling pathways than previously appreciated and show that PMCs integrate a complex set of both generalized and spatially localized cues in assembling the endoskeleton. [ABSTRACT FROM AUTHOR]

Somitogenesis and subsequent axial skeletal development is regulated by the interaction of pathways that determine the periodicity of somite formation, rostrocaudal somite polarity and segment identity. Here we use a hypomorphic mutant mouse line to demonstrate that Supt20 ( Suppressor of Ty20 ) is required for development of the axial skeleton. Supt20 hypomorphs display fusions of the ribs and vertebrae at lower thoracic levels along with anterior homeotic transformation of L1 to T14. These defects are preceded by reduction of the rostral somite and posterior shifts in Hox gene expression. While cycling of Notch target genes in the posterior presomitic mesoderm (PSM) appeared normal, expression of Lfng was reduced. In the anterior PSM, Mesp2 expression levels and cycling were unaffected; yet, expression of downstream targets such as Lfng, Ripply2 , Mesp1 and Dll3 in the prospective rostral somite was reduced accompanied by expansion of caudal somite markers such as EphrinB2 and Hes7. Supt20 interacts with the Gcn5-containing SAGA histone acetylation complex. Gcn5 hypomorphic mutant embryos show similar defects in axial skeletal development preceded by posterior shift of Hoxc8 and Hoxc9 gene expression. We demonstrate that Gcn5 and Supt20 hypomorphs show similar defects in rostral-caudal somite patterning potentially suggesting shared mechanisms. [ABSTRACT FROM AUTHOR]

In the sea urchin embryo, primary mesenchyme cells (PMCs) adhere to one another and fuse via filopodia, forming cable-like structures within which skeletal rods are deposited. Although this process was first described more than a century ago, molecules that participate in PMC adhesion and fusion have not been identified. Here we show that KirrelL, a PMC-specific, Ig domain-containing transmembrane protein, is essential for PMC fusion, probably by mediating filopodial adhesions that are a pre-requisite for subsequent membrane fusion. We show that KirrelL is not required for PMC specification, migration, or for direct filopodial contacts between PMCs. In the absence of KirrelL, however, filopodial contacts do not result in fusion. kirrelL is a member of a family of closely related, intronless genes that likely arose through an echinoid-specific gene expansion, possibly via retrotransposition. Our findings are significant in that they establish a direct linkage between the transcriptional network deployed in the PMC lineage and an effector molecule required for a critically important PMC morphogenetic process. In addition, our results point to a conserved role for Ig domain-containing adhesion proteins in facilitating cell fusion in both muscle and non-muscle cell lineages during animal development. [ABSTRACT FROM AUTHOR]

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24 hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos. [ABSTRACT FROM AUTHOR]

Asymmetric cell division (ACD) is universally required for the development of multicellular organisms. Unlike animal cells, plant cells have a rigid cellulosic extracellular matrix, the cell wall, which provides physical support and forms communication routes. This fundamental difference leads to some unique mechanisms in plants for generating asymmetries during cell division. However, plants also utilize intrinsically polarized proteins to regulate asymmetric signaling and cell division, a strategy similar to the differentiation mechanism found in animals. Current progress suggests that common regulatory modes, i.e. protein spontaneous clustering and cytoskeleton reorganization, underlie protein polarization in both animal and plant cells. Despite these commonalities, it is important to note that intrinsic mechanisms in plants are heavily influenced by extrinsic cues. To control physical asymmetry in cell division, although our understanding is fragmentary thus far, plants might have evolved novel polarization strategies to orientate cell division plane. Recent studies also suggest that the phytohormone auxin, one of the most pivotal small molecules in plant development, regulates ACD in plants. [ABSTRACT FROM AUTHOR]

