Mechanical regulation of substrate adhesion and de-adhesion drives a cell-contractile wave during Drosophila tissue morphogenesis.

During morphogenesis, mechanical forces induce large-scale deformations; yet, how forces emerge from cellular contractility and adhesion is unclear. In Drosophila embryos, a tissue-scale wave of actomyosin contractility coupled with adhesion to the surrounding vitelline membrane drives polarized tissue invagination. We show that this process emerges subcellularly from the mechanical coupling between myosin II activation and sequential adhesion/de-adhesion to the vitelline membrane. At the wavefront, integrin clusters anchor the actin cortex to the vitelline membrane and promote activation of myosin II, which in turn enhances adhesion in a positive feedback. Following cell detachment, cortex contraction and advective flow amplify myosin II. Prolonged contact with the vitelline membrane prolongs the integrin-myosin II feedback, increases integrin adhesion, and thus slows down cell detachment and wave propagation. The angle of cell detachment depends on adhesion strength and sets the tensile forces required for detachment. Thus, we document how the interplay between subcellular mechanochemical feedback and geometry drives tissue morphogenesis.

Serotonin signaling regulates actomyosin contractility during morphogenesis in evolutionarily divergent lineages.

Serotonin is a neurotransmitter that signals through 5-HT receptors to control key functions in the nervous system. Serotonin receptors are also ubiquitously expressed in various organs and have been detected in embryos of different organisms. Potential morphogenetic functions of serotonin signaling have been proposed based on pharmacological studies but a mechanistic understanding is still lacking. Here, we uncover a role of serotonin signaling in axis extension of Drosophila embryos by regulating Myosin II (MyoII) activation, cell contractility and cell intercalation. We find that serotonin and serotonin receptors 5HT2A and 5HT2B form a signaling module that quantitatively regulates the amplitude of planar polarized MyoII contractility specified by Toll receptors and the GPCR Cirl. Remarkably, serotonin signaling also regulates actomyosin contractility at cell junctions, cellular flows and epiblast morphogenesis during chicken gastrulation. This phylogenetically conserved mechanical function of serotonin signaling in regulating actomyosin contractility and tissue flow reveals an ancestral role in morphogenesis of multicellular organisms.

Growth anisotropy of the extracellular matrix shapes a developing organ.

Final organ size and shape result from volume expansion by growth and shape changes by contractility. Complex morphologies can also arise from differences in growth rate between tissues. We address here how differential growth guides the morphogenesis of the growing Drosophila wing imaginal disc. We report that 3D morphology results from elastic deformation due to differential growth anisotropy between the epithelial cell layer and its enveloping extracellular matrix (ECM). While the tissue layer grows in plane, growth of the bottom ECM occurs in 3D and is reduced in magnitude, thereby causing geometric frustration and tissue bending. The elasticity, growth anisotropy and morphogenesis of the organ are fully captured by a mechanical bilayer model. Moreover, differential expression of the Matrix metalloproteinase MMP2 controls growth anisotropy of the ECM envelope. This study shows that the ECM is a controllable mechanical constraint whose intrinsic growth anisotropy directs tissue morphogenesis in a developing organ.

Curvature gradient drives polarized tissue flow in the Drosophila embryo.

Tissue flow during morphogenesis is commonly driven by local constriction of cell cortices, which is caused by the activation of actomyosin contractility. This can lead to long-range flows due to tissue viscosity. However, in the absence of cell-intrinsic polarized forces or polarity in forces external to the tissue, these flows must be symmetric and centered around the region of contraction. Polarized tissue flows have been previously demonstrated to arise from the coupling of such contractile flows to points of increased friction or adhesion to external structures. However, we show with experiments and modeling that the onset of polarized tissue flow in early Drosophila morphogenesis occurs independent of adhesion and is instead driven by a geometric coupling of apical actomyosin contractility to tissue curvature. Particularly, the onset of polarized flow is driven by a mismatch between the position of apical myosin activation and the position of peak curvature at the posterior pole of the embryo. Our work demonstrates how genetic and geometric information inherited from the mother interact to create polarized flow during embryo morphogenesis.

