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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, β-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 β-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 β-H-Spectrin nor Big bang require microtubules for their localisation. β-H-Spectrin is instead recruited via binding to apicalmedial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of β-H-Spectrin (β-H33) displaces endogenous β-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. [ABSTRACT FROM AUTHOR]

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 RNAsequencing 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 depositedNMs 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 andROCK2) inhibition inmouse 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. [ABSTRACT FROM AUTHOR]

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-taggedWtip 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 Cterminal 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cell delamination is a conserved morphogenetic process important for the generation of cell diversity and maintenance of tissue homeostasis. Here, we used Drosophila embryonic neuroblasts as a model to study the apical constriction process during cell delamination. We observe dynamic myosin signals both around the cell adherens junctions and underneath the cell apical surface in the neuroectoderm. On the cell apical cortex, the nonjunctional myosin forms flows and pulses, which are termed medial myosin pulses. Quantitative differences in medial myosin pulse intensity and frequency are crucial to distinguish delaminating neuroblasts from their neighbors. Inhibition of medial myosin pulses blocks delamination. The fate of a neuroblast is set apart from that of its neighbors by Notch signaling-mediated lateral inhibition. When we inhibit Notch signaling activity in the embryo, we observe that small clusters of cells undergo apical constriction and display an abnormal apical myosin pattern. Together, these results demonstrate that a contractile actomyosin network across the apical cell surface is organized to drive apical constriction in delaminating neuroblasts. [ABSTRACT FROM AUTHOR]

Early in the development of the central nervous system, progenitor cells undergo a shape change, called apical constriction, that triggers the neural plate to form a tubular structure. How apical constriction in the neural plate is controlled and how it contributes to tissue morphogenesis are not fully understood. In this study, we show that intracellular calcium ions (Ca2+) are required for Xenopus neural tube formation and that there are two types of Ca2+- concentration changes, a single-cell and a multicellular wave-like fluctuation, in the developing neural plate. Quantitative imaging analyses revealed that transient increases in Ca2+ concentration induced cortical F-actin remodeling, apical constriction and accelerations of the closing movement of the neural plate. We also show that extracellular ATP and N-cadherin (cdh2) participate in the Ca2+-induced apical constriction. Furthermore, our mathematical model suggests that the effect of Ca2+ fluctuations on tissue morphogenesis is independent of fluctuation frequency and that fluctuations affecting individual cells are more efficient than those at the multicellular level. We propose that distinct Ca2+ signaling patterns differentially modulate apical constriction for efficient epithelial folding and that this mechanism has a broad range of physiological outcomes. [ABSTRACT FROM AUTHOR]

In lung development, the apically constricted columnar epithelium forms numerous buds during the pseudoglandular stage. Subsequently, these epithelial cells change shape into the flat or cuboidal pneumocytes that form the air sacs during the canalicular and saccular (canalicular-saccular) stages, yet the impact of cell shape on tissue morphogenesis remains unclear. Here, we show that the expression of Wnt components is decreased in the canalicular-saccular stages, and that genetically constitutive activation of Wnt signaling impairs air sac formation by inducing apical constriction in the epithelium as seen in the pseudoglandular stage. Organ culture models also demonstrate that Wnt signaling induces apical constriction through apical actomyosin cytoskeletal organization. Mathematical modeling reveals that apical constriction induces bud formation and that loss of apical constriction is required for the formation of an air sac-like structure. We identify MAP/microtubule affinity-regulating kinase 1 (Mark1) as a downstream molecule of Wnt signaling and show that it is required for apical cytoskeletal organization and bud formation. These results suggest that Wnt signaling is required for bud formation by inducing apical constriction during the pseudoglandular stage, whereas loss of Wnt signaling is necessary for air sac formation in the canalicular-saccular stages. [ABSTRACT FROM AUTHOR]

The Müllerian duct (MD) and Wolffian duct (WD) are embryonic tubular tissues giving rise to female and male reproductive tracts, respectively. In amniote embryos, both MD and WD emerge in both sexes, but subsequently degenerate in the males and females, respectively. Here, by using MD-specific gene manipulations in chicken embryos, we identify the molecular and cellular mechanisms that link early MD specification to tubular invagination. Early (pre-) specification of MD precursors in the coelomic epithelium requires BMP signaling and its downstream target Pax2 in a WD-independent process. Subsequently, the BMP/Pax2 axis induces Lim1 expression, a hallmark of MD specification, for which FGF/ERK and WD-derived signals are also required. Finally, the sequential actions of the BMP/Pax2 and FGF/Lim1 axes culminate in epithelial invagination to form a tubular structure driven by an apical constriction, where apical accumulation of phospho-myosin light chain is positively regulated by FGF/ERK signaling. Our study delineates mechanisms governing the early formation of the MD, and also serves as a model of how an epithelial cell sheet is transformed to a tubular structure, a process seen in a variety of developmental contexts. [ABSTRACT FROM AUTHOR]

