The Interactive Fly

Genes involved in tissue and organ development

Dorsal closure

  • Transiently reorganized microtubules are essential for zippering during dorsal closure in Drosophila
  • Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure
  • Mechanical control of global cell behaviour during dorsal closure in Drosophila
  • Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure
  • Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure
  • Temporal and molecular hierarchies in the link between death, delamination and dorsal closure
  • Talin autoinhibition is required for morphogenesis
  • Bazooka inhibits aPKC to limit antagonism of actomyosin networks during amnioserosa apical constriction
  • High plasticity in epithelial morphogenesis during insect dorsal closure
  • Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure
  • The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development
  • Pathway to a phenocopy: Heat stress effects in early embryogenesis
  • Emergent material properties of developing epithelial tissues
  • Quantifying dorsal closure in three dimensions
  • Cell boundary elongation by non-autonomous contractility in cell oscillation
  • The actin cable is dispensable in directing dorsal closure dynamics but neutralizes mechanical stress to prevent scarring in the Drosophila embryo
  • Basal cell-extracellular matrix adhesion regulates force transmission during tissue morphogenesis
  • Cell-cell and cell-ECM adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure
  • Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo
  • Novel interplay between JNK and Egfr signaling in Drosophila dorsal closure
  • Activation and synchronization of the oscillatory morphodynamics in multicellular monolayer
    Genes affecting cell motility in dorsal closure

    Transiently reorganized microtubules are essential for zippering during dorsal closure in Drosophila

    There is emerging evidence that microtubules in nondividing cells can be employed to remodel the intracellular space. This study demonstrates an essential role for microtubules in dorsal closure (DC), which occurs toward the end of Drosophila melanogaster embryogenesis. Dorsal closure is a morphogenetic process similar to wound healing, whereby a gap in the epithelium is closed through the coordinated action of different cell types. Surprisingly, this complex process requires microtubule function exclusively in epithelial cells and only for the last step, the zippering, which seals the gap. Preceding zippering, the epithelial microtubules reorganize to attain an unusual spatial distribution, which is described with subcellular resolution in the intact, living organism. This study provides a clearly defined example where cells of a developing organism transiently reorganize their microtubules to fulfill a specialized morphogenetic task (Jankovics, 2006).

    Using time-lapse imaging of DC-stage embryos expressing GFP-tagged forms of tubulin or the plus end tracking protein EB1, it is shown that during DC, antiparallel MTs form stable bundles that align parallel to the D/V cell axis at the apical cell cortex. Within bundles, the MTs remain highly dynamic, which allows them to grow into the cellular protrusions that form at the dorsal surface of the dorsal-most row of epithelial cells (DME) cells. Surprisingly, elimination of these MTs by injection of MT depolymerizing drugs into the embryo or by expressing an MT-severing protein Spastin in a subset of epithelial cells inhibited exclusively the zippering process that completes DC. All other essential processes, the convergence of the two lateral epithelial cell layers, D/V cell polarization of the DMEs, or their actin-based dorsal constriction were not altered (Jankovics, 2006).

    How could MT function be linked to zippering at the molecular level? One obvious scenario is that MTs are required for the local delivery of adhesion proteins. MT-based, localized delivery of factors was already shown to be crucial for proper morphogenesis in fission yeast cells and for a number of processes during early Drosophila development. However, zippering is not a simple one-step process. It is proposed to start with the interdigitation of the cell protrusions that form at the dorsal side of the DME cells and that establish the first contacts between equivalent cells of the two opposing epithelial cell layers. Consistent with this, the protrusions are essential for zippering. The initial cell-cell contacts subsequently develop into the known cell-adhesion structures. The possibility for initial interdigitation occurs at the anterior and posterior ends of the dorsal opening where the two cell layers meet. As this possibility also exists in embryos that cannot zipper due to a lack of MTs, it is conceivable that MTs are required for the interdigitation of protrusions. Intriguingly, the absence of MTs considerably affects the number and appearance of cellular protrusions known to be essential for zippering, which provides another possible explanation for the inability of these cells to interact with each other. An MT-mediated increase in protrusion formation could provide DME cells with sufficient interactive surface or interaction time to enable interdigitation. Consistent with this, the stripes of cells lacking MTs in Spastin-overexpression experiments were unable to zipper on their own but eventually managed to establish cell adhesion when forced into close proximity by zippering of the neighboring wild-type cells. To understand MT function during zippering, one may therefore need to ask how MTs modify cell protrusions. As protrusions can still form in the absence of MTs, these do not seem to control the on/off activity of the protrusion-forming machinery but may rather modulate its activity. MTs were previously shown to affect protrusion formation in several cultured cell types but the molecular mechanisms are not clear. It was speculated that MTs may modulate the actin machinery by delivering regulatory factors such as guanine nucleotide exchange factors or GTPase-activating proteins that modulate the actin organizing activities of the Rac1, Cdc42, and Rho GTPases. It is also possible that growing MTs produce pushing forces that support protrusion growth (Jankovics, 2006).

    Why do MTs reorganize in such a specific way? It was not possible to answer this question. If delivery of adhesion factors or promoting protrusion formation were indeed the critical functions of MTs, then their orientation parallel to the D/V cell axis would certainly improve delivery to the relevant site. However, because of the antiparallel arrangement, transport of factors is bidirectional, and therefore the factors would also be delivered to the wrong cell ends. In addition, MTs reorganize in all epithelial cells, although most of them are not involved in zippering and therefore do not need delivery of the proposed factors. It is possible that MTs reorganize to fulfill additional, nonessential functions that optimize the DC process. For example, the MTs are required for proper epithelial cell morphology. During convergence, these cells gradually elongate along the D/V axis while thinning out in the apical-basal direction. Elongation coincides with the D/V alignment of the MTs, implying that MT reorganization may be the consequence of cell shape changes. However, cells lacking MTs cannot maintain their shape after an initial elongation phase, suggesting that proper cell morphology is dependent on MT function. Notably, this is not the consequence of defects in polarization, since cell polarity is not affected in the absence of MTs. The role of epithelial cell shape changes in DC is not clear. Their dorsalward stretching may contribute to gap closure, but recent data indicated that it is not essential. The finding that the shape abnormalities resulting from MT depletion did not affect convergence of epithelial cell layers is consistent with this view (Jankovics, 2006).

    These results provide further evidence that MT reorganization is not only crucial when cells change from an interphase to a mitotic state but also when they change behavior during development. It is important to understand how such rearrangements are controlled at the molecular level and also how cell-type-specific differences are achieved. DC in Drosophila provides an excellent experimental system in which the molecular mechanisms controlling MT organization can be studied in vivo by live imaging with appropriate subcellular resolution in combination with classical genetics (Jankovics, 2006).

    Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure

    Dorsal closure is a paradigm epithelial fusion episode that occurs late in Drosophila embryogenesis and leads to sealing of a midline hole by bonding of two opposing epithelial sheets. The leading edge epithelial cells express filopodia and fusion is dependent on interdigitation of these filopodia to prime formation of adhesions. Since the opposing epithelia are molecularly patterned there must exist some mechanism for accurately aligning the two sheets across this fusion seam. To address this, a fly was generated in which RFP-Moesin and GFP-Moesin are expressed in mutually exclusive stripes within each segment using the engrailed and patched promoters. Mutually exclusive interactions were observed between the filopodia of engrailed and patched cells. Interactions between filopodia from matching cells leads to formation of tethers between them, and these tethers can pull misaligned epithelial sheets into alignment. Filopodial matching also occurs during repair of laser wounds in the ventral epithelium, and so this behaviour is not restricted to leading edge cells during dorsal closure. Finally, the behaviour was characterised of a patched-expressing cell that was observed within the engrailed region of segments A1-A5; evidence is provided that this cell contributes to cell matching (Millard, 2008).

    What is the molecular basis of cell matching? The data are consistent with matching being based on just two sets of molecular interactions, one allowing anterior (A) compartment cells to recognise one another and the other performing the same function for posterior (P) compartment cells. An obvious possibility is that the molecules that mediate cell matching during DC are the same as those that maintain the integrity of these compartments throughout the epithelium. Alternatively, there could be a different set of recognition molecules present exclusively at the leading edge to mediate cell-cell matching. Filopodia are also observed during the healing of wounds in the ventral epithelium and it was reasoned that these wound filopodia should exhibit matching behaviour if the molecules that mediate matching are present throughout the epithelium. Laser wounds were made to the ventral epithelium across en stripes such that the wound edge consisted of both en-RFP-Moesin and ptc-GFP-Moesin cells. On healing of these wounds, repeated interactions were observed between the filopodia of matching cells, but not between mismatching cells. In an example, a number of filopodial tethers formed between ptc cells on opposite sides of the wound as closure proceeded. Near the end of closure, a filopodial tether formed between en cells on opposite sides of the wound, leading to fusion of these cells and regeneration of the en stripe. This sequence of events was observed in six out of ten similar wounds. In the remaining wounds, the tethers between ptc cells became permanent adhesions before the en cells were close enough to form tethers and hence the en stripe was not regenerated. These data suggest that both A and P compartment cells away from the leading edge can carry out filopodial matching analogous to that occurring during DC, and hence the adhesion molecules that mediate the process are not leading edge specific (Millard, 2008).

    The data demonstrate that specific recognition events ensure the accuracy of fusion during DC. Filopodia facilitate matching by allowing a cell to search for its match and also to pull misaligned sheets into alignment. This explains why genetic interventions that abolish filopodia lead to an increase in mismatching. It appears that at least two recognition processes act during DC, one for P compartments and one for A compartment cells, but these recognition events are not segment-specific, since fusions can occur between matching compartments from different segments. Filopodial matching is also observed during healing of wounds in the ventral epithelium, suggesting that the molecules mediating recognition are found throughout the epithelium. These data are consistent with the notion that the adhesion molecules that mediate filopodial matching during DC are the same as those that ensure compartment integrity throughout the epithelium; however, the identity of these molecules is currently unknown. Experimental and modelling studies have shown that cells can sort based on differential levels of just one adhesion molecule, and it has been hypothesised that a single adhesion molecule might be responsible for compartmental segregation. The data suggest that, at least during filopodial matching, this is not the case, since observe specific recognition events were observed for both A and P compartments and neither compartment is obviously dominant in the matching process. It is of course possible that multiple mechanisms contribute to cell matching and segregation, perhaps with different adhesion molecules governing the rapid, transient associations between filopodia and the long-lived adhesions that hold cells together permanently. Whereas segregation between leading edge A and P compartment cells is absolute at the parasegment boundary, reproducibly a single A compartment ptc cell is seen to move into the P compartment at the segment boundary. This might suggest that differences in adhesive properties between cells either side of the segment boundary are small, permitting a degree of mixing. However, during DC, the misplaced ptc cells are consistently able to recognise and specifically fuse with matching cells in the opposing epithelial sheet, indicating adhesive properties distinct from their neighbours. When the arrangement of the misplaced ptc cells is disrupted, it can result in severe mismatches; therefore, correct positioning of these cells is clearly important in epithelial sheet alignment. These cells occupy a unique and defined position in each segment and might assist the matching process by acting as a 'keystone' that helps to ensure precise alignment within the segment (Millard, 2008).

