Myocardin-related transcription factors regulate morphogenetic events in vertebrate embryos by controlling F-actin organization and apical constriction.

Myocardin-related transcription factors (Mrtfa and Mrtfb), also known as megakaryoblastic leukemia proteins (Mkl1/MAL and Mkl2), associate with serum response factor (Srf) to regulate transcription in response to actin dynamics, however, the functions of Mrtfs in early vertebrate embryos remain largely unknown. Here we document the requirement of Mrtfs for blastopore closure at gastrulation and neural plate folding in Xenopus early embryos. Both stimulation and inhibition of Mrtf activity caused similar gross morphological phenotypes, yet the effects on F-actin distribution and cell behavior were different. Suppressing Mrtf-dependent transcription reduced overall F-actin levels and inhibited apical constriction during gastrulation and neurulation. By contrast, constitutively active Mrtf caused tricellular junction remodeling and induced apical constriction in superficial ectoderm. The underlying mechanism appeared distinct from the one utilized by known apical constriction inducers. We propose that the regulation of apical constriction is among the primary cellular responses to Mrtf. Our findings highlight a dedicated role of specific transcription factors, Mrtfs, in early morphogenetic processes.

Imaging Planar Cell Polarity Proteins in Xenopus Neuroectoderm.

Planar cell polarity (PCP) refers to coordinated cell polarization in the plane of the tissue. Genetic studies in Drosophila identified several core PCP genes, whose products function together in a signaling pathway that regulates cell shape, epithelial tissue organization and remodeling during morphogenesis. PCP is detected by the asymmetric distribution of core PCP proteins at different borders of epithelial cells. Believed to be critical for signaling, this segregation is studied by a variety of techniques, such as direct immunostaining and imaging of fluorescent PCP protein fusions or fluorescence recovery after photobleaching (FRAP). All of the above techniques can be applied to the analysis of the Xenopus neural plate to study the dynamics of tissue polarization, making this system one of the best vertebrate PCP models. This chapter describes how to image PCP proteins in Xenopus neuroectoderm for both fixed and live samples. These robust cellular techniques will contribute to mechanistic studies of PCP in vertebrate embryos.

Identification of the centrosomal maturation factor SSX2IP as a Wtip-binding partner by targeted proximity biotinylation.

Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains associates with the actin-binding protein Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. By contrast, the N-terminal domain localizes to apical junctions in the ectoderm and basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Targeted proximity biotinylation (TPB) using anti-GFP antibody fused to the biotin ligase BirA identified SSX2IP as a candidate protein that binds GFP-WtipN. SSX2IP, also known as Msd1 or ADIP, is a component of cell junctions, centriolar satellite protein and a targeting factor for ciliary membrane proteins. WtipN physically associated with SSX2IP and the two proteins readily formed mixed aggregates in overexpressing cells. By contrast, we observed only partial colocalization of full length Wtip and SSX2IP, suggesting that Wtip adopts a 'closed' conformation in the cell. Furthermore, the double depletion of Wtip and SSX2IP in early embryos uncovered the functional interaction of the two proteins during neural tube closure. Our results suggest that the association of SSX2IP and Wtip is essential for cell junction remodeling and morphogenetic processes that accompany neurulation. We propose that TPB can be a general approach that is applicable to other GFP-tagged proteins.

The dorsal blastopore lip is a source of signals inducing planar cell polarity in the Xenopus neural plate.

Coordinated polarization of cells in the tissue plane, known as planar cell polarity (PCP), is associated with a signaling pathway critical for the control of morphogenetic processes. Although the segregation of PCP components to opposite cell borders is believed to play a critical role in this pathway, whether PCP derives from egg polarity or preexistent long-range gradient, or forms in response to a localized cue, remains a challenging question. Here we investigate the Xenopus neural plate, a tissue that has been previously shown to exhibit PCP. By imaging Vangl2 and Prickle3, we show that PCP is progressively acquired in the neural plate and requires a signal from the posterior region of the embryo. Tissue transplantations indicated that PCP is triggered in the neural plate by a planar cue from the dorsal blastopore lip. The PCP cue did not depend on the orientation of the graft and was distinct from neural inducers. These observations suggest that neuroectodermal PCP is not instructed by a preexisting molecular gradient but induced by a signal from the dorsal blastopore lip.

