Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis

Regulation of epithelial cell shape, for example, changes in relative sizes of apical, basal, and lateral membranes, is a key mechanism driving morphogenesis. However, it is unclear how epithelial cells control the size of their membranes. In the epithelium of the Drosophila melanogaster ovary, cuboidal precursor cells transform into a squamous epithelium through a process that involves lateral membrane shortening coupled to apical membrane extension. In this paper, we report a mutation in the gene Tao, which resulted in the loss of this cuboidal to squamous transition. We show that the inability of Tao mutant cells to shorten their membranes was caused by the accumulation of the cell adhesion molecule Fasciclin 2, the Drosophila N-CAM (neural cell adhesion molecule) homologue. Fasciclin 2 accumulation at the lateral membrane of Tao mutant cells prevented membrane shrinking and thereby inhibited morphogenesis. In wild-type cells, Tao initiated morphogenesis by promoting Fasciclin 2 endocytosis at the lateral membrane. Thus, we identify here a mechanism controlling the morphogenesis of a squamous epithelium.     

Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis

Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA–Rho kinase–myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear–centrosomal orientation and lumen initiation during 3D epithelial morphogenesis.     

Multifaceted control of E-cadherin dynamics by Adaptor Protein Complex 1 during epithelial morphogenesis

Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargoes. We examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. We found that AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell–extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. We discovered a distinct pool of AP-1 that localizes at the subapical adherens junctions. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. We then provide evidence that E-cadherin hyperinternalization acts upstream of cell death in a potential tumor-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell–cell adhesion.     

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.     

EGFR controls IQGAP basolateral membrane localization and mitotic spindle orientation during epithelial morphogenesis

Establishing the correct orientation of the mitotic spindle is an essential step in epithelial cell division in order to ensure that epithelial tubules form correctly during organ development and regeneration. While recent findings have identified some of the molecular mechanisms that underlie spindle orientation, many aspects of this process remain poorly understood. Here, we have used the 3D‐MDCK model system to demonstrate a key role for a newly identified protein complex formed by IQGAP1 and the epithelial growth factor receptor (EGFR) in controlling the orientation of the mitotic spindle. IQGAP1 is a scaffolding protein that regulates many cellular pathways, from cell‐cell adhesion to microtubule organization, and its localization in the basolateral membrane ensures correct spindle orientation. Through its IQ motifs, IQGAP1 binds to EGFR, which is responsible for maintaining IQGAP1 in the basolateral membrane domain. Silencing IQGAP1, or disrupting the basolateral localization of either IQGAP1 or EGFR, results in a non‐polarized distribution of NuMA, mitotic spindle misorientation and defects in single lumen formation.     

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.     

The Cdc42 GEF Intersectin 2 controls mitotic spindle orientation to form the lumen during epithelial morphogenesis

Epithelial organs are made of tubes and cavities lined by a monolayer of polarized cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. The Rho guanosine triphosphatase (GTPase) Cdc42, which is a master regulator of cell polarity, regulates the formation of the central lumen in epithelial morphogenesis. However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the three-dimensional Madin–Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localizes to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects.     

Active Rab11 and functional recycling endosome are required for E-cadherin trafficking and lumen formation during epithelial morphogenesis

The correct targeting and trafficking of the adherens junction protein epithelial cadherin (E-cadherin) is a major determinant for the acquisition of epithelial cell polarity and for the maintenance of epithelial integrity. The compartments and trafficking components required to sort and transport E-cadherin to the basolateral cell surface remain to be fully defined. On the basis of previous data, we know that E-cadherin is trafficked via the recycling endosome (RE) in nonpolarized and newly polarized cells. Here we explore the role of the RE throughout epithelial morphogenesis in MDCK monolayers and cysts. Time-lapse microscopy in live cells, altering RE function biochemically, and expressing a dominant-negative form of Rab11 (DN-Rab11), each showed that the RE is always requisite for E-cadherin sorting and trafficking. The RE remained important for E-cadherin trafficking in MDCK cells from a nonpolarized state through to fully formed, polarized epithelial monolayers. During the development of epithelial cysts, DN-Rab11 disrupted E-cadherin targeting and trafficking, the subapical localization of pERM and actin, and cyst lumen formation. This final effect demonstrated an early and critical interdependence of Rab11 and the RE for E-cadherin targeting, apical membrane formation, and cell polarity in cysts.     

Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis

Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin–Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.     

Targeting of the RhoGEF Ect2 to the equatorial membrane controls cleavage furrow formation during cytokinesis

In animal cells, formation of the cytokinetic furrow requires activation of the GTPase RhoA by the guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cortex during anaphase is not understood. Here, we show that Ect2 concentrates at the equatorial membrane during cytokinesis in live cells. Ect2 membrane association requires a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids. Both guanine nucleotide exchange function and membrane targeting of Ect2 are essential for RhoA activation and cleavage furrow formation in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2's spindle midzone anchor complex, and is temporally coordinated with chromosome segregation through the activation state of CDK1. We propose that targeting of Ect2 to the equatorial membrane represents a key step in the delivery of the cytokinetic signal to the cortex.     

PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42

Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation.     

Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis

Many organs consist of tubes of epithelial cells enclosing a central lumen. How the space of this lumen is generated is a key question in morphogenesis. Two predominant mechanisms of de novo lumen formation have been observed: hollowing and cavitation. In hollowing, the lumen is formed by exocytosis and membrane separation, whereas, in cavitation, the lumen is generated by apoptosis of cells in the middle of the structure [1, 2]. Using MDCK cells in three-dimensional cultures, we found an inverse correlation between polarization efficiency and apoptosis. When cells were grown in collagen, where cells polarized slowly, apoptosis was needed for lumen formation. However, in the presence of Matrigel, which allowed rapid polarization, lumens formed without apoptosis. If polarization in Matrigel was perturbed by blocking formation of the apical surface by RNAi of Cdc42, lumens formed by apoptosis. In a complementary approach, we plated cells at high density so that aggregates formed with little polarity. These aggregates required apoptosis to form lumens, whereas cells plated at low density formed cysts with rapidly polarizing cells and did not need apoptosis to form lumens. The mechanism of lumen formation in the 3D-MDCK model can shift between hollowing and cavitation, depending on cell polarization.     

Regulation of cell polarity during epithelial morphogenesis

Epithelial cells have an apical surface facing a lumen or outside of the organism, and a basolateral surface facing other cells and extracellular matrix. The identity of the apical surface is determined by phosphatidylinositol 4,5-bisphosphate, while phosphatidylinositol 3,4,5-trisphophosphate determines the identity of the basolateral surface. The Par3/Par6/atypical protein kinase C complex, as well as the Crumbs and Scribble complexes, controls epithelial polarity. Par4 and AMP kinase regulate polarity during conditions of energy depletion. Lumens are formed in hollow cysts and tubules by fusions of apical vesicles, such as the vacuolar apical compartment, with the plasma membrane. The polarity of individual cells is oriented and coordinated with other cells by interactions with the extracellular matrix.     

Muscle contraction controls skeletal morphogenesis through regulation of chondrocyte convergent extension

Convergent extension driven by mediolateral intercalation of chondrocytes is a key process that contributes to skeletal growth and morphogenesis. While progress has been made in deciphering the molecular mechanism that underlies this process, the involvement of mechanical load exerted by muscle contraction in its regulation has not been studied. Using the zebrafish as a model system, we found abnormal pharyngeal cartilage morphology in both chemically and genetically paralyzed embryos, demonstrating the importance of muscle contraction for zebrafish skeletal development. The shortening of skeletal elements was accompanied by prominent changes in cell morphology and organization. While in control the cells were elongated, chondrocytes in paralyzed zebrafish were smaller and exhibited a more rounded shape, confirmed by a reduction in their length-to-width ratio. The typical columnar organization of cells was affected too, as chondrocytes in various skeletal elements exhibited abnormal stacking patterns, indicating aberrant intercalation. Finally, we demonstrate impaired chondrocyte intercalation in growth plates of muscle-less Sp(d) mouse embryos, implying the evolutionary conservation of muscle force regulation of this essential morphogenetic process.Our findings provide a new perspective on the regulatory interaction between muscle contraction and skeletal morphogenesis by uncovering the role of muscle-induced mechanical loads in regulating chondrocyte intercalation in two different vertebrate models.     

A conserved Pbx-Wnt-p63-Irf6 regulatory module controls face morphogenesis by promoting epithelial apoptosis

Morphogenesis of mammalian facial processes requires coordination of cellular proliferation, migration, and apoptosis to develop intricate features. Cleft lip and/or palate (CL/P), the most frequent human craniofacial birth defect, can be caused by perturbation of any of these programs. Mutations of?WNT, P63, and IRF6 yield CL/P in humans and mice; however, how these genes are regulated remains elusive. We generated mouse lines lacking Pbx genes in cephalic ectoderm and demonstrated that they exhibit fully penetrant CL/P and perturbed Wnt signaling. We also characterized a midfacial regulatory element that Pbx proteins bind to control the expression of Wnt9b-Wnt3, which in turn regulates p63. Altogether, we establish a Pbx-dependent Wnt-p63-Irf6 regulatory module in midfacial ectoderm that is conserved within mammals. Dysregulation of this network leads to localized suppression of midfacial apoptosis and CL/P. Ectopic Wnt ectodermal expression in Pbx mutants rescues the clefting, opening avenues for tissue repair.