    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, it is not known how individual cell behaviours are transformed into tissue movements. To approach this question a novel image-analysis method was applied to measure strain rates in local domains of cells, and a kinematic analysis of DC was performed. This study reveals spatial and temporal differences in the rate of apical constriction of AS cells. A slow phase of DC is found, 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. This analysis was applied to the study of mutant situations, and a particular genotype was associated with quantitative and reproducible changes in the rate of cell contraction and hence in the overall rate of the process. This mutant analysis reveals the contribution of mechanical elements to the rate and pattern of DC (Gorfinkiel, 2009).

    The process of dorsal closure provides a good system to explore the relationship between cell biology and tissue mechanics and the way this informs morphogenesis. There are sound descriptions of the molecular and cellular events underpinning the process, a large collection of mutants that interfere with the different stages, and some of the macroscopic forces underlying DC have been identified (Hutson, 2003; Kiehart, 2000; Toyama, 2008). Notwithstanding this, most of the studies have focused on the consequence that the loss of a gene has for the process. For this reason, an understanding of how individual cell behaviours contribute to the global patterning in the wild type is lacking. This study has begun to approach this problem by focusing on the AS, a genetically homogeneous tissue, the contraction of which provides a component of the force that drives closure (Gorfinkiel, 2009).

    This study reveals that AS cell contraction is patterned in space and in time. At present there are no reports of differences in gene expression between different AS cells during DC, and therefore it is unlikely that the complex dynamics that are observed can be related to patterned gene activity. On the basis of these results, in particular the strong correlation between the zippering speed and the rate of contraction of the AS, the possibility is favoured that the behaviour of the AS during DC is governed by mechanical interactions with its environment. Additional evidence for the role of cell mechanics in the process comes from the AP differences in the rate of contraction of AS cells and their patterns of shape changes, as well as the timing of individual contractions in the ML and AP axes, which are indicative of different stresses along the AP axis of the embryo. Thus, although AS cell contraction is triggered by Dpp signalling (Fernández, 2007), the current work suggests that this might create a plastic state that is patterned by the integration, at the single cell level, of global mechanical cues. A role for mechanical cues in determining cell behaviour and cell fate has been shown for different cell types in cell culture. The morphogenetic alterations discussed above, together with quantitative changes in the geometry of cell-cell contacts during DC suggest that adhesive and cytoskeletal properties of AS cells are modulated during the process through a combination of chemical and mechanical signals (Gorfinkiel, 2009).

    The two phases of activity of the AS cells revealed by this analysis are divided by the onset of epidermal zippering during which the filopodial activity developed by dorsal-most epidermal cells makes a significant contribution to the fusion and matching of the opposite epidermal flanks (Jacinto, 2000; Jankovics, 2006; Millard, 2008). This analysis shows that perturbing the zippering process, through interference of microtubule dynamics in the epidermis, affects the rate of contraction of AS cells, and, reciprocally, that perturbing the dynamics of AS contraction through inhibition of apoptosis-mediated extrusion affects the zippering rate. These observations support an interdependence between these two processes that is manifest in the correlation between the zippering rate and the dynamics of the rate of apical AS cell contraction. Recent work from Edwards, Kiehart and colleagues (Toyama, 2008) has also shown that inhibiting or enhancing apoptosis in the AS changes the kinematic properties of the closure, i.e. the rate of closure, the rate of zipping and the force produced by the AS. However, in contrast to the current interpretation, the authors suggest a direct and causal relationship between apoptosis and the force generation in the AS. In the light of the current results, it is surmised that the changes in the force-generating capability of AS cells observed in their experiments are a secondary consequence of the effects of apoptosis on the zippering, which thus feeds back onto the AS. Cell extrusion is indeed an important contributor to the normal rates of DC, but this work suggests that it is an event that responds to, rather than directs, the strains of the whole AS (Gorfinkiel, 2009).

    This study also demonstrates that assigning functions to particular genes during morphogenesis might be misleading. As is shown in this study, the phenotype of a mutant during dorsal closure is not a direct consequence of the loss of function of a particular gene but the outcome that this loss of function has on a series of related cellular activities. In higher-order processes, such as morphogenesis, the result of a mutation is an array of perturbations acting at different levels of organization, which makes it difficult to infer a direct causal relationship between the mutated gene and the terminal phenotype. Acknowledging this, the system-level analysis performed in this study shows a correlation of a particular genotype with a reproducible change in the dynamics of the process, revealing the contribution of each gene product to the overall function (Gorfinkiel, 2009).

    Morphogenetic processes require complex spatiotemporal integration of cellular activities that results in patterns of activity at the tissue level. The behaviour of the AS during DC provides a simple system in which to begin to unravel the nature and levels of this integration and the manner in which chemical, mechanical and genetic inputs pattern the behaviour of an epithelium. To do a method is used that allows measurement of the deformation of individual cells and of tissue domains (Blanchard, 2009), and use was made of mutants to perturb the system in a defined and controlled manner. This study reveals that regular patterns of tissue behaviour emerge from the short-range coordinated behaviour of individual cells upon which chemical signals and mechanical constraints are impressed from surrounding tissues. These results highlight the importance of looking at the dynamics of cell populations, which cannot be obtained from the behaviour of individual cells (Gorfinkiel, 2009).

    Physical models have recently been applied to recapitulate the appearance of higher-order tissue architectures in different epithelia from the mechanical properties of individual cells. Kinematic quantitative analyses like the one presented in this study do provide the basis on which to build computational simulations of morphogenetic processes that will allow integration of the activity of genes, signals and mechanical properties into the behaviour of tissues (Gorfinkiel, 2009).

    Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure

    Dorsal closure (DC) is a tissue-modeling process in the developing Drosophila embryo during which an epidermal opening is closed. It begins with the appearance of a supracellular actin cable (AC) that surrounds the opening and provides a contractile force. Amnioserosa cells that fill the opening produce an additional critical force pulling on the surrounding epidermal tissue. This force is not gradual but pulsed and occurs long before dorsal closure starts. Quantitative analysis, combined with laser cutting experiments and simulations, reveals that tension-based dynamics and cell coupling control the force pulses. These constitutively pull the surrounding epidermal tissue dorsally, but the displacement is initially transient. It is translated into dorsal-ward movement only with the help of the actin cable, which acts like a ratchet, counteracting ventral-ward epidermis relaxation after force pulses. This work uncovers a sophisticated mechanism of cooperative force generation between two major forces driving morphogenesis (Solon, 2009).

    This work has addressed the cooperative nature of two main forces driving dorsal closure. The results suggest a model that is in contrast to two popular views of DC mechanics. First, they show that force production by AS cells is constitutive and independent of the signal that triggers DC. The previous view that AS cells start to produce their pulling force at the onset of DC was based on the observation that only then do they begin to reduce their apical surfaces, a reduction that was consequently interpreted as being the force-producing process. The data of this study suggest that this reduction in surface area is passive and a consequence of closure, which globally reduces AS tissue surface and therefore prevents AS cells from expanding back to their original size after a contraction (Solon, 2009).

    The second modification to the current picture is that the AC acts like a ratchet supporting the AS-mediated force pulses rather than contributing to dorsal-ward LE displacement as an autonomous purse string. AC tension builds up slowly, becoming prominent only much later in the DC process. Efficient DC, however, starts with the first signs of an AC, when the latter is unlikely to produce sufficient force for purse-string activity. This view is also supported by the overall geometry of the AC; its contractile forces act in the anterior-posterior direction, parallel to the LE, and will produce a dorsal-ward force component only because of AC curvature. There is evidence that this force component is not sufficient to independently pull on the epidermis because releasing tension by cutting the AS tissue leads to an initial ventral-ward relaxation of the AC. In this model, the AC does not have to exert much force, because it merely has to shift the preexisting equilibrium of forces between AS and epidermal tissue to counteract the ventral-ward relaxation of the LE after a force pulse. Simulations demonstrate that this scenario is indeed possible (Solon, 2009).

    To understand how the AC exerts the proposed ratchet-like function at the molecular level, it will be necessary to understand the behavior of the major AC components that build up tension, actin and the motor protein myosin. The details of subcellular actin/myosin organization are not known, but it is possible to speculate how they may work to create a ratchet-like function. It is important to keep in mind that the AC is a supracellular structure made of many smaller ACs, which form inside the cells of the LE. These produce individual forces that are transmitted via intercellular bonds. Every cell can therefore act as an individual unit. If an AS cell next to the LE contracts, it will not only pull the LE dorsally but will also compress regions of the LE that are in contact with it. This compressive force will act in the anterior/posterior direction and will therefore slightly shorten the LE and as a consequence locally reduce AC tension. The involved LE cells can then compensate for this tension loss by condensing their actin/myosin network along the compression. This can be achieved by myosin-mediated sliding of actin filaments. When the AS cell relaxes, AC tension will increase again because the epidermal tissue will now try to pull the LE back to its original position, which requires it to stretch. This cannot happen if the condensed actin/myosin network does not relax. In this system, every force pulse would locally increase AC tension, which fits well with the observed gradual increase of AC tension over time (Solon, 2009).

    At the mechanistic level, the major difference between this model and the previous purse-string model is that the AC manages to shorten only during dorsal-ward LE displacement, when it is supported by compressive forces, whereas in between force pulses it merely maintains its state. It is possible that later in the process, the AC is sufficiently enforced to produce an independent purse-string activity. Unfortunately, during these stages the zippering process creates additional pulling forces, which hamper the respective analysis (Solon, 2009).

    Another important question arising from these observations is how AS cell pulsing is produced and controlled at the molecular level. It is conceivable that this may also involve actin and myosin function. It has been shown that Myosin II is critical for AS cell activity during DC, which is consistent with this view. What regulates the pulsed activity of these molecules remains to be shown. In the simplest case one can imagine a cell-autonomous mechanism that leads to regular pulses. Simulations show that such a mechanism works in principle but it cannot reproduce the full behavior of the in vivo system. In contrast, in vivo behavior can be convincingly reconstituted by introducing a tension-based mechanism in the simulations, where contraction initiation simply depends on cells being critically stretched by contracting neighbors. In particular, this model reproduces pulsing arrest in cells surrounding a laser-induced cut. It also reproduces the coupling behavior observed between AS cells in vivo. Such coupling could be achieved by a mechano-sensitive signaling system. At this stage, the action of a complex reaction-diffusion signaling network controlling cell pulsing cannot be fully excluded. However, no evidence was found that would indicate the existence of such a network, such as wave-like propagation of pulsing or any other regularity (Solon, 2009).