Cryosectioning and Immunostaining of Xenopus Embryonic Tissues.

The Xenopus embryo is a classical vertebrate model for molecular, cellular, and developmental biology. Despite many advantages of this organism, such as large egg size and external development, imaging of early embryonic stages is challenging because of nontransparent cytoplasm. Staining and imaging of thin tissue sections is one way to overcome this limitation. Here we describe a step-by-step protocol that combines cryosectioning of gelatin-embedded embryos with immunostaining and imaging. The purpose of this protocol is to examine various cellular and tissue markers after the manipulation of protein function. This protocol can be performed within a 2-d period and allows detection of many antigens by immunofluorescence.

Pinhead antagonizes Admp to promote notochord formation.

Dorsoventral patterning of a vertebrate embryo critically depends on the activity of Smad1 that mediates signaling by BMP proteins, anti-dorsalizing morphogenetic protein (Admp), and their antagonists. Pinhead (Pnhd), a cystine-knot-containing secreted protein, is expressed in the ventrolateral mesoderm during Xenopus gastrulation; however, its molecular targets and signaling mechanisms have not been fully elucidated. Our mass spectrometry-based screen of the gastrula secretome identified Admp as Pnhd-associated protein. We show that Pnhd binds Admp and inhibits its ventralizing activity by reducing Smad1 phosphorylation and its transcriptional targets. Importantly, Pnhd depletion further increased phospho-Smad1 levels in the presence of Admp. Furthermore, Pnhd synergized with Chordin and a truncated BMP4 receptor in the induction of notochord markers in ectoderm cells, and Pnhd-depleted embryos displayed notochord defects. Our findings suggest that Pnhd binds and inactivates Admp to promote notochord development. We propose that the interaction between Admp and Pnhd refines Smad1 activity gradients during vertebrate gastrulation.

Pinhead signaling regulates mesoderm heterogeneity via the FGF receptor-dependent pathway.

Among the three embryonic germ layers, the mesoderm plays a central role in the establishment of the vertebrate body plan. The mesoderm is specified by secreted signaling proteins from the FGF, Nodal, BMP and Wnt families. No new classes of extracellular mesoderm-inducing factors have been identified in more than two decades. Here, we show that the pinhead (pnhd) gene encodes a secreted protein that is essential for the activation of a subset of mesodermal markers in the Xenopus embryo. RNA sequencing revealed that many transcriptional targets of Pnhd are shared with those of the FGF pathway. Pnhd activity was accompanied by Erk phosphorylation and required FGF and Nodal but not Wnt signaling. We propose that during gastrulation Pnhd acts in the marginal zone to contribute to mesoderm heterogeneity via an FGF receptor-dependent positive feedback mechanism.

Par3 interacts with Prickle3 to generate apical PCP complexes in the vertebrate neural plate.

Vertebrate neural tube formation depends on the coordinated orientation of cells in the tissue known as planar cell polarity (PCP). In the Xenopus neural plate, PCP is marked by the enrichment of the conserved proteins Prickle3 and Vangl2 at anterior cell boundaries. Here we show that the apical determinant Par3 is also planar polarized in the neuroepithelium, suggesting a role for Par3 in PCP. Consistent with this hypothesis, interference with Par3 activity inhibited asymmetric distribution of PCP junctional complexes and caused neural tube defects. Importantly, Par3 physically associated with Prickle3 and promoted its apical localization, whereas overexpression of a Prickle3-binding Par3 fragment disrupted PCP in the neural plate. We also adapted proximity biotinylation assay for use in Xenopus embryos and show that Par3 functions by enhancing the formation of the anterior apical PCP complex. These findings describe a mechanistic link between the apical localization of PCP components and morphogenetic movements underlying neurulation.