RacGAP1 is one of the two components of the centralspindlin complex essential for orchestrating cytokinesis in all animal cells. We report here that the early arrest mutant ogre is a maternal and zygotic loss of function mutation in the zebrafish homolog of racgap1 . Like the other model organisms in which racgap1 is mutated, cells in the mutant stop dividing. In vivo cell recordings reveal that gradual loss of wild-type RacGAP1 leads progressively from a failure of abscission, then to cleavage furrow ingression, and finally complete absence of furrow formation. Despite the lack of cytokinesis, gross patterning occurs overtly normally in ogre mutants and cells continue to cycle slowly, some even attaining four or eight nuclei. Many multinucleate cells differentiate and survive, but the majority of cells enter apoptosis that we demonstrate is due to cumulative rounds of defective cytokinesis. Investigation of the cells that differentiate in the mutant indicate that RacGAP1 is also needed for long-term survival of motoneurons and the cytoskeletal organization of sensory axons. We conclude that while RacGAP1 function is crucial for cytokinesis and its activity at different levels controls different aspects of cytokinesis, these defects have occluded other critical roles of this interesting protein. [ABSTRACT FROM AUTHOR]

During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7 , which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures. [ABSTRACT FROM AUTHOR]

Apical-basal and planar cell polarities are hallmarks of metazoan epithelia required to separate internal and external environments and to regulate trans- and intracellular transport, cytoskeletal organization, and morphogenesis. Mechanisms of cell polarization have been intensively studied in bilaterian model organisms, particularly in early embryos and cultured cells, while cell polarity in pre-bilaterian tissues is poorly understood. Here, we have studied apical-basal and planar polarization in regenerating (aggregating) clusters of epitheliomuscular cells of Hydra , a simple representative of the ancestral, pre-bilaterian phylum Cnidaria. Immediately after dissociation, single epitheliomuscular cells do not exhibit cellular polarity, but they polarize de novo during aggregation. Reestablishment of the Hydra -specific epithelial bilayer is a result of short-range cell sorting. In the early phase of aggregation, apical-basal polarization starts with an enlargement of the epithelial apical-basal diameter and by the development of belt-like apical septate junctions. Specification of the basal pole of epithelial cells occurs shortly later and is linked to synthesis of mesoglea, development of hemidesmosome-like junctions, and formation of desmosome-like junctions connecting the basal myonemes of neighbouring cells. Planar polarization starts, while apical-basal polarization is already ongoing. It is executed gradually starting with cell-autonomous formation, parallelization, and condensation of myonemes at the basal end of each epithelial cell and continuing with a final planar alignment of epitheliomuscular cells at the tissue level. Our findings reveal that epithelial polarization in Hydra aggregates occurs in defined steps well accessible by histological and ultrastructural techniques and they will provide a basis for future molecular studies. [ABSTRACT FROM AUTHOR]

The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling – pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate. [ABSTRACT FROM AUTHOR]

Intramembranous ossification is a complex multi-step process which relies on extensive interactions among bone cells and surrounding tissues. The embryonic vasculature is essential in regulating endochondral ossification; however, its role during intramembranous ossification remains poorly understood, and in vivo studies are lacking. Previous research from our lab on the development of the intramembranous scleral ossicles has demonstrated an intriguing pattern of vascular development in which the areas of future osteogenesis remain avascular until after bone induction has occurred. Such avascular zones are located directly beneath each of the conjunctival papillae, epithelial structures which provide osteogenic signals to the underlying mesenchyme. Here we provide a high-resolution map of the developing vasculature from the time of ossicle induction to mineralization using a novel technique. We show that vegfa is expressed by the papillae and nearby mesenchymal tissue throughout HH 34–37, when vascular growth is taking place, and is down-regulated thereafter. Localized inhibition of Vegf results in expansion of the avascular zone surrounding the implanted papilla and mispatterning of the scleral ossicles. These results demonstrate that Vegf signaling could provide important insights into the complex relationship between bone and vasculature during intramembranous bone development. [ABSTRACT FROM AUTHOR]

