A mechanical wave travels along a genetic guide to drive the formation of an epithelial furrow during Drosophila gastrulation.

Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.

Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation.

Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much is now understood of the molecular mechanism responsible for apical constriction and of the tissue-scale integration of the ensuing in-plane deformations, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, we use the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging leading to tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, we now challenge this idea and show that embryo-scale force balance at the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

Composite morphogenesis during embryo development.

Morphogenesis drives the formation of functional living shapes. Gene expression patterns and signaling pathways define the body plans of the animal and control the morphogenetic processes shaping the embryonic tissues. During embryogenesis, a tissue can undergo composite morphogenesis resulting from multiple concomitant shape changes. While previous studies have unraveled the mechanisms that drive simple morphogenetic processes, how a tissue can undergo multiple and simultaneous changes in shape is still not known and not much explored. In this chapter, we focus on the process of concomitant tissue folding and extension that is vital for the animal since it is key for embryo gastrulation and neurulation. Recent pioneering studies focus on this problem highlighting the roles of different spatially coordinated cell mechanisms or of the synergy between different patterns of gene expression to drive composite morphogenesis.

A two-tier junctional mechanism drives simultaneous tissue folding and extension.

During embryo development, tissues often undergo multiple concomitant changes in shape. It is unclear which signaling pathways and cellular mechanisms are responsible for multiple simultaneous tissue shape transformations. We focus on the process of concomitant tissue folding and extension that is key during gastrulation and neurulation. We use the Drosophila embryo as model system and focus on the process of mesoderm invagination. Here, we show that the prospective mesoderm simultaneously folds and extends. We report that mesoderm cells, under the control of anterior-posterior and dorsal-ventral gene patterning synergy, establish two sets of adherens junctions at different apical-basal positions with specialized functions: while apical junctions drive apical constriction initiating tissue bending, lateral junctions concomitantly drive polarized cell intercalation, resulting in tissue convergence-extension. Thus, epithelial cells devise multiple specialized junctional sets that drive composite morphogenetic processes under the synergistic control of apparently orthogonal signaling sources.

Cellular and Supracellular Planar Polarity: A Multiscale Cue to Elongate the Drosophila Egg Chamber.

Tissue elongation is known to be controlled by oriented cell division, elongation, migration and rearrangement. While these cellular processes have been extensively studied, new emerging supracellular mechanisms driving tissue extension have recently been unveiled. Tissue rotation and actomyosin contractions have been shown to be key processes driving Drosophila egg chamber elongation. First, egg chamber rotation facilitates the dorsal-ventral alignment of the extracellular matrix and of the cell basal actin fibers. Both fiber-like structures form supracellular networks constraining the egg growth in a polarized fashion thus working as 'molecular corsets'. Second, the supracellular actin fiber network, powered by myosin periodic oscillation, contracts anisotropically driving tissue extension along the egg anterior-posterior axis. During both processes, cellular and supracellular planar polarity provide a critical cue to control Drosophila egg chamber elongation. Here we review how different planar polarized networks are built, maintained and function at both cellular and supracellular levels in the Drosophila ovarian epithelium.

Cell intercalation in a simple epithelium.

Cell intercalation is a key topological transformation driving tissue morphogenesis, homeostasis and diseases such as cancer cell invasion. In recent years, much work has been undertaken to better elucidate the fundamental mechanisms controlling intercalation. Cells often use protrusions to propel themselves in between cell neighbours, resulting in topology changes. Nevertheless, in simple epithelial tissues, formed by a single layer of densely packed prism-shaped cells, topology change takes place in an astonishing fashion: cells exchange neighbours medio-laterally by conserving their apical-basal architecture and by maintaining an intact epithelial layer. Medio-lateral cell intercalation in simple epithelia is thus an exemplary case of both robustness and plasticity. Interestingly, in simple epithelia, cells use a combinatory set of mechanisms to ensure a topological transformation at the apical and basal sides. This article is part of the discussion meeting issue 'Contemporary morphogenesis'.

A Cdc42-mediated supracellular network drives polarized forces and Drosophila egg chamber extension.

Actomyosin supracellular networks emerge during development and tissue repair. These cytoskeletal structures are able to generate large scale forces that can extensively remodel epithelia driving tissue buckling, closure and extension. How supracellular networks emerge, are controlled and mechanically work still remain elusive. During Drosophila oogenesis, the egg chamber elongates along the anterior-posterior axis. Here we show that a dorsal-ventral polarized supracellular F-actin network, running around the egg chamber on the basal side of follicle cells, emerges from polarized intercellular filopodia that radiate from basal stress fibers and extend penetrating neighboring cell cortexes. Filopodia can be mechanosensitive and function as cell-cell anchoring sites. The small GTPase Cdc42 governs the formation and distribution of intercellular filopodia and stress fibers in follicle cells. Finally, our study shows that a Cdc42-dependent supracellular cytoskeletal network provides a scaffold integrating local oscillatory actomyosin contractions at the tissue scale to drive global polarized forces and tissue elongation.