    The simulations also offer an explanation for the sequential pulsation arrest observed in vivo. DC progression reduces the overall surface of the AS tissue. As a consequence, individual cells can no longer expand their surfaces to the initial size after a contraction, which is reflected by the observed decrease in pulsing amplitude. The simulations show how this quickly leads to a critical loss of surface tension and the arrest of pulsations in the entire tissue. In vivo, AS cells sequentially arrest in a contracted state, which increases the available space for the remaining AS cells, thus compensating for the surface loss and maintaining tissue tension. The simulations show that in a tension-based system, this mechanism will extend AS cell force production. A recent paper added further support to this model. It showed that apoptosis of about 10% of the AS cells contributes to closing efficiency by up to 50% (Toyama, 2008). It is clear that the elimination of AS cells also creates additional space and preserves tissue tension. Apoptosis could thus represent a second mechanism acquired to prolong pulsed force generation by AS cells. Accordingly, this study found that apoptosis starts late only in DC, at a stage when the AS tissue has significantly decreased in size. How apoptosis and the sequential arrest of pulsing are controlled remains to be determined. The latter could involve the diffusible signaling molecule Dpp, which is secreted by the cells of the LE. A Dpp gradient originating from the LE could in principle generate the observed, sequential pulsation arrest, which may explain the behavioral defects of AS cells in Dpp mutants (Fernandez, 2007; Wada, 2007; Solon, 2009).

    This work describes a mechanism for displacing and shaping tissues that combines a field of pulsing cells with a ratchet-like function. This may represent a more general principle by which other tissues can also be remodeled; the pulsing cells generate a constitutive force and the location of the ratchet mechanism determines which tissue is affected in what way by the force. A recent paper describing Drosophila gastrulation suggested a similar principle mediating the first step of epidermal tissue invagination. There, a stepwise contraction of the apical cell surfaces occurs, which is maintained by a ratchet mechanism that is intrinsic to the cell and prevents cell relaxation after each contraction step (Martin, 2009). It will be interesting to investigate the dynamics of further tissue movements with the appropriate time resolution to see whether this does indeed represent a general mechanism for the directed movement of cell sheets, one of the hallmarks of morphogenesis (Solon, 2009).

    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 this study shows 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 are 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. These 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 (Blanchard, 2010).

    The process of DC relies on the coordination of the elongation of epidermal cells and contraction of the AS. Throughout the early stages of this process the AS cells exhibit fluctuations of their apical area. This fluctuating behaviour is driven by transient actin-myosin accumulations at the apical cortex of cells, involving the assembly, contraction and disassembly of large-scale actin and myosin structures. The formation and disassembly of foci could result from the self-organising properties of myosin and actin and the presence of specific regulators, which are known to spontaneously form active networks, asters and rings in vitro. Tension generated by the contraction of neighbouring cells, the sudden loss of tension due to detachment of actin from the cell membrane, spontaneous catastrophic collapse, and the stretching of the apical membrane resulting in the influx of ions such as calcium, could all contribute to the timing and location of fluctuations (Blanchard, 2010).

    Differences in the absolute and relative strengths of actin-myosin structures in distinct subcellular populations provide an explanation for the patterns that were observed in wild-type and mutant embryos. The observations show that throughout DC the amounts of myosin increase in two subcellular populations: in a cortical ring and in a medial apical network. The amount of myosin at cell-cell junctions determines the shape of cell membranes: low levels lead to wiggly membranes and increasing levels of junctional myosin lead to the straightening of the membranes. The amount of actin-myosin in the medial population determines the fluctuating behaviour of cells: low levels lead to low-frequency fluctuations and no tissue contraction, as seen in the early phase of DC; intermediate levels lead to high-frequency fluctuations and slow tissue contraction, as seen in the slow phase of DC; and high levels lead to a coherent, but dynamic, sheet of actin-myosin across cells that was strongly contractile, as seen in the wild-type fast phase (Blanchard, 2010).

    Increasing the amounts of both junctional and medial myosin in ASGal4/UAS-ctMLCK embryos led to premature apical contraction, precocious straight cell membranes and isotropic cell shapes, which are a signature of high cortical tension. By contrast, in ASGal4/UAS-DiaCA embryos, expressing an activated form of Diaphanous, low junctional myosin levels throughout DC gave rise to wiggly apical membranes. However, these cells showed precocious apical contraction and no discernible apical shape fluctuations. An increase in myosin levels was observed at a sub-apical position, which suggests that in these cells contraction is not only apical but spans the sub-apical and lateral axis. This sub-apical and lateral contraction of AS cells could be an impediment to apical shape fluctuations, as apical contraction must be accommodated by basal or lateral expansion (Blanchard, 2010).

    Overactivating myosin led to apical blebbing in AS cells. Blebbing results from an increase in the ratio of cortical tension to cortex-membrane adhesion and is associated with transient actin-myosin accumulations similar to those observed in this study. No evidence was found for blebs in AS cells in wild-type embryos. It is suggested that dynamic actin-myosin structures are the more general property of cells, which in combination with high cortical tension or weak cortex-membrane adhesion can lead to blebs (Blanchard, 2010).

    Previous results have shown that neighbours oscillate mainly in anti-phase (Solon, 2009). The current results reveal a more subtle picture, with anti-phase correlation predominantly in one orientation and in-phase correlation perpendicular to this. This combination results in interesting patterns, with rows or patches of cells that become synchronised for short periods of time and represent an emergent property of the system. It is expected that some sort of multicellular pattern is inevitable because of the requirement to maintain a coherent epithelium while cells fluctuate. However, the patterns also suggest that cell fluctuations can become entrained locally and for short periods. Analysis of the dynamics of cell shape suggests that cell contraction is the active process, but that fluctuating behaviour of a cell can be influenced and the timing of active contraction altered by the forces generated by immediate neighbours and more distant cells. Thus, the organisation of apical contractions and expansions at the multicellular scale arises from the feedback in both directions between intrinsic cell behaviour and mechanical context (Blanchard, 2010).

    This study was undertaken to understand how changes in actin-myosin behaviour at a subcellular scale resulted in the patterned contraction of the AS. There is a gradual increase in the rate of contraction of individual cells that strongly accelerates with the onset of zippering behaviour. These changes were correlated primarily with a shortening of fluctuation cycle length. The current results suggest that these changes result from an increase in both apical medial and junctional myosin levels. The overall increase in myosin levels and the formation of a continuous actin-myosin network could provide the molecular basis for the transition of the AS to a more solid tissue (Blanchard, 2010).

    What causes the increase in apical myosin levels? One possibility is that it is induced by a chemical or mechanical signal from the epidermis. A radial gradient of fluctuations (Solon, 2009) and of the rate of contraction of AS cells (Gorfinkiel, 2009) suggests that the epidermis is providing some information for the patterned contraction of the AS. However, the analysis of tkv mutants shows that even when the mechanical properties of dorsal-most epidermal cells have been altered and Dpp signalling is compromised, AS cells change their fluctuation behaviour in a similar pattern to the wild type and finally contract. This reveals that several processes that are individually redundant ensure DC. DC could result from an AS-autonomous programme of increasing medial and junctional myosin (and/or actin), through changes in the dynamics of actin and myosin activity, of intracellular trafficking or of cell adhesion. Alternatively, apical myosin might increase as a result of the fluctuating behaviour of AS cells and the build-up of tension due to neighbour contractions. Whatever the mechanism, it is likely that an increase in myosin activity involves activation of the Rho GTPase, which has a central role both in integrating mechanical and structural cues and in regulating myosin-based tension (Blanchard, 2010).

    AS cells fluctuate at low frequencies for a long period during early DC without any tissue contraction. High-frequency fluctuations drive moderate cell and tissue contraction during the slow phase, before they disappear in the transition to rapid tissue contraction. This raises the question of whether fluctuations have a function, as it is at least theoretically possible that contraction could be achieved in a smooth manner. One can speculate that cell fluctuations would be a way to maintain a basic level of cell activity that could be turned easily into morphogenetically relevant behaviours. Cell fluctuations could, more simply, be an epiphenomenon of the self-organised dynamics of actin and myosin. Alternatively, pulsatile contraction might ensure that differences in apical tension are equilibrated between neighbouring cells, ensuring coordination in the contraction of cells across the tissue. This analysis of neighbour relations suggests that fluctuations allow for a certain degree of coordination between cells. A combination of empirical investigation and modelling will be crucial to understand the importance of fluctuations per se during morphogenesis (Blanchard, 2010).

    Temporal and molecular hierarchies in the link between death, delamination and dorsal closure

    One process that occurs during dorsal closure is cell delamination, the seemingly stochastic, rapid apical constriction of cells that culminates in their extrusion from the ectodermal layer. Their number (between 10% and 30%) and position is variable and unpredictable. Extruded cells are engulfed by hemocytes. This behaviour is thought to contribute to up to one-third of the force generated in the amnioserosa for dorsal closure and exhibits a preferential occurrence at the anterior canthus. Its suppression by caspase inhibition has led to the suggestion that ¬Ďapoptosis¬í triggers delamination. This study explored whether cell delamination in the amnioserosa, a seemingly stochastic event that results in the extrusion of a small fraction of cells and known to provide a force for dorsal closure, is contingent upon the receipt of an apoptotic signal. Through the analysis of mutant combinations and the profiling of apoptotic signals in situ, spatial, temporal and molecular hierarchies were establish in the link between death and delamination. Although an apoptotic signal is necessary and sufficient to provide cell-autonomous instructions for delamination, its induction during natural delamination occurs downstream of mitochondrial fragmentation. It was further shown that apoptotic regulators can influence both delamination and dorsal closure cell non-autonomously, presumably by influencing tissue mechanics. The spatial heterogeneities in delamination frequency and mitochondrial morphology suggest that mechanical stresses may underlie the activation of the apoptotic cascade through their influence on mitochondrial dynamics. These results document the temporal propagation of an apoptotic signal in the context of cell behaviours that accomplish morphogenesis during development. They highlight the importance of mitochondrial dynamics and tissue mechanics in its regulation. Together, they provide novel insights into how apoptotic signals can be deployed to pattern tissues (Muliyil, 2011).

    These results establish the necessity and utility of apoptotic signals in driving cellular delamination in the amnioserosa and in patterning the spatiotemporal dynamics of closure. They invoke the induction of pro-apoptotic genes and thus go beyond earlier observations that inferred the role of an apoptotic cascade through the effects of caspase suppression. The results also provide mechanistic insights into the mode of action of the apoptotic cascade by demonstrating cell-autonomous effects of pro-apoptotic genes and caspase activity (DIAP1 overexpression) on the rates of apical constriction. This suggests that apoptotic regulators must regulate cytoskeletal organisation and cell mechanics. A question that arises is whether both classes of regulators function in the linear hierarchy that was delineated or whether functions independent of the apoptotic cascade contribute to their role in driving delamination. The analysis of the molecular hierarchy shows that caspase activation induced by reaper upregulation is a necessary downstream event. Its late activation in delaminating cells, however, raises the issue of whether it is necessary for apical constriction or just for cell extrusion. Although the complete suppression of delamination by p35 overexpression precludes the analysis of constriction rates, this analysis reveals an absence of rosette patterns that characterise delamination rather than the presence of constricted cells that fail to extrude. This suggests that caspase activation must also be necessary for apical constriction. One explanation is that this marked upregulation of the cascade triggers the almost abrupt transition in cell behaviour, characterised by the rapid fall in cell area in a delaminating cell. This is consistent with the higher rates of decrease in cell area with increases in the amounts of caspases/Reaper. Although the phenotypes associated with DIAP1 overexpression also support a role for caspases in cell constriction, caspase-independent functions of DIAP1 have been reported to influence actin organisation in Drosophila border cells. Thus, apoptotic signals must impinge on a distinct set of regulators of the actin cytoskeleton to facilitate apical constriction and tissue contraction. Caspase activation may also regulate adhesion to facilitate extrusion. Indeed, the adherens junction component armadillo/β-catenin is a caspase substrate during cell death in Drosophila and mammals (Muliyil, 2011).