Mechanochemical Principles of Spatial and Temporal Patterns in Cells and Tissues.

Patterns are ubiquitous in living systems and underlie the dynamic organization of cells, tissues, and embryos. Mathematical frameworks have been devised to account for the self-organization of biological patterns, most famously the Turing framework. Patterns can be defined in space, for example, to form stripes; in time, such as during oscillations; or both, to form traveling waves. The formation of these patterns can have different origins: purely chemical, purely mechanical, or a combination of the two. Beyond the variety of molecular implementations of such patterns, we emphasize the unitary principles associated with them, across scales in space and time, within a general mechanochemical framework. We illustrate where such mechanisms of pattern formation arise in biological systems from cellular to tissue scales, with an emphasis on morphogenesis. Our goal is to convey a picture of pattern formation that draws attention to the principles rather than solely to specific molecular mechanisms.

In search of conserved principles of planar cell polarization.

The making of an embryo and its internal organs entails the spatial coordination of cellular activities. This manifests during tissue morphogenesis as cells change shape, rearrange and divide along preferential axis and during cell differentiation. Cells live in a polarized field and respond to it by polarizing their cellular activities in the plane of the tissue by a phenomenon called planar cell polarization. This phenomenon is ubiquitous in animals and depends on a few conserved planar cell polarity (PCP) pathways. All PCP pathways share two essential characteristics: the existence of local interactions between protein complexes present at the cell surface leading to their asymmetric distribution within cells; a supracellular graded cue that aligns these cellular asymmetries at the tissue level. Here, we discuss the potential common principles of planar cell polarization by comparing the local and global mechanisms employed by the different PCP pathways identified so far. The focus of the review is on the logic of the system rather than the molecules per se.

Forward and feedback control mechanisms of developmental tissue growth.

The size and proportions of animals are tightly controlled during development. How this is achieved remains poorly understood. The control of organ size entails coupling of cellular growth and cell division on one hand, and the measure of organ size on the other. In this review we focus on three layers of growth control consisting of genetic patterning, notably chemical gradients, mechanics and energetics which are complemented by a systemic control unit that modulates growth in response to the nutritional conditions and coordinates growth between different organs so as to maintain proportions. Growth factors, often present as concentration dependent chemical gradients, are positive inducers of cellular growth that may be considered as deterministic cues, hence acting as organ-intrinsic controllers of growth. However, the exponential growth dynamics in many developing tissues necessitate more stringent growth control in the form of negative feedbacks. Feedbacks endow biological systems with the capacity to quickly respond to perturbations and to correct the growth trajectory to avoid overgrowth. We propose to integrate chemical, mechanical and energetic control over cellular growth in a framework that emphasizes the self-organizing properties of organ-autonomous growth control in conjunction with systemic organ non-autonomous feedback on growth.

Formation of polarized contractile interfaces by self-organized Toll-8/Cirl GPCR asymmetry.

Interfaces between cells with distinct genetic identities elicit signals to organize local cell behaviors driving tissue morphogenesis. The Drosophila embryonic axis extension requires planar polarized enrichment of myosin-II powering oriented cell intercalations. Myosin-II levels are quantitatively controlled by GPCR signaling, whereas myosin-II polarity requires patterned expression of several Toll receptors. How Toll receptors polarize myosin-II and how this involves GPCRs remain unknown. Here, we report that differential expression of a single Toll receptor, Toll-8, polarizes myosin-II through binding to the adhesion GPCR Cirl/latrophilin. Asymmetric expression of Cirl is sufficient to enrich myosin-II, and Cirl localization is asymmetric at Toll-8 expression boundaries. Exploring the process dynamically, we reveal that Toll-8 and Cirl exhibit mutually dependent planar polarity in response to quantitative differences in Toll-8 expression between neighboring cells. Collectively, we propose that the cell surface protein complex Toll-8/Cirl self-organizes to generate local asymmetric interfaces essential for planar polarization of contractility.

Distinct actin-dependent nanoscale assemblies underlie the dynamic and hierarchical organization of E-cadherin.

