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).

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).

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. Yhis 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).

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).

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-Dia^CA 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).

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 ‘apoptosis’ 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).