Prickle and Ror modulate Dishevelled-Vangl interaction to regulate non-canonical Wnt signaling during convergent extension.

Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and "core members" of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/ Dishevelled (Dvl) and Van Gogh-like (Vangl)/ Prickel (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/ Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane but simultaneously keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz. PK inhibits the transition, and functionally synergize with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition, and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly, but can be cofractionated with Vangl. We propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serves two functions: 1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and 2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt induced dimerization of Fz and Ror.

Recognition of H2AK119ub plays an important role in RSF1-regulated early Xenopus development.

Polycomb group (PcG) proteins are key regulators of gene expression and developmental programs via covalent modification of histones, but the factors that interpret histone modification marks to regulate embryogenesis are less studied. We previously identified Remodeling and Spacing Factor 1 (RSF1) as a reader of histone H2A lysine 119 ubiquitination (H2AK119ub), the histone mark deposited by Polycomb Repressive Complex 1 (PRC1). In the current study, we used Xenopus laevis as a model to investigate how RSF1 affects early embryonic development and whether recognition of H2AK119ub is important for the function of RSF1. We showed that knockdown of Xenopus RSF1, rsf1, not only induced gastrulation defects as reported previously, but specific targeted knockdown in prospective neural precursors induced neural and neural crest defects, with reductions of marker genes. In addition, similar to knockdown of PRC1 components in Xenopus, the anterior-posterior neural patterning was affected in rsf1 knockdown embryos. Binding of H2AK119ub appeared to be crucial for rsf1 function, as a construct with deletion of the UAB domain, which is required for RSF1 to recognize the H2AK119ub nucleosomes, failed to rescue rsf1 morphant embryos and was less effective in interfering with early Xenopus development when ectopically expressed. Furthermore, ectopic deposition of H2AK119ub on the Smad2 target gene gsc using a ring1a-smad2 fusion protein led to ectopic recruitment of RSF1. The fusion protein was inefficient in inducing mesodermal markers in the animal region or a secondary axis when expressed in the ventral tissues. Taken together, our results reveal that rsf1 modulates similar developmental processes in early Xenopus embryos as components of PRC1 do, and that RSF1 acts at least partially through binding to the H2AK119ub mark via the UAB domain during development.

The RhoGEF protein Plekhg5 regulates medioapical and junctional actomyosin dynamics of apical constriction during Xenopus gastrulation.

Apical constriction results in apical surface reduction in epithelial cells and is a widely used mechanism for epithelial morphogenesis. Both medioapical and junctional actomyosin remodeling are involved in apical constriction, but the deployment of medial versus junctional actomyosin and their genetic regulation in vertebrate embryonic development have not been fully described. In this study, we investigate actomyosin dynamics and their regulation by the RhoGEF protein Plekhg5 in Xenopus bottle cells. Using live imaging and quantitative image analysis, we show that bottle cells assume different shapes, with rounding bottle cells constricting earlier in small clusters followed by fusiform bottle cells forming between the clusters. Both medioapical and junctional actomyosin signals increase as surface area decreases, though correlation of apical constriction with medioapical actomyosin localization appears to be stronger. F-actin bundles perpendicular to the apical surface form in constricted cells, which may correspond to microvilli previously observed in the apical membrane. Knockdown of plekhg5 disrupts medioapical and junctional actomyosin activity and apical constriction but does not affect initial F-actin dynamics. Taking the results together, our study reveals distinct cell morphologies, uncovers actomyosin behaviors, and demonstrates the crucial role of a RhoGEF protein in controlling actomyosin dynamics during apical constriction of bottle cells in Xenopus gastrulation.

Ash2l, an obligatory component of H3K4 methylation complexes, regulates neural crest development.