    The results also provide evidence for cell non-autonomous regulation of delamination by components of the apoptotic cascade. Further support for this comes from ongoing observations that caspase inhibition influences actin organisation in the entire amnioserosa. It is speculated that the influence of low undetectable levels of caspase activation not restricted to delaminating cells, regulates tissue mechanics in the amnioserosa and through it also influences cell delamination. The results also show that non-autonomous influences on delamination can originate in the epidermis. Uncovering the molecular players that underlie both autonomous and non-autonomous effects of apoptotic signals on cell behaviour will be interesting avenues to pursue (Muliyil, 2011).

    Temporal and epistatic analysis position mitochondrial fragmentation upstream of the induction of pro-apoptotic genes and caspase activation both during delamination and degeneration. This is the first time that the sequence of propagation of an apoptotic signal has been elucidated in the context of cell behaviour in vivo. Mitochondrial fragmentation is thus the earliest indicator of the cellular commitment to delamination. Other studies have placed mitochondrial fragmentation downstream of the pro-apoptotic genes reaper and hid. What, if not the pro-apoptotic genes, then triggers mitochondrial fragmentation in the amnioserosa? Two recent reports have documented the ability of chemical and radiation injuries to trigger changes in mitochondrial morphology and lead to the induction of apoptosis. An attractive candidate for the trigger in the amnioserosa, consistent with the spatial heterogeneities in delamination frequency and mitochondrial morphology observed, is mechanical stress. Two sets of observations support this. First, not all cells that overexpress pro-apoptotic genes delaminate, and the anterior predominance of such events is maintained. This suggests that although an apoptotic signal is necessary, it must cooperate with other permissive signals to accomplish delamination. Second, the studies of native dorsal closure uncover spatial heterogeneities in mitochondrial morphology. Two features characterise this heterogeneity: (1) the early abundance of cells with predominantly fragmented mitochondria in the anterior AS and (2) their delayed transition to tubular/hyperfused morphologies prior to degeneration compared with the posterior. It has been suggested in a different context that low levels of chemical stress can induce hyperfusion as a means of countering stress (through optimisation of mitochondrial ATP production), whereas higher magnitudes of stress lead to fragmentation and apoptosis. A similar reasoning (with the substitution of chemical stresses by mechanical stresses) might underlie the spatial heterogeneities in delamination frequency. Specifically, high magnitudes of stress locally (from head involution) might be responsible for increased mitochondrial fragmentation and subsequent delamination, whereas prolonged lower levels of stresses (from the leading edge) may drive hyperfusion and subsequent degeneration of the amnioserosa. Adhesion anisotropies resulting from differences in the substrate (yolk anteriorly and hindgut posteriorly) could additionally contribute to the force anisotropies between the anterior and posterior amnioserosa (Muliyil, 2011).

    Taken together, the results reveal that apoptotic regulators contribute multiple forces to dorsal closure. In the amnioserosa, they act locally to drive delamination but also globally to maintain tissue tension. The latter is attributed to the low levels of caspase activation and pro-apoptotic gene induction. This provides a permissive environment for mitochondrial fragmentation and the subsequent marked upregulation of the cascade in delaminating cells. Additionally, they contribute to forces generated in the epidermis. This is best inferred from anti-apoptotic perturbations. In hid mutants, the rates of dorsal closure are higher despite the absence of delaminations in the amnioserosa. Conversely, delamination in the amnioserosa is 'upregulated' when either caspases or hid is downregulated in the epidermis, but their effects on closure rates are different. These non-autonomous effects must reflect the feedback regulation of forces generated in the epidermis and in the amnioserosa. That multiple forces contribute to dorsal closure and can feedback regulate each other has been long appreciated. These studies identify apoptotic signals as crucial regulators of the balance of forces that drive dorsal closure. Uncovering the basis for feedback regulation and the force hierarchies that lend dorsal closure resilience will be interesting. A recent study reported on a novel, non-apoptotic role for an epidermal caspase, caspase 8: its effect on interleukin signalling resulted in the recapitulation of a wound healing response when deleted in the skin. In light of the above observations, it is interesting that in this analysis of dorsal closure, which recapitulates wound closure, some perturbations that suppress apoptosis also resulted in accelerated closure (Muliyil, 2011).

    These explorations demonstrate the primacy of mitochondrial fragmentation in the induction of apoptotic signalling and uncover the complex relationships between death signals, delamination and dorsal closure. Furthermore, they illustrate how an apoptotic signal is deployed multiple times in the same tissue to accomplish heterogeneity in cell behaviour and have helped identify some of the cellular properties they modulate. Understanding the triggers for mitochondrial fragmentation and the precise outcomes and mechanisms of apoptotic signals on cell biological attributes of delaminating cells will be interesting avenues to explore (Muliyil, 2011).

    Talin autoinhibition is required for morphogenesis

    The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that could autoinhibit its activity. However, the functional consequence of such an interaction has not been previously explored in vivo. This study demonstrates that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, evidence is presented that talin autoinhibition is regulated by Rap1-dependent signaling. Based on these data, it is proposed that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis (Ellis, 2013).

    Overall, this study identifies an important role for the regulation of talin function through autoinhibition. Failure to autoinhibit talin impairs morphogenetic processes, but this is not due to defects in integrin-mediated attachment to the ECM or in the assembly of the adhesion complex. Thus, it is unlikely that the FERM domain mutation E1777A, which completely blocks autoinhibition, blocks integrin-mediated cell-ECM attachment in a dominant-negative fashion. An alternative explanation for the phenotype is that the E1777A mutant behaves like a gain-of-function allele of talin and that the morphogenetic defects that were observe are due to too much rather than too little adhesion. This would not be the first time such a phenomenon has been observed; for example, overexpression of integrins in either the wing or the muscle gives rise to phenotypes identical to those found in integrin-null mutants. How could the E1777A mutation give rise to stronger adhesion? It was shown that this mutation enhances the recruitment and colocalization of talin and integrin at sites of adhesion. Importantly, it was shown that the E1777A mutation effectively reduces talin turnover at sites of adhesion. Indeed, the data fit with a gain-of-function model: blocking talin autoinhibition leads to increased integrin-mediated adhesion, and this impairs morphogenetic processes that require cyclic adhesion assembly and disassembly. Further consistent with this model is the observation that adhesion at myotendinous junctions (MTJs), a non-morphogenetic context, is not perturbed upon blocking autoinhibition of talin. The possibility cannot be excluded that E1777A may confer its effect on talin function through a means other than disruption of autoinhibition. Encouragingly, however, homology modeling and NMR analyses strongly suggest that the fly protein behaves much as the mammalian homolog does (Ellis, 2013).

    How does prevention of autoinhibition stabilize integrin-mediated adhesion? This study shows that autoinhibition regulates talin recruitment to adhesions through a RIAM-Rap1-dependent mechanism. Interestingly, the E1777A autoinhibition mutant talin is more strongly recruited to adhesions than WT talin; this enhanced recruitment occurs independent of RIAM-Rap1 activity. Thus, it is possible that constitutive relief of autoinhibition works to stabilize and promote adhesion by enhancing recruitment of the talin molecule to adhesions, thus bypassing the need of the RIAM-Rap1 pathway for recruitment. At the membrane, adhesion strengthening may occur via talin's scaffolding function, as talin can interact with multiple components of the integrin adhesion complex IAC, and these interactions may increase and/or change when talin assumes a more extended conformation. Another possibility, consistent with structural studies, is that relief of autoinhibition frees up the FERM/IBS-1 domain of talin such that it can activate integrins. It is predicted that mutations in talin that block IBS-1-mediated integrin activation would lead to more dynamic adhesions, and this is indeed what was observed. According to the model, talin recruitment is determined by the sum of interactions that a single molecule can make with other IAC components at any one time. For example, the autoinhibited form of talin relies on Rap1/RIAM for efficient recruitment, even though it may still bind integrin through its free IBS-2 domain; both mechanisms may contribute to targetting of talin to adhesions. It is speculated that relief of autoinhibition makes the IBS-1 available, as well as the many other binding sites for IAC components that are found in the talin rod domain (e.g., vinculin binding sites), thereby substantially increasing the number of possible interactions that can lead to talin recruitment to the IAC (Ellis, 2013).

    There are likely to be multiple avenues leading to relief of talin autoinhibition. Recent superresolution studies provided elegant evidence that autoinhibition is primarily relieved within adhesion complexes, implicating the need for a mechanism to specifically recruit autoinhibited talin to adhesions. This study has showm that forcing talin to remain in an open, nonautoinhibited conformation gives rise to very similar phenotypes as activating the RIAM-Rap1 pathway (RIAM is Rap1 interacting adaptor protein). Based on the results, it is proposed that RIAM-Rap1 brings autoinhibited talin to the membrane where autoinhibition can subsequently be relieved, possibly through electrostatic interactions with the membrane/ PIP2. RIAM-Rap1 has a previously established role in mediating the recruitment of talin to sites of adhesion, but it has recently been demonstrated that the requirement for RIAM-Rap1 is context dependent. Structural and biochemical studies have revealed that the binding of talin to either RIAM or vinculin is mutually exclusive and likely dependent on force. Moreover, in cell culture, vinculin-stimulated integrin activation is RIAM-Rap1 independent, raising the possibility that more mature adhesions might not need RIAM-Rap1 to promote talin activation in this case. Along similar lines, this study demonstrated that RIAM-Rap1 activity is dispensable for recruitment of a nonautoinhibited talin molecule (Ellis, 2013).

    In summary, the results suggest that talin autoinhibition confers a switch through which fine control of integrin-mediated adhesion can be exerted in vivo. The findings also reveal RIAM-Rap1-mediated regulation of integrin adhesion is an important modulator of morphogenesis, and evidence is provided for an autoinhibition-based pathway for control of talin function through RIAM-Rap1. Furthermore, this study exemplifies how subtle tuning of adhesion complex composition and stability elicits different adhesive functions and cellular behaviors during development (Ellis, 2013).

    Bazooka inhibits aPKC to limit antagonism of actomyosin networks during amnioserosa apical constriction

    Cell shape changes drive tissue morphogenesis during animal development. An important example is the apical cell constriction that initiates tissue internalisation. Apical constriction can occur through a phase of cyclic assembly and disassembly of apicomedial actomyosin networks, followed by stabilisation of these networks. Delayed negative-feedback mechanisms typically underlie cyclic behaviour, but the mechanisms regulating cyclic actomyosin networks remain obscure, as do mechanisms that transform overall network behaviour. This study shows that a known inhibitor of apicomedial actomyosin networks in Drosophila amnioserosa cells, the Par-6-aPKC complex, is recruited to the apicomedial domain by actomyosin networks during dorsal closure of the embryo. This finding establishes an actomyosin-aPKC negative-feedback loop in the system. Additionally, aPKC was found to recruit Bazooka to the apicomedial domain, and phosphorylates Bazooka for a dynamic interaction. Remarkably, stabilising aPKC-Bazooka interactions can inhibit the antagonism of actomyosin by aPKC, suggesting that Bazooka acts as an aPKC inhibitor, and providing a possible mechanism for delaying the actomyosin-aPKC negative-feedback loop. These data also implicate an increasing degree of Par-6-aPKC-Bazooka interactions as dorsal closure progresses, potentially explaining a developmental transition in actomyosin behaviour from cyclic to persistent networks. This later impact of aPKC inhibition is supported by mathematical modelling of the system. Overall, this work illustrates how shifting chemical signals can tune actomyosin network behaviour during development (David, 2013).