Cell and tissue manipulation with ultrashort infrared laser pulses in light-sheet microscopy.

Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology. Precise spatio-temporal manipulation of biological entities is often required for a deeper functional understanding of the underlying biological process. Here we present a home-built integrated framework and optical design that combines three-dimensional light-sheet imaging over time with precise spatio-temporal optical manipulations induced by short infrared laser pulses. We demonstrate their potential for sub-cellular ablation of neurons and nuclei, tissue cauterization and optogenetics by using the Drosophila melanogaster and zebrafish model systems.

Embryo-scale tissue mechanics during Drosophila gastrulation movements.

Morphogenesis of an organism requires the development of its parts to be coordinated in time and space. While past studies concentrated on defined cell populations, a synthetic view of the coordination of these events in a whole organism is needed for a full understanding. Drosophila gastrulation begins with the embryo forming a ventral furrow, which is eventually internalized. It is not understood how the rest of the embryo participates in this process. Here we use multiview selective plane illumination microscopy coupled with infrared laser manipulation and mutant analysis to dissect embryo-scale cell interactions during early gastrulation. Lateral cells have a denser medial-apical actomyosin network and shift ventrally as a compact cohort, whereas dorsal cells become stretched. We show that the behaviour of these cells affects furrow internalization. A computational model predicts different mechanical properties associated with tissue behaviour: lateral cells are stiff, whereas dorsal cells are soft. Experimental analysis confirms these properties in vivo.

Local and tissue-scale forces drive oriented junction growth during tissue extension.

Convergence-extension is a widespread morphogenetic process driven by polarized cell intercalation. In the Drosophila germ band, epithelial intercalation comprises loss of junctions between anterior-posterior neighbours followed by growth of new junctions between dorsal-ventral neighbours. Much is known about how active stresses drive polarized junction shrinkage. However, it is unclear how tissue convergence-extension emerges from local junction remodelling and what the specific role, if any, of junction growth is. Here we report that tissue convergence and extension correlate mostly with new junction growth. Simulations and in vivo mechanical perturbations reveal that junction growth is due to local polarized stresses driven by medial actomyosin contractions. Moreover, we find that tissue-scale pulling forces at the boundary with the invaginating posterior midgut actively participate in tissue extension by orienting junction growth. Thus, tissue extension is akin to a polarized fluid flow that requires parallel and concerted local and tissue-scale forces to drive junction growth and cell-cell displacement.

Noninvasive In Toto Imaging of the Thymus Reveals Heterogeneous Migratory Behavior of Developing T Cells.

The migration of developing T cells (thymocytes) between distinct thymic microenvironments is crucial for their development. Ex vivo studies of thymus tissue explants suggest two distinct migratory behaviors of thymocytes in the thymus. In the cortex, thymocytes exhibit a stochastic migration, whereas medullary thymocytes show confined migratory behavior. Thus far, it has been difficult to follow all thymocytes in an entire thymus and relate their differentiation steps to their migratory dynamics. To understand the spatial organization of the migratory behavior and development of thymocytes in a fully functional thymus, we developed transgenic reporter lines for the chemokine receptors ccr9a and ccr9b, as well as for rag2, and used them for noninvasive live imaging of the entire thymus in medaka (Oryzias latipes). We found that the expression of these two chemokine receptors in the medaka juvenile thymus defined two spatially distinct subpopulations of thymocytes. Landmark events of T cell development including proliferation, somatic recombination, and thymic selection can be mapped to subregions of the thymus. The migratory behavior of thymocytes within each of the subpopulations is equally heterogeneous, and specific migratory behaviors are not associated with particular domains in the thymus. During the period when thymocytes express rag2 their migratory behavior was more homogeneous. Therefore, the migratory behavior of thymocytes is partly correlated with their developmental stage rather than being defined by their spatial localization.

Physical models of mesoderm invagination in Drosophila embryo.

The invagination of the mesoderm in the Drosophila melanogaster embryo is an intensely studied example of epithelial folding. Several theoretical studies have explored the conditions and mechanisms needed to reproduce the formation of the invagination in silico. Here we discuss the aspects of epithelial folding captured by these studies, and compare the questions addressed, the approaches used, and the answers provided.

A model of epithelial invagination driven by collective mechanics of identical cells.

We propose a 2D mechanical model of a tubular epithelium resembling the early Drosophila embryo. The model consists of a single layer of identical cells with energy associated with the tension of cell cortex. Depending on the relative tension of the apical, basal, and lateral sides of the cells, tissue thickness, and the degree of external constraint, the minimal-energy states of the epithelial cross section include circular shapes as well as a range of inward-buckled shapes. Some of the solutions are characterized by a single deep groove, which shows that an epithelium consisting of cells of identical mechanical properties can infold. This is consistent with what is seen in embryos of certain Drosophila mutants. To ensure that the infolding occurs at a predetermined section of the epithelium, we extend the model by increasing the cross-sectional area of a subset of cells, which is consistent with observations in wild-type embryos. This variation of cell parameters across the epithelium is sufficient to make it fold at a specific site. The model explores previously untested minimal conditions for tissue invagination and is devoid of specificity needed to accurately describe an in vivo situation in Drosophila.

