Adherens junctions as molecular regulators of emergent tissue mechanics.

Tissue and organ development during embryogenesis relies on the collective and coordinated action of many cells. Recent studies have revealed that tissue material properties, including transitions between fluid and solid tissue states, are controlled in space and time to shape embryonic structures and regulate cell behaviours. Although the collective cellular flows that sculpt tissues are guided by tissue-level physical changes, these ultimately emerge from cellular-level and subcellular-level molecular mechanisms. Adherens junctions are key subcellular structures, built from clusters of classical cadherin receptors. They mediate physical interactions between cells and connect biochemical signalling to the physical characteristics of cell contacts, hence playing a fundamental role in tissue morphogenesis. In this Review, we take advantage of the results of recent, quantitative measurements of tissue mechanics to relate the molecular and cellular characteristics of adherens junctions, including adhesion strength, tension and dynamics, to the emergent physical state of embryonic tissues. We focus on systems in which cell-cell interactions are the primary contributor to morphogenesis, without significant contribution from cell-matrix interactions. We suggest that emergent tissue mechanics is an important direction for future research, bridging cell biology, developmental biology and mechanobiology to provide a holistic understanding of morphogenesis in health and disease.

The front and rear of collective cell migration.

Collective cell migration has a key role during morphogenesis and during wound healing and tissue renewal in the adult, and it is involved in cancer spreading. In addition to displaying a coordinated migratory behaviour, collectively migrating cells move more efficiently than if they migrated separately, which indicates that a cellular interplay occurs during collective cell migration. In recent years, evidence has accumulated confirming the importance of such intercellular communication and exploring the molecular mechanisms involved. These mechanisms are based both on direct physical interactions, which coordinate the cellular responses, and on the collective cell behaviour that generates an optimal environment for efficient directed migration. The recent studies have described how leader cells at the front of cell groups drive migration and have highlighted the importance of follower cells and cell-cell communication, both between followers and between follower and leader cells, to improve the efficiency of collective movement.

Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.

Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.

A ß-catenin chromobody-based probe highlights endothelial maturation during vascular morphogenesis in vivo.

Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as versatile probes for live cell imaging of endogenous proteins. However, how these chromobodies behave in vivo and how accurately they monitor tissue changes remain poorly explored. Here, we generated an endothelial-specific ß-catenin chromobody-derived probe and analyzed its expression pattern during cardiovascular development in zebrafish. Using high-resolution confocal imaging, we show that the chromobody signal correlates with the localization of ß-catenin in the nucleus and at cell-cell junctions, and thereby can be used to assess endothelial maturation. Loss of Cadherin 5 strongly affects the localization of the chromobody at the cell membrane, confirming the cadherin-based adherens junction role of ß-catenin. Furthermore, using a genetic model to block blood flow, we observed that cell junctions are compromised in most endothelial cells but not in the endocardium, highlighting the heterogeneous response of the endothelium to the lack of blood flow. Overall, our data further expand the use of chromobodies for in vivo applications and illustrate their potential to monitor tissue morphogenesis at high resolution.

A mechanical transition from tension to buckling underlies the jigsaw puzzle shape morphogenesis of histoblasts in the Drosophila epidermis.

The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. We report a striking morphological transition of histoblasts during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and we show that the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. Our model is supported by laser dissections and optical tweezer experiments together with computer simulations. Our analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, we propose a possible mechanism for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology.

Planar cell polarity links axes of spatial dynamics in neural-tube closure.

Neural-tube closure is a critical step of embryogenesis, and its failure causes serious birth defects. Coordination of two morphogenetic processes--convergent extension and neural-plate apical constriction--ensures the complete closure of the neural tube. We now provide evidence that planar cell polarity (PCP) signaling directly links these two processes. In the bending neural plates, we find that a PCP-regulating cadherin, Celsr1, is concentrated in adherens junctions (AJs) oriented toward the mediolateral axes of the plates. At these AJs, Celsr1 cooperates with Dishevelled, DAAM1, and the PDZ-RhoGEF to upregulate Rho kinase, causing their actomyosin-dependent contraction in a planar-polarized manner. This planar-polarized contraction promotes simultaneous apical constriction and midline convergence of neuroepithelial cells. Together our findings demonstrate that PCP signals confer anisotropic contractility on the AJs, producing cellular forces that promote the polarized bending of the neural plate.