    These data outline a regulatory circuit for guiding amnioserosa apical constriction. The circuit controls both the localisation and activity of its components. In terms of protein localisation, it was found that amnioserosa actomyosin networks recruit the Par proteins to the apicomedial domain. Although Par protein puncta are not continually dependent on the actomyosin networks, their numbers build over developmental time, apparently owing to the cumulative effect of multiple rounds of actomyosin network assembly. The networks appear to impact aPKC directly, and in turn, aPKC recruits Baz to the apical domain. This recruitment depends on the C-terminal aPKC-binding region of Baz, which aPKC phosphorylates for a dynamic relationship with Baz in the apical domain of amnioserosa cells (David, 2013).

    Par-6-aPKC activity inhibits amnioserosa actomyosin networks (David, 2010), and the recruitment of aPKC by the networks implicates a negative-feedback loop. As delayed negative feedback tied to a continual input signal can produce an oscillatory output, the actomyosin-aPKC negative-feedback loop might explain how aPKC regulates actomyosin network assembly-disassembly cycles (David, 2010). However, apical populations of Par-6-aPKC puncta are not fully recruited and fully removed with each actomyosin cycle, suggesting additional mechanisms. Importantly, Par-6-aPKC activity can be tempered by Baz. Thus, aPKC inhibition by Baz might delay the actomyosin-aPKC negative-feedback loop during early DC, promoting the actomyosin assembly-disassembly cycles. As DC proceeds, the additive effects of actomyosin assembly-disassembly cycles could increase apical Par protein levels; additionally, the gradual apical constriction of the cells decreases their apical surface areas and could thus increase apical surface Par protein concentrations. It is proposed that a gradual increase to apicomedial aPKC-Baz interactions inhibits aPKC and thus leads to the stabilisation of actomyosin networks. Simulations indicate that this transition in network behaviour can occur abruptly following incremental reductions to myosin inhibition during earlier DC. It is proposed that Baz acts as a competitive inhibitor to reduce aPKC phosphorylation of cytoskeletal regulators. This idea is consistent with reports of Par-3 inhibiting aPKC in kinase assays in vitro. However, Baz is also known to promote aPKC localisation in the epidermis and amnioserosa. Thus, Baz appears to both promote and inhibit aPKC activity, potentially forming a paradoxical circuit (or incoherent feed-forward loop) in which Baz and aPKC promote each other's recruitment, and in which Baz competitively inhibits aPKC activity. Significantly, Baz has multiple binding sites for the Par-6-aPKC complex [Par-6 binds Baz PDZ1; aPKC binds Baz PDZ2-3; aPKC binds the Baz C-terminal aPKC-binding region], suggesting cooperative binding and that Baz interactions with the Par-6-aPKC complex are stronger than those between the Par-6-aPKC complex and its cytoskeleton targets. Notably, this study found that Baz apical surface levels are ~66% lower than those of Par-6, suggesting that the inhibitory effect of Baz must be dynamic; Baz cannot simply sequester all Par-6-aPKC complexes by outnumbering them. The inhibitory effect must also depend on phosphatases because aPKC interactions with Baz are weakened following phosphorylation (Morais-de-Sá, 2010). Baz/Par-3 is known to be regulated by Protein phosphatase 1 and Protein phosphatase 2A with Protein phosphatase 1 de-phosphorylating the aPKC phosphorylation site of Par-3. Thus, Baz may act as a strong and dynamic inhibitor of Par-6-aPKC to buffer and eventually overcome the actomyosin-aPKC negative-feedback loop (David, 2013).

    A crucial unknown is the identity of the cytoskeletal target(s) of aPKC. Cytoskeletal targets of aPKC have been identified but have not been examined during amnioserosa apical constriction. In mammalian cells, Par-6-aPKC can phosphorylate Smurf1, an E3 ubiquitin ligase, in turn leading to RhoA degradation in cellular protrusions (Wang, 2003). During dendritic spine morphogenesis, Par-6-aPKC acts though p190RhoGAP to inhibit RhoA (Zhang, 2008). As well, aPKC phosphorylation of Rho kinase leads to its cortical dissociation in mammalian cell culture (Ishiuchi, 2011), and apparently during salivary gland tubulogenesis in Drosophila (Röper, 2012). Of note, the persistent Par-6-aPKC puncta could actively downregulate actomyosin activity, or prolong the lull between actomyosin activations, or do both. Another question is how actomyosin networks recruit aPKC. The recruitment of Par proteins by actomyosin networks has been documented during Drosophila cellularisation and C. elegans one-cell polarisation, and Baz and aPKC have been shown to co-immunoprecipitate with myosin regulatory light chain from Drosophila egg chambers, but specific linkages have yet to be identified. Defining further components of the actomyosin-aPKC negative-feedback loop will be crucial for understanding its regulation and its effects on actomyosin network dynamics. In particular, despite identifying a potential delay mechanism for the loop, it is unclear how the loop and the delay mechanism could translate into oscillatory network behaviour. Perhaps the cytoskeletal target(s) of aPKC are co-recruited with the assembling networks, which in combination with the buffering effect of Baz, could delay their phosphorylation by aPKC. It is also possible that the clustering of Par protein puncta with each network assembly event could somehow modify the Baz buffering effect (David, 2013).

    Another unanswered question is the influence of circumferential anchors for Baz or Par-6-aPKC, as weakening of these anchors could contribute to apicomedial Par protein accumulation over DC. Echinoid (Ed), a transmembrane AJ-associated protein that can directly bind Baz, is normally lost from the amnioserosa during DC. It is hypothesised that this loss might promote the loss of Baz from AJs and its apicomedial accumulation. However, ectopic expression of Ed in the amnioserosa leading to circumferential Ed levels higher than those seen in the epidermis had no apparent effect on apicomedial Baz localisation. Thus, differences in Ed expression alone cannot account for the differential localisation of Par proteins between the amnioserosa and epidermis. It is possible that the effects of actomyosin can overpower ectopic Ed, or that other changes to the apical circumference of amnioserosa cells are involved. More generally, other Par protein interaction partners should be considered. For example, Baz and Stardust also interact and, together with Crumbs and Patj, they form the apical Crumbs complex (Tepass, 2012). Recent results suggest Patj can activate myosin by suppressing myosin light chain phosphatase. Intriguingly, amnioserosa BazS980A apical surface puncta also recruit Patj, suggesting that this pathway might contribute to myosin activity as well (David, 2013).

    In summary, the data argue that the differential regulation of amnioserosa actomyosin networks by Baz and Par-6-aPKC can be explained by a single pathway in which Baz inhibits Par-6-aPKC antagonism of the cytoskeletal networks. It was also found that the actomyosin networks recruit aPKC, forming a negative-feedback loop. It is proposed that the inhibition of aPKC by Baz delays the negative feedback at earlier DC for cycling actomyosin networks, and with increased inhibition of aPKC by later DC, the actomyosin networks persist. These findings provide an example of how chemical signalling, and changes to this signalling, can modify the behaviour of actomyosin networks during embryo development (David, 2013).

    High plasticity in epithelial morphogenesis during insect dorsal closure
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    Insect embryos complete the outer form of the body via dorsal closure (DC) of the epidermal flanks, replacing the transient extraembryonic (EE) tissue. Cell shape changes and morphogenetic behavior are well characterized for DC in Drosophila, but these data represent a single species with a secondarily reduced EE component (the amnioserosa) that is not representative across the insects. This study examined DC in the red flour beetle, Tribolium castaneum, providing the first detailed, functional analysis of DC in an insect with complete EE tissues (distinct amnion and serosa). Surprisingly, it was found that differences between Drosophila and Tribolium DC are not restricted to the EE tissue, but also encompass the dorsal epidermis, which differs in cellular architecture and method of final closure (zippering). EE tissue complement was experimentally manipulated via RNAi for Tc-zen1, eliminating of the serosa and allow examination of viable DC in a system with a single EE tissue (the amnion). It was found that the EE domain is particularly plastic in morphogenetic behavior and tissue structure. In contrast, embryonic features and overall kinetics are robust to Tc-zen1(RNAi) manipulation in Tribolium and conserved with a more distantly related insect, but remain substantially different from Drosophila. Although correct DC is essential, plasticity and regulative, compensatory capacity have permitted DC to evolve within the insects. Thus, DC does not represent a strong developmental constraint on the nature of EE development, a property that may have contributed to the reduction of the EE component in the fly lineage (Panfilio, 2013; full text of article).

    Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

    Ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. Ca2+ was found to be sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca2+ leads to cell contraction. Furthermore, endogenous Ca2+ fluxes correlate with cell contraction in the amnioserosa (AS) during closure, whereas the chelation of Ca2+ slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca2+ and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. Two channel subunits, ripped pocket and dtrpA1 (TrpA1), were identified that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. These results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo (Hunter, 2014).

    These data provide evidence that ion channels function in closure to regulate ion flux in individual cells in the AS and leading edge (LE), leading to Ca2+-dependent cell contractility. Intracellular ion flux via mechanically gated ion channels (MGCs) can promote cytoskeletal and junction organization in cell culture. The findings that localization of the Ca2+ reporter C2:GFP correlates with AS cell contraction and that elevated Ca2+ induces AS cell contraction (via uncaging NP EGTA AM or spontaneous flashes) support a role for Ca2+-dependent contractility in closure. Nevertheless, the observed correlation between perimeter shortening (contraction) and increases in free Ca2+ is by no means perfect. It is hypothesized that there are several reasons for this lack of tight correlation. First, although the data suggest that Ca2+ plays a role in regulating actomyosin contractility, there are other regulators of this important process, and small GTPases (especially Rac and Rho) are sure to play a regulatory role. Ca2+ signaling must be integrated into the context of other signaling pathways and programs of gene expression regulating morphogenesis. Second, individual cell behavior must be considered in the context of the AS cell sheet, in which the behavior of a cell perimeter is profoundly influenced by the behavior of the cells to which it is attached. It is possible that passive perimeter shortening on one side of a given cell is actively driven by contractility in its neighbor. Finally, two-dimensional changes in cell shape, as observed in a given optical section or series of optical sections, must be considered in the context of the three-dimensional nature of cells. It is hypothesized that cell volume does not fluctuate rapidly because of the relative incompressibility of cellular constituents and because the cell does not rapidly lose or gain volume. Thus, cell volume acts as a buffer and changes in crosssectional area (e.g., measured at the level of junctional belts) may be the consequence of contractile activities functioning elsewhere in the cell. Complete understanding of how MGCs and Ca2+-mediated contraction are integrated into cellular homeostasis and morphogenesis requires a more complete picture of how other signaling pathways contribute to changes in cell shape. Moreover, it will require more complete imaging sets, with higher temporal and spatial resolution, of the three-dimensional changes that occur during morphogenesis, even in relatively simple morphogenetic movements such as dorsal closure. The advent of new biosensors and high-speed imaging techniques place the technologies required for such investigations of morphogenesis within the realm of possibility (Hunter, 2014).

