The cell in the age of the genomic revolution: Cell Regulatory Networks.

Over the last few years an intense activity in the areas of advanced microscopy and quantitative cell biology has put the focus on the morphogenetic events that shape embryos. The interest in these processes is taking place against the backdrop of genomic studies, particularly of global patterns of gene expression at the level of single cells, which cannot fully account for the way cells build tissues and organs. Here we discuss the need to integrate the activity of genes with that of cells and propose the need to develop a framework, based on cellular processes and cell interactions, that parallels that which has been created for gene activity in the form of Gene Regulatory Networks (GRNs). We begin to do this by suggesting elements for building Cell Regulatory Networks (CRNs). In the same manner that GRNs create schedules of gene expression that result in the emergence of cell fates over time, CRNs create tissues and organs i.e. space. We also suggest how GRNs and CRNs might interact in the building of embryos through feedback loops involving mechanics and tissue tectonics.

From pulsatile apicomedial contractility to effective epithelial mechanics.

We review recent developments in the understanding of the biomechanics of apicomedial actomyosin and how its contractility can tense and deform tissue. Myosin pulses are driven by a biochemical oscillator but how they are modulated by the mechanical context remains unclear. On the other hand, the emergence of tissue behaviour is highly dependent on the material properties of actin, on how strongly components are connected and on the influence of neighbouring tissues. We further review the use of constitutive equations in exploring the mechanics of epithelial apices dominated by apicomedial Myosin contractility.

Integration of actomyosin contractility with cell-cell adhesion during dorsal closure.

In this work, we combine genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure during morphogenesis of the amnioserosa, the main force-generating tissue during the dorsal closure in Drosophila We show that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. By contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, we show that in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis.

α-Catenin stabilises Cadherin-Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction.

We have investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end, we have analysed the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. We find that α-Catenin regulates pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin (Drosophila E-Cadherin, also known as Shotgun) at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, we uncover a genetic interaction between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue.

From actomyosin oscillations to tissue-level deformations.

Pulsatile actomyosin contractility driving cell shape oscillations is a common feature of actomyosin networks present in a variety of tissues undergoing morphogenetic processes. The origin of this oscillatory dynamics, how it is stabilized over time to give rise to net cell shape changes and how it is spatially coordinated across a tissue, are questions that have being extensively investigated in recent years. In this work, I review how genetics, cell biology, and quantitative and theoretical approaches have started to give a comprehensive understanding of these problems revealing that both biochemical and mechanical regulation play an important role in the emergence, coordination and stabilization of this activity.

Emergent material properties of developing epithelial tissues.

BACKGROUND: Force generation and the material properties of cells and tissues are central to morphogenesis but remain difficult to measure in vivo. Insight is often limited to the ratios of mechanical properties obtained through disruptive manipulation, and the appropriate models relating stress and strain are unknown. The Drosophila amnioserosa epithelium progressively contracts over 3 hours of dorsal closure, during which cell apices exhibit area fluctuations driven by medial myosin pulses with periods of 1.5-6 min. Linking these two timescales and understanding how pulsatile contractions drive morphogenetic movements is an urgent challenge. RESULTS: We present a novel framework to measure in a continuous manner the mechanical properties of epithelial cells in the natural context of a tissue undergoing morphogenesis. We show that the relationship between apicomedial myosin fluorescence intensity and strain during fluctuations is consistent with a linear behaviour, although with a lag. We thus used myosin fluorescence intensity as a proxy for active force generation and treated cells as natural experiments of mechanical response under cyclic loading, revealing unambiguous mechanical properties from the hysteresis loop relating stress to strain. Amnioserosa cells can be described as a contractile viscoelastic fluid. We show that their emergent mechanical behaviour can be described by a linear viscoelastic rheology at timescales relevant for tissue morphogenesis. For the first time, we establish relative changes in separate effective mechanical properties in vivo. Over the course of dorsal closure, the tissue solidifies and effective stiffness doubles as net contraction of the tissue commences. Combining our findings with those from previous laser ablation experiments, we show that both apicomedial and junctional stress also increase over time, with the relative increase in apicomedial stress approximately twice that of other obtained measures. CONCLUSIONS: Our results show that in an epithelial tissue undergoing net contraction, stiffness and stress are coupled. Dorsal closure cell apical contraction is driven by the medial region where the relative increase in stress is greater than that of stiffness. At junctions, by contrast, the relative increase in the mechanical properties is the same, so the junctional contribution to tissue deformation is constant over time. An increase in myosin activity is likely to underlie, at least in part, the change in medioapical properties and we suggest that its greater effect on stress relative to stiffness is fundamental to actomyosin systems and confers on tissues the ability to regulate contraction rates in response to changes in external mechanics.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

Interactions between the amnioserosa and the epidermis revealed by the function of the u-shaped gene.

