Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling

Remodeling of epithelial sheets plays important roles in animal morphogenesis. Shroom3 is known to regulate the apical constriction of epithelial cells. Here, we show that Shroom3 binds ROCKs and recruits them to the epithelial apical junctions. We identified the Shroom3-binding site (RII-C1) on ROCKs, and found that RII-C1 could antagonize the Shroom3-ROCK interaction, interfering with the action of Shroom3 on cell morphology. In the invaginating neural plate/tube, Shroom3 colocalized with ROCKs at the apical junctions; Shroom3 depletion or RII-C1 expression in the tube removed these apically localized ROCKs, and concomitantly blocked neural tube closure. Closing neural plate exhibited peculiar cell assemblies, including rosette formation, as well as a planar-polarized distribution of phosphorylated myosin regulatory light chain, but these were abolished by ROCK inhibition or RII-C1 expression. These results demonstrate that the Shroom3-ROCK interaction is crucial for the regulation of epithelial and neuroepithelial cell arrangement and remodeling.     

aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development

Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. We show that atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, we find a balance of microtubule and actin interactions controls AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost.     

Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse

We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.     

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.     

Mechanical roles of apical constriction,cell elongation,and cell migration during neural tube formation in Xenopus

Neural tube closure is an important and necessary process during the development of the central nervous system. The formation of the neural tube structure from a flat sheet of neural epithelium requires several cell morphogenetic events and tissue dynamics to account for the mechanics of tissue deformation. Cell elongation changes cuboidal cells into columnar cells, and apical constriction then causes them to adopt apically narrow, wedge-like shapes. In addition, the neural plate in Xenopus is stratified, and the non-neural cells in the deep layer (deep cells) pull the overlying superficial cells, eventually bringing the two layers of cells to the midline. Thus, neural tube closure appears to be a complex event in which these three physical events are considered to play key mechanical roles. To test whether these three physical events are mechanically sufficient to drive neural tube formation, we employed a three-dimensional vertex model and used it to simulate the process of neural tube closure. The results suggest that apical constriction cued the bending of the neural plate by pursing the circumference of the apical surface of the neural cells. Neural cell elongation in concert with apical constriction further narrowed the apical surface of the cells and drove the rapid folding of the neural plate, but was insufficient for complete neural tube closure. Migration of the deep cells provided the additional tissue deformation necessary for closure. To validate the model, apical constriction and cell elongation were inhibited in Xenopus laevis embryos. The resulting cell and tissue shapes resembled the corresponding simulation results.

     

Seven-Pass Transmembrane Cadherins: Roles and Emerging Mechanisms in Axonal and Dendritic Patterning

The Flamingo/Celsr seven-transmembrane cadherins represent a conserved subgroup of the cadherin superfamily involved in multiple aspects of development. In the developing nervous system, Fmi/Celsr control axonal blueprint and dendritic morphogenesis from invertebrates to mammals. As expected from their molecular structure, seven-transmembrane cadherins can induce cell–cell homophilic interactions but also intracellular signaling. Fmi/Celsr is known to regulate planar cell polarity (PCP) through interactions with PCP proteins. In the nervous system, Fmi/Celsr can function in collaboration with or independently of other PCP genes. Here, we focus on recent studies which show that seven-transmembrane cadherins use distinct molecular mechanisms to achieve diverse functions in the development of the nervous system.     

Mitotic internalization of planar cell polarity proteins preserves tissue polarity

Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. Here, we show that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division.     

MAGI1 recruits Dll1 to cadherin-based adherens junctions and stabilizes it on the cell surface