    GsMTx4 is the most specific pharmacological reagent for manipulating MGC activity in vitro and in vivo, and this study reports its use during Drosophila embryogenesis. Acute, bimodal effects of GsMTx4 on closure are consistent with the presence of MGCs that ultimately pass Ca2+ ions. At the microM concentrations of GsMTx4 experienced by cells at or near the site of injection, increases in cellular free Ca2+ are followed by constriction over the course of tens of seconds. By contrast, the long-term effects of low concentrations of GsMTx4, which cells experience after the bolus of peptide diffuses away from the injection site, appear to be inhibition of closure via the failure of key actomyosin structures and activities. It is hypothesized that GsMTx4 affects MGC activity by modifying the thickness or curvature of the lipid bilayer in which these channels are embedded, consistent with known mechanisms of GsMTx4 action. Studies in cell culture demonstrate that loss of MGC function by pharmacological inhibitors or targeted mutations in channel subunits leads to defects in actomyosin contractile behaviors. Nevertheless, it cannot be ruled out that possible indirect effects of MGC inhibition obscure specific and direct long-term effects of MGC inhibition (e.g. the effect of membrane thickness and curvature on non-MGC membrane proteins during development or secondary consequences of inhibiting RPK and dTRPA1) (Hunter, 2014).

    Long-term phenotypes due to GsMTx4-mediated MGC inhibition are recapitulated by RNAi expression or mutational analysis that disrupt the function of specific channels. Congenital loss of channel expression is a long-term effect, and disrupting expression of rpk or dtrpA1 in embryos leads to closure defects. Discrepancies in phenotypes may be the consequence of multiple channels functioning in closure or to differences in the timing and pattern of knockdown or inhibition. Whereas in the current experiments, the expression knockdown of a single channel subunit tissue specifically (via RNAi) or in the embryo as a whole prior to closure (via mutant allele), an advantage of pharmacological inhibitors is acute delivery before or during closure. Indeed, phenotypes were observed consistent with genetic knockdown when GsMTx4 was knocked down prior to closure: defects in AS shape, canthus and purse string formation and failure to close. It is speculated that the embryo can compensate for the congenital loss of a single channel subunit (as in the case of dtrpA1) in ways not possible when drug is applied acutely or when RNAi knocks down channel function (less acutely than the drug, more acutely than inherited mutant alleles). The regulation of contractility via ion channels during closure appears to be both cell-autonomous and non-cell-autonomous (Hunter, 2014).

    Specifically, the loss of leading edge cell elongation and their purse strings when channel subunits are targeted in the AS indicates that robust channel activity in the AS is required for normal cell shape changes in both the AS (i.e. cell-autonomous) and in leading edge cells (i.e. non-cell-autonomous). Actomyosin contractility during closure can act non-cell-autonomously, implicating positive reinforcement of force-producing activities or structures between and within embryonic tissues: clones of cells expressing myosin because of a transgenic mosaic effect contract (cell-autonomous effect) but stretch neighboring cells (non-cell-autonomous effect). It is hypothesized that channel activity contributes to tension at a single-cell level in the AS, and that tension in the AS, exerted on the LE, is required for wild-type actomyosin-dependent structures and cell shapes in the leading edge of the LE (Hunter, 2014).

    Verification of a mechanical circuit(s) regulated by MGCs requires that which channels are involved can be unequivocally established, and the gating mechanisms of each channel(s) be determined in the embryonic epithelia. The sensitivity of DEG/ENaC and TRPA1 homologs to applied force has been studied in other systems, but is unknown for dorsal closure tissues. Future studies should include electrophysiological recordings, but such methods have not yet been developed for analysis of Drosophila embryonic epithelial cells. These studies could be key for understanding how a Na+-permeable channel (RPK) contributes to Ca2+ flux. Although its ability to conduct Ca2+ or associate with Ca2+ channel subunits is unknown, RPK is involved in Ca2+-dependent processes such as Drosophila oocyte activation and the response to gentle touch in larvae . This study implicates ion flux and MGCs in the molecular mechanisms that regulate closure. Force sensing by MGCs could constitute a rapid means of affecting cell behaviors in order to adapt to acute changes during closure. For example, at the level of apical junctions, individual AS cells change shape dramatically, whereas the overall area of the AS decreases slowly and monotonically. Based on the current observations, it is hypothesized that MGCs function in a mechanical circuit(s) to coordinate forces across the embryo. Similar feedback loops are proposed for the oscillatory behavior of other mechanically coupled, contractile cells. Given that morphogenesis throughout Drosophila development requires the assembly and regulation of force-producing structures, it will be interesting to determine how other morphogenetic processes are affected by channel inhibition (Hunter, 2014).

    The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development

    Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization, and can stimulate filopodia in cultured cells. However, redundancy in mammals and the Diaphanous role in cytokinesis limit analysis of whether and how they regulate protrusions during development. This study used two tissues driving Drosophila dorsal closure, migratory leading-edge (LE) and non-migratory amnioserosal (AS) cells, as models to define how cells shape distinct protrusions during morphogenesis. Non-migratory AS cells were found to produce filopodia that are morphologically and dynamically distinct from those of LE cells. It was hypothesized that differing Enabled and/or Diaphanous activity drive these differences. Combining gain- and loss-of-function with quantitative approaches revealed Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative 'fingerprint', the protrusive profile, which the data suggest is characteristic of each actin regulator. The data suggest LE protrusiveness is primarily Enabled-driven, while Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence (Nowotarski, 2014).

    Pathway to a phenocopy: Heat stress effects in early embryogenesis

    Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants - having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from non-specific heat stress to phenocopied abnormalities is unknown. Drosophila embryos subjected to 30-min, 38- degrees C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 microm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. It is concluded that the pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity - i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types (Crews, 2015).

    Emergent material properties of developing epithelial tissues

    This study measures the mechanical properties of epithelial cells during dorsal closure. The relationship between apicomedial myosin fluorescence intensity and strain during fluctuations is shown to be consistent with a linear behaviour. Myosin fluorescence intensity was used as a proxy for active force generation. This study established 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 these findings with those from previous laser ablation experiments, it was shown that both apicomedial and junctional stress also increase over time, with the relative increase in apicomedial stress approximately twice that of other obtained measures. These 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 it is suggested 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 (Machado, 2015).

    Quantifying dorsal closure in three dimensions

    Dorsal closure is an essential stage of Drosophila embryogenesis and is a powerful model system for morphogenesis, wound healing, and tissue biomechanics. During closure two flanks of lateral epidermis close an eye-shaped dorsal opening that is filled with amnioserosa. The two flanks of lateral epidermis are zipped together at each canthus ("corner" of the eye). Actomyosin-rich purse strings are localized at each of the two leading edges of lateral epidermis ("lids" of the eye). This study reports that each purse string indents the dorsal surface at each leading edge. The amnioserosa tissue bulges outward during the early-to-mid stages of closure to form a remarkably smooth, asymmetric dome indicative of an isotropic and uniform surface tension. Internal pressure of the embryo and tissue elastic properties help shape the dorsal surface (Li , 2016).

    Cell boundary elongation by non-autonomous contractility in cell oscillation

    This study explored the dynamics of the amnioserosa, which is known to exhibit cell shape oscillation, as a model system to study the subcellular-level mechanics that spatiotemporally evolve during Drosophila dorsal closure. It was shown that cell boundary elongation occurs through a combination of a non-autonomous active process and an autonomous process. The former is driven by a transient change in the level of non-muscle myosin II in the neighboring cells that pull the vertices, whereas the latter is governed by the relaxation of junctional tension. By monitoring cell boundary deformation during live imaging, junctional tension at the specific phase of cell boundary oscillation, e.g., contraction or elongation, was probed by laser ablation. Junctional tension during boundary elongation is lower than during the other phase of oscillation. The tension measurements were extended to non-invasively estimate a tension map across the tissue, and a correlation between junctional tension and vinculin dynamics at the cell junction was found. The study proposes that the medial actomyosin network is used as an entity to both contract and elongate the cell boundary. Moreover, these findings raise a possibility that the level of vinculin at the cell boundary could be used to approximate junctional tension in vivo (Hara, 2016).

    The actin cable is dispensable in directing dorsal closure dynamics but neutralizes mechanical stress to prevent scarring in the Drosophila embryo

    The actin cable is a supracellular structure that embryonic epithelia produce to close gaps. However, the action of the cable remains debated. This study has addressed the function of the cable using Drosophila dorsal closure, a paradigm to understand wound healing. First, the actin cytoskeleton scaffold protein Zasp52 was shown to be specifically required for actin cable formation. Next, Zasp52 loss of function to dissect the mechanism of action of the cable. Surprisingly, closure dynamics are perfect in Zasp52 mutants: the cable is therefore dispensable for closure, even in the absence of the amnioserosa. Conversely, it was observed that the cable protects cellular geometries from robust morphogenetic forces that otherwise interfere with closure: the absence of cable results in defects in epithelial organization that lead to morphogenetic scarring. It is proposed that the cable prevents morphogenetic scarring by stabilizing cellular interactions rather than by acting on closure dynamics (Ducuing, 2016).

    Basal cell-extracellular matrix adhesion regulates force transmission during tissue morphogenesis
    Tissue morphogenesis requires force-generating mechanisms to organize cells into complex structures. Although many such mechanisms have been characterized, little is known about how forces are integrated across developing tissues. This study provides evidence that integrin-mediated cell-extracellular matrix (ECM) adhesion modulates the transmission of apically generated tension during dorsal closure (DC) in Drosophila. Integrin-containing adhesive structures (see Myospheroid) resembling focal adhesions were identified on the basal surface of the amnioserosa (AS), an extraembryonic epithelium essential for DC. Genetic modulation of integrin-mediated adhesion results in defective DC. Quantitative image analysis and laser ablation experiments reveal that basal cell-ECM adhesions provide resistance to apical cell displacements and force transmission between neighboring cells in the AS. Finally, the study provides evidence for integrin-dependent force transmission to the AS substrate. Overall, these data indicate that integrins regulate force transmission within and between cells, thereby playing an essential role in transmitting tension in developing tissues (Goodwin, 2016).

    During morphogenesis, cells undergo complex rearrangements to generate tissues and organs. Forces generated through the actin cytoskeleton and transmitted through cell-cell adhesion receptors govern the changes in cell shape and position that drive morphogenetic events. Interactions between cells and their extracellular environment, mediated by cell to extracellular matrix (ECM) adhesions, also play an important role in establishing appropriate tissue mechanics during development (Goodwin, 2016).