Dorsal closure (DC) is an essential step during Drosophila development whereby a hole is sealed in the dorsal epidermis and serves as a model for cell sheet morphogenesis and wound healing. It involves the orchestrated interplay of transcriptional networks and dynamic regulation of cell machinery to bring about shape changes, mechanical forces, and emergent properties. Here we provide insight into the regulation of dorsal closure by describing novel autonomous and non-autonomous roles for U-shaped (Ush) in the amnioserosa, the epidermis, and in mediation of communication between the tissues. We identified Ush by gene expression microarray analysis of Dpp signaling targets and show that Ush mediates some DC functions of Dpp. By selectively restoring Ush function in either the AS or the epidermis in ush mutants, we show that the AS makes a greater (Ush-dependent) contribution to closure than the epidermis. A signal from the AS induces epidermal cell elongation and JNK activation in the DME, while cable formation requires Ush on both sides of the leading edge, i.e. in both the AS and epidermis. Our study demonstrates that the amnioserosa and epidermis communicate at several steps during the process: sometimes the epidermis instructs the amnioserosa, other times the AS instructs the epidermis, and still other times they appear to collaborate.

Dynamics of actomyosin contractile activity during epithelial morphogenesis.

In the past few years, advances in microscopy and quantitative image analysis have lead to a completely new understanding of the processes underlying the cell shape changes and cell rearrangements that drive tissue morphogenesis. In a handful of tissues so far, though the number will surely increase rapidly, it has been shown that cell behaviour is not continuous but proceeds in pulses driven by the contractile activity of dynamic cortical actomyosin networks. The patterns and dynamics of temporary subcellular contractile foci, driven by local increases in actin and myosin, are remarkably similar in disparate tissues. Cells in all tissues display a similar range of intervals between contractions, with increasing frequencies associated with stronger tissue morphogenesis. Contractile foci appear to flow within cells with speeds that are consistent across tissues. We highlight the difference between contractile tension and stiffness, the latter being a requirement for any ratchet mechanism that stabilises contraction to produce effective tissue morphogenesis. At least two different types of ratchet mechanism are discussed, with the stiffness conferred either by a more stable actomyosin population at cell-cell junctions or through cortical actomyosin forming a quasi-stable supra-cellular network. Pulsatile contractions, polarized cell organization and various stiffening ratchet mechanisms combine to provide a rich variety of options for robust epithelial tissue remodelling.

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium.

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.

Endocytic and recycling endosomes modulate cell shape changes and tissue behaviour during morphogenesis in Drosophila.

During development tissue deformations are essential for the generation of organs and to provide the final form of an organism. These deformations rely on the coordination of individual cell behaviours which have their origin in the modulation of subcellular activities. Here we explore the role endocytosis and recycling on tissue deformations that occur during dorsal closure of the Drosophila embryo. During this process the AS contracts and the epidermis elongates in a coordinated fashion, leading to the closure of a discontinuity in the dorsal epidermis of the Drosophila embryo. We used dominant negative forms of Rab5 and Rab11 to monitor the impact on tissue morphogenesis of altering endocytosis and recycling at the level of single cells. We found different requirements for endocytosis (Rab5) and recycling (Rab11) in dorsal closure, furthermore we found that the two processes are differentially used in the two tissues. Endocytosis is required in the AS to remove membrane during apical constriction, but is not essential in the epidermis. Recycling is required in the AS at early stages and in the epidermis for cell elongation, suggesting a role in membrane addition during these processes. We propose that the modulation of the balance between endocytosis and recycling can regulate cellular morphology and tissue deformations during morphogenesis.

Integrative approaches to morphogenesis: lessons from dorsal closure.

Although developmental biology has been dominated by the genetic analysis of embryonic development, in recent years genetic tools have been combined with new approaches such as imaging of live processes, automated and quantitative image analysis, mechanical perturbation and mathematical modeling, to study the principles underlying the formation of organisms. Here we focus on recent work carried out on Dorsal Closure, a morphogenetic process during Drosophila embryogenesis, to illustrate how this multidisciplinary approach is yielding new and unexpected insights into how cells organize themselves through the activity of their molecular components to give rise to the stereotyped and macroscopic movements observed during development.

Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure.

Fluctuations in the shape of amnioserosa (AS) cells during Drosophila dorsal closure (DC) provide an ideal system with which to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behaviour emerges from the activity of individual cells. Using quantitative image analysis we show that apical shape fluctuations are driven by the medial cytoskeleton, with periodic foci of contractile myosin and actin travelling across cell apices. Shape changes were mostly anisotropic and neighbouring cells were often, but transiently, organised into strings with parallel deformations. During the early stages of DC, shape fluctuations with long cycle lengths produced no net tissue contraction. Cycle lengths shortened with the onset of net tissue contraction, followed by a damping of fluctuation amplitude. Eventually, fluctuations became undetectable as AS cells contracted rapidly. These transitions were accompanied by an increase in apical myosin, both at cell-cell junctions and medially, the latter ultimately forming a coherent, but still dynamic, sheet across cells. Mutants with increased myosin activity or actin polymerisation exhibited precocious cell contraction through changes in the subcellular localisation of myosin. thick veins mutant embryos, which exhibited defects in the actin cable at the leading edge, showed similar timings of fluctuation damping to the wild type, suggesting that damping is an autonomous property of the AS. Our results suggest that cell shape fluctuations are a property of cells with low and increasing levels of apical myosin, and that medial and junctional myosin populations combine to contract AS cell apices and drive DC.