Fluid- and gas-transporting tubular organs are critical to metazoan development and homeostasis. Tubulogenesis involves cell polarization and morphogenesis to specify the luminal, adhesive, and basal cell domains and to establish an open lumen. We explore a requirement for Talin, a cytoplasmic integrin adapter, during Drosophila melanogaster embryonic heart tube development. Talin marks the presumptive luminal domain and is required to orient and develop an open luminal space within the heart. Genetic analysis demonstrates that loss of zygotic or maternal-and-zygotic Talin disrupts heart cell migratory dynamics, morphogenesis, and polarity. Talin is essential for subsequent polarization of luminal determinants Slit, Robo, and Dystroglycan as well as stabilization of extracellular and intracellular integrin adhesion factors. In the absence of Talin function, mini-lumens enriched in luminal factors form in ectopic locations. Rescue experiments performed with mutant Talin transgenes suggest that actin-binding is required for normal lumen formation, but not for initial heart cell polarization. We propose that Talin provides instructive cues to position the luminal domain and coordinate the actin cytoskeleton during Drosophila heart lumen development. [ABSTRACT FROM AUTHOR]

dKDM5/LID regulates transcription of essential developmental genes and, thus, is required for different developmental processes. Here, we report the essential contribution of dKDM5/LID to hematopoiesis in Drosophila . Our results show that dKDM5/LID is abundant in hemocytes and that its depletion induces over-proliferation and differentiation defects of larval hemocytes and disrupts organization of the actin cytoskeleton. We also show that dKDM5/LID regulates expression of key factors of hematopoietic development. In particular, dKDM5/LID depletion up-regulates expression of several transcription factors involved in hemocytes proliferation and differentiation as well as of several small-GTPases that link signaling effectors to actin cytoskeleton formation and dynamics. [ABSTRACT FROM AUTHOR]

Species-specific symmetry-breaking events at the left–right organizer (LRO) drive an evolutionarily-conserved cascade of gene expression in the lateral plate mesoderm that is required for the asymmetric positioning of organs within the body cavity. The mechanisms underlying the transfer of the left and right laterality information from the LRO to the lateral plate mesoderm are poorly understood. Here, we investigate the role of Claudin-10, a tight junction protein, in facilitating the transfer of left–right identity from the LRO to the lateral plate mesoderm. Claudin-10 is asymmetrically expressed on the right side of the chick LRO, Hensen’s node. Gain- and loss-of-function studies demonstrated that right-sided expression of Claudin-10 is essential for normal rightward heart tube looping, the first morphological asymmetry during organogenesis. Manipulation of Claudin-10 expression did not perturb asymmetric gene expression at Hensen’s node, but did disrupt asymmetric gene expression in the lateral plate mesoderm. Bilateral expression of Claudin-10 at Hensen’s node prevented expression of Nodal , Lefty-2 and Pitx2c in the left lateral plate mesoderm, while morpholino knockdown of Claudin-10 inhibited expression of Snail1 in the right lateral plate mesoderm. We also determined that amino acids that are predicted to affect ion selectivity and protein interactions that bridge Claudin-10 to the actin cytoskeleton were essential for its left–right patterning function. Collectively, our data demonstrate a novel role for Claudin-10 during the transmission of laterality information from Hensen’s node to both the left and right sides of the embryo and demonstrate that tight junctions have a critical role during the relay of left–right patterning cues from Hensen’s node to the lateral plate mesoderm. [ABSTRACT FROM AUTHOR]

Epithelial sheet movement is an essential morphogenetic process during mouse embryonic eyelid closure in which Mitogen-Activated Protein 3 Kinase 1 (MAP3K1) and c-Jun play a critical role. Here we show that MAP3K1 associates with the cytoskeleton, activates Jun N-terminal kinase (JNK) and actin polymerization, and promotes the eyelid inferior epithelial cell elongation and epithelium protrusion. Following epithelium protrusion, c-Jun begins to express and acts to promote ERK phosphorylation and migration of the protruding epithelial cells. Homozygous deletion of either gene causes defective eyelid closure, but non-allelic non-complementation does not occur between Map3k1 and c-Jun and the double heterozygotes have normal eyelid closure. Results from this study suggest that MAP3K1 and c-Jun signal through distinct temporal-spatial pathways and that productive epithelium movement for eyelid closure requires the consecutive action of MAP3K1-dependent cytoskeleton reorganization followed by c-Jun-mediated migration. [ABSTRACT FROM AUTHOR]