Prickle2 regulates apical junction remodeling and tissue fluidity during vertebrate neurulation.

The process of folding the flat neuroectoderm into an elongated neural tube depends on tissue fluidity, a property that allows epithelial deformation while preserving tissue integrity. Neural tube folding also requires the planar cell polarity (PCP) pathway. Here, we report that Prickle2 (Pk2), a core PCP component, increases tissue fluidity by promoting the remodeling of apical junctions (AJs) in Xenopus embryos.

Mechanical control of neural plate folding by apical domain alteration.

Vertebrate neural tube closure is associated with complex changes in cell shape and behavior, however, the relative contribution of these processes to tissue folding is not well understood. At the onset of Xenopus neural tube folding, we observed alternation of apically constricted and apically expanded cells. This apical domain heterogeneity was accompanied by biased cell orientation along the anteroposterior axis, especially at neural plate hinges, and required planar cell polarity signaling.

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.

Mechanical control of neural plate folding by apical domain alteration.

Vertebrate neural tube closure is associated with complex changes in cell shape and behavior, however, the relative contribution of these processes to tissue folding is not well understood. In this study, we evaluated morphology of the superficial cell layer in the neural plate. At the stages corresponding to the onset of tissue folding, we observed the alternation of cells with apically constricting and apically expanding apical domains.

Lmo7 recruits myosin II heavy chain to regulate actomyosin contractility and apical domain size in Xenopus ectoderm.

Apical constriction, or a reduction in size of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction; however, the detailed analysis of molecular mechanisms is still pending. Here, we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells.

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.

Analysis of Planar Cell Polarity Complexes by Proximity Biotinylation in Xenopus Embryos.

Understanding signaling processes operating in cells during development and disease requires extensive knowledge of protein interactions. Proximity-dependent biotinylation mediated by a promiscuous bacterial biotin ligase is a sensitive approach for evaluating protein interactions under physiological conditions. This technique allows for assessing protein association when conventional pull-down assays are not applicable due to high background or transient nature of the interaction.

Frizzled3 inhibits Vangl2-Prickle3 association to establish planar cell polarity in the vertebrate neural plate.

The orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here, we use proximity biotinylation and crosslinking approaches to show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3, also known as Fzd3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue.

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.

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.

Cryosectioning and Immunostaining of Embryonic Tissues.

The 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.

Rspo2 inhibits TCF3 phosphorylation to antagonize Wnt signaling during vertebrate anteroposterior axis specification.

The Wnt pathway activates target genes by controlling the β-catenin-T-cell factor (TCF) transcriptional complex during embryonic development and cancer. This pathway can be potentiated by R-spondins, a family of proteins that bind RNF43/ZNRF3 E3 ubiquitin ligases and LGR4/5 receptors to prevent Frizzled degradation. Here we demonstrate that, during Xenopus anteroposterior axis specification, Rspo2 functions as a Wnt antagonist, both morphologically and at the level of gene targets and pathway mediators.

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 gastrulation; however, its molecular targets and signaling mechanisms have not been fully elucidated. Our mass spectrometry-based screen of the gastrula identified Admp as Pnhd-associated protein.

Xenopus neural tube closure: A vertebrate model linking planar cell polarity to actomyosin contractions.

Planar cell polarity (PCP) refers to the coordinated polarization of cells within the plane of a tissue. PCP is a controlled by a group of conserved proteins organized in a specific signaling pathway known as the PCP pathway. A hallmark of PCP signaling is the asymmetric localization of "core" PCP protein complexes at the cell cortex, although endogenous PCP cues needed to establish this asymmetry remain unknown.

Planar cell polarity pathway in kidney development, function and disease.

Planar cell polarity (PCP) refers to the coordinated orientation of cells in the tissue plane. Originally discovered and studied in Drosophila melanogaster, PCP is now widely recognized in vertebrates, where it is implicated in organogenesis. Specific sets of PCP genes have been identified.

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.

Rspo2 antagonizes FGF signaling during vertebrate mesoderm formation and patterning.

R-spondins are a family of secreted proteins that play important roles in embryonic development and cancer. R-spondins have been shown to modulate the Wnt pathway; however, their involvement in other developmental signaling processes have remained largely unstudied. Here, we describe a novel function of Rspo2 in FGF pathway regulation Overexpressed Rspo2 inhibited elongation of ectoderm explants and Erk1 activation in response to FGF.

Isolation of nanobodies against Xenopus embryonic antigens using immune and non-immune phage display libraries.

The use of Xenopus laevis as a model for vertebrate developmental biology is limited by a lack of antibodies specific for embryonic antigens. This study evaluated the use of immune and non-immune phage display libraries for the isolation of single domain antibodies, or nanobodies, with specificities for Xenopus embryonic antigens. The immune nanobody library was derived from peripheral blood lymphocyte RNA obtained from a llama immunized with Xenopus gastrula homogenates.

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 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.

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.

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 early embryos. Targeted depletion of Wtip inhibited apical constriction in neuroepithelial cells and elicited neural tube defects.

At the Crossroads between Cell Polarity and Adhesion in Neocortical Development.

In this issue of Developmental Cell, Jossin et al. (2017) show that the tumor suppressor protein Lgl1 interacts with N-cadherin to stabilize apical junctions in brain stem cells. Neural progenitors lacking Lgl1 had decreased N-cadherin internalization and abnormal cell junctions, generating an ectopic neuronal layer that resembles cerebral cortex abnormalities in humans.

Wnt proteins can direct planar cell polarity in vertebrate ectoderm.

The coordinated orientation of cells across the tissue plane, known as planar cell polarity (PCP), is manifested by the segregation of core PCP proteins to different sides of the cell. Secreted Wnt ligands are involved in many PCP-dependent processes, yet whether they act as polarity cues has been controversial. We show that in early ectoderm, the Prickle3/Vangl2 complex was polarized to anterior cell edges and this polarity was disrupted by several Wnt antagonists.

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.