RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration.

Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.

ß-H-Spectrin is a key component of an apical-medial hub of proteins during cell wedging in tube morphogenesis.

Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. We previously identified that interplay between the apical-medial actomyosin, which drives apical constriction, and the underlying longitudinal microtubule array has a key role during tube budding of salivary glands in the Drosophila embryo. At this microtubule-actomyosin interface, a hub of proteins accumulates, and we have shown before that this hub includes the microtubule-actin crosslinker Shot and the microtubule minus-end-binding protein Patronin. Here, we identify two actin-crosslinkers, ß-heavy (H)-Spectrin (also known as Karst) and Filamin (also known as Cheerio), and the multi-PDZ-domain protein Big bang as components of the protein hub. We show that tissue-specific degradation of ß-H-Spectrin leads to reduction of apical-medial F-actin, Shot, Patronin and Big bang, as well as concomitant defects in apical constriction, but that residual Patronin is still sufficient to assist microtubule reorganisation. We find that, unlike Patronin and Shot, neither ß-H-Spectrin nor Big bang require microtubules for their localisation. ß-H-Spectrin is instead recruited via binding to apical-medial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of ß-H-Spectrin (ß-H-33) displaces endogenous ß-H-Spectrin and leads to strong morphogenetic defects. This protein hub therefore requires the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining apical constriction during tubulogenesis.

A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di-phosphorylated myosin light chain (ppMLC)-driven contraction of actomyosin-based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC-triggered system at TJ-associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule-associated proteins in the AJC-enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ-, but not at AJ-, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ-associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule-facilitated manner. Our results uncovered a hitherto unknown microtubule-LUZP1 association at TJ-associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure.

Neural tube closure (NTC) is a fundamental process during vertebrate development and is indispensable for the formation of the central nervous system. Here, using Xenopus laevis embryos, live imaging, single-cell tracking, optogenetics and loss-of-function experiments, we examine the roles of convergent extension and apical constriction, and define the role of the surface ectoderm during NTC. We show that NTC is a two-stage process with distinct spatiotemporal contributions of convergent extension and apical constriction at each stage. Convergent extension takes place during the first stage and is spatially restricted at the posterior tissue, whereas apical constriction occurs during the second stage throughout the neural plate. We also show that the surface ectoderm is mechanically coupled with the neural plate and its movement during NTC is driven by neural plate morphogenesis. Finally, we show that an increase in surface ectoderm resistive forces is detrimental for neural plate morphogenesis.

The cell polarity determinant Dlg1 facilitates epithelial invagination by promoting tissue-scale mechanical coordination.

Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains unknown whether additional mechanical inputs are required for apical constriction-mediated folding. Using Drosophila mesoderm invagination as a model, we identified an important role for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ('flanking cells') in facilitating epithelial folding. We found that depletion of the basolateral determinant Dlg1 disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wild-type embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. Our findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry.

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

Ascidian gastrulation and blebbing activity of isolated endoderm blastomeres.

Gastrulation is the first dynamic cell movement during embryogenesis. Endoderm and mesoderm cells are internalized into embryos during this process. Ascidian embryos provide a simple system for studying gastrulation in chordates. Gastrulation starts in spherical late 64-cell embryos with 10 endoderm blastomeres. The mechanisms of gastrulation in ascidians have been investigated, and a two-step model has been proposed. The first step involves apical constriction of endoderm cells, followed by apicobasal shortening in the second step. In this study, isolated ascidian endoderm progenitor cells displayed dynamic blebbing activity at the gastrula stage, although such a dynamic cell-shape change was not recognized in toto. Blebbing is often observed in migrating animal cells. In ascidians, endoderm cells displayed blebbing activity, while mesoderm and ectoderm cells did not. The timing of blebbing of isolated endoderm cells coincided with that of cell invagination. The constriction rate of apical surfaces correlated with the intensity of blebbing activity in each endoderm-lineage cell. Fibroblast growth factor (FGF) signaling was both necessary and sufficient for inducing blebbing activity, independent of cell fate specification. In contrast, the timing of initiation of blebbing and intensity of blebbing response to FGF signaling were controlled by intrinsic cellular factors. It is likely that the difference in intensity of blebbing activity between the anterior A-line and posterior B-line cells could account for the anteroposterior difference in the steepness of the archenteron wall. Inhibition of zygotic transcription, FGF signaling, and Rho kinase, all of which suppressed blebbing activity, resulted in incomplete apical constriction and failure of the eventual formation of cup-shaped gastrulae. Blebbing activity was involved in the progression and maintenance of apical constriction, but not in apicobasal shortening in whole embryos. Apical constriction is mediated by distinct blebbing-dependent and blebbing-independent mechanisms. Surface tension and consequent membrane contraction may not be the sole mechanical force for apical constriction and formation of cup-shaped gastrulae. The present study reveals the hidden cellular potential of endodermal cells during gastrulation and discusses the possible roles of blebbing in the invagination process.

Mechano-chemical feedback mediated competition for BMP signalling leads to pattern formation.