The vertebrate nervous system develops from embryonic neural plate and neural crest. Although genetic mechanisms governing vertebrate neural development have been investigated in depth, epigenetic regulation of this process remains less understood. Redundancy of epigenetic factors and early lethality of animals deficient in critical epigenetic components pose major challenges in characterization of epigenetic factors in vertebrate neural development. In this study, we use the amphibian model Xenopus laevis to investigate the roles of non-redundant, obligatory components of all histone H3K4 activating methylation complexes (COMPASS, also known as SET1/MLL complexes) in early neural development. The two genes that we focus on, Ash2l and Dpy30, regulate mesendodermal differentiation in mouse embryonic stem cells and cause early embryonic lethality when removed from mouse embryos. Using targeted knockdown of the genes in dorsal ectoderm of Xenopus that gives rise to future nervous system, we show here that ash2l and dpy30 are required for neural and neural crest marker expression in Xenopus late neurula embryos but are dispensable for early neural and neural plate border gene expression. Co-immunoprecipitation assays reveal that Dpy30 and Ash2L associate with the neural plate border transcription factors, such as Msx1 and Tfap2a. Chromatin immunoprecipitation (ChIP) assay further demonstrates that Ash2L and the H3K4me3 active histone mark accumulate at the promoter regions of the neural crest gene sox10 in a Tfap2a-dependent manner. Collectively, our data suggest that Ash2l and Dpy30 interact with specific transcription factors to recruit COMPASS complexes to the regulatory regions of neural crest specification genes to control their expression and influence development of the nervous system during vertebrate embryogenesis.

Assays for Apical Constriction Using the Xenopus Model.

Apical constriction refers to the active, actomyosin-driven process that reduces apical cell surface area in epithelial cells. Apical constriction is utilized in epithelial morphogenesis during embryonic development in multiple contexts, such as gastrulation, neural tube closure, and organogenesis. Defects in apical constriction can result in congenital birth defects, yet our understanding of the molecular control of apical constriction is relatively limited. To uncover new genetic regulators of apical constriction and gain mechanistic insight into the cell biology of this process, we need reliable assay systems that allow real-time observation and quantification of apical constriction as it occurs and permit gain- and loss-of-function analyses to explore gene function and interaction during apical constriction. In this chapter, we describe using the early Xenopus embryo as an assay system to investigate molecular mechanisms involved in apical constriction during both gastrulation and neurulation.

Imaging of dynamic actin remodeling reveals distinct behaviors of head and trunk mesoderm in gastrulating Xenopus laevis.

Gastrulation involves coordinated movements of cells, facilitating mesoderm and endoderm internalization and proper patterning of tissues across the germ layers. In Xenopus laevis, head mesoderm migrates collectively along the blastocoel roof fibronectin network towards the animal pole. Meanwhile, the trunk mesodermal cells migrate over each other in convergent thickening and convergent extension movements elongating the body axis. The behaviors of cells in these regions are investigated mainly in tissue explants taken from the respective head or trunk mesodermal regions. How cells behave at the transitional zone between these territories is not described in detail. To learn about cell behaviors around this junction, we imaged cell movements in an explant that encompassed the head and trunk mesoderm. We observed that head mesoderm migration on fibronectin employed lamellipodial protrusions at the leading edge and dynamic actin remodeling in the trailing cells. Trunk mesodermal cells underwent mediolateral cell elongation and intercalation to form the notochord. Lateral edges of the notochord were defined before the anterior edge. Our movie reveals distinct mesodermal cell behaviors occurring simultaneously in different regions of gastrulating embryos. This study highlights the power of applying modern microscopy tools to revisit classical experiments, permitting a greater understanding of the cellular dynamics that shape the embryo.

Heterochromatin protein 1 beta regulates neural and neural crest development by repressing pluripotency-associated gene pou5f3.2/oct25 in Xenopus.

BACKGROUND: Heterochromatin protein 1 (HP1) is associated with and plays a role in compact chromatin conformation, but the function of HP1 in vertebrate embryogenesis is not understood completely. RESULTS: Here, we explore the activity of HP1 in early neural development in the frog Xenopus laevis. We show that the three isoforms of HP1, HP1α, ß, and γ, are expressed in similar patterns in the neural and neural crest derivatives in early embryos. Despite this, knockdown of HP1ß and HP1γ, but not HP1α, in presumptive neural tissues leads to head defects. Late pan-neural markers and neural crest specifier genes are reduced, but early neural and neural plate border genes are less affected in the morphant embryos. Further investigation reveals that neuronal differentiation is impaired and a pluripotency-associated gene, pou5f3.2/oct25, is expanded in HP1ß morphants. Ectopic expression of pou5f3.2/oct25 mimics the effect of HP1ß knockdown on marker expression, whereas simultaneous knockdown of HP1ß and pou5f3.2/oct25 partially rescues expression of these genes. CONCLUSION: Taken together, the data suggest that HP1ß regulates transition from precursor to more differentiated cell types during neural and neural crest development in Xenopus, and it does so at least partially via repression of the pluripotency-associated transcription regulator pou5f3.2/oct25.