Origin and function of fluctuations in cell behaviour and the emergence of patterns.

Morphogenesis is the process whereby cells assemble into tissues and organs. Recent studies of this process have revealed heterogeneity of individual cell behaviours that contrasts with the deterministic activity of tissues as a whole. Here we review these observations and suggest that fluctuations and heterogeneities are a central substrate for morphogenesis and that there might exist mechanisms dedicated to the averaging of these fluctuations to ensure robust and reproducible behaviours at the tissue level.

Quantitative approaches in developmental biology.

The tissues of a developing embryo are simultaneously patterned, moved and differentiated according to an exchange of information between their constituent cells. We argue that these complex self-organizing phenomena can only be fully understood with quantitative mathematical frameworks that allow specific hypotheses to be formulated and tested. The quantitative and dynamic imaging of growing embryos at the molecular, cellular and tissue level is the key experimental advance required to achieve this interaction between theory and experiment. Here we describe how mathematical modelling has become an invaluable method to integrate quantitative biological information across temporal and spatial scales, serving to connect the activity of regulatory molecules with the morphological development of organisms.

Mechanical control of global cell behaviour during dorsal closure in Drosophila.

Halfway through embryonic development, the epidermis of Drosophila exhibits a gap at the dorsal side covered by an extraembryonic epithelium, the amnioserosa (AS). Dorsal closure (DC) is the process whereby interactions between the two epithelia establish epidermal continuity. Although genetic and biomechanical analysis have identified the AS as a force-generating tissue, we do not know how individual cell behaviours are transformed into tissue movements. To approach this question we have applied a novel image-analysis method to measure strain rates in local domains of cells and performed a kinematic analysis of DC. Our study reveals spatial and temporal differences in the rate of apical constriction of AS cells. We find a slow phase of DC, during which apical contraction of cells at the posterior end predominates, and a subsequent fast phase, during which all the cells engage in the contraction, which correlates with the zippering process. There is a radial gradient of AS apical contraction, with marginal cells contracting earlier than more centrally located cells. We have applied this analysis to the study of mutant situations and associated a particular genotype with quantitative and reproducible changes in the rate of cell contraction and hence in the overall rate of the process. Our mutant analysis reveals the contribution of mechanical elements to the rate and pattern of DC.

Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation.

The dynamic reshaping of tissues during morphogenesis results from a combination of individual cell behaviors and collective cell rearrangements. However, a comprehensive framework to unambiguously measure and link cell behavior to tissue morphogenesis is lacking. Here we introduce such a kinematic framework, bridging cell and tissue behaviors at an intermediate, mesoscopic, level of cell clusters or domains. By measuring domain deformation in terms of the relative motion of cell positions and the evolution of their shapes, we characterized the basic invariant quantities that measure fundamental classes of cell behavior, namely tensorial rates of cell shape change and cell intercalation. In doing so we introduce an explicit definition of cell intercalation as a continuous process. We mapped strain rates spatiotemporally in three models of tissue morphogenesis, gaining insight into morphogenetic mechanisms. Our quantitative approach has broad relevance for the precise characterization and comparison of morphogenetic phenotypes.

Requirements for adherens junction components in the interaction between epithelial tissues during dorsal closure in Drosophila.

Dynamic interactions between epithelial sheets are a regular feature of morphogenetic processes. Dorsal closure in Drosophila relies on the coordinated movements of two epithelia, the epidermis and the amnioserosa, and provides an excellent model system for a genetic and cell biological approach. Here, we have analyzed the contribution of junctional organization of these epithelia to dorsal closure. We observe a stringent requirement for adherens junctions at the leading edge, the interface between the amnioserosa and the epidermis, for the transmission of the forces generated during the process. We also find that interactions between Armadillo and E-cadherin play an important role in maintaining the adhesion at the leading edge, revealing the particular dynamics of this interface. Our results show that regulated cell adhesion is a crucial element of the interactions that shape epithelial sheets in morphogenetic processes.

Mechanisms of Hedgehog gradient formation and interpretation.

Morphogens are molecules that spread from localized sites of production, specifying distinct cell outcomes at different concentrations. Members of the Hedgehog (Hh) family of signaling molecules act as morphogens in different developmental systems. If we are to understand how Hh elicits multiple responses in a temporally and spatially specific manner, the molecular mechanism of Hh gradient formation needs to be established. Moreover, understanding the mechanisms of Hh signaling is a central issue in biology, not only because of the role of Hh in morphogenesis, but also because of its involvement in a wide range of human diseases. Here, we review the mechanisms affecting the dynamics of Hh gradient formation, mostly in the context of Drosophila wing development, although parallel findings in vertebrate systems are also discussed.