The zonula adherens matura redefines the apical junction of intestinal epithelia.

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

The Dilute domain in Canoe is not essential for linking cell junctions to the cytoskeleton but supports morphogenesis robustness.

Robust linkage between adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The Drosophila multidomain protein Canoe and its mammalian homolog afadin are crucial for this, as in their absence many events of morphogenesis fail. To define the mechanism of action for Canoe, we are taking it apart. Canoe has five folded protein domains and a long intrinsically disordered region. The largest is the Dilute domain, which is shared by Canoe and myosin V. To define the roles of this domain in Canoe, we combined biochemical, genetic and cell biological assays. AlphaFold was used to predict its structure, providing similarities and contrasts with Myosin V. Biochemical data suggested one potential shared function - the ability to dimerize. We generated Canoe mutants with the Dilute domain deleted (CnoΔDIL). Surprisingly, they were viable and fertile. CnoΔDIL localized to adherens junctions and was enriched at junctions under tension. However, when its dose was reduced, CnoΔDIL did not provide fully wild-type function. Furthermore, canoeΔDIL mutants had defects in the orchestrated cell rearrangements of eye development. This reveals the robustness of junction-cytoskeletal connections during morphogenesis and highlights the power of natural selection to maintain protein structure.

Neurogenesis redirects ß-catenin from adherens junctions to the nucleus to promote axonal growth.

Here, we show that, in the developing spinal cord, after the early Wnt-mediated Tcf transcription activation that confers dorsal identity to neural stem cells, neurogenesis redirects ß-catenin from the adherens junctions to the nucleus to stimulate Tcf-dependent transcription in a Wnt-independent manner. This new ß-catenin activity regulates genes implicated in several aspects of contralateral axon growth, including axon guidance and adhesion. Using live imaging of ex-vivo chick neural tube, we showed that the nuclear accumulation of ß-catenin and the rise in Tcf-dependent transcription both initiate before the dismantling of the adherens junctions and remain during the axon elongation process. Notably, we demonstrated that ß-catenin activity in post-mitotic cells depends on TCF7L2 and is central to spinal commissural axon growth. Together, our results reveal Wnt-independent Tcf/ß-catenin regulation of genes that control the growth and guidance of commissural axons in chick spinal cord.

Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development.

BMP signaling is crucial to blood vessel formation and function, but how pathway components regulate vascular development is not well-understood. Here, we find that inhibitory SMAD6 functions in endothelial cells to negatively regulate ALK1-mediated responses, and it is required to prevent vessel dysmorphogenesis and hemorrhage in the embryonic liver vasculature. Reduced Alk1 gene dosage rescued embryonic hepatic hemorrhage and microvascular capillarization induced by Smad6 deletion in endothelial cells in vivo. At the cellular level, co-depletion of Smad6 and Alk1 rescued the destabilized junctions and impaired barrier function of endothelial cells depleted for SMAD6 alone. Mechanistically, blockade of actomyosin contractility or increased PI3K signaling rescued endothelial junction defects induced by SMAD6 loss. Thus, SMAD6 normally modulates ALK1 function in endothelial cells to regulate PI3K signaling and contractility, and SMAD6 loss increases signaling through ALK1 that disrupts endothelial cell junctions. ALK1 loss-of-function also disrupts vascular development and function, indicating that balanced ALK1 signaling is crucial for proper vascular development and identifying ALK1 as a 'Goldilocks' pathway in vascular biology that requires a certain signaling amplitude, regulated by SMAD6, to function properly.

Src42A is required for E-cadherin dynamics at cell junctions during Drosophila axis elongation.