Mammalian brain development requires coordination between neural precursor proliferation, differentiation and cellular organization to create the intricate neuronal networks of the adult brain. Here, we examined the role of the atypical cadherins Fat1 and Fat4 in this process. We show that mutation of Fat1 in mouse embryos causes defects in cranial neural tube closure, accompanied by an increase in the proliferation of cortical precursors and altered apical junctions, with perturbations in apical constriction and actin accumulation. Similarly, knockdown of Fat1 in cortical precursors by in utero electroporation leads to overproliferation of radial glial precursors. Fat1 interacts genetically with the related cadherin Fat4 to regulate these processes. Proteomic analysis reveals that Fat1 and Fat4 bind different sets of actin-regulating and junctional proteins. In vitro data suggest that Fat1 and Fat4 form cis-heterodimers, providing a mechanism for bringing together their diverse interactors. We propose a model in which Fat1 and Fat4 binding coordinates distinct pathways at apical junctions to regulate neural progenitor proliferation, neural tube closure and apical constriction. [ABSTRACT FROM AUTHOR]

Apical constriction (AC) is a widely utilized mechanism of cell shape change whereby epithelial cells transform from a cylindrical to conical shape, which can facilitatemorphogeneticmovements during embryonic development. Invertebrate epithelial cells undergoing AC depend on the contraction of apical cortex-spanning actomyosin filaments that generate force on the apical junctions and pull them toward the middle of the cell, effectively reducing the apical circumference. A current challenge is to determine whether these mechanisms are conserved in vertebrates and to identify the molecules responsible for linking apical junctions with the AC machinery. Utilizing the developing mouse eye as a model, we have uncovered evidence that lens placode AC may be partially dependent on apically positioned myosin-containing filaments associated with the zonula adherens. In addition we found that, among several junctional components, p120-catenin genetically interacts with Shroom3, a protein required for AC during embryonic morphogenesis. Further analysis revealed that, similar to Shroom3, p120-catenin is required for AC of lens cells. Finally, we determined that p120-catenin functions by recruiting Shroom3 to adherens junctions. Together, these data identify a novel role for p120-catenin during AC and further define the mechanisms required for vertebrate AC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Apical constriction typically accompanies inward folding of an epithelial sheet. In recent years there has been progress in understanding mechanisms of apical constriction and their contribution to morphogenetic processes. Sea urchin embryos form a specialized region of ectoderm, the ciliary band, which is a strip of epithelium, three to five cells wide, encircling the oral ectoderm and functioning in larval swimming and feeding. Ciliary band cells exhibit distinctive apical-basal elongation, have narrow apices bearing a cilium, and are planar polarized, so that cilia beat away from the mouth. Here, we show that filamentous actin and phosphorylated myosin light chain are uniquely distributed in ciliary band cells. Inhibition of myosin phosphorylation or actin polymerization perturbs this distribution and blocks apical constriction. During ciliary band formation, Sp-Ephrin and Sp-Eph expression overlap in the presumptive ciliary band. Knockdown of Sp-Eph or Sp-Ephrin, or treatment with an Eph kinase inhibitor interferes with actomyosin networks, accumulation of phosphorylated FAK (pY397FAK), and apical constriction. The cytoplasmic domain of Sp-Eph, fused to GST and containing a single amino acid substitution reported as kinase dead, will pull down pY397FAK from embryo lysates. As well, pY397FAK colocalizes with Sp-Eph in a JNK-dependent, planar polarized manner on latitudinal apical junctions of the ciliary band and this polarization is dissociable from apical constriction. We propose that Sp-Eph and pY397FAK function together in an apical complex that is necessary for remodeling actomyosin to produce centripetal forces causing apical constriction. Morphogenesis of ciliary band cells is a unique example of apical constriction in which receptor-mediated cell shape change produces a strip of specialized tissue without an accompanying folding of epithelium. [ABSTRACT FROM AUTHOR]