Cortical forces in cell shape changes and tissue morphogenesis.

Cortical forces drive a variety of cell shape changes and cell movements during tissue morphogenesis. While the molecular components underlying these forces have been largely identified, how they assemble and spatially and temporally organize at cell surfaces to promote cell shape changes in developing tissues are open questions. We present here different key aspects of cortical forces: their physical nature, some rules governing their emergence, and how their deployment at cell surfaces drives important morphogenetic movements in epithelia. We review a wide range of literature combining genetic/molecular, biophysical and modeling approaches, which explore essential features of cortical force generation and transmission in tissues.

Planar polarized actomyosin contractile flows control epithelial junction remodelling.

Force generation by Myosin-II motors on actin filaments drives cell and tissue morphogenesis. In epithelia, contractile forces are resisted at apical junctions by adhesive forces dependent on E-cadherin, which also transmits tension. During Drosophila embryonic germband extension, tissue elongation is driven by cell intercalation, which requires an irreversible and planar polarized remodelling of epithelial cell junctions. We investigate how cell deformations emerge from the interplay between force generation and cortical force transmission during this remodelling in Drosophila melanogaster. The shrinkage of dorsal-ventral-oriented ('vertical') junctions during this process is known to require planar polarized junctional contractility by Myosin II (refs 4, 5, 7, 12). Here we show that this shrinkage is not produced by junctional Myosin II itself, but by the polarized flow of medial actomyosin pulses towards 'vertical' junctions. This anisotropic flow is oriented by the planar polarized distribution of E-cadherin complexes, in that medial Myosin II flows towards 'vertical' junctions, which have relatively less E-cadherin than transverse junctions. Our evidence suggests that the medial flow pattern reflects equilibrium properties of force transmission and coupling to E-cadherin by α-Catenin. Thus, epithelial morphogenesis is not properly reflected by Myosin II steady state distribution but by polarized contractile actomyosin flows that emerge from interactions between E-cadherin and actomyosin networks.

Repression of Wasp by JAK/STAT signalling inhibits medial actomyosin network assembly and apical cell constriction in intercalating epithelial cells.

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

Closing in on mechanisms of tissue morphogenesis.

It remains largely unknown how large-scale tissue movements during development emerge from the interplay of different tensile forces associated with actomyosin networks. Solon et al. (2009) now report that a ratchet-like mechanism drives the movement of epithelial sheets during dorsal closure in embryos of the fruit fly Drosophila.

Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis.

The morphogenesis of developing embryos and organs relies on the ability of cells to remodel their contacts with neighbouring cells. Using quantitative modelling and laser nano-dissection, we probed the mechanics of a morphogenetic process, the elongation of Drosophila melanogaster embryos, which results from polarized cell neighbour exchanges. We show that anisotropy of cortical tension at apical cell junctions is sufficient to drive tissue elongation. We estimated its value through comparisons between in silico and in vivo data using various tissue descriptors. Nano-dissection of the actomyosin network indicates that tension is anisotropically distributed and depends on myosin II accumulation. Junction relaxation after nano-dissection also suggests that cortical elastic forces are dominant in this process. Interestingly, fluctuations in vertex position (points where three or more cells meet) facilitate neighbour exchanges. We delineate the contribution of subcellular tensile activity polarizing junction remodelling, and the permissive role of vertex fluctuations during tissue elongation.

A two-tiered mechanism for stabilization and immobilization of E-cadherin.

Epithelial tissues maintain a robust architecture which is important for their barrier function, but they are also remodelled through the reorganization of cell-cell contacts. Tissue stability requires intercellular adhesion mediated by E-cadherin, in particular its trans-association in homophilic complexes supported by actin filaments through beta- and alpha-catenin. How alpha-catenin dynamic interactions between E-cadherin/beta-catenin and cortical actin control both stability and remodelling of adhesion is unclear. Here we focus on Drosophila homophilic E-cadherin complexes rather than total E-cadherin, including diffusing 'free' E-cadherin, because these complexes are a better proxy for adhesion. We find that E-cadherin complexes partition in very stable microdomains (that is, bona fide adhesive foci which are more stable than remodelling contacts). Furthermore, we find that stability and mobility of these microdomains depend on two actin populations: small, stable actin patches concentrate at homophilic E-cadherin clusters, whereas a rapidly turning over, contractile network constrains their lateral movement by a tethering mechanism. alpha-Catenin controls epithelial architecture mainly through regulation of the mobility of homophilic clusters and it is largely dispensable for their stability. Uncoupling stability and mobility of E-cadherin complexes suggests that stable epithelia may remodel through the regulated mobility of very stable adhesive foci.