Regulation of neural crest development by the formin family protein Daam1.

The neural crest (NC) multipotent progenitor cells form at the neural plate border and migrate to diverse locations in the embryo to differentiate into many cell types. NC is specified by several embryonic pathways, however the role of noncanonical Wnt signaling in this process remains poorly defined. Daam1 is a formin family protein that is present in embryonic ectoderm at the time of NC formation and can mediate noncanonical Wnt signaling. Our interference experiments indicated that Daam1 is required for NC gene activation. To further study the function of Daam1 in NC development we used a transgenic reporter Xenopus line, in which GFP transcription is driven by sox10 upstream regulatory sequences. The activation of the sox10:GFP reporter in a subset of NC cells was suppressed after Daam1 depletion and in embryos expressing N-Daam1, a dominant interfering construct. Moreover, N-Daam1 blocked reporter activation in neuralized ectodermal explants in response to Wnt11, but not Wnt8 or Wnt3a, confirming that the downstream pathways are different. In complementary experiments, a constitutively active Daam1 fragment expanded the NC territory, but this gain-of-function activity was eliminated in a construct with a point mutation in the FH2 domain that is critical for actin polymerization. These observations suggest a new role of Daam1 and actin remodeling in NC specification.

The Ajuba family protein Wtip regulates actomyosin contractility during vertebrate neural tube closure.

Ajuba family proteins are implicated in the assembly of cell junctions and have been reported to antagonize Hippo signaling in response to cytoskeletal tension. To assess the role of these proteins in actomyosin contractility, we examined the localization and function of Wtip, a member of the Ajuba family, in Xenopus early embryos. Targeted in vivo depletion of Wtip inhibited apical constriction in neuroepithelial cells and elicited neural tube defects. Fluorescent protein-tagged Wtip showed predominant punctate localization along the cell junctions in the epidermis and a linear junctional pattern in the neuroectoderm. In cells undergoing Shroom3-induced apical constriction, the punctate distribution was reorganized into a linear pattern. Conversely, the linear junctional pattern of Wtip in neuroectoderm changed to a more punctate distribution in cells with reduced myosin II activity. The C-terminal fragment of Wtip physically associated with Shroom3 and interfered with Shroom3 activity and neural fold formation. We therefore propose that Wtip is a tension-sensitive cytoskeletal adaptor that regulates apical constriction during vertebrate neurulation.This article has an associated First Person interview with the first author of the paper.

Prickle3 synergizes with Wtip to regulate basal body organization and cilia growth.

PCP proteins maintain planar polarity in many epithelial tissues and have been implicated in cilia development in vertebrate embryos. In this study we examine Prickle3 (Pk3), a vertebrate homologue of Drosophila Prickle, in Xenopus gastrocoel roof plate (GRP). GRP is a tissue equivalent to the mouse node, in which cilia-generated flow promotes left-right patterning. We show that Pk3 is enriched at the basal body of GRP cells but is recruited by Vangl2 to anterior cell borders. Interference with Pk3 function disrupted the anterior polarization of endogenous Vangl2 and the posterior localization of cilia in GRP cells, demonstrating its role in PCP. Strikingly, in cells with reduced Pk3 activity, cilia growth was inhibited and γ-tubulin and Nedd1 no longer associated with the basal body, suggesting that Pk3 has a novel function in basal body organization. Mechanistically, this function of Pk3 may involve Wilms tumor protein 1-interacting protein (Wtip), which physically associates with and cooperates with Pk3 to regulate ciliogenesis. We propose that, in addition to cell polarity, PCP components control basal body organization and function.

The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development.