    Integrin heterodimers are the main family of cell-ECM adhesion receptors in metazoans. They are composed of an α and a β subunit and connect to the actin cytoskeleton through an intracellular adhesion complex comprising many adapter and signaling proteins that modulate integrin receptor function. Understanding of integrin biology has largely been obtained from study of focal adhesions (FAs), prominent integrin-mediated adhesion structures observed in 2D cell culture. These studies have demonstrated that integrins can be regulated not only by biochemical signals but also by extracellular and/or intracellular mechanical cues. In FAs, integrins are key regulatory hubs for both sensing and responding to changes in the mechanical properties of the cellular microenvironment; this function impinges greatly on cell behavior. Translating the insights generated from the study of FAs in 2D culture into 3D models has proved to be contentious because FAs do not form as readily in 3D, and by some accounts do not form at all. Therefore, identification of contexts in intact living organisms in which FAs form and affect cell behavior is of great interest for corroborating and building upon mechanisms delineated in cell culture (Goodwin, 2016).

    Studies of morphogenetic movements that occur during Drosophila development have provided many insights into the role of mechanical forces in shaping developing tissues, as well as elucidation of the molecular mechanisms that underlie tissue biomechanics. One such process, dorsal closure (DC), is a well-studied integrin-dependent morphogenetic event that occurs midway through fly embryogenesis to create a continuous epidermal sheet over the dorsal surface of the embryo. An extraembryonic epithelium called the amnioserosa (AS) contracts and ingresses, allowing the lateral epidermis from opposing sides of the embryo to migrate toward the dorsal midline. Integrin adhesion complexes form in both tissues, and are thought to carry out a variety of functions. In particular, integrins mediate attachment between the AS and the underlying yolk cell through adhesion to a layer of laminin-rich ECM; disruption of laminin phenocopies loss of integrin function, suggesting that integrin-dependent cell-ECM adhesion is required for this process. However, the specific role of integrins in the regulation of tissue biomechanics during DC has not been addressed. Since integrins are known to transduce traction forces that are generated by actomyosin networks in spreading and migrating cells, it is possible that they may play a similar role in force transmission during DC (Goodwin, 2016).

    The role of mechanical forces driving DC is well established. Forces originating within the AS alone are sufficient to drive tissue closure (Wells, 2014). AS contractile forces are generated through cell death and extrusion, which reduces AS surface area, as well as by apical ratcheting, generated by coordinated pulses of medial actomyosin networks. Ratcheting drives apical oscillations and constriction of individual AS cells and generates tension in the apical plane of the tissue. Mechanical forces generated in the leading-edge epidermis also contribute to closure. A contractile actin cable assembles at the leading edge of the epidermis and forms a purse-string-like structure that generates tension to help contract the hole. Cells in the leading-edge epidermis also elongate to provide a counteractive pulling force away from the dorsal midline. Later in closure, projections formed at the leading edge participate in zippering, a process hypothesized to provide additional pulling forces for closure. The essential requirement for forces contributed from the AS versus the leading edge remains a somewhat contentious issue; it is likely an optimized balance of forces originating from both tissues that leads to efficient DC. However, in neither tissue has the question of how forces are transmitted and coordinated between cells been addressed. Cell-ECM adhesions have been shown to regulate the balance between force transmission across cells and to the substrate in culture models. Given the precise spatiotemporal patterning of forces required for DC and the known requirement for integrins in the AS, it was hypothesized that integrins may act in a similar capacity during DC to facilitate controlled propagation of forces generated across the apical plane of the tissue between neighboring cells (Goodwin, 2016).

    By focusing on subcellular regulation of integrins in the AS, and through a combination of quantitative live imaging, genetics, and biophysical approaches, this study identified adhesive structures and describes a role for cell-ECM adhesion in regulating local cell displacements in the AS. Perturbation of integrin function was found to lead to changes in force transmission across the apical plane of cells. Furthermore, evidence is provided for integrin-dependent mechanical coupling between the AS and its substrate. Based on these results, it is proposed that the apical pole of AS cells is tethered to the substrate via apical-basal mechanical coupling and cell-ECM adhesions; thus, apically generated forces that drive cell oscillations are subject to passive resistance from basal adhesions to the ECM. Finally, evidence is provided that tethering at cell-ECM adhesions may resist large-scale cell displacements during tissue contraction in DC. These findings highlight the importance of integrin-mediated coordination of mechanical forces during collective cell behaviors that drive tissue morphogenesis (Goodwin, 2016).

    This work uncovers an essential role for integrin-dependent cell-ECM adhesion in the coordination of forces during tissue morphogenesis. It significantly enhances understanding of how integrin function in the AS contributes to DC. Specifically, deviations in either direction from wild-type levels of cell-ECM adhesion are sufficient to disrupt DC. Through identification of focal adhesion-like structures (FALS) and characterization of their morphology and behavior, subcellular insights were gained into how cell-ECM adhesions regulate displacement of AS cells. Furthermore, evidence is provided that FALS morphology dictates tissue response to ablation, implicating cell-ECM adhesion in the efficiency of apical force transmission across AS cells. Genetic perturbation of cell-ECM adhesion leads to altered force transmission and, consequently, tissue response to ablation and cell displacement are misregulated. Finally, evidence is provided for integrin-dependent force transmission to the ECM by demonstrating that cell-substrate coupling is lost in the absence of FALS. Therefore, it is concluded that cell-ECM adhesion plays a critical role in modulating the transmission of cell-generated forces between neighboring cells and to the substrate (Goodwin, 2016).

    This study identifies FALS, integrin-dependent adhesive structures that in many ways resemble FAs. In particular, like FAs, FALS are force responsive. Previous studies have demonstrated that integrins play a conserved role in force sensing in Drosophila ( Pines, 2012). Since the AS is a morphogenetically active epithelial tissue, FALS represent a particularly attractive system in which to study FAs in the context of an intact, developing organism. Links between FAs and cell behavior are well studied in cell culture models. In the AS, cells exhibit non-directional mobility as a result of neighboring cell deformation, and the extent to which they move is negatively correlated with the amount of cell-ECM adhesion. However, the motion that was observe is distinct from crawling cell behaviors that are used to generate directed cell migration in cell culture. This analysis of cell-substrate mechanical coupling shows that substrate movement is positively correlated with cell movement, suggesting that the substrate is deformed in the same direction as cell movement. This is the opposite of what is observed in crawling cells, where cells pull themselves over the substrate, deforming the substrate in the opposite direction to cell movement (Goodwin, 2016).

    The data support the hypothesis that cell-ECM adhesion plays a key role in transmitting tension between AS cells, since tissue response to laser-induced release of tension was perturbed when cell-ECM adhesion was modulated. This finding is in agreement with recent work which showed that clusters of cells in culture exhibit greater correlation between cell-cell forces in opposing cell junctions and, therefore, more force transmission across cells when integrin-mediated adhesion is downregulated (Ng, 2014). Cell culture models have also been used to examine the relationship between traction exerted at cell-ECM adhesions and tension experienced at cell-cell adhesions (Maruthamuthu, 2011). In cell pairs the ratio between traction and tension has been shown to be constant, and thus changes to traction result in changes to tension. However, as the number of cell contacts increases, this ratio changes: traction exerted by cells adhering to multiple other cells decreases, while tension at cell-cell interfaces is enhanced (Maruthamuthu, 2011). How the relationship between forces exerted by cells on the ECM and tension experienced across cell membranes changes in the context of a tissue remains unclear. This study examined the effects of changing cell-ECM adhesion on stresses experienced at cell-cell contacts within a simple epithelium. While it was not possible to measure the magnitude of tension across cells or of traction exerted by cells within an intact, living embryo, it was found that changes to cell-ECM adhesion (and thus potentially to traction forces) result in differential responses to laser ablation at the level of cell-cell contacts. These findings suggest that in the context of a living tissue, there may be a strong relationship between the traction exerted by cells and the tension experienced across their junctions, as observed in cell culture models (Goodwin, 2016).

    By tracking the movement of FALS during cell oscillations and laser ablation experiments, this study provides evidence that cell-ECM adhesion can resist apically generated forces. Specifically, it was found that wild-type variations in FALS morphology can predict differences in apical behaviors: in the presence of larger FALS, cell membranes exhibit less displacement during oscillations and less recoil during laser ablation experiments. These data provide strong evidence for two main conclusions: firstly, that the apical and basal parts of the cell are mechanically coupled, and secondly, that FALS act as tethers to the ECM to resist apically generated forces. It is proposed that this mechanical coupling is achieved through the actin cytoskeleton, given that both cell-cell and cell-ECM adhesion associated with F-actin, and that F-actin is present along the entire cortex of AS cells. However, it is also possible that other cytoskeletal components play a role. For example, microtubules mediate flattening and elongation of AS cells in early embryogenesis, and in Caenorhabditis elegans, morphogenesis of adjacent tissues is mechanically coupled via hemidesmosomes and intermediate filaments. Apical-basal mechanical coupling in AS cells could be achieved through the combined efforts of different cytoskeletal networks; future studies will be needed to characterize the entity that mediates this mechanical coupling (Goodwin, 2016).

    If cell-ECM adhesions provide tethering to the ECM, then AS cells would exert forces on their substrate, leading to deformation. This was visualized by developing a tool inspired by TFM to measure deformations of the underlying yolk cell membrane. The movements of AS cells and the yolk membrane were found to be correlated, suggesting that cells are mechanically coupled to the underlying substrate. Furthermore, this correlation is lost in the absence of integrins, as predicted by downregulating cell-ECM adhesion in multicellular clusters. These findings confirm that cell-generated forces pull and deform the underlying substrate in vivo, and that tethers connected to the ECM through FALS are required for mechanical coupling between cells and the substrate. Overall, these findings suggest that a balance of cell-cell and cell-ECM force transmission previously described in cell culture may play a role in the generation of tension across AS cells (Goodwin, 2016).

    Based on these results, a model is proposed whereby integrins act as tethers to the ECM and resist apically generated forces, thereby mediating force transmission between neighboring cells and to the substrate. As a result, cell-ECM adhesion controls displacement and mechanics of AS cells, which may have consequences for tissue contraction and closure. DC requires precise timing and coordination of many different cell behaviors; disrupting any one of these can lead to closure defects. By examining large-scale cell movements in the AS, it was found that cell displacement toward the dorsal midline during the slow phase of contraction is increased in mys / mutants. Previous studies of mys / mutants revealed that in the slow phase of closure, inner cells of the AS contract more rapidly than in the wild-type. It is speculated that this increased rate of contraction could be due to a loss of basal tethering; this would also lead to increased cell displacement toward the midline. Evidently this increased rate of contraction is not maintained, as mys / embryos experience delayed or failed closure; defects later in closure likely arise from loss of adhesion between tissue layers, leading to tears between the AS and the epidermis. When cell-ECM adhesion is increased, more tethering results in a downregulation of cell-cell force transmission, which could hinder contraction of the AS and lead to delayed or failed DC. In line with this, it was found that cell displacement toward the dorsal midline is reduced in Talin(E1777A) and Rap1-CA mutant embryos (Goodwin, 2016).