The type II integral ER membrane protein VAP-B homolog in C. elegans is cleaved to release the N-terminal MSP domain to signal non-cell-autonomously.

VAMP/synaptobrevin-associated protein B (VAP-B) is a type II ER membrane protein, but its N-terminal MSP domain (MSPd) can be cleaved and secreted. Mutations preventing the cleavage and secretion of MSPd have been implicated in cases of human neurodegenerative diseases. The site of VAP cleavage and the tissues capable in releasing the processed MSPd are not understood. In this study, we analyze the C. elegans VAP-B homolog, VPR-1, for its processing and secretion from the intestine. We show that intestine-specific expression of an N-terminally FLAG-tagged VPR-1 rescues underdeveloped gonad and sterility defects in vpr-1 null hermaphrodites. Immunofluorescence studies reveal that the tagged intestinal expressed VPR-1 is present at the distal gonad. Mass spectrometry analysis of a smaller product of the N-terminally tagged VPR-1 identifies a specific cleavage site at Leu156. Mutation of the leucine results in loss of gonadal MSPd signal and reduced activity of the mutant VPR-1. Thus, we report for the first time the cleavage site of VPR-1 and provide direct evidence that intestinally expressed VPR-1 can be released and signal in the distal gonad. These results establish the foundation for further exploration of VAP cleavage, MSPd secretion, and non-cell-autonomous signaling in development and diseases.

STRAP regulates alternative splicing fidelity during lineage commitment of mouse embryonic stem cells.

Alternative splicing (AS) is involved in cell fate decisions and embryonic development. However, regulation of these processes is poorly understood. Here, we have identified the serine threonine kinase receptor-associated protein (STRAP) as a putative spliceosome-associated factor. Upon Strap deletion, there are numerous AS events observed in mouse embryoid bodies (EBs) undergoing a neuroectoderm-like state. Global mapping of STRAP-RNA binding in mouse embryos by enhanced-CLIP sequencing (eCLIP-seq) reveals that STRAP preferably targets transcripts for nervous system development and regulates AS through preferred binding positions, as demonstrated for two neuronal-specific genes, Nnat and Mark3. We have found that STRAP involves in the assembly of 17S U2 snRNP proteins. Moreover, in Xenopus, loss of Strap leads to impeded lineage differentiation in embryos, delayed neural tube closure, and altered exon skipping. Collectively, our findings reveal a previously unknown function of STRAP in mediating the splicing networks of lineage commitment, alteration of which may be involved in early embryonic lethality in mice.

The transcription factor Hypermethylated in Cancer 1 (Hic1) regulates neural crest migration via interaction with Wnt signaling.

The transcription factor Hypermethylated in Cancer 1 (HIC1) is associated with both tumorigenesis and the complex human developmental disorder Miller-Dieker Syndrome. While many studies have characterized HIC1 as a tumor suppressor, HIC1 function in development is less understood. Loss-of-function mouse alleles show embryonic lethality accompanied with developmental defects, including craniofacial abnormalities that are reminiscent of human Miller-Dieker Syndrome patients. However, the tissue origin of the defects has not been reported. In this study, we use the power of the Xenopus laevis model system to explore Hic1 function in early development. We show that hic1 mRNA is expressed throughout early Xenopus development and has a spatial distribution within the neural plate border and in migrating neural crest cells in branchial arches. Targeted manipulation of hic1 levels in the dorsal ectoderm that gives rise to neural and neural crest tissues reveals that both overexpression and knockdown of hic1 result in craniofacial defects with malformations of the craniofacial cartilages. Neural crest specification is not affected by altered hic1 levels, but migration of the cranial neural crest is impaired both in vivo and in tissue explants. Mechanistically, we find that Hic1 regulates cadherin expression profiles and canonical Wnt signaling. Taken together, these results identify Hic1 as a novel regulator of the canonical Wnt pathway during neural crest migration.