Cell shape changes drive tissue morphogenesis during animal development. An important example is the apical cell constriction that initiates tissue internalisation. Apical constriction can occur through a phase of cyclic assembly and disassembly of apicomedial actomyosin networks, followed by stabilisation of these networks. Delayed negative-feedback mechanisms typically underlie cyclic behaviour, but the mechanisms regulating cyclic actomyosin networks remain obscure, as do mechanisms that transform overall network behaviour. Here, we show that a known inhibitor of apicomedial actomyosin networks in Drosophila amnioserosa cells, the Par-6- aPKC complex, is recruited to the apicomedial domain by actomyosin networks during dorsal closure of the embryo. This finding establishes an actomyosin-aPKC negative-feedback loop in the system. Additionally, we find that aPKC recruits Bazooka to the apicomedial domain, and phosphorylates Bazooka for a dynamic interaction. Remarkably, stabilising aPKC-Bazooka interactions can inhibit the antagonism of actomyosin by aPKC, suggesting that Bazooka acts as an aPKC inhibitor, and providing a possible mechanism for delaying the actomyosin-aPKC negative-feedback loop. Our data also implicate an increasing degree of Par-6-aPKCBazooka interactions as dorsal closure progresses, potentially explaining a developmental transition in actomyosin behaviour from cyclic to persistent networks. This later impact of aPKC inhibition is supported by mathematical modelling of the system. Overall, this work illustrates how shifting chemical signals can tune actomyosin network behaviour during development. [ABSTRACT FROM AUTHOR]

During development, morphogenetic processes require a precise coordination of cell differentiation, cell shape changes and, often, cell migration. Yet, how pattern information is used to orchestrate these different processes is still unclear. During lateral line (LL) morphogenesis, a group of cells simultaneously migrate and assemble radially organized cell clusters, termed rosettes, that prefigure LL sensory organs. This process is controlled by Fibroblast growth factor (FGF) signalling, which induces cell fate changes, cell migration and cell shape changes. However, the exact molecular mechanisms induced by FGF activation that mediate these changes on a cellular level are not known. Here, we focus on the mechanisms by which FGFs control apical constriction and rosette assembly. We show that apical constriction in the LL primordium requires the activity of non-muscle myosin. We demonstrate further that shroom3, a well-known regulator of non-muscle myosin activity, is expressed in the LL primordium and that its expression requires FGF signalling. Using gain- and loss-of-function experiments, we demonstrate that Shroom3 is the main organizer of cell shape changes during rosette assembly, probably by coordinating Rho kinase recruitment and non-muscle myosin activation. In order to quantify morphogenesis in the LL primordium in an unbiased manner, we developed a unique trainable 'rosette detector'. We thus propose a model in which Shroom3 drives rosette assembly in the LL downstream of FGF in a Rho kinase- and non-muscle myosindependent manner. In conclusion, we uncovered the first mechanistic link between patterning and morphogenesis during LL sensory organ formation. [ABSTRACT FROM AUTHOR]

Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, we are making use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Here, we use live imaging to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, we demonstrate that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, we show that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs rok- and Drakdependent AJ remodelling while also promoting neurogenesis. In conclusion, our work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation. [ABSTRACT FROM AUTHOR]

Many morphogenetic movements during development require the formation of transient intermediates called rosettes. Within rosettes, cells are polarized with apical ends constricted towards the rosette center and nuclei basally displaced. Whereas the polarity and cytoskeletal machinery establishing these structures has been extensively studied, the extracellular cues and intracellular signaling cascades that promote their formation are not well understood. We examined how extracellular Fibroblast growth factor (Fgf) signals regulate rosette formation in the zebrafish posterior lateral line primordium (pLLp), a group of ∼100 cells that migrates along the trunk during embryonic development to form the lateral line mechanosensory system. During migration, the pLLp deposits rosettes from the trailing edge, while cells are polarized and incorporated into nascent rosettes in the leading region. Fgf signaling was previously shown to be crucial for rosette formation in the pLLp. We demonstrate that activation of Fgf receptor (Fgfr) induces intracellular Ras-MAPK, which is required for apical constriction and rosette formation in the pLLp. Inhibiting Fgfr-Ras-MAPK leads to loss of apically localized Rho-associated kinase (Rock) 2a, which results in failed actomyosin cytoskeleton activation. Using mosaic analyses, we show that a cell-autonomous Ras-MAPK signal is required for apical constriction and Rock2a localization. We propose a model whereby activated Fgfr signals through Ras-MAPK to induce apical localization of Rock2a in a cell-autonomous manner, activating the actomyosin network to promote apical constriction and rosette formation in the pLLp. This mechanism presents a novel cellular strategy for driving cell shape changes. [ABSTRACT FROM AUTHOR]

