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).

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).