Src kinases are important regulators of cell adhesion. Here, we have explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. We find that, during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, we show that Src42A acts in concert with Abl kinase, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time.

Mechanisms of stretch-mediated skin expansion at single-cell resolution.

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.

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.

Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells.

Collective cell migration is the coordinated movement of multiple cells connected by cadherin-based adherens junctions and is essential for physiological and pathological processes. Cadherins undergo dynamic intracellular trafficking, and their surface level is determined by a balance between endocytosis, recycling and degradation. However, the regulatory mechanism of cadherin turnover in collective cell migration remains elusive. In this study, we show that the Bin/amphiphysin/Rvs (BAR) domain protein pacsin 2 (protein kinase C and casein kinase substrate in neurons protein 2) plays an essential role in collective cell migration by regulating N-cadherin (also known as CDH2) endocytosis in human cancer cells. Pacsin 2-depleted cells formed cell-cell contacts enriched with N-cadherin and migrated in a directed manner. Furthermore, pacsin 2-depleted cells showed attenuated internalization of N-cadherin from the cell surface. Interestingly, GST pull-down assays demonstrated that the pacsin 2 SH3 domain binds to the cytoplasmic region of N-cadherin, and expression of an N-cadherin mutant defective in binding to pacsin 2 phenocopied pacsin 2 RNAi cells both in cell contact formation and N-cadherin endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration, highlighting pacsin 2 as a possible therapeutic target for cancer metastasis.

Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis.

During Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs.

Neurotensin modulates ovarian vascular permeability via adherens junctions.

Neurotensin (NTS) is a 13-amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of NTS in the ovulatory follicle of the cynomolgus macaque impairs ovulation and induces follicular vascular dysregulation, with excessive pooling of red blood cells in the follicle antrum. We hypothesize that NTS is an essential intrafollicular regulator of vascular permeability. In the present study, follicle injection of the NTS receptor antagonist SR142948 also resulted in vascular dysregulation. To measure vascular permeability changes in vitro, primary macaque ovarian microvascular endothelial cells (mOMECs) were enriched from follicle aspirates and studied in vitro. When treated with NTS, permeability of mOMECs decreased. RNA sequencing (RNA-Seq) of mOMECs revealed high mRNA expression of the permeability-regulating adherens junction proteins N-cadherin (CDH2) and K-cadherin (CDH6). Immunofluorescent detection of CDH2 and CDH6 confirmed expression and localized these cadherins to the cell-cell boundaries, consistent with function as components of adherens junctions. mOMECs did not express detectable levels of the typical vascular endothelial cadherin, VE-cadherin (CDH5) as determined by RNA-Seq, qPCR, western blot, and immunofluorescence. Knockdown of CDH2 or CDH6 via siRNA abrogated the NTS effect on mOMEC permeability. Collectively, these data suggest that NTS plays an ovulation-critical role in vascular permeability maintenance, and that CDH2 and CDH6 are involved in the permeability modulating effect of NTS on the ovarian microvasculature. NTS can be added to a growing number of angiogenic regulators which are critical for successful ovulation.

Neurogenesis at the brain-cerebrospinal fluid interface.

Cerebral cortical progenitor cells can be classified into several different types, and each progenitor type integrates cell-intrinsic and cell-extrinsic cues to regulate neurogenesis. On one hand, cell-intrinsic mechanisms that depend upon appropriate apical-basal polarity are established by adherens junctions and apical complex proteins and are particularly important in progenitors with apical processes contacting the lateral ventricle. The apical protein complexes themselves are concentrated at the ventricular surface, and apical complex proteins regulate mitotic spindle orientation and cell fate. On the other hand, remarkably little is known about how cell-extrinsic cues signal to progenitors and couple with cell-intrinsic mechanisms to instruct neurogenesis. Recent research shows that the cerebrospinal fluid, which contacts apical progenitors at the ventricular surface and bathes the apical complex of these cells, provides growth- and survival-promoting cues for neural progenitor cells in developing and adult brain. This review addresses how the apical-basal polarity of progenitor cells regulates cell fate and allows progenitors to sample diffusible signals distributed by the cerebrospinal fluid. We also review several classes of signaling factors that the cerebrospinal fluid distributes to the developing brain to instruct neurogenesis.