The PDZ-GEF protein Dizzy (Dzy) and its downstream GTPase Rap1 have pleiotropic roles during development of the Drosophila embryo. Here, we show that maternally provided Dzy and Rap1 first function during ventral furrow formation (VFF) where they are critical to guarantee rapid apical cell constrictions. Contraction of the apical actomyosin filament system occurs independently of Dzy and Rap1, but loss of Dzy results in a delayed establishment of the apical adherens junction (AJ) belt, whereas in the absence of Rap1 only a fragmentary apical AJ belt is formed in the epithelium. The timely establishment of apical AJs appears to be essential for coupling actomyosin contractions to cell shape change and to assure completion of the ventral furrow. Immediately after VFF, the downregulation of Dzy and Rap1 is necessary to allow normal mesodermal development to continue after the epithelial-to-mesenchymal transition, as overexpression of Dzy or of constitutively active Rap1 compromises mesodermal migration and monolayer formation. We propose that Dzy and Rap1 are crucial factors regulating the dynamics of AJs during gastrulation. [ABSTRACT FROM AUTHOR]

Epithelial invagination is a common feature of embryogenesis. An example of invagination morphogenesis occurs during development of the early eye when the lens placode forms the lens pit. This morphogenesis is accompanied by a columnar-to-conical cell shape change (apical constriction or AC) and is known to be dependent on the cytoskeletal protein Shroom3. Because Shroom3-induced AC can be Rock1/2 dependent, we hypothesized that during lens invagination, RhoA, Rock and a RhoA guanine nucleotide exchange factor (RhoA-GEF) would also be required. In this study, we show that Rock activity is required for lens pit invagination and that RhoA activity is required for Shroom3-induced AC. We demonstrate that RhoA, when activated and targeted apically, is sufficient to induce AC and that RhoA plays a key role in Shroom3 apical localization. Furthermore, we identify Trio as a RhoA-GEF required for Shroom3-dependent AC in MDCK cells and in the lens pit. Collectively, these data indicate that a Trio-RhoA-Shroom3 pathway is required for AC during lens pit invagination. [ABSTRACT FROM AUTHOR]

Many epithelial tissues undergo extensive remodelling during morphogenesis. How their epithelial features, such as apicobasal polarity or adhesion, are maintained and remodelled and how adhesion and polarity proteins contribute to morphogenesis are two important questions in development. Here, we approach these issues by investigating the role of the apical determinant protein Crumbs (Crb) during the morphogenesis of the embryonic Drosophila tracheal system. Crb accumulates differentially throughout tracheal development and is required for different tracheal events. The earliest requirement for Crb is for tracheal invagination, which is preceded by an enhanced accumulation of Crb in the invagination domain. There, Crb, acting in parallel with the epidermal growth factor receptor (Egfr) pathway, is required for tracheal cell apical constriction and for organising an actomyosin complex, which we propose is mediated by Crb recruitment of moesin (Moe). The ability of a Crb isoform unable to rescue polarity in crb mutants to otherwise rescue their invagination phenotype, and the converse inability of a FERM-binding domain mutant Crb to rescue faulty invagination, support our hypothesis that it is the absence of Crb-dependent Moe enrichment, and not the polarity defect, that mainly underlies the crb invagination phenotype. This hypothesis is supported by the phenotype of lethal giant larvae (lgl); crb double mutants. These results unveil a link between Crb and the organisation of the actin cytoskeleton during morphogenesis. [ABSTRACT FROM AUTHOR]

Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates. [ABSTRACT FROM AUTHOR]

Neural tube formation is one of the most dynamic morphogenetic processes of vertebrate development. However, the molecules regulating its initiation are mostly unknown. Here, we demonstrated that nectin-2, an immunoglobulin-like cell adhesion molecule, is involved in the neurulation of Xenopus embryos in cooperation with N-cadherin. First, we found that, at the beginning of neurulation, nectin-2 was strongly expressed in the superficial cells of neuroepithelium. The knockdown of nectin-2 impaired neural fold formation by attenuating F-actin accumulation and apical constriction, a cell-shape change that is required for neural tube folding. Conversely, the overexpression of nectin-2 in non-neural ectoderm induced ectopic apical constrictions with accumulated F-actin. However, experiments with domain-deleted nectin-2 revealed that the intracellular afadin-binding motif, which links nectin-2 and F-actin, was not required for the generation of the ectopic apical constriction. Furthermore, we found that nectin-2 physically interacts with N-cadherin through extracellular domains, and they cooperatively enhanced apical constriction by driving the accumulation of F-actin at the apical cell surface. Interestingly, the accumulation of N-cadherin at the apical surface of neuroepithelium was dependent on the presence of nectin-2, but that of nectin-2 was not affected by depletion of N-cadherin. We propose a novel mechanism of neural tube morphogenesis regulated by the two types of cell adhesion molecules. [ABSTRACT FROM AUTHOR]