A YWHAZ Variant Associated With Cardiofaciocutaneous Syndrome Activates the RAF-ERK Pathway.

Cardiofaciocutaneous (CFC) syndrome is a genetic disorder characterized by distinctive facial features, congenital heart defects, and skin abnormalities. Several germline gain-of-function mutations in the RAS/RAF/MEK/ERK pathway are associated with the disease, including KRAS, BRAF, MEK1, and MEK2. CFC syndrome thus belongs to a group of disorders known as RASopathies, which are all caused by pathogenic mutations in various genes encoding components of the RAS pathway. We recently identified novel variants in YWHAZ, a 14-3-3 family member, in individuals with a phenotype consistent with CFC that may potentially be deleterious and disease-causing. In the current study, we take advantage of the vertebrate model Xenopus laevis to analyze the functional consequence of a particular YWHAZ variant, S230W, and investigate the molecular mechanisms underlying its activity. We show that compared with wild type YWHAZ, the S230W variant induces severe embryonic defects when ectopically expressed in early Xenopus embryos. The S230W variant also rescues the defects induced by a dominant negative FGF receptor more efficiently and enhances Raf-stimulated Erk phosphorylation to a higher level than wild type YWHAZ. Although neither YWHAZ nor the variant promotes membrane recruitment of Raf proteins, the variant binds to more Raf and escapes phosphorylation by casein kinase 1a. Our data provide strong support to the hypothesis that the S230W variant of YWHAZ is a gain-of-function mutation in the RAS-ERK pathway and may underlie a CFC phenotype.

The RhoGEF protein Plekhg5 regulates apical constriction of bottle cells during gastrulation.

Apical constriction regulates epithelial morphogenesis during embryonic development, but how this process is controlled is not understood completely. Here, we identify a Rho guanine nucleotide exchange factor (GEF) gene plekhg5 as an essential regulator of apical constriction of bottle cells during Xenopus gastrulation. plekhg5 is expressed in the blastopore lip and its expression is sufficient to induce ectopic bottle cells in epithelia of different germ layers in a Rho-dependent manner. This activity is not shared by arhgef3, which encodes another organizer-specific RhoGEF. Plekhg5 protein is localized in the apical cell cortex via its pleckstrin homology domain, and the GEF activity enhances its apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation. Knockdown of plekhg5 inhibits activin-induced bottle cell formation and endogenous blastopore lip formation in gastrulating frog embryos. Apical accumulation of actomyosin, apical constriction and bottle cell formation fail to occur in these embryos. Taken together, our data indicate that transcriptional regulation of plekhg5 expression at the blastopore lip determines bottle cell morphology via local polarized activation of Rho by Plekhg5, which stimulates apical actomyosin activity to induce apical constriction.

Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.

Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.

MicroRNA-31 is required for astrocyte specification.

Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis.

Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing.

Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.

Bimodal regulation of Dishevelled function by Vangl2 during morphogenesis.

Convergent extension (CE) is a fundamental morphogenetic mechanism that underlies numerous processes in vertebrate development, and its disruption can lead to human congenital disorders such as neural tube closure defects. The dynamic, oriented cell intercalation during CE is regulated by a group of core proteins identified originally in flies to coordinate epithelial planar cell polarity (PCP). The existing model explains how core PCP proteins, including Van Gogh (Vang) and Dishevelled (Dvl), segregate into distinct complexes on opposing cell cortex to coordinate polarity among static epithelial cells. The action of core PCP proteins in the dynamic process of CE, however, remains an enigma. In this report, we show that Vangl2 (Vang-like 2) exerts dual positive and negative regulation on Dvl during CE in both the mouse and Xenopus. We find that Vangl2 binds to Dvl to cell-autonomously promote efficient Dvl plasma membrane recruitment, a pre-requisite for PCP activation. At the same time, Vangl2 inhibits Dvl from interacting with its downstream effector Daam1 (Dishevelled associated activator of morphogenesis 1), and functionally suppresses Dvl â†’ Daam1 cascade during CE. Our finding uncovers Vangl2-Dvl interaction as a key bi-functional switch that underlies the central logic of PCP signaling during morphogenesis, and provides new insight into PCP-related disorders in humans.

Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing.

During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed signaling and cytoskeleton regulators in different embryonic regions of Xenopus gastrulae and imply their functions in regulating cell fates and/or behaviors during gastrulation.

Agonists and Antagonists of TGF-ß Family Ligands.

The discovery of the transforming growth factor ß (TGF-ß) family ligands and the realization that their bioactivities need to be tightly controlled temporally and spatially led to intensive research that has identified a multitude of extracellular modulators of TGF-ß family ligands, uncovered their functions in developmental and pathophysiological processes, defined the mechanisms of their activities, and explored potential modulator-based therapeutic applications in treating human diseases. These studies revealed a diverse repertoire of extracellular and membrane-associated molecules that are capable of modulating TGF-ß family signals via control of ligand availability, processing, ligand-receptor interaction, and receptor activation. These molecules include not only soluble ligand-binding proteins that were conventionally considered as agonists and antagonists of TGF-ß family of growth factors, but also extracellular matrix (ECM) proteins and proteoglycans that can serve as "sink" and control storage and release of both the TGF-ß family ligands and their regulators. This extensive network of soluble and ECM modulators helps to ensure dynamic and cell-specific control of TGF-ß family signals. This article reviews our knowledge of extracellular modulation of TGF-ß growth factors by diverse proteins and their molecular mechanisms to regulate TGF-ß family signaling.

Molecular model for force production and transmission during vertebrate gastrulation.

Vertebrate embryos undergo dramatic shape changes at gastrulation that require locally produced and anisotropically applied forces, yet how these forces are produced and transmitted across tissues remains unclear. We show that depletion of myosin regulatory light chain (RLC) levels in the embryo blocks force generation at gastrulation through two distinct mechanisms: destabilizing the myosin II (MII) hexameric complex and inhibiting MII contractility. Molecular dissection of these two mechanisms demonstrates that normal convergence force generation requires MII contractility and we identify a set of molecular phenotypes correlated with both this failure of convergence force generation in explants and of blastopore closure in whole embryos. These include reduced rates of actin movement, alterations in C-cadherin dynamics and a reduction in the number of polarized lamellipodia on intercalating cells. By examining the spatial relationship between C-cadherin and actomyosin we also find evidence for formation of transcellular linear arrays incorporating these proteins that could transmit mediolaterally oriented tensional forces. These data combine to suggest a multistep model to explain how cell intercalation can occur against a force gradient to generate axial extension forces. First, polarized lamellipodia extend mediolaterally and make new C-cadherin-based contacts with neighboring mesodermal cell bodies. Second, lamellipodial flow of actin coalesces into a tension-bearing, MII-contractility-dependent node-and-cable actin network in the cell body cortex. And third, this actomyosin network contracts to generate mediolateral convergence forces in the context of these transcellular arrays.

Animal Cap Assay for TGF-ß Signaling.

TGF-ß signals regulate a variety of processes during early vertebrate development, from stem cell maintenance and differentiation to tissue patterning and organogenesis. Detailed understanding of how this signaling pathway operates and what genes control activities of the signaling components of the pathway is therefore important for us to comprehend temporal- and tissue-specific TGF-ß functions in vertebrate embryogenesis. Xenopus model system has been employed extensively in research on TGF-ß signals, and much insight about TGF-ß signaling mechanisms has been gained from these studies. Besides using whole embryos, explants from the ectodermal region of Xenopus, also known as animal caps, are used widely in investigations of the activities of an array of signal transducers as well as regulators of the pathway. This chapter introduces methods for dissection of animal caps and analyses of TGF-ß signaling effects on animal caps.