    Animal morphogenesis and particularly DC are dependent upon the cellular mechanisms that generate biomechanical forces and, subsequently, on the proper translation of these forces into changes in tissue architecture. This study provides insight into how forces are transmitted within and between cells. It is proposed that cell-ECM adhesion must be fine-tuned to allow for proper force transmission, which could play a role in establishing optimal levels of tension across the AS during tissue contraction. Given the ubiquitous presence of cell-ECM adhesions in epithelia, the ability of integrins to modulate force transmission may represent a fundamental, conserved feature of animal development (Goodwin, 2016).

    Cell-cell and cell-ECM adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure

    Tissue morphogenesis relies on the coordinated action of actin networks, cell-cell adhesions, and cell-ECM adhesions. Such coordination can be achieved through crosstalk between cell-cell and cell-ECM adhesions. Drosophila Dorsal Closure (DC), a morphogenetic process wherein an extra-embryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell-cell and cell-ECM adhesions. However, whether the function of these two types of adhesion is coordinated during DC is not known. This study analyzed possible interdependence between cell-cell and cell-ECM adhesions during DC, and its effect on the actomyosin network. Loss of cell-ECM adhesion was found to result in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa; and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell-cell adhesion caused an upregulation of cell-ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. These results show how interdependence between cell-cell and cell-ECM adhesions is important in regulating cell behaviours, force generation and force transmission critical for tissue morphogenesis (Goodwin, 2017).

    Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo

    During Dorsal closure (DC), JNK (JUN N-terminal Kinase) signalling controls leading edge (LE) differentiation generating local forces and cell shape changes essential for DC. The LE represents a key morphogenetic domain in which, in addition to JNK, a number of signalling pathways converges and interacts (anterior/posterior -AP- determination; segmentation genes, such as Wingless; Decapentaplegic). To better characterize properties of the LE morphogenetic domain, this study sought out new JNK target genes through a genomic approach: 25 were identified of which 8 are specifically expressed in the LE, similarly to decapentaplegic or puckered. Quantitative in situ gene profiling of this new set of LE genes reveals complex patterning of the LE along the AP axis, involving a three-way interplay between the JNK pathway, segmentation and HOX genes. Patterning of the LE into discrete domains appears essential for coordination of tissue sealing dynamics. Loss of anterior or posterior HOX gene function leads to strongly delayed and asymmetric DC, due to incorrect zipping in their respective functional domain. Therefore, in addition to significantly increasing the number of JNK target genes identified so far, the results reveal that the LE is a highly heterogeneous morphogenetic organizer, sculpted through crosstalk between JNK, segmental and AP signalling. This fine-tuning regulatory mechanism is essential to coordinate morphogenesis and dynamics of tissue sealing (Rousset, 2017).

    This identification of several new JNK target genes during DC and analysis of their quantitative expression patterns uncovers the complex transcriptional response taking place in the LE morphogenetic domain. Results reveal an intricate regulatory network integrating multiple signalling layers. In this process, AP positional information and JNK signalling cooperate to generate a highly patterned, yet apparently smooth and regular LE. Mutant analysis shows that LE partitioning into discrete domains is important to control the coordination, and hence the dynamics of the whole closure process (Rousset, 2017).

    The LE is a major component of DC, being the site of JNK activity and actin cable assembly; it also provides an active boundary with the amnioserosa, driving epidermal spreading and seamless tissue sealing. Therefore, it is important to determine its morphogenetic and signalling features and how these are dynamically controlled. To this end, a new set of target genes was identified whose expression in the dorsal ectoderm is dependent on JNK activity during DC. Transcriptome analysis allowed identification of 1648 independent genes which are up- or down-regulated in JNK activated embryos. Filtering of this large set yielded a group of 194 genes whose expression was analysed by quantitative in situ hybridization under different genetic conditions. Transcriptional profiling unveiled 31 Drosophila JNK target genes, of which only a fraction were already known, including jra/jun, reaper, Zasp52 and scab. Amongst novel targets were also Scaf and Rab30 the roles of which during DC have previously been described. Two categories of JNK target genes were distinguished: genes that are specifically expressed in the LE and genes whose expression is more ubiquitous in the dorsal ectoderm. Genes belonging to the latter category may play a general role in the ectoderm under the control of different pathways, for example in the case of Rab30. In contrast, LE-specific genes likely play a specific role during DC, as is the case for puc, dpp and scaf. However, it is also possible that some of the new genes, despite being expressed in the embryo in a JNK-dependent manner, are not involved in DC. These target genes thus remain under the control of JNK, but are functionally ‘silent’ during DC. This behaviour is best illustrated by reaper, whose expression is JNK-dependent in the embryo, but which does not seem to have any function in the LE, acting only later during development or at the adult stage (Rousset, 2017).

    Surprisingly, quantitative analysis of LE-specific gene expression profiles showed a variety of previously uncharacterized expression patterns along the LE, with two levels of regulation, AP and segmental. These observations reveal a new property of the LE which appears highly patterned along the AP axis, contrasting with the homogenous and linear structure previously envisioned. In addition, the higher order regulation that emerges from these results provides every LE cell with its own identity through the cross-talk between JNK, AP and segmental information. Such cell-level patterning through signalling crosstalk is likely essential for coordination and robustness of closure as well as segment matching. In this view, recent work showed that Wg and JNK interact at the LE to control the formation of specific mixer cells at segment boundaries (Rousset, 2017).

    Previous work showed that, instead of acting independently, HOX and segmentation genes can be coupled to regulate target genes in the embryo. This study revealed an additional layer of regulation involving the 'morphogenetic' JNK signalling pathway. During DC, JNK acts as a tissue-specific switch whose activity can be regulated by HOX and segmentation pathways, providing positional information an 'onion-like' regulatory model allows for several levels of regulation/information to pile up in order to regulate individual cellular behaviours important for tissue morphogenesis. Each layer can act positively or negatively on LE target gene expression, generating a complex repertoire of regulatory pathways. Distinct categories of expression profiles were identified in this study through the analysis of individual target genes, with the likelihood of more gene-specific patterns to be discovered. For example, the same HOX gene (abd-A or Abd-B) can have activating or repressive activity according to the target gene, as is the case for the transcription factor En. Molecular functional characterization of cis-regulatory elements controlling LE gene expression will bring a more detailed view of how transcription factor complexes are formed, how specificity of DNA recognition is achieved and how activating or repressive activities are regulated to generate LE patterning (Rousset, 2017).

    scaf proves to be a remarkable case among the JNK target genes, showing the different levels of regulation that can be integrated into a single promoter. Not only is it strongly expressed in the LE in a JNK-dependent manner, but it is also regulated by both the segmentation gene en and the HOX genes. In particular scaf displays a transcriptional response induced by all the trunk HOX genes tested, being positively controlled by Scr, Antp, Ubx and Abd-B and negatively by abd-A. It can therefore be considered as a general HOX target gene, i.e. regulated by most Hox paralogs, as previously defined. Another example of a general target is the Drosophila gene optix, which is activated by the head HOX genes labial and Deformed (Dfd) and inhibited by the trunk HOX genes. Nonetheless the general HOX target genes do not represent the majority. A genomic analysis in the Drosophila embryo identified more than 1500 genes regulated by at least one of the six HOX paralogs tested (Dfd, Scr, Antp, Ubx, abd-A, Abd-B). Only 1.3% of these genes are regulated by the six paralogs and 1.5% by the five paralogs that were used in this study. Interestingly more than 40% of the ~1500 HOX target genes are also present in the JNK genomic data set that was obtained. This strong overlap well reflects the fact that the LE runs along most of the body AP axis encompassing the thorax and abdomen. More importantly, it also indicates that AP patterning plays a crucial role in the regulation of DC, as shown in this study (Rousset, 2017).

    Live imaging and mathematical modelling revealed asymmetries in the geometry and zipping process along the AP axis; these can be attributed to local constraints induced by head involution and apoptosis. Head involution is concomitant with DC and induces tension in the anterior part of the embryo, explaining why the DC phenotypes are almost exclusively observed in the anterior part, leading to the so-called 'anterior-open phenotype'. The exception to this rule is the experimental manipulation of the posterior zipping rate through localized laser ablation of the amnioserosa close to the canthus, which induces a strong delay of posterior closure. The results with the abd-A and Abd-B mutants show that posterior delay can also be obtained in genetically-perturbed embryos. However, while anterior zipping is slightly up-regulated when posterior zipping is laser-targeted, it was shown that the anterior speed of closure is diminished in the Abd-B embryo. Thus, compensatory mechanisms may only appear when tissue integrity is severely impaired. Apoptosis was also proposed to participate in the asymmetric properties of DC. Delamination of apoptotic cells in the anterior amnioserosa produces forces that are responsible for a higher rate of anterior zipping. However, the phenotype that was observed with the abd-A or Abd-B mutation cannot be attributed to defects in this mechanism, as the rate of apoptosis is already very low in the posterior amnioserosa. In summary, the data reveal a genetic control of zipping through precise transcriptional regulation in the LE. Overall, this work provides a framework for apprehending how the HOX selector genes and their cofactors collaborate with other signalling pathways to generate specific transcriptional responses allowing morphogenetic patterning and proper coordinated development (Rousset, 2017).

    Novel interplay between JNK and Egfr signaling in Drosophila dorsal closure

    Dorsal closure (DC) is a developmental process in which two contralateral epithelial sheets migrate to seal a large hole in the dorsal ectoderm of the Drosophila embryo. Two signaling pathways act sequentially to orchestrate this dynamic morphogenetic process. First, c-Jun N-terminal kinase (JNK) signaling activity in the dorsal-most leading edge (LE) cells of the epidermis induces expression of decapentaplegic (dpp). Second, Dpp, a secreted TGF-beta homolog, triggers cell shape changes in the adjacent, ventrally located lateral epidermis, that guide the morphogenetic movements and cell migration mandatory for DC. This study uncovered a cell non-autonomous requirement for the Epidermal growth factor receptor (Egfr) pathway in the lateral epidermis for sustained dpp expression in the LE. Specifically, it was demonstrated that Egfr pathway activity in the lateral epidermis prevents expression of the gene scarface (scaf), encoding a secreted antagonist of JNK signaling. In embryos with compromised Egfr signaling, upregulated Scaf causes reduction of JNK activity in LE cells, thereby impeding completion of DC. These results identify a new developmental role for Egfr signaling in regulating epithelial plasticity via crosstalk with the JNK pathway (Kushnir, 2017).

    Activation and synchronization of the oscillatory morphodynamics in multicellular monolayer

    Oscillatory morphodynamics provides necessary mechanical cues for many multicellular processes. Owing to their collective nature, these processes require robustly coordinated dynamics of individual cells, which are often separated too distantly to communicate with each other through biomaterial transportation. Although it is known that the mechanical balance generally plays a significant role in the systems' morphologies, it remains elusive whether and how the mechanical components may contribute to the systems' collective morphodynamics. This paper analyzed the collective oscillations in the Drosophila amnioserosa tissue to elucidate the regulatory roles of the mechanical components. The tensile stress was identified as the key activator that switches the collective oscillations on and off. This regulatory role is shown analytically using the Hopf bifurcation theory. The physical properties of the tissue boundary are directly responsible for synchronizing the oscillatory intensity and polarity of all inner cells and for orchestrating the spatial oscillation patterns in the tissue (Lin, 2017).

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    Genes involved in tissue development

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