Cadherins are transmembrane adhesion proteins required for the formation of cohesive tissues.1-4 Intracellular interactions of E-cadherin with the Catenin family proteins, α- and ß-catenin, facilitate connections with the cortical actomyosin network. This is necessary for maintaining the integrity of cell-cell adhesion in epithelial tissues.5-11 The supra-molecular architecture of E-cadherin is an important feature of its adhesion function; cis and trans interactions of E-cadherin are deployed12-15 to form clusters, both in cis and trans.11,16-21 Studies in Drosophila embryo have also shown that Drosophila E-cadherin (dE-cad) is organized as finite-sized dynamic clusters that localize with actin patches at cell-cell junctions, in continuous exchange with the extra-junctional pool of dE-cad surrounding the clusters.11,19 Here, we use the ectopic expression of dE-cad in larval hemocytes, which lack endogenous dE-cad to recapitulate functional cell-cell junctions in a convenient model system. We find that, while dE-cad at cell-cell junctions in hemocytes exhibits a clustered trans-paired organization similar to that reported previously in embryonic epithelial tissue, extra-junctional dE-cad is also organized as relatively immobile nanoclusters as well as more loosely packed diffusive oligomers. Oligomers are promoted by cis interactions of the ectodomain, and their growth is counteracted by the activity of cortical actomyosin. Oligomers in turn promote assembly of dense nanoclusters that require cortical actomyosin activity. Thus, cortical actin activity remodels oligomers and generates nanoclusters. The requirement for dynamic actin in the organization of dE-cad at the nanoscale may provide a mechanism to dynamically tune junctional strength.

Programmed and self-organized flow of information during morphogenesis.

How the shape of embryos and organs emerges during development is a fundamental question that has fascinated scientists for centuries. Tissue dynamics arise from a small set of cell behaviours, including shape changes, cell contact remodelling, cell migration, cell division and cell extrusion. These behaviours require control over cell mechanics, namely active stresses associated with protrusive, contractile and adhesive forces, and hydrostatic pressure, as well as material properties of cells that dictate how cells respond to active stresses. In this Review, we address how cell mechanics and the associated cell behaviours are robustly organized in space and time during tissue morphogenesis. We first outline how not only gene expression and the resulting biochemical cues, but also mechanics and geometry act as sources of morphogenetic information to ultimately define the time and length scales of the cell behaviours driving morphogenesis. Next, we present two idealized modes of how this information flows - how it is read out and translated into a biological effect - during morphogenesis. The first, akin to a programme, follows deterministic rules and is hierarchical. The second follows the principles of self-organization, which rests on statistical rules characterizing the system's composition and configuration, local interactions and feedback. We discuss the contribution of these two modes to the mechanisms of four very general classes of tissue deformation, namely tissue folding and invagination, tissue flow and extension, tissue hollowing and, finally, tissue branching. Overall, we suggest a conceptual framework for understanding morphogenetic information that encapsulates genetics and biochemistry as well as mechanics and geometry as information modules, and the interplay of deterministic and self-organized mechanisms of their deployment, thereby diverging considerably from the traditional notion that shape is fully encoded and determined by genes.

Deterministic and Stochastic Rules of Branching Govern Dendrite Morphogenesis of Sensory Neurons.

Dendrite morphology is necessary for the correct integration of inputs that neurons receive. The branching mechanisms allowing neurons to acquire their type-specific morphology remain unclear. Classically, axon and dendrite patterns were shown to be guided by molecules, providing deterministic cues. However, the extent to which deterministic and stochastic mechanisms, based upon purely statistical bias, contribute to the emergence of dendrite shape is largely unknown. We address this issue using the Drosophila class I vpda multi-dendritic neurons. Detailed quantitative analysis of vpda dendrite morphogenesis indicates that the primary branch grows very robustly in a fixed direction, though secondary branch numbers and lengths showed fluctuations characteristic of stochastic systems. Live-tracking dendrites and computational modeling revealed how neuron shape emerges from few local statistical parameters of branch dynamics. We report key opposing aspects of how tree architecture feedbacks on the local probability of branch shrinkage. Child branches promote stabilization of parent branches, although self-repulsion promotes shrinkage. Finally, we show that self-repulsion, mediated by the adhesion molecule Dscam1, indirectly patterns the growth of secondary branches by spatially restricting their direction of stable growth perpendicular to the primary branch. Thus, the stochastic nature of secondary branch dynamics and the existence of geometric feedback emphasize the importance of self-organization in neuronal dendrite morphogenesis.

Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability.

Tissue remodelling during Drosophila embryogenesis is notably driven by epithelial cell contractility. This behaviour arises from the Rho1-Rok-induced pulsatile accumulation of non-muscle myosin II pulling on actin filaments of the medioapical cortex. While recent studies have highlighted the mechanisms governing the emergence of Rho1-Rok-myosin II pulsatility, little is known about how F-actin organization influences this process. Here, we show that the medioapical cortex consists of two entangled F-actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1-dependent manner. The other forms a persistent and homogeneous network independent of Rho1. We identify the formin Frl (also known as Fmnl) as a critical nucleator of the persistent network, since modulating its level in mutants or by overexpression decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue-scale morphogenetic defects.

Distinct RhoGEFs Activate Apical and Junctional Contractility under Control of G Proteins during Epithelial Morphogenesis.

Small RhoGTPases direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of actomyosin contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. We examined this general problem in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein-coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (Gα and Gßγ) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized. However, the mechanisms responsible for polarizing Rho1 activity are unclear. We report that distinct guanine exchange factors (GEFs) activate Rho1 in these two cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1 but is recruited both medial-apically and at junctions by Gα12/13-GTP, also called Concertina (Cta) in Drosophila. On the other hand, Dp114RhoGEF (Dp114), a newly characterized RhoGEF, is required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions. Its localization is restricted to adherens junctions and is under Gß13F/Gγ1 control. Furthermore, Gß13F/Gγ1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. Finally, we found that Dp114RhoGEF is absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results clarify the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia.

Genetic induction and mechanochemical propagation of a morphogenetic wave.

Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. Here we report two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1-MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1-MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. We find that MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation.

Distinct contributions of tensile and shear stress on E-cadherin levels during morphogenesis.

During epithelial morphogenesis, cell contacts (junctions) are constantly remodeled by mechanical forces that work against adhesive forces. E-cadherin complexes play a pivotal role in this process by providing persistent cell adhesion and by transmitting mechanical tension. In this context, it is unclear how mechanical forces affect E-cadherin adhesion and junction dynamics. During Drosophila embryo axis elongation, Myosin-II activity in the apico-medial and junctional cortex generates mechanical forces to drive junction remodeling. Here we report that the ratio between Vinculin and E-cadherin intensities acts as a ratiometric readout for these mechanical forces (load) at E-cadherin complexes. Medial Myosin-II loads E-cadherin complexes on all junctions, exerts tensile forces, and increases levels of E-cadherin. Junctional Myosin-II, on the other hand, biases the distribution of load between junctions of the same cell, exerts shear forces, and decreases the levels of E-cadherin. This work suggests distinct effects of tensile versus shear stresses on E-cadherin adhesion.

Strengthen Your Junctions to Resist the Force.

In this issue of Developmental Cell, Acharya et al. (2018) uncover a mechanosensitive pathway that activates RhoA in epithelial cells exposed to acute tensile stress. This pathway serves to preserve tissue integrity via regulation of tensile strength, the threshold force above which adherens junctions rupture.

Quantitative Control of GPCR Organization and Signaling by Endocytosis in Epithelial Morphogenesis.

Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. We show that GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, we probe the surface of the plasma membrane, and we show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is regulated by the kinase Gprk2 and ß-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, we find higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. We identify that dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo.

Viscoelastic Dissipation Stabilizes Cell Shape Changes during Tissue Morphogenesis.

Tissue morphogenesis relies on the production of active cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to our understanding of morphogenesis. Here, we use myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from myosin II pulses, we find that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which we use to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by myosin II pulses. We test this estimate by applying time-controlled forces on junctions with optical tweezers. Finally, we show that actin turnover participates in dissipation, as reducing it pharmacologically increases the reversibility of contractile events. Our results argue that active junctional deformation is stabilized by actin-dependent dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.