Cell-shape change in epithelial structures is fundamental to animal morphogenesis. Recent studies identified myosin-II as the major generator of driving forces for cell-shape changes during morphogenesis. Lulu (Epb41l5) is a major regulator of morphogenesis, although the downstream molecular and cellular mechanisms remain obscure in mammals. In Drosophila and zebrafish, Lulu proteins were reported to negatively regulate Crumbs, an apical domain regulator, thus regulating morphogenesis. In this study, we show that mammalian Lulu activates myosin-II, thus regulating epithelial cell shape. In our experiments, Lulu expression in epithelial cells resulted in apical constriction and lateral elongation in the cells, accompanied by upregulation of myosin-II. The inhibition of myosin-II activity almost completely blocked this Lulu-driven cell-shape change. We further found that Rock participates in the myosin-II activation. Additionally, RNAi-mediated depletion of Lulu in epithelial cells resulted in disorganization of myosin-II and a concomitant loss of proper lateral domain organization in the cells. From these results, we propose that Lulu regulates epithelial cell shape by controlling myosin-II activity. [ABSTRACT FROM AUTHOR]

Organ morphogenesis requires cooperation between cells, which determine their course of action based upon location within a tissue. Just as important, cells must synchronize their activities, which requires awareness of developmental time. To understand how cells coordinate behaviors in time and space, we analyzed Drosophila egg chamber development. We found that the transcription factor Tramtrack69 (TTK69) controls the fates and shapes of all columnar follicle cells by integrating temporal and spatial information, restricting characteristic changes in morphology and expression that occur at stage 10B to appropriate domains. TTK69 is required again later in oogenesis: it controls the volume of the dorsal-appendage (DA) tubes by promoting apical re-expansion and lateral shortening of DA-forming follicle cells. We show that TTK69 and Notch compete to repress each other's expression and that a local Ecdysone signal is required to shift the balance in favor of TTK69. We hypothesize that TTK69 then cooperates with spatially restricted co-factors to define appropriate responses to a globally available (but as yet unidentified) temporal signal that initiates the S10B transformations. [ABSTRACT FROM AUTHOR]

Tissue morphogenesis requires stereotyped cell shape changes, such as apical cell constriction in the mesoderm and cell intercalation in the ventrolateral ectoderm of Drosophila. Both processes require force generation by an actomyosin network. The subcellular localization of Myosin-II (Myo-II) dictates these different morphogenetic processes. In the intercalating ectoderm Myo-II is mostly cortical, but in the mesoderm Myo-II is concentrated in a medial meshwork. We report that apical constriction is repressed by JAK/STAT signalling in the lateral ectoderm independently of Twist. Inactivation of the JAK/STAT pathway causes germband extension defects because of apical constriction ventrolaterally. This is associated with ectopic recruitment of Myo-II in a medial web, which causes apical cell constriction as shown by laser nanosurgery. Reducing Myo-II levels rescues the JAK/STAT mutant phenotype, whereas overexpression of the Myo-II heavy chain (also known as Zipper), or constitutive activation of its regulatory light chain, does not cause medial accumulation of Myo-II nor apical constriction. Thus, JAK/STAT controls Myo-II localization by additional mechanisms. We show that regulation of actin polymerization by Wasp, but not by Dia, is important in this process. Constitutive activation of Wasp, a branched actin regulator, causes apical cell constriction and promotes medial 'web' formation. Wasp is inactivated at the cell cortex in the germband by JAK/STAT signalling. Lastly, wasp mutants rescue the normal cortical enrichment of Myo-II and inhibit apical constriction in JAK/STAT mutants, indicating that Wasp is an effector of JAK/STAT signalling in the germband. We discuss possible models for the role of Wasp activity in the regulation of Myo-II distribution. [ABSTRACT FROM AUTHOR]