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

Cell-center-based model for simulating three-dimensional monolayer tissue deformation.

The shape of the epithelial monolayer can be depicted as a curved tissue in three-dimensional (3D) space, where individual cells are tightly adhered to one another. The 3D morphogenesis of these tissues is governed by cell dynamics, and a variety of mathematical modeling and simulation studies have been conducted to investigate this process. One promising approach is the cell-center model, which can account for the discreteness of cells. The cell nucleus, which is considered to correspond to the cell center, can be observed experimentally. However, there has been a shortage of cell-center models specifically tailored for simulating 3D monolayer tissue deformation. In this study, we developed a mathematical model based on the cell-center model to simulate 3D monolayer tissue deformation. Our model was confirmed by simulating the in-plane deformation, out-of-plane deformation, and invagination due to apical constriction.

Scribble mutation disrupts convergent extension and apical constriction during mammalian neural tube closure.

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.

Coordination of cytoskeletal dynamics and cell behaviour during Drosophila abdominal morphogenesis.

During morphogenesis, cells exhibit various behaviours, such as migration and constriction, which need to be coordinated. How this is achieved remains elusive. During morphogenesis of the Drosophila adult abdominal epidermis, larval epithelial cells (LECs) migrate directedly before constricting apically and undergoing apoptosis. Here, we study the mechanisms underlying the transition from migration to constriction. We show that LECs possess a pulsatile apical actomyosin network, and that a change in network polarity correlates with behavioural change. Exploring the properties of the contractile network, we find that cell contractility, as determined by myosin activity, has an impact on the behaviour of the network, as well as on cytoskeletal architecture and cell behaviour. Pulsed contractions occur only in cells with intermediate levels of contractility. Furthermore, increasing levels of the small Rho GTPase Rho1 disrupts pulsing, leading to cells that cycle between two states, characterised by a junctional cortical and an apicomedial actin network. Our results highlight that behavioural change relies on tightly controlled cellular contractility. Moreover, we show that constriction can occur without pulsing, raising questions why constricting cells pulse in some contexts but not in others.

ßH-spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination.

Actomyosin-mediated apical constriction drives a wide range of morphogenetic processes. Activation of myosin-II initiates pulsatile cycles of apical constrictions followed by either relaxation or stabilization (ratcheting) of the apical surface. While relaxation leads to dissipation of contractile forces, ratcheting is critical for the generation of tissue-level tension and changes in tissue shape. How ratcheting is controlled at the molecular level is unknown. Here, we show that the actin crosslinker ßH-spectrin is upregulated at the apical surface of invaginating mesodermal cells during Drosophila gastrulation. ßH-spectrin forms a network of filaments which co-localize with medio-apical actomyosin fibers, in a process that depends on the mesoderm-transcription factor Twist and activation of Rho signaling. ßH-spectrin knockdown results in non-ratcheted apical constrictions and inhibition of mesoderm invagination, recapitulating twist mutant embryos. ßH-spectrin is thus a key regulator of apical ratcheting during tissue invagination, suggesting that actin cross-linking plays a critical role in this process.

Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure.

Cellular generation of mechanical forces required to close the presumptive spinal neural tube, the 'posterior neuropore' (PNP), involves interkinetic nuclear migration (INM) and apical constriction. Both processes change the apical surface area of neuroepithelial cells, but how they are biomechanically integrated is unknown. Rho kinase (Rock; herein referring to both ROCK1 and ROCK2) inhibition in mouse whole embryo culture progressively widens the PNP. PNP widening is not caused by increased mechanical tension opposing closure, as evidenced by diminished recoil following laser ablation. Rather, Rock inhibition diminishes neuroepithelial apical constriction, producing increased apical areas in neuroepithelial cells despite diminished tension. Neuroepithelial apices are also dynamically related to INM progression, with the smallest dimensions achieved in cells positive for the pan-M phase marker Rb phosphorylated at S780 (pRB-S780). A brief (2 h) Rock inhibition selectively increases the apical area of pRB-S780-positive cells, but not pre-anaphase cells positive for phosphorylated histone 3 (pHH3+). Longer inhibition (8 h, more than one cell cycle) increases apical areas in pHH3+ cells, suggesting cell cycle-dependent accumulation of cells with larger apical surfaces during PNP widening. Consequently, arresting cell cycle progression with hydroxyurea prevents PNP widening following Rock inhibition. Thus, Rock-dependent apical constriction compensates for the PNP-widening effects of INM to enable progression of closure.This article has an associated First Person interview with the first authors of the paper.

Proliferation-independent role of NF2 (merlin) in limiting biliary morphogenesis.

