Junctionally restricted RhoA activity is necessary for apical constriction during phase 2 inner ear placode invagination

After induction, the inner ear is transformed from a superficially located otic placode into an epithelial vesicle embedded in the mesenchyme of the head. Invagination of this epithelium is biphasic: phase 1 involves the expansion of the basal aspect of the otic cells, and phase 2, the constriction of their apices. Apical constriction is important not only for otic invagination, but also the invagination of many other epithelia; however, its molecular basis is still poorly understood. Here we show that phase 2 otic morphogenesis, like phase 1 morphogenesis, results from the activation of myosin-II. However unlike the actin depolymerising activity observed basally, active myosin-II results in actomyosin contractility. Myosin-II activation is triggered by the accumulation of the planar cell polarity (PCP) core protein, Celsr1 in apical junctions (AJ). Apically polarized Celsr1 orients and recruits the Rho Guanine exchange factor (GEF) ArhGEF11 to apical junctions, thus restricting RhoA activity to the junctional membrane where it activates the Rho kinase ROCK. We suggest that myosin-II and RhoA activation results in actomyosin dependent constriction in an apically polarised manner driving otic epithelium invagination.     

SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of beta(H)-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 alpha-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of alpha-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.     

folded gastrulation, cell shape change and the control of myosin localization

The global cell movements that shape an embryo are driven by intricate changes to the cytoarchitecture of individual cells. In a developing embryo, these changes are controlled by patterning genes that confer cell identity. However, little is known about how patterning genes influence cytoarchitecture to drive changes in cell shape. In this paper, we analyze the function of the folded gastrulation gene (fog), a known target of the patterning gene twist. Our analysis of fog function therefore illuminates a molecular pathway spanning all the way from patterning gene to physical change in cell shape. We show that secretion of Fog protein is apically polarized, making this the earliest polarized component of a pathway that ultimately drives myosin to the apical side of the cell. We demonstrate that fog is both necessary and sufficient to drive apical myosin localization through a mechanism involving activation of myosin contractility with actin. We determine that this contractility driven form of localization involves RhoGEF2 and the downstream effector Rho kinase. This distinguishes apical myosin localization from basal myosin localization, which we find not to require actinomyosin contractility or FOG/RhoGEF2/Rho-kinase signaling. Furthermore, we demonstrate that once localized apically, myosin continues to contract. The force generated by continued myosin contraction is translated into a flattening and constriction of the cell surface through a tethering of the actinomyosin cytoskeleton to the apical adherens junctions. Our analysis of fog function therefore provides a direct link from patterning to cell shape change.     

Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase

Coordinated cell movements shape simple epithelia into functional tissues and organs during embryogenesis. Regulators and effectors of the small GTPase Rho have been shown to be essential for epithelial morphogenesis in cell culture; however, the mechanism by which Rho GTPase and its downstream effectors control coordinated movement of epithelia in a developing tissue or organ is largely unknown. Here, we show that Rho1 GTPase activity is required for the invagination of Drosophila embryonic salivary gland epithelia and for directed migration of the internalized gland. We demonstrate that the absence of zygotic function of Rho1 results in the selective loss of the apical proteins, Crumbs (Crb), Drosophila atypical PKC and Stardust during gland invagination and that this is partially due to reduced crb RNA levels and apical localization. In parallel to regulation of crb RNA and protein, Rho1 activity also signals through Rho-kinase (Rok) to induce apical constriction and cell shape change during invagination. After invagination, Rho-Rok signaling is required again for the coordinated contraction and dorsal migration of the proximal half of the gland. We also show that Rho1 activity is required for proper development of the circular visceral mesoderm upon which the gland migrates. Our genetic and live-imaging analyses provide novel evidence that the proximal gland cells play an essential and active role in salivary gland migration that propels the entire gland to turn and migrate posteriorly.     

Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis

Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis.     

LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

LKB1 is a Ser/Thr kinase that plays an important role in controlling both energy metabolism and cell polarity in metazoan organisms. LKB1 is also a tumor suppressor, and homozygous, inactivating mutations are found in a wide range of human cancers. In lung cancer, inactivating mutations are found in 10 to 50% of cases, but the consequences of functional loss in this context are poorly understood. We report here that LKB1 is required for the maturation of apical junctions in the human bronchial epithelial cell line 16HBE14o- (16HBE). This activity is dependent on an interaction with the Rho guanine nucleotide exchange factor p114RhoGEF but is independent of LKB1 kinase activity. Together, LKB1 and p114RhoGEF control RhoA activity in these cells to promote apical junction assembly.     

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help us understand animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. Here, we report the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. We find that Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, our data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction.     

DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo

Morphogenesis of the Drosophila melanogaster embryo is associated with a dynamic reorganization of the actin cytoskeleton that is mediated by small GTPases of the Rho family. Often, Rho1 controls different aspects of cytoskeletal function in parallel, requiring a complex level of regulation. We show that the guanine triphosphate (GTP) exchange factor DRhoGEF2 is apically localized in epithelial cells throughout embryogenesis. We demonstrate that DRhoGEF2, which has previously been shown to regulate cell shape changes during gastrulation, recruits Rho1 to actin rings and regulates actin distribution and actomyosin contractility during nuclear divisions, pole cell formation, and cellularization of syncytial blastoderm embryos. We propose that DRhoGEF2 activity coordinates contractile actomyosin forces throughout morphogenesis in Drosophila by regulating the association of myosin with actin to form contractile cables. Our results support the hypothesis that specific aspects of Rho1 function are regulated by specific GTP exchange factors.     

RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane.     

Rho signaling pathway and apical constriction in the early lens placode

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368-379, 2011.     

The role of the actomyosin cytoskeleton in coordination of tissue growth during Drosophila oogenesis

The Drosophila egg chamber is an organ composed of a somatic epithelium that covers a germline cyst. After egg-chamber formation, the germline cells grow rapidly without dividing while the surface of the epithelium expands by cell proliferation [1, 2]. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. Here we identify a role for the actomyosin cytoskeleton in this process. We show that myosin activity is restricted to the epithelium's apical surface, which is facing the growing cyst. We demonstrate that the epithelium collapses in the absence of myosin activity and show that the force that deforms the epithelium originates from the growing cyst. Thus, myosin activity maintains epithelial shape by balancing the force emanating from cyst growth. Further, our data indicate that cyst growth induces cell division in the epithelium. In addition, we show how apical restriction of myosin activity is controlled. Myosin is activated at the apical cortex by localized Rho kinase and inhibited at the basolateral cortex by PP1beta9C. In addition, our data indicate that active myosin is apically anchored by the Baz/Par-6/aPKC complex.     

The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.     

Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.     

Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.     

A Trio-RhoA-Shroom3 pathway is required for apical constriction and epithelial invagination

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.     

Monoclonal antibodies as probes of epithelial membrane polarization

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen.     

Lamellipodia-based migrations of larval epithelial cells are required for normal closure of the adult epidermis of Drosophila

Cell migrations are an important feature of animal development. They are, furthermore, essential to wound healing and tumour progression. Despite recent progress, it is still mysterious how cell migration is spatially and temporally regulated during morphogenesis and how cell migration is coordinated with other cellular behaviours to shape tissues and organs. The formation of the abdominal epithelium of Drosophila during metamorphosis provides an attractive system to study morphogenesis. Here, the diploid adult histoblasts replace the polyploid larval epithelial cells (LECs). Using in vivo 4D microscopy, I show that, besides apical constriction and apoptosis, the LECs undergo extensive coordinated migrations. The migrations follow a transition from a stationary (epithelial) to a migratory mode. The migratory behaviour is stimulated by autocrine Dpp signalling. Directed apical lamellipodia-like protrusions propel the cells. Initially, planar cell polarity determines the orientation of LEC migration. While LECs are migrating they also constrict apically, and changes in activity of the small GTPase Rho1 can favour one behaviour over the other. This study shows that the LECs play a more active role in morphogenesis than previously thought, with their migrations contributing to abdominal closure. It furthermore provides insights into how the migratory behaviour of cells is regulated during morphogenesis.