The planar cell polarity (PCP) pathway orients cells in diverse epithelial tissues in Drosophila and vertebrate embryos and has been implicated in many human congenital defects and diseases, such as ciliopathies, polycystic kidney disease and malignant cancers. During vertebrate gastrulation and neurulation, PCP signaling is required for convergent extension movements, which are primarily driven by mediolateral cell intercalations, whereas the role for PCP signaling in radial cell intercalations has been unclear. In this study, we examine the function of the core PCP proteins Vangl2, Prickle3 (Pk3) and Disheveled in the ectodermal cells, which undergo radial intercalations during Xenopus gastrulation and neurulation. In the epidermis, multiciliated cell (MCC) progenitors originate in the inner layer, but subsequently migrate to the embryo surface during neurulation. We find that the Vangl2/Pk protein complexes are enriched at the apical domain of intercalating MCCs and are essential for the MCC intercalatory behavior. Addressing the underlying mechanism, we identified KIF13B, as a motor protein that binds Disheveled. KIF13B is required for MCC intercalation and acts synergistically with Vangl2 and Disheveled, indicating that it may mediate microtubule-dependent trafficking of PCP proteins necessary for cell shape regulation. In the neural plate, the Vangl2/Pk complexes were also concentrated near the outermost surface of deep layer cells, suggesting a general role for PCP in radial intercalation. Consistent with this hypothesis, the ectodermal tissues deficient in Vangl2 or Disheveled functions contained more cell layers than normal tissues. We propose that PCP signaling is essential for both mediolateral and radial cell intercalations during vertebrate morphogenesis. These expanded roles underscore the significance of vertebrate PCP proteins as factors contributing to a number of diseases, including neural tube defects, tumor metastases, and various genetic syndromes characterized by abnormal migratory cell behaviors.

Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling.

The vertebrate neural tube forms as a result of complex morphogenetic movements, which require the functions of several core planar cell polarity (PCP) proteins, including Vangl2 and Prickle. Despite the importance of these proteins for neurulation, their subcellular localization and the mode of action have remained largely unknown. Here we describe the anteroposterior planar cell polarity (AP-PCP) of the cells in the Xenopus neural plate. At the neural midline, the Vangl2 protein is enriched at anterior cell edges and that this localization is directed by Prickle, a Vangl2-interacting protein. Our further analysis is consistent with the model, in which Vangl2 AP-PCP is established in the neural plate as a consequence of Wnt-dependent phosphorylation. Additionally, we uncover feedback regulation of Vangl2 polarity by Myosin II, reiterating a role for mechanical forces in PCP. These observations indicate that both Wnt signaling and Myosin II activity regulate cell polarity and cell behaviors during vertebrate neurulation.

Neural crest specification by inhibition of the ROCK/Myosin II pathway.

Neural crest is a population of multipotent progenitor cells that form at the border of neural and non-neural ectoderm in vertebrate embryos, and undergo epithelial-mesenchymal transition and migration. According to the traditional view, the neural crest is specified in early embryos by signaling molecules including BMP, FGF, and Wnt proteins. Here, we identify a novel signaling pathway leading to neural crest specification, which involves Rho-associated kinase (ROCK) and its downstream target nonmuscle Myosin II. We show that ROCK inhibitors promote differentiation of human embryonic stem cells (hESCs) into neural crest-like progenitors (NCPs) that are characterized by specific molecular markers and ability to differentiate into multiple cell types, including neurons, chondrocytes, osteocytes, and smooth muscle cells. Moreover, inhibition of Myosin II was sufficient for generating NCPs at high efficiency. Whereas Myosin II has been previously implicated in the self-renewal and survival of hESCs, we demonstrate its role in neural crest development during ESC differentiation. Inhibition of this pathway in Xenopus embryos expanded neural crest in vivo, further indicating that neural crest specification is controlled by ROCK-dependent Myosin II activity. We propose that changes in cell morphology in response to ROCK and Myosin II inhibition initiate mechanical signaling leading to neural crest fates.

Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation.