Delta-Notch signaling plays an essential role in cell fate determination in many tissue types, including the central nervous system. Although the signaling mechanism of Notch has been extensively studied, the behaviors of its ligands are not well understood. In the present study, we found that, in the developing neural tube, Dll1(Delta-like 1) was mainly localized on the processes extending from nascent neurons toward both the pia and the ventricle and accumulated at apical termini, where adherens junctions (AJs) were formed. To understand the mechanism of Dll1 localization, we searched for binding proteins for Dll1 and identified a scaffolding molecule, MAGI1. In the developing spinal cord, MAGI1 mRNA was highly expressed in the ventricular zone, where Dll1 mRNA was expressed. MAGI1 protein accumulated at the AJs formed around the termini of apically extending processes and was partially colocalized with Dll1. MAGI1 bound not only to Dll1 but also to N-cadherin-beta-catenin complexes. In cultured AJ-forming fibroblasts, MAGI1 was localized at AJs, and Dll1 was recruited to these AJs through binding to MAGI1. In addition, Dll1 was stabilized on the cell surface by MAGI1. Taken together, these results suggest that Dll1 is presented on the surface of AJs formed at the apical termini of processes through interaction with MAGI1 to activate Notch on neighboring cells in the developing central nervous system.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.     

Genetic interactions between planar cell polarity genes cause diverse neural tube defects in mice

Neural tube defects (NTDs) are among the commonest and most severe forms of developmental defect, characterized by disruption of the early embryonic events of central nervous system formation. NTDs have long been known to exhibit a strong genetic dependence, yet the identity of the genetic determinants remains largely undiscovered. Initiation of neural tube closure is disrupted in mice homozygous for mutations in planar cell polarity (PCP) pathway genes, providing a strong link between NTDs and PCP signaling. Recently, missense gene variants have been identified in PCP genes in humans with NTDs, although the range of phenotypes is greater than in the mouse mutants. In addition, the sequence variants detected in affected humans are heterozygous, and can often be detected in unaffected individuals. It has been suggested that interactions between multiple heterozygous gene mutations cause the NTDs in humans. To determine the phenotypes produced in double heterozygotes, we bred mice with all three pairwise combinations of Vangl2^Lp, Scrib^Crc and Celsr1^Crsh mutations, the most intensively studied PCP mutants. The majority of double-mutant embryos had open NTDs, with the range of phenotypes including anencephaly and spina bifida, therefore reflecting the defects observed in humans. Strikingly, even on a uniform genetic background, variability in the penetrance and severity of the mutant phenotypes was observed between the different double-heterozygote combinations. Phenotypically, Celsr1^Crsh;Vangl2^Lp;Scrib^Crc triply heterozygous mutants were no more severe than doubly heterozygous or singly homozygous mutants. We propose that some of the variation between double-mutant phenotypes could be attributed to the nature of the protein disruption in each allele: whereas Scrib^Crc is a null mutant and produces no Scrib protein, Celsr1^Crsh and Vangl2^Lp homozygotes both express mutant proteins, consistent with dominant effects. The variable outcomes of these genetic interactions are of direct relevance to human patients and emphasize the importance of performing comprehensive genetic screens in humans.KEY WORDS: Neural tube defects, Planar cell polarity, Genetic interactions, Craniorachischisis, Multiple heterozygosity     

Neural tube closure requires Dishevelled-dependent convergent extension of the midline

In Xenopus, Dishevelled (Xdsh) signaling is required for both neural tube closure and neural convergent extension, but the connection between these two morphogenetic processes remains unclear. Indeed normal neurulation requires several different cell polarity decisions, any of which may require Xdsh signaling. In this paper we address two issues: (1) which aspects of normal neurulation require Xdsh function; and (2) what role convergent extension plays in the closure of the neural tube. We show that Xdsh signaling is not required for neural fold elevation, medial movement or fusion. Disruption of Xdsh signaling therefore provides a specific tool for uncoupling convergent extension from other processes of neurulation. Using disruption of Xdsh signaling, we demonstrate that convergent extension is crucial to tube closure. Targeted injection revealed that Xdsh function was required specifically in the midline for normal neural tube closure. We suggest that the inherent movement of the neural folds can accomplish only a finite amount of medial progress and that convergent extension of the midline is necessary to reduce the distance between the nascent neural folds, allowing them to meet and fuse. Similar results with Xenopus strabismus implicate the planar cell polarity (PCP) signaling cascade in neural convergent extension and tube closure. Together, these data demonstrate that PCP-mediated convergent extension movements are crucial to proper vertebrate neurulation.     

Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension

Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar-polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin pattern influence the multiple cell behaviors, including cell intercalation, cell shape changes, and apical cell area fluctuations, that simultaneously occur during morphogenesis. Here, we developed two optogenetic tools, optoGEF and optoGAP, to activate or deactivate Rho1 signaling, respectively. We used these tools to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation and analyzed the effects on contractile cell behaviors. We show that uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar-polarized pattern of myosin at cell junctions on the timescale of 3–5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation but have distinct effects on cell area fluctuations and cell packings that are linked with changes in the medial and junctional myosin pools. These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar-polarized myosin pattern in the germband during axis elongation. Moreover, our results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles not only in organizing the cell rearrangements that are known to directly contribute to axis elongation but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.     

Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles

Mammalian body hairs align along the anterior-posterior (A-P) axis and offer a striking but poorly understood example of global cell polarization, a phenomenon known as planar cell polarity (PCP). We have discovered that during embryogenesis, marked changes in cell shape and cytoskeletal polarization occur as nascent hair follicles become anteriorly angled, morphologically polarized and molecularly compartmentalized along the A-P axis. Hair follicle initiation coincides with asymmetric redistribution of Vangl2, Celsr1 and Fzd6 within the embryonic epidermal basal layer. Moreover, loss-of-function mutations in Vangl2 and Celsr1 show that they have an essential role in hair follicle polarization and orientation, which develop in part through non-autonomous mechanisms. Vangl2 and Celsr1 are both required for their planar localization in vivo, and physically associate in a complex in vitro. Finally, we provide in vitro evidence that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).     

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.     

The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear

Background

The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins.

Results

We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2 ^Lp and Celsr1 ^Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants.

Conclusions

The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7.     

Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with β- and α-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-ΔPR1–2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.     

Distinct functions for Rho1 in maintaining adherens junctions and apical tension in remodeling epithelia

Maintenance and remodeling of adherens junctions (AJs) and cell shape in epithelia are necessary for the development of functional epithelia and are commonly altered during cancer progression/metastasis. Although formation of nascent AJs has received much attention, whether shared mechanisms are responsible for the maintenance and remodeling of AJs in dynamic epithelia, particularly in vivo, is not clear. Using clonal analysis in the postmitotic Drosophila melanogaster pupal eye epithelium, we demonstrate that Rho1 is required to maintain AJ integrity independent of its role in sustaining apical cell tension. Rho1 depletion in a remodeling postmitotic epithelium disrupts AJs but only when depleted in adjacent cells. Surprisingly, neither of the Rho effectors, Rok or Dia, is necessary downstream of Rho1 to maintain AJs; instead, Rho1 maintains AJs by inhibiting Drosophila epithelial cadherin endocytosis in a Cdc42/Par6-dependent manner. In contrast, depletion of Rho1 in single cells decreases apical tension, and Rok and myosin are necessary, while Dia function also contributes, downstream of Rho1 to sustain apical cell tension.     

Multifaceted role of Rho, Rac, Cdc42 and Ras in intercellular junctions, lessons from toxins

Tight junctions (TJs) and adherens junctions (AJs) are dynamic structures linked to the actin cytoskeleton, which control the paracellular permeability of epithelial and endothelial barriers. TJs and AJs are strictly regulated in a spatio-temporal manner by a complex signaling network, including Rho/Ras-GTPases, which have a pivotal role. Rho preferentially regulates TJs by controlling the contraction of apical acto-myosin filaments, whereas Rac/Cdc42 mainly coordinate the assembly-disassembly of AJ components. However, a subtle balance of Rho/Ras-GTPase activity and interplay between these molecules is required to maintain an optimal organization and function of TJs and AJs. Conversely, integrity of intercellular junctions generates signals through Rho-GTPases, which are involved in the regulation of multiple cellular processes. Rho/Ras-GTPases and the control of intercellular junctions are the target of various bacterial toxins responsible for severe diseases in man and animals, and are part of their mechanism of action. This review focuses on the regulation of TJs and AJs by Rho/Ras-GTPases through molecular approaches and bacterial toxins.