Juvenile hormone promotes paracellular transport of yolk proteins via remodeling zonula adherens at tricellular junctions in the follicular epithelium.

Juvenile hormone (JH) acts as a gonadotrophic hormone stimulating insect vitellogenesis and oogenesis. Paracellular transport of yolk proteins through intercellular channels (patency) in the follicular epithelium is a developmentally regulated and evolutionarily conserved process during vitellogenesis. However, the mechanisms underlying patency opening are poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH-regulated remodeling of zonula adherens (ZA), the belt-like adherens junction maintaining physical linking between follicle cells controlled the opening of patency. JH triggered phosphorylation of Partitioning defective protein 3 (Par3) via a signaling cascade including G protein-coupled receptor (GPCR), small GTPase Cell division cycle 42 (Cdc42) and atypical Protein kinase C (aPKC). Par3 phosphorylation resulted in its disassociation from ß-Catenin, the cytoplasmic partner of ZA core component E-Cadherin. Release of Par3 from the ß-Catenin/E-Cadherin complex caused ZA disassembly at tricellular contacts, consequently leading to patency enlargement. This study provides new insight into how JH stimulates insect vitellogenesis and egg production via inducing the opening of paracellular route for vitellogenin transport crossing the follicular epithelium barrier.

Myosin V facilitates polarised E-cadherin secretion.

E-cadherin has a fundamental role in epithelial tissues by providing cell-cell adhesion. Polarised E-cadherin exocytosis to the lateral plasma membrane is central for cell polarity and epithelial homeostasis. Loss of E-cadherin secretion compromises tissue integrity and is a prerequisite for metastasis. Despite this pivotal role of E-cadherin secretion, the transport mechanism is still unknown. Here we identify Myosin V as the motor for E-cadherin secretion. Our data reveal that Myosin V and F-actin are required for the formation of a continuous apicolateral E-cadherin belt, the zonula adherens. We show by live imaging how Myosin V transports E-cadherin vesicles to the plasma membrane, and distinguish two distinct transport tracks: an apical actin network leading to the zonula adherens and parallel actin bundles leading to the basal-most region of the lateral membrane. E-cadherin secretion starts in endosomes, where Rab11 and Sec15 recruit Myosin V for transport to the zonula adherens. We also shed light on the endosomal sorting of E-cadherin by showing how Rab7 and Snx16 cooperate in moving E-cadherin into the Rab11 compartment. Thus, our data help to understand how polarised E-cadherin secretion maintains epithelial architecture and prevents metastasis.

Rap1 regulates lumen continuity via Afadin in renal epithelia.

The continuity of a lumen within an epithelial tubule is critical for its function. We previously found that the F-actin binding protein Afadin is required for timely lumen formation and continuity in renal tubules formed from the nephrogenic mesenchyme in mice. Afadin is a known effector and interactor of the small GTPase Rap1, and in the current study, we examine the role of Rap1 in nephron tubulogenesis. Here, we demonstrate that Rap1 is required for nascent lumen formation and continuity in cultured 3D epithelial spheroids and in vivo in murine renal epithelial tubules derived from the nephrogenic mesenchyme, where its absence ultimately leads to severe morphogenetic defects in the tubules. By contrast, Rap1 is not required for lumen continuity or morphogenesis in renal tubules derived from the ureteric epithelium, which differ in that they form by extension from a pre-existing tubule. We further demonstrate that Rap1 is required for correct localization of Afadin to adherens junctions both in vitro and in vivo. Together, these results suggest a model in which Rap1 localizes Afadin to junctional complexes, which in turn regulates nascent lumen formation and positioning to ensure continuous tubulogenesis.