The architecture of individual cells and cell collectives enables functional specification, a prominent example being the formation of epithelial tubes that transport fluid or gas in many organs. The intrahepatic bile ducts (IHBDs) form a tubular network within the liver parenchyma that transports bile to the intestine. Aberrant biliary 'neoductulogenesis' is also a feature of several liver pathologies including tumorigenesis. However, the mechanism of biliary tube morphogenesis in development or disease is not known. Elimination of the neurofibromatosis type 2 protein (NF2; also known as merlin or neurofibromin 2) causes hepatomegaly due to massive biliary neoductulogenesis in the mouse liver. We show that this phenotype reflects unlimited biliary morphogenesis rather than proliferative expansion. Our studies suggest that NF2 normally limits biliary morphogenesis by coordinating lumen expansion and cell architecture. This work provides fundamental insight into how biliary fate and tubulogenesis are coordinated during development and will guide analyses of disease-associated and experimentally induced biliary pathologies.

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.

Secretory pathway calcium ATPase 1 (SPCA1) controls mouse neural tube closure by regulating cytoskeletal dynamics.

Neural tube closure relies on the apical constriction of neuroepithelial cells. Research in frog and fly embryos has found links between the levels of intracellular calcium, actomyosin dynamics and apical constriction. However, genetic evidence for a role of calcium in apical constriction during mammalian neurulation is still lacking. Secretory pathway calcium ATPase (SPCA1) regulates calcium homeostasis by pumping cytosolic calcium into the Golgi apparatus. Loss of function in Spca1 causes cranial exencephaly and spinal cord defects in mice, phenotypes previously ascribed to apoptosis. However, our characterization of a novel allele of Spca1 revealed that neurulation defects in Spca1 mutants are not due to cell death, but rather to a failure of neuroepithelial cells to apically constrict. We show that SPCA1 influences cell contractility by regulating myosin II localization. Furthermore, we found that loss of Spca1 disrupts actin dynamics and the localization of the actin remodeling protein cofilin 1. Taken together, our results provide evidence that SPCA1 promotes neurulation by regulating the cytoskeletal dynamics that promote apical constriction and identify cofilin 1 as a downstream effector of SPCA1 function.

Membrane trafficking in morphogenesis and planar polarity.

Our understanding of how membrane trafficking pathways function to direct morphogenetic movements and the planar polarization of developing tissues is a new and emerging field. While a central focus of developmental biology has been on how protein asymmetries and cytoskeletal force generation direct cell shaping, the role of membrane trafficking in these processes has been less clear. Here, we review recent advances in Drosophila and vertebrate systems in our understanding of how trafficking events are coordinated with planar cytoskeletal function to drive lasting changes in cell and tissue topologies. We additionally explore the function of trafficking pathways in guiding the complex interactions that initiate and maintain core PCP (planar cell polarity) asymmetries and drive the generation of systematically oriented cellular projections during development.

Apical constriction is necessary for crypt formation in small intestinal organoids.

Small intestinal organoids have become an important tool to study crypt homeostasis, cell fate dynamics and tissue biomechanics. Yet, the mechanisms that drive the budding of crypts from the smooth organoid epithelium remain incompletely understood. Locally enhanced proliferation has been suggested to induce tissue buckling and crypt initiation. Here we report that changes in cell morphology play a crucial role in crypt formation. Crypt formation is preceded by local epithelial thickening, apicobasal elongation, and apical narrowing, resulting in a wedge-like cell-shape, followed by apical evagination and crypt outgrowth. Myosin II activity is found to coincide with apical constriction of cells, while inhibition of myosin suppresses apical constriction and bud formation. The data suggest that myosin-driven apical constriction is a key driving force of bud initiation in small intestinal organoids.

Formin homology 2 domain-containing 3 (Fhod3) controls neural plate morphogenesis in mouse cranial neurulation by regulating multidirectional apical constriction.

Neural tube closure requires apical constriction during which contraction of the apical F-actin network forces the cell into a wedged shape, facilitating the folding of the neural plate into a tube. However, how F-actin assembly at the apical surface is regulated in mammalian neurulation remains largely unknown. We report here that formin homology 2 domain-containing 3 (Fhod3), a formin protein that mediates F-actin assembly, is essential for cranial neural tube closure in mouse embryos. We found that Fhod3 is expressed in the lateral neural plate but not in the floor region of the closing neural plate at the hindbrain. Consistently, in Fhod3-null embryos, neural plate bending at the midline occurred normally, but lateral plates seemed floppy and failed to flex dorsomedially. Because the apical accumulation of F-actin and constriction were impaired specifically at the lateral plates in Fhod3-null embryos, we concluded that Fhod3-mediated actin assembly contributes to lateral plate-specific apical constriction to advance closure. Intriguingly, Fhod3 expression at the hindbrain was restricted to neuromeric segments called rhombomeres. The rhombomere-specific accumulation of apical F-actin induced by the rhombomere-restricted expression of Fhod3 was responsible for the outward bulging of rhombomeres involving apical constriction along the anteroposterior axis, as rhombomeric bulging was less prominent in Fhod3-null embryos than in the wild type. Fhod3 thus plays a crucial role in the morphological changes associated with neural tube closure at the hindbrain by mediating apical constriction not only in the mediolateral but also in the anteroposterior direction, thereby contributing to tube closure and rhombomere segmentation, respectively.

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.