Core planar cell polarity (PCP) proteins are well known to regulate polarity in Drosophila and vertebrate epithelia; however, their functions in vertebrate morphogenesis remain poorly understood. In this study, we describe a role for PCP signaling in the process of apical constriction during Xenopus gastrulation. The core PCP protein Vangl2 is detected at the apical surfaces of cells at the blastopore lip, and it functions during blastopore formation and closure. Further experiments show that Vangl2, as well as Daam1 and Rho-associated kinase (Rock), regulate apical constriction of bottle cells at the blastopore and ectopic constriction of ectoderm cells triggered by the actin-binding protein Shroom3. At the blastopore lip, Vangl2 is required for the apical accumulation of the recycling endosome marker Rab11. We also show that Rab11 and the associated motor protein Myosin V play essential roles in both endogenous and ectopic apical constriction, and might be involved in Vangl2 trafficking to the cell surface. Overexpression of Rab11 RNA was sufficient to partly restore normal blastopore formation in Vangl2-deficient embryos. These observations suggest that Vangl2 affects Rab11 to regulate apical constriction during blastopore formation.

Role of Rab11 in planar cell polarity and apical constriction during vertebrate neural tube closure.

Epithelial folding is a critical process underlying many morphogenetic events including vertebrate neural tube closure, however, its spatial regulation is largely unknown. Here we show that during neural tube formation Rab11-positive recycling endosomes acquire bilaterally symmetric distribution in the Xenopus neural plate, being enriched at medial apical cell junctions. This mediolateral polarization was under the control of planar cell polarity (PCP) signalling, was necessary for neural plate folding and was accompanied by the polarization of the exocyst component Sec15. Our further experiments demonstrate that similar PCP-dependent polarization of Rab11 is essential for ectopic apical constriction driven by the actin-binding protein Shroom and during embryonic wound repair. We propose that anisotropic membrane trafficking has key roles in diverse morphogenetic behaviours of individual cells and propagates in a tissue by a common mechanism that involves PCP.

GEF-H1 functions in apical constriction and cell intercalations and is essential for vertebrate neural tube closure.

Rho family GTPases regulate many morphogenetic processes during vertebrate development including neural tube closure. Here we report a function for GEF-H1/Lfc/ArhGEF2, a RhoA-specific guanine nucleotide exchange factor that functions in neurulation in Xenopus embryos. Morpholino-mediated depletion of GEF-H1 resulted in severe neural tube defects, which were rescued by GEF-H1 RNA. Lineage tracing of GEF-H1 morphants at different developmental stages revealed abnormal cell intercalation and apical constriction, suggesting that GEF-H1 regulates these cell behaviors. Molecular marker analysis documented defects in myosin II light chain (MLC) phosphorylation, Rab11 and F-actin accumulation in GEF-H1-depleted cells. In gain-of-function studies, overexpressed GEF-H1 induced Rho-associated kinase-dependent ectopic apical constriction - marked by apical accumulation of phosphorylated MLC, γ-tubulin and F-actin in superficial ectoderm - and stimulated apical protrusive activity of deep ectoderm cells. Taken together, our observations newly identify functions of GEF-H1 in morphogenetic movements that lead to neural tube closure.

Lulu regulates Shroom-induced apical constriction during neural tube closure.

Apical constriction is an essential cell behavior during neural tube closure, but its underlying mechanisms are not fully understood. Lulu, or EPB4.1l5, is a FERM domain protein that has been implicated in apical constriction and actomyosin contractility in mouse embryos and cultured cells. Interference with the function of Lulu in Xenopus embryos by a specific antisense morpholino oligonucleotide or a carboxy-terminal fragment of Lulu impaired apical constriction during neural plate hinge formation. This effect was likely due to lack of actomyosin contractility in superficial neuroectodermal cells. By contrast, overexpression of Lulu RNA in embryonic ectoderm cells triggered ectopic apico-basal elongation and apical constriction, accompanied by the apical recruitment of F-actin. Depletion of endogenous Lulu disrupted the localization and activity of Shroom3, a PDZ-containing actin-binding protein that has also been implicated in apical constriction. Furthermore, Lulu and Shroom3 RNAs cooperated in triggering ectopic apical constriction in embryonic ectoderm. Our findings reveal that Lulu is essential for Shroom3-dependent apical constriction during vertebrate neural tube closure.

Neural crest specification by noncanonical Wnt signaling and PAR-1.

Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/ß-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize ß-catenin has remained unclear. Our gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of ß-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate.