The Interactive Fly

Genes involved in tissue and organ development


Drosophila mesoderm migration behaviour during gastrulation

folded gastrulation, cell shape change and the control of myosin localization

Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2

Pulsed contractions of an actin-myosin network drive apical constriction

Heterotrimeric G protein signaling governs the cortical stability during apical constriction in Drosophila gastrulation

Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation

Passive mechanical forces control cell-shape change during Drosophila ventral furrow formation

Embryo-scale tissue mechanics during Drosophila gastrulation movements

Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis

Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids

Actomyosin meshwork mechanosensing enables tissue shape to orient cell force

Dynamic control of dNTP synthesis in early embryos

Genes affecting gastrulation

Drosophila mesoderm migration behaviour during gastrulation

Mesoderm migration is a pivotal event in the early embryonic development of animals. One of the best-studied examples occurs during Drosophila gastrulation. Here, mesodermal cells invaginate, undergo an epithelial-to-mesenchymal transition (EMT), and spread out dorsally over the inner surface of the ectoderm. Although several genes required for spreading have been identified, the inability to visualise mesodermal cells in living embryos has hampered gathering of information about the cell rearrangements involved. Several mechanisms, such as chemotaxis towards a dorsally expressed attractant, differential affinity between mesodermal cells and the ectoderm, and convergent extension, have been proposed. This study resolved the behaviour of Drosophila mesodermal cells in live embryos using photoactivatable-GFP fused to alpha-Tubulin (PAGFP-Tub). By photoactivating presumptive mesodermal cells before gastrulation, it was possible to observe their migration over non-fluorescent ectodermal cells. The outermost (outer) cells, which are in contact with the ectoderm, migrate dorsolaterally as a group but can be overtaken by more internal (inner) cells. Using laser-photoactivation of individual cells, it was then shown that inner cells adjacent to the center of the furrow migrate dorsolaterally away from the midline to reach dorsal positions, while cells at the center of the furrow disperse randomly across the mesoderm, before intercalating with outer cells. These movements are dependent on the FGF receptor Heartless. The results indicate that chemotactic movement and differential affinity are the primary drivers of mesodermal cell spreading. These characterisations pave the way for a more detailed analysis of gene function during early mesoderm development (Murray, 2007).

Using a combination of whole mesoderm and single-cell photoactivation this study has observed the combination of cell behaviours employed by Drosophila mesodermal cells to form a monolayer, providing insights into the mechanisms responsible for this important part of gastrulation. The first observation was that outer cells moved dorsolaterally over the ectoderm. Although this is not unexpected, it nevertheless confirms a central prediction of the chemoattraction model: that cells migrate in a dorsolateral direction. Remarkably, it was then observed that inner cells are able to overtake outer cells to achieve a more dorsal position. Single-cell labelling then showed that these inner cells were likely to have originated from a position adjacent to the centre of the ventral furrow. Significantly, inner lateral (IL) cell progeny invariably move away from the midline, suggesting that they receive a directional guidance cue from the dorsal region of the ectoderm, again consistent with a chemoattraction model (Murray, 2007).

A complication in the simple chemoattraction model is that the two likely chemoattractants, Pyr and Ths, are initially expressed in quite broad lateral domains. During mesoderm migration, however, pyr expression does become restricted to the more dorsal parts of the ectoderm, whereas ths is expressed in a complementary fashion in the ventral regions of the neurogenic ectoderm. It has been suggested that the two ligands may have different binding affinities, and that the refinement of Pyr expression to more dorsal positions could guide mesodermal cells dorsally. An alternative is that those regions of the ectoderm that are not yet covered with mesodermal cells, such as the dorsal ectoderm, are highly attractive to mesodermal cells simply because the FGF ligands that they are producing are not being bound and internalised by outer cells already in contact with the ectoderm (Murray, 2007).

An alternative to chemoattraction that has been suggested is that FGFR activation is permissive rather than instructive and simply imparts a degree of motility to cells, allowing them to disperse until they are able to contact the ectoderm. This motility, combined with a steric hindrance effect, in which cells tended to move into unoccupied territory, could theoretically achieve a monolayer in the absence of directional cues. It would be expected, however, that if IL cell progeny were simply made motile and moved randomly, that cells adjacent to the midline would sometimes cross the midline to contact the ectoderm on the opposing side. This was never observed (Murray, 2007).

The movement of inner cells past the lateralmost outer cells is also consistent with the differential affinity model, according to which mesodermal cells form strong adhesions with the ectoderm. Cells not already in contact with the ectoderm would either intercalate between existing outer cells, or, as seen here, move past them. The fact that intercalation was not seen suggests either that outer cells adhere strongly to the ectoderm and do not easily move apart, or, again, that outer cells are masking FGF produced in the ectoderm. If a differential affinity model is active, the most likely candidate adhesion molecules would be integrins, which are expressed at the interface of the mesoderm and ectoderm, although there is, as yet, no published evidence for a functional role for integrins in this process (Murray, 2007).

During the initial migration of outer cells over the ectoderm it was found that cells maintained their position relative to their immediate neighbours. This result supports the argument against the convergent extension model. If convergent extension was a primary driving force behind lateral spreading, one would expect to see widespread intercalation throughout the mesoderm as inner cells pushed in between existing outer cells. This was not observed, although the possibility cannot be ruled out that some degree of intercalation does occur during this migration phase. Intercalation does, however, appear to play a part during the later stages of the formation of the monolayer, where inner medial (IM) cell progeny are seen appearing at the ectoderm. The timing of this event, at around the time of the second mitosis, suggests that the sudden lateral spreading that accompanies the second mitotic wave (50 minutes of development) may be due to the intercalation of a pool of inner cells. One possibility is that the adhesion between the mesodermal cells and the surrounding cells, both mesodermal and ectodermal, is decreased as they go through mitosis, permitting the inner cells access to their preferred position in association with the ectoderm. Thus, although a general convergent extension is not in evidence, intercalation does appear to contribute to mesoderm spreading (Murray, 2007).

On the basis of these observations, the following model of mesoderm cell behaviour following ventral furrow formation is presented. Following the breakdown of the epithelium, the first division results in a rapid spreading down onto the ectoderm, presumably due to decreased adhesion between mesodermal cells. Cells that are thereby placed in contact with the ectoderm start to polarise and proceed to migrate dorsolaterally as a group. Outer cells form a strong adhesive contact with the ectoderm, which prevents inner cells from intercalating between them and instead forces inner cells either to take up positions that outer cells vacate near the midline or move past them to more dorsal positions. Inner lateral cells receive a directional cue from the dorsal ectoderm guiding them laterally, over the outer cells. In this manner, by the time of the second mitosis the ectoderm is largely covered by mesodermal cells. Inner medial cell progeny that have failed to contact the ectoderm during the initial spreading are prevented from doing so by cells already strongly adhered to the ectoderm until the time of the second division. The second division then allows the remaining inner cells to contact the ectoderm. This intercalation produces a rapid lateral extension followed by a general retraction as the cells exit mitosis and re-establish adhesive contacts, with the ectoderm finally forming the monolayer (Murray, 2007).

The combination of behaviours observed may represent the most efficient way to rapidly spread one tissue over another. The tendency for cells to migrate dorsolaterally helps to constantly make space for those cells placed nearer the midline. If cells that contacted the ectoderm never moved away, it would mean that internal cells would have to travel further and further dorsally to find space on the ectoderm. In a similar manner, if chemotaxis towards a dorsally placed attractant was the only mechanism operating, one might expect that cells would continue moving dorsally, even if this resulted in an excess of cells in dorsal positions and a deficit closer to the midline. The tendency of mesodermal cells to develop and maintain a strong adhesive contact with the ectoderm would help ensure that all parts of the ectoderm remain covered. Finally, having a period of intercalation serves to give any remaining inner cells a chance to finally contact the ectoderm (Murray, 2007).

The resolution of mesodermal cell behaviour described in this study will make it possible analysis in greater detail of the migration defects in mutants such as htl and pebble. It will also make it possible to test whether cell rearrangements are normal in those situations in which directional information is lost, but in which spreading still occurs (e.g. rescue with activated Htl, or widespread, non-localised expression of FGF ligands). Finally, it will be of interest to determine whether the behaviors observed are typical of mesoderm migration in other systems. In mouse embryos, mesodermal cells emanating from the primitive streak migrate out over the basal surface of the primitive ectoderm to eventually form the mesodermal layer of cells. The cell rearrangements that occur during this process are not known. Photoactivatable GFP, which has provided such a versatile analysis tool here, could be applied to cultured mouse embryos to resolve these events (Murray, 2007).

folded gastrulation, cell shape change and the control of myosin localization

The global cell movements that shape an embryo are driven by intricate changes to the cytoarchitecture of individual cells. In a developing embryo, these changes are controlled by patterning genes that confer cell identity. However, little is known about how patterning genes influence cytoarchitecture to drive changes in cell shape. This paper analyzes the function of the folded gastrulation gene (fog), a known target of the patterning gene twist. Analysis of fog function therefore illuminates a molecular pathway spanning all the way from patterning gene to physical change in cell shape. Secretion of Fog protein is apically polarized, making this the earliest polarized component of a pathway that ultimately drives myosin to the apical side of the cell. fog is both necessary and sufficient to drive apical myosin localization through a mechanism involving activation of myosin contractility with actin. This contractility driven form of localization involves RhoGEF2 and the downstream effector Rho kinase. This distinguishes apical myosin localization from basal myosin localization; the latter does not require actinomyosin contractility or FOG/RhoGEF2/Rho-kinase signaling. Furthermore, once localized apically, myosin continues to contract. The force generated by continued myosin contraction is translated into a flattening and constriction of the cell surface through a tethering of the actinomyosin cytoskeleton to the apical adherens junctions. Therefore, this analysis of fog function provides a direct link from patterning to cell shape change (Dawes-Hoang, 2005).

Investigation of fog function began with an analysis of Fog protein distribution within the cells of the ventral furrow and posterior midgut. In both cases Fog protein was found to be present in a characteristically punctate pattern; the protein is distributed unevenly within the cells. The distribution of Fog is polarized with more Fog puncta present on the apical compared with the basal side of the cells. This punctate staining is consistent with the localization of signaling molecules to vesicles involved in both signal production and reception. To investigate this possibility further, distribution of Fog was examined in embryos carrying a temperature-sensitive mutation in the gene shibire, which encodes the Drosophila homolog of dynamin. At the non-permissive temperature, this mutation blocks endocytosis, and exocytosis is also compromised. When embryos are shifted to the non-permissive temperature during early gastrulation (earlier shifts severely disrupt the process of cellularization) the Fog protein is already being made and some protein may already be undergoing endocytosis. However, the localization of Fog in these embryos is still clearly disrupted, with much less punctate staining and a decrease in apical polarization. This suggests that the punctate staining of Fog in normal embryos may arise from localization to vesicles derived through endocytosis, and this supports the hypothesis that fog encodes a secreted protein. The apical polarization of Fog therefore raises the possibility that apical secretion and reception of Fog signal may provide a mechanism for restricting Fog function to the apical side of the cell (Dawes-Hoang, 2005).

To understand the molecular basis of the control of the cytoskeleton by Fog, changes in myosin II dynamics were investigated in fog mutant embryos. Analysis of myosin dynamics is easiest in the posterior midgut where fog is the primary pathway controlling cell constriction and the geometry of the egg enables visualization of a myosin lightchain-GFP fusion (sqhGFP) in time-lapse movies of living embryos. During gastrulation myosin localizes to the apical side of cells throughout the posterior midgut primordium of control embryos. However, in fog mutant embryos of the same age, the apical localization of myosin is severely disrupted and is restricted to just a few cells underlying the pole cells. Analysis of myosin localization in fixed embryos also reveals a disruption to apical localization, both in the posterior midgut and the ventral furrow of fog mutants. This is consistent with previous data showing that myosin is also disrupted in concertina (cta) mutants. In the ventral furrow only a subset of cells (39%) localizes myosin apically in fog114 mutant embryos. The fog114 allele is an RNA null. The patchiness of this defect in the ventral furrow of fog114 mutants therefore probably reflects the redundancy with additional pathways that control cell shape change in these cells and/or a small maternal contribution of fog (Dawes-Hoang, 2005).

To determine whether fog is not only necessary but also sufficient to localize myosin to the apical side of cells, a UASfog transgene was constructed. Despite high levels of fog expression from this transgene during cellularization, there is no apparent change in myosin localization. Myosin localizes normally to the cellularization front and the subsequent basal loss of myosin in the ventral most cells and the increased depth of cellularization in these cells that occurs in normal embryos also occur in these fog-overexpressing embryos (Dawes-Hoang, 2005).

The first effects of fog expression are seen at the onset of gastrulation. In embryos uniformly expressing fog the apical localization of myosin now occurs in all cells instead of being restricted to a ventral domain. In the ventral cells of these fog-overexpressing embryos, the apical localization of myosin precedes the apical localization in the more lateral and dorsal cells and reaches a higher level. It is also a higher level than in the ventral cells of control embryos. It is unclear whether this reflects higher levels of fog expression in the ventral cells (owing to both endogenous and UASfog expression) or whether it reflects an earlier or increased competence of these ventral cells to react to fog signal. The apical localization of myosin in the lateral and dorsal cells of fog-overexpressing embryos continues throughout gastrulation and occurs without any concomitant reduction in levels of basally localized myosin. This raises the possibility that the apical and basal localizations of myosin may be independently controlled (Dawes-Hoang, 2005).

Not all fog-overexpressing embryos show the same degree of ectopic apical myosin localization in lateral and dorsal cells. Furthermore, limited apical myosin staining is occasionally seen in control embryos. This variability was quantified over five separate experiments. During cellularization, onset of gastrulation and later gastrulation 0%, 73% and 84% of fog-overexpressing embryos show ectopic apical myosin compared with 0%, 8% and 22% of controls respectively (Dawes-Hoang, 2005).

In wild-type embryos myosin accumulates apically in all cells after the completion of ventral furrow invagination, at the onset of germ band extension. Therefore, apical accumulation of myosin in dorsal and lateral cells of apparently gastrulating embryos may occur as the result of a delay in ventral furrow formation. To investigate this possibility, time-lapse movies of gastrulating embryos were followed and morphology was examined in precisely timed embryo collections. In both cases, a slight delay was found in the completion of ventral furrow formation in fog-overexpressing embryos compared with controls. In equivalently aged collections, only 32% of control embryos were undergoing ventral furrow formation compared with 45% of fog-overexpressing embryos. This implies that fog-overexpressing embryos take about 1.4 times longer to complete ventral furrow formation than control embryos. However, this is considerably less than the ~3.5 times delay that would be required to account for the large difference seen in apical myosin localization between the UASfog-expressing embryos and controls. Assuming that if the process were to take twice as long in UASfog embryos this would account for 50% of the embryos showing apical myosin simply because they are in fact older, it is estimated that the process would have to be ~3.5 times as long to account for the actual increased numbers of embryos seen (Dawes-Hoang, 2005).

Therefore fog-overexpressing embryos show a consistent increase in apical myosin staining in the lateral and dorsal cells of gastrulating embryos when compared with controls, and this increase is too large to be explained by the slight delay in gastrulation. It is concluded that fog signaling is both necessary and sufficient to localize myosin II to the apical side of cells (Dawes-Hoang, 2005).

It is possible that fog provides a signal to localize or transport myosin apically, and myosin is then activated to interact and contract with actin. An intriguing alternative, however, is that fog itself may be activating myosin contractility, initiating an active motor-driven mechanism of myosin localization. To help distinguish between these two possibilities, a form of myosin was constructed that is no longer able to interact or contract with actin and it was asked if this form of myosin was still able to localize normally (Dawes-Hoang, 2005).

Myosin is a hexamer comprising two myosin heavy chains (MHCs), two essential light chains and two regulatory light chains (RLCs). It is the globular head domain of the MHC subunits that interacts directly with actin and contains the region of ATPase activity that drives this actin-based motor. In addition, the ATPase activity and strength of actin binding can be modified through phosphorylation of the regulatory light chains, while the coiled-coil tail domains of the MHCs are required for assembly of multiple myosin molecules into organized filaments (Dawes-Hoang, 2005).

A myosin-YFP transgene (mYFP-myosin IIDN) was constructed in which the YFP moiety has replaced the actin-binding motor head domain of the myosin heavy chain, zipper. Based on equivalent modifications in Dictyostelium, mYFP-myosin IIDN homodimers should completely lack actin binding and contractility, and the 'single headed' wild-type myosin/mYFP-myosin IIDN heterodimers should have severely decreased actin binding and contractility. Consistent with this, it was found that YFP-containing myosin isolated from mYFP-myosin IIDN expressing Drosophila embryos shows reduced actin binding when compared with wild-type myosin in a standard spin down assay. However, no dominant-negative activity of this transgene during embryogenesis was detected, presumably because of the high levels of endogenous myosin (Dawes-Hoang, 2005).

To analyze the localization of this mYFP-myosin IIDN, the Gal4 system was used to express the transgene uniformly in embryos that also carry wild-type copies of zipper. For comparison the following were examined: (1) a fully functional myosin-GFP fusion, in which GFP is fused to the myosin light chain, sqhGFP, and (2) the endogenous myosin II of wild-type embryos. No differences were found between the localization patterns of sqhGFP and endogenous myosin, and only the endogenous myosin will be referred to (Dawes-Hoang, 2005).

When cells divide during later stages of development, the non-functional mYFP-myosin IIDN shows a localization similar to endogenous myosin. Both localize to the contractile ring as it forms, constricts and then disappears following the completion of cell cleavage. Similarly, during cellularization, mYFP-myosin IIDN localizes to the cellularization front in a manner similar to endogenous myosin. As reported for sqhGFP, the mYFP-myosin IIDN tends to form aggregates in the interior of the embryo. In time-lapse movies, the aggregates associate with the cellularization front, which 'clears' them from the outer edges of the embryos as cellularization proceeds, but they do not fully integrate into the regular hexagonal array of mYFP-myosin IIDN associated with the advancing furrows (Dawes-Hoang, 2005).

The first differences between functional and non-functional myosin are observed at the onset of gastrulation. Unlike endogenous myosin, mYFP-myosin IIDN fails to localize apically at the onset of ventral furrow formation and throughout later stages of apical constriction and invagination. The ability of these cells to undergo normal ventral furrow formation despite a lack of apically localized mYFP-myosin IIDN presumably reflects the activity of endogenous zipper. Both endogenous myosin and mYFP-myosin IIDN are lost from the basal side of the invaginating ventral furrow cells. This basal loss is slightly delayed and patchy for mYFP-myosin IIDN, but otherwise proceeds normally (Dawes-Hoang, 2005).

The requirement for actin binding and subsequent actin-dependent contractile activity therefore appears to distinguish two functionally different modes of myosin localization: an actin-independent mode of localization during cellularization and cytokinesis, and a second mode during gastrulation where localization to the apical side of the cell is dependent upon actin binding/contractility. It is possible that the mYFP-myosin IIDN is defective in ways other than its ability to interact with actin. However, equivalent constructs in Dictyostelium do not effect any other aspects of myosin function, including RLC phosphorylation or filament assembly. Therefore, although such secondary effects can not be entirely ruled out, the defects seen are most likely a result of the inability to interact with actin and at the very least distinguish two different types of myosin localization to the apical and basal sides of the cell. They also highlight the potential importance of actin-myosin interaction and contractility as a target for fog signaling (Dawes-Hoang, 2005).

The components acting downstream of fog to mediate its effects on the cytoskeleton are largely unknown. One candidate, RhoGEF2 (a guanine nucleotide exchange factor that promotes Rho activation) has been shown to be required for ventral furrow formation and can genetically interact with a fog transgene. However, embryos mutant for RhoGEF2 have a much more severe disruption of ventral furrow formation than embryos mutant for fog and the point at which the products of these two genes interact on a mechanistic or subcellular level is unknown. Recent studies have shown a requirement for RhoGEF2 in controlling actin dynamics/stability during cellularization and have also shown a disruption to myosin localization at gastrulation. Therefore the re-localization of myosin during cellularization and gastrulation was analyzed in RhoGEF2 mutants and previous studies were extended by looking at a potential downstream effector of RhoGEF2 signaling (Dawes-Hoang, 2005).

Embryos mutant for RhoGEF2 localize myosin normally to the forming cellularization front. However, unlike fog mutants, the RhoGEF2 embryos show defects in cellularization, including an irregular, wavy cellularization front. This implies that although RhoGEF2 function is not required to localize myosin to the cellularization front it is required to maintain the normal structure of the cellularization front and that the presence of myosin is not itself sufficient to maintain a straight cellularization front. This is consistent with previous studies of RhoGEF2 mutants and a potential role in controlling actin but not myosin dynamics (Dawes-Hoang, 2005 and references therein).

However, despite the defects during early cellularization, RhoGEF2 mutant embryos that reach the end of cellularization look remarkably normal. The irregularity of the cellularization front recovers, particularly in the ventral cells, and both the increased cell depth and basal loss of myosin occur normally in these cells. However, in RhoGEF2 embryos, precisely staged for the onset of gastrulation, there is an absolute failure to re-localize myosin to the apical side of the ventral cells, despite a normal loss of myosin from the basal side of these cells. This is consistent with independent mechanisms controlling the basal loss and apical accumulation of myosin during gastrulation and demonstrates an absolute requirement for RhoGEF2 in apical myosin localization. It also confirms the previous report of RhoGEF2 being required for apical myosin in ventral furrow cells (Nikolaidou, 1994; Dawes-Hoang, 2005).

RhoGEF2 interacts with myosin in other systems through the Rho-kinase family of Ser/Thr kinases that inhibit myosin phosphatase and also directly phosphorylate myosin. Both these activities led to activation of actin binding by myosin and increased actomyosin based contractility. Additional myosin activators include MLCK and citron kinase but the extent to which these different activators play specific or overlapping roles with Rho-kinase is unclear, and the role of any of these myosin activators during Drosophila gastrulation is not known (Dawes-Hoang, 2005).

Therefore embryos were produced mutant for Drosophila Rho-kinase (Drok) by making germline clones of two Drok alleles, both of which produced similar phenotypes. Myosin localizes to the cellularization front of Drok mutant embryos but often does so unevenly and, as for RhoGEF2, the cellularization front is 'wavy'. Unlike the RhoGEF2 mutant embryos, the nuclei of Drok mutant embryos have striking defects, including displacement into the interior of the embryo leaving reduced numbers at the cortex: these remaining nuclei are often of increased size and irregular morphology. It is unclear to what extent these nuclear phenotypes may represent an earlier defect during cell-cycle/nuclear division (Dawes-Hoang, 2005).

Despite these defects, many Drok mutant embryos complete cellularization and though the increased depth of cellularization in ventral cells is difficult to discern, basal loss of myosin proceeds normally. However, Drok mutant embryos show a complete failure to localize myosin to the apical side of the ventral cells at the onset of gastrulation. At later stages of gastrulation, the outer layer of wild-type embryos consists of a single cell layered epithelium that folds in specific locations during germband extension. In Drok mutant embryos this morphology is severely disrupted and the outer epithelium becomes multilayered and irregular, containing large often rounded cells. Drok is therefore required to maintain epithelial integrity (Dawes-Hoang, 2005).

Both Drok and RhoGEF2 mutant embryos show defects during cellularization and then fail to localize myosin to the apical side of ventral cells at gastrulation. However, it is unlikely that the earlier cellularization defects are what prevent the later apical myosin localization because many other cellularization mutants, such as nullo, display severe cellularization defects but still go on to localize myosin to the apical side of ventral cells at the onset of gastrulation. The failure of Drok and RhoGEF2 mutant embryos to localize myosin apically during gastrulation therefore probably reflects a direct requirement for both these genes in the apical localization of myosin. Despite these disruptions to gastrulation, Drok embryos do still produce Fog protein that is as punctate and apically concentrated as in wild-type embryos. This is therefore consistent with a model whereby Drok driven activation of myosin contractility drives myosin apically in response to fog and RhoGEF2 signaling (Dawes-Hoang, 2005).

To examine the morphological consequences of fog-induced myosin re-localization, scanning electron microscopy (SEM) was performed on embryos overexpressing fog. A range of phenotypes was seen consistent with previous reports in which fog was expressed from a heat-shock promoter. It is difficult to predict the types of defects to expect in fog overexpressing embryos, as ventral furrow cells already express fog and cells outside the ventral furrow may require additional factors for full shape changes. Furthermore, early defects may lead to non-specific later defects by the end of gastrulation. However, apical flattening is the very first effect seen, coincident with the apical re-localization of myosin and this raises the issue of how these two processes are connected (Dawes-Hoang, 2005).

This connection is likely to require adherens junctions that anchor the actin-myosin cytoskeleton to the cell membrane and hold the cells of an epithelium together. Previous studies have concentrated on the role of junctions in cell polarity and maintaining integrity of epithelial sheets, or in cell rearrangements that do not involve changes in cell shape. Much less is understood about the role of adherens junctions in specific aspects of cell shape change (Dawes-Hoang, 2005).

Therefore the behavior of adherens junctions was analyzed in fog-overexpressing embryos. At the completion of cellularization the embryo consists of a single layered epithelium with the basal junctions of cellularization located just apical to the myosin rich cellularization front and the newly forming adherens junctions located about 6 µm in from the apical surface of the embryo. At the onset of ventral furrow formation, adherens junctions in the ventral most region of the embryo shift to a completely apical location as the cell surfaces flatten, whereas the junctions in more lateral cells maintain their sub-apical position. These relative positions are maintained during the phase of apical constriction as basal junctions gradually disappear. When fog is expressed throughout the embryo the apical shift of adherens junctions occurs normally in the ventral furrow region, but under these conditions also occurs in more lateral and dorsal cells and these junctions are more tightly condensed than the equivalent junctions of control embryos. The apical localization of myosin seen in fog-overexpressing embryos therefore correlates with an apical shift in adherens junctions (Dawes-Hoang, 2005).

The adherens junctions are possibly being pulled into an apical position because of forces generated by contractile myosin that has been apically re-localized in response to fog signal. To investigate the connection between myosin contractility and adherens junctions, myosin localization was examined in embryos that lack adherens junctions (Dawes-Hoang, 2005).

It is not possible to examine embryos totally lacking junctional components such as Armadillo (Arm) because the maternally supplied components are required earlier during oogenesis. To get around this problem use was made of the effects of nullo protein. Expression of nullo during late cellularization completely blocks the formation of apical spot junctions. To confirm that results using this technique are due to the lack of adherens junctions and not to additional effects of nullo expression, the analysis was repeated with embryos made from arm043A01 germline clones. The arm043A0 allele is of the 'medium class' of arm alleles, lacking the last few Arm repeats and the entire C terminus. Germline clones of this class of alleles produce sufficient levels of Arm function to enable a few eggs to complete oogenesis but subsequent function of Arm in the embryo is severely compromised and these embryos fail to assemble apical adherens junctions. The same results were found using both techniques (Dawes-Hoang, 2005).

In both cases, myosin localizes normally to the basal cellularization front and to the apical surface of cells in the ventral furrow. This implies that functional Arm-containing junctions are not required for myosin to become localized within the cell. However, subsequent events are affected. As ventral furrow cells of wild-type embryos undergo apical constriction, myosin is seen throughout the apical surface of cells, but in embryos lacking junctions to tether the actin-myosin network, myosin appears to contract into the center or side of the cell forming a tight 'ball' of presumably contracted myosin. The most likely explanation of these results is that myosin contractility is normal in cells lacking adherens junctions but when myosin is no longer tethered to junctions it contracts without being able to exert force on the plasma membrane. As a result, these cells are unable to flatten or constrict their apical surfaces. These results suggest that apically localized myosin is contractile and that this contractility alone is not sufficient to result in changes in cell shape but must be tethered to the apical adherens junctions to elicit apical flattening and constriction (Dawes-Hoang, 2005).

Adherens junctions are also known to play an important role in establishing and maintaining apicobasal polarity in epithelial cells. However, the results demonstrate that the polarizing signal for the apical activation of myosin is not dependent upon any polarizing influence emanating from intact apical adherens junctions. This is consistent with the idea that it is the Fog protein, through its apical secretion and reception, that provides the polarizing signal for myosin activation and that this process is independent of intact adherens junctions (Dawes-Hoang, 2005).

Thus, this study demonstrates that fog signal is both necessary and sufficient to trigger the relocalization of myosin to the apical side of the cell. This raises the possibility that a secreted signal is used as a means of producing a polarized response. In this case, secreting a signaling molecule on the apical side of the cell could be used to ensure an apically localized response to that signal. In support of this model, it was found that Fog protein is indeed apically concentrated and therefore comprises the earliest apically polarized component of this pathway. It will be interesting to see if the fog independent, parallel pathway of apical myosin recruitment uses a similar mechanism (Dawes-Hoang, 2005).

It was also demonstrated that apical myosin localization requires the ability of myosin to interact and/or contract with actin. Furthermore, it was shown that fog signaling results in a shift of adherens junctions from their usual apicolateral position to a more apical position and that these junctions are necessary to translate contractile forces into physical changes in cell shape (Dawes-Hoang, 2005).

Taken together these data suggest the following model. Expression of the patterning gene twi in the prospective mesoderm cells results in activation of fog transcription. The resulting Fog protein is then secreted from the apical surface of the cells and this signal activates fog receptors. The degree to which this activation is paracrine versus autocrine has yet to be determined. The apically activated receptors trigger a transduction pathway involving the G-alpha subunit, Concertina, and the Rho activator RhoGEF2. A downstream target of this pathway is Rho-kinase, which in turn activates the ability of myosin to interact and contract with actin in this sub-apical region of the cell. A localized source of activated actin-myosin contractility initiates an active motor-driven mechanism of myosin localization that concentrates contractile myosin to the apical side of the cell. This actin-myosin network is tethered to the cell surface through adherens junctions. Contraction of this network therefore puts tension on the junctions, pulling them into a completely apical location and flattening the domed apical surface in the process. Continued contraction exerts further tension and ultimately pulls the junctions together so much that the entire apical cell surface constricts. Intriguingly, RhoGEF2 protein can associate with the tips of microtubules in cultured cells. The extent to which this may add to a polarization of the fog pathway during gastrulation and how this ties in with the above model will therefore be interesting avenues for further investigation. It will also be important to examine any changes to the actin and microtubule organization of these cells (Dawes-Hoang, 2005).

Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2

A hallmark of epithelial invagination is the constriction of cells on their apical sides. During Drosophila gastrulation, apical constrictions under the control of the transcription factor Twist lead to the invagination of the mesoderm. Twist-controlled G protein signaling is involved in mediating the invagination but is not sufficient to account for the full activity of Twist. A Twist target was identified, the transmembrane protein T48, which acts in conjunction with G protein signaling to orchestrate shape changes. Together with G protein signaling, T48 recruits adherens junctions and the cytoskeletal regulator RhoGEF2 to the sites of apical constriction, ensuring rapid and intense changes in cell shape (Kolsch, 2007).

Apical constriction of cells can contribute to the invagination of epithelia, such as during gastrulation or organogenesis, and the closure of wounds. In the Drosophila embryo, apical constrictions occur along the ventral side of the blastoderm epithelium, leading to the formation of the ventral furrow and the invagination of the mesoderm. Proteins necessary for the mechanics of these cell shape changes include the Rho guanosine 5'-triphosphate-exchange factor RhoGEF2 and a heterotrimeric G protein. Whereas RhoGEF2 is essential for furrow formation, disruption of the heterotrimeric G protein, such as by loss of its α subunit Concertina (Cta), leads to a delay but no lasting defects in mesoderm morphogenesis. These maternally supplied proteins must be activated under the control of the zygotic genome in the embryo (Kolsch, 2007).

Twist is the zygotic transcriptional activator that is essential for the cell shape changes that produce the ventral furrow. One of its targets is the transcriptional repressor Snail, which is also essential for mesodermal morphogenesis (Kolsch, 2007).

However, the cell biological events responsible for the cell shape changes must ultimately be regulated by targets that are not transcription factors. Of the known Twist targets, only one, folded gastrulation (fog), is involved in mediating shape changes. Mutants in fog, which codes for a secreted peptide, show the same defects as embryos lacking Cta. Fog is therefore thought to act in the same pathway as Cta, which is referred to as Fog/Cta signaling (Kolsch, 2007).

Fog/Cta signaling is thought to cause changes in the actin cytoskeleton in conjunction with RhoGEF2. Recruitment of myosin from basal to apical in constricting ventral cells is partly dependent on Fog/Cta and absolutely dependent on RhoGEF2. Furthermore, the mammalian homologs of RhoGEF2 and Cta interact. Finally, binding of Drosophila RhoGEF2 to microtubules by means of EB1 is disrupted by activated Cta. Given that myosin recruitment and apical constriction are reduced but not abolished in the absence of Fog/Cta, there must be other factors regulated by Twist that explain its effects on apical constriction (Kolsch, 2007).

In a screen for genes that mediate the zygotic control of gastrulation, the region uncovered by the chromosomal deficiency Df(3R)TlP was found to be necessary for the proper formation of the ventral furrow. Phenotypic analysis and molecular mapping of a set of overlapping deficiencies identified the gene T48 as being responsible for the defects seen in Df(3R)TlP. T48 is expressed in the mesoderm. It codes for a predicted protein with a signal peptide and a potential transmembrane domain. When an internally hemagglutinin-tagged T48 protein (T48HA) was expressed in embryos, it localized at the peripheries of blastoderm cells, consistent with a close association with or insertion into the plasma membrane. Optical cross-sections showed that T48HA is targeted to the apical membrane (Kolsch, 2007).

No other structural motifs are recognizable in the protein. However, the C-terminal amino acid sequence -Ile-Thr-Thr-Glu-Leu (-ITTEL) conforms to the class I consensus for peptides that interact with PDZ domains. T48 has no obvious human ortholog but shows some similarity to the intracellular part of Fras1, which also has a PDZ-binding motif. To find candidates for PDZ domains that might interact with T48, the putative PDZ-binding sequence was analyzed with an algorithm designed to determine the PDZ domains that show the optimal fit for any given peptide. Of the predicted interactors, RhoGEF2 was particularly interesting in view of its role in ventral furrow formation. Furthermore, the mammalian ortholog of RhoGEF2 has been shown to bind to Plexin-B1 by means of a PDZ-binding motif (-Val-Thr-Asp-Leu) very similar to that of T48 (Kolsch, 2007).

Whether the C terminus of T48 is indeed able to interact with RhoGEF2 was tested. A 35S-labeled C-terminal peptide of T48 preferentially coprecipitated with the PDZ domain of RhoGEF2 rather than those of other PDZ domain-containing proteins, in contrast to Crumbs, which was used as a control and which preferentially coprecipitated with PDZ domains from its physiological interaction partner Stardust, as well as Bazooka. In Schneider S2 cells, a green fluorescent protein (GFP)-tagged RhoGEF2 PDZ domain or full-length RhoGEF2 was localized in the cytoplasm or formed intracellular aggregates when expressed alone, but localized to the plasma membrane when coexpressed with T48. In both assays, the interaction required the presence of the -ITTEL motif and was not seen with other PDZ domains. Thus, T48 interacts with RhoGEF2 by means of its PDZ-binding motif and is able to enrich RhoGEF2 to the plasma membrane (Kolsch, 2007).

To understand the function of T48 during gastrulation, the subcellular localization of RhoGEF2 and its dependence on T48 were studied in the developing embryo. Before gastrulation, the apical surfaces of the blastoderm epithelium are dome shaped and the developing adherens junctions are located subapically. RhoGEF2 is associated with the basally located furrow canals, whereas Armadillo is found just below this site and at a subapical position of the lateral cell membranes (Kolsch, 2007).

After cellularization was completed, these distributions changed specifically in ventral cells. Even before morphological changes occurred, RhoGEF2 and Armadillo disappeared from the basal ends. Subsequently, Armadillo disappeared from its subapical site and accumulated apically. A weak association of RhoGEF2 with the apical plasma membrane was seen at this stage (Kolsch, 2007).

As cells begin to flatten apically, high levels of both RhoGEF2 and Armadillo accumulate apically. Although they concentrated in the same region of the cell, Armadillo was restricted to the cell junctions, whereas RhoGEF2 was often more enriched between these sites. Notably, movement of the adherens junctions occurred not only in constricting cells but also in the more lateral mesodermal cells that flattened and became stretched on their apical sides (Kolsch, 2007).

To examine whether these processes depend on T48, stage-selected T48 mutant embryos were stained. Loss of RhoGEF2 and Armadillo from the basal side was unaffected in these embryos, as was the apical concentration of Armadillo. The cells flatten apically and lengthen, but the absence of constrictions results in a thick placode rather than an indentation. Localization of RhoGEF2 to the apical membrane is slightly delayed and possibly reduced. T48 therefore contributes to but is not essential for the recruitment of RhoGEF2 to the apical membrane. This is consistent with the observation that furrow formation is not completely abolished, but only delayed or weakened. Therefore other mechanisms were examined that might participate in RhoGEF2 localization (Kolsch, 2007).

As in the case of T48, mutations in the Fog/Cta pathway delay but do not abolish apical constriction and furrow formation. It was therefore considered whether Fog/Cta signaling might cooperate with T48 to recruit RhoGEF2. In embryos lacking Cta, the recruitment of RhoGEF2 was weakened. Combining mutations in cta and T48 resulted in much more notable effects. These cta,T48 embryos failed to make a furrow; the lack of apical constrictions was mirrored by a failure to accumulate RhoGEF2 apically. Thus, T48 and Fog/Cta signaling act in parallel to concentrate RhoGEF2 apically (Kolsch, 2007).

Severe defects were also observed in the behavior of the adherens junctions in the double-mutant embryos. Armadillo staining disappeared from its tight subapical localization but did not reaccumulate apically. Thus, movement of the junctions is not simply mediated by a tensile force from the constricting actin cytoskeleton: an independent step of at least partial disassembly must occur. It is speculated that this might be controlled by Snail, which regulates the disassembly of cell junctions in vertebrates. It was found that the disassembly of Armadillo from the subapical position was indeed blocked in snail (but not in twist) mutant embryos. Thus, Snail acts in parallel to Twist to direct the disassembly of subapical junctions, a process to which currently unknown Twist targets may also contribute (Kolsch, 2007).

Having observed that T48 and Fog/Cta activation are required for the apical localization of RhoGEF2 and Armadillo, whether T48, like Fog/Cta signaling, was able to trigger their relocalization in other cells was also tested. Ubiquitous expression of T48 in the embryo led to a concentration of RhoGEF2 at the apical membranes of lateral cells. Armadillo localization in ectodermal cells was no longer restricted to a distinct subapical domain but extended to the apical end of the lateral membranes in many cells. When T48 was coexpressed with activated Cta, this effect was slightly enhanced, and some embryos showed morphological defects (Kolsch, 2007).

With T48, a missing factor has been found in the control cascade from transcriptional regulation by Twist to the cell biological mediators of furrow morphogenesis. Two Twist targets, Fog and T48, appear to act in separate pathways that converge on RhoGEF2, which integrates the signal to activate myosin and modify the actin cytoskeleton. This model shows the maternally supplied RhoGEF2 is largely attached to microtubules by means of EB1. The onset of Twist expression has two effects. Fog is synthesized, which triggers the activation of Cta. This in turn releases RhoGEF2 from the microtubules that, by analogy to its vertebrate homologs, may bind to Cta through its RGS domain, allowing some myosin activation and constriction. In parallel, T48 is synthesized and targeted to the apical membrane, where it acts to concentrate RhoGEF2 through its PDZ-binding motif. In the absence of Fog-mediated displacement of RhoGEF2 from EB1, T48 can probably still recruit sufficient freely diffusible RhoGEF2 to allow slow constriction. Only when both mechanisms fail are the downstream events of constriction and junction reassembly abolished completely (Kolsch, 2007).

The utilization of Gα12/13 proteins and a microtubule-bound RhoGEF have also been reported in vertebrate gastrulation. The absence of an obvious homolog of T48 in vertebrates might suggest that this element of the control mechanism is unique to Drosophila gastrulation. However, the PDZ-binding motif in Plexin-B1 is similar to that of T48 and acts during neuronal growth cone remodeling by recruiting PDZ-RhoGEF. Therefore, this mechanism of controlling cell shape may operate in a variety of systems (Kolsch, 2007).

Pulsed contractions of an actin-myosin network drive apical constriction

Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. This study shows, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. These results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally (Martin, 2009).

During Drosophila gastrulation, apical constriction of ventral cells facilitates the formation of a ventral furrow and the subsequent internalization of the presumptive mesoderm. Although myosin is known to localize to the apical cortex of constricting ventral furrow cells, it is not known how myosin produces force to drive constriction. Understanding this mechanism requires a quantitative analysis of cell and cytoskeletal dynamics. Methods were developed to reveal and quantify apical cell shape with Spider-GFP, a green fluorescent protein (GFP)-tagged membrane-associated protein that outlines individual cells. Ventral cells were constricted to about 50% of their initial apical area before the onset of invagination and continued to constrict during invagination. Although the average apical area steadily decreased at a rate of about 5 microm2 min-1, individual cells showed transient pulses of rapid constriction that exceeded 10-15 microm2 min-1. During the initial 2 min of constriction, weak constriction pulses were often interrupted by periods of cell stretching. However, at 2 min, constriction pulses increased in magnitude and cell shape seemed to be stabilized between pulses, leading to net constriction. These two phases probably correspond to the 'slow/apical flattening' and 'fast/stochastic' phases that have been described previously. Overall, cells underwent an average of 3.2 ± 1.2 constriction pulses over 6 min, with an average interval of 82.8 ± 48 s between pulses (mean ± s.d., n = 40 cells, 126 pulses). Constriction pulses were mostly asynchronous between adjacent cells. As a consequence, cell apices between constrictions seemed to be pulled by their constricting neighbours. Thus, apical constriction occurs by means of pulses of rapid constriction interrupted by pauses during which cells must stabilize their constricted state before reinitiating constriction (Martin, 2009).

To determine how myosin might generate force during pulsed constrictions, myosin and cell dynamics were simultaneously imaged by using myosin regulatory light chain (spaghetti squash, or squ) fused to mCherry (Myosin-mCherry) and Spider-GFP. Discrete myosin spots and fibres present on the apical cortex formed a network that extended across the tissue. These myosin structures were dynamic, with apical myosin spots repeatedly increasing in intensity and moving together (at about 40 nm s-1) to form larger and more intense myosin structures at the medial apical cortex. This process, which is referred to as myosin coalescence, resulted in bursts of myosin accumulation that were correlated with constriction pulses. The peak rate of myosin coalescence preceded the peak constriction rate by 5-10 s, suggesting that myosin coalescence causes apical constriction. Between myosin coalescence events, myosin structures, including fibres, remained present on the cortex, possibly maintaining cortical tension between constriction pulses. Contrary to the purse-string model, no significant myosin accumulation was seen at cell-cell junctions. To confirm that constriction involved medial myosin coalescence and not contraction of a circumferential purse-string, constriction rate was correlated with myosin intensity at either the medial or junctional regions of the cell. Apical constriction was correlated more significantly with medial myosin, suggesting that, in contrast to the purse-string model, constriction is driven by contractions at the medial apical cortex (Martin, 2009).

Myosin coalescence resembled contraction of a cortical actin-myosin network. Therefore, to determine whether apical constriction is driven by pulsed contractions of the actin-myosin network, the organization of the cortical actin cytoskeleton was examined. In fibroblasts and keratocytes, actin network contraction bundles actin filaments into fibre-like structures. Consistent with this expectation was the identification of an actin filament meshwork underlying the apical cortex in which prominent actin-myosin fibres spanning the apical cortex appeared specifically in constricting cells. An actin-myosin network contraction model would predict that myosin coalescence results from myosin spots exerting traction on each other through the cortical actin network. To test whether myosin coalescence requires an intact actin network, the actin network was disrupted with cytochalasin D (CytoD). Disruption of the actin network with CytoD resulted in apical myosin spots that localized together with actin structures and appeared specifically in ventral cells. Myosin spots in CytoD-injected embryos showed more rapid movement than those in control-injected embryos, suggesting that apical myosin spots in untreated embryos are constrained by the cortical actin network. Although myosin movement was uninhibited in CytoD-treated embryos, myosin spots failed to coalesce and cells failed to constrict. Because myosin coalescence requires an intact actin network, it is proposed that pulses of myosin coalescence represent contractions of the actin-myosin network (Martin, 2009).

Because actin-myosin contractions occurred at the medial apical cortex, it was unclear how the actin-myosin network was coupled to adherens junctions. Therefore E-Cadherin-GFP and Myosin-mCherry were imaged to examine the relationship between myosin and adherens junctions. Before apical constriction, adherens junctions are present about 4 microm below the apical cortex. As apical constriction initiated, these subapical adherens junctions gradually disappeared and adherens junctions simultaneously appeared apically at the same level as myosin. This apical redistribution of adherens junctions occurred at specific sites along cell edges (midway between vertices). As apical constriction initiated, these sites bent inwards. This bending depended on the presence of an intact actin network, which is consistent with contraction of the actin-myosin network generating force to pull junctions. Indeed, myosin spots undergoing coalescence were observed to lead adherens junctions as they transiently bent inwards. Thus, pulsed contraction of the actin-myosin network at the medial cortex seems to pull the cell surface inwards at discrete adherens junction sites, resulting in apical constriction (Martin, 2009).

The transcription factors Twist and Snail regulate the apical constriction of ventral furrow cells. Snail is a transcriptional repressor whose target or targets are currently unknown, whereas Twist enhances snail expression and activates the expression of fog and t48, which are thought to activate the Rho1 GTPase and promote myosin contractility. To examine the mechanism of pulsed apical constriction further, how Twist and Snail regulate myosin dynamics was tested. In contrast to wild-type ventral cells, in which myosin was concentrated on the apical cortex, twist and snail mutants accumulated myosin predominantly at cell junctions, similarly to lateral cells. These ventral cells failed to constrict productively, which supported the cortical actin-myosin network contraction model, rather than the purse-string model, for apical constriction. twist and snail mutants differentially affected the coalescence of the minimal myosin that did localize to the apical cortex. Although myosin coalescence was inhibited in snail mutants, it still occurred in twist mutants, as did pulsed constrictions. This difference was also observed when Snail or Twist activity was knocked down by RNA-mediated interference. However, the magnitude of constriction pulses in twistRNAi embryos was greater than that of twist mutant embryos, suggesting that the low level of Twist activity present in twistRNAi embryos enhances contraction efficiency by activating the expression of snail or other transcriptional targets. Myosin coalescence was inhibited in snail twist double mutants, demonstrating that the pulsed constrictions in twist mutants required snail expression. Thus, the expression of snail, not twist, initiates the actin-myosin network contractions that power constriction pulses (Martin, 2009).

Net apical constriction was inhibited in both snailRNAi and twistRNAi embryos. It was therefore asked why the pulsed contractions that were observed in twistRNAi embryos failed to constrict cells. Using Spider-GFP to visualize cell outlines, it was found that although constriction pulses were inhibited in snailRNAi embryos, constriction pulses still occurred in twistRNAi embryos. However, the constricted state of cells in twistRNAi embryos was not stabilized between pulses, resulting in fluctuations in apical area with little net constriction. This stabilization defect was not due to lower snail activity, because these fluctuations continued when snail expression was driven independently of twist by using the P[sna] transgene. Although the frequency and magnitude of constriction pulses in such embryos were similar to those in control embryos, stretching events were significantly higher in twistRNAi; P[sna] embryos, suggesting a defect in maintaining cortical tension. This defect might result from a failure to establish a dense actin meshwork, because both twist mutants and twistRNAi embryos had a more loosely arranged apical meshwork of actin spots and fibres than constricting wild-type cells did. twist expression therefore stabilizes the constricted state of cells between pulsed contractions (Martin, 2009).

Thus, a 'ratchet' model is proposed for apical constriction, in which phases of actin-myosin network contraction and stabilization are repeated to constrict the cell apex incrementally. In contrast to the purse-string model, it was found that apical constriction is correlated with pulses of actin-myosin network contraction that occur on the apical cortex. Pulsed cortical contractions could allow dynamic rearrangements of the actin network to optimize force generation as cells change shape. Because contractions are asynchronous, cells must resist pulling forces from adjacent cells between contractions. A cortical actin-myosin meshwork seems to provide the cortical tension necessary to stabilize apical cell shape and promote net constriction. The transcription factors Snail and Twist are critical for the contraction and stabilization phases of constriction, respectively. Thus, Snail and Twist activities are temporally coordinated to drive productive apical constriction. Despite the dynamic nature of the contractions in individual cells, the behaviour of the system at the tissue level is continuous, in a similar manner to convergent extension in Xenopus. Pulsed contraction may therefore represent a conserved cellular mechanism that drives precise tissue-level behaviour (Martin, 2009).

Heterotrimeric G protein signaling governs the cortical stability during apical constriction in Drosophila gastrulation

During gastrulation in Drosophila melanogaster, coordinated apical constriction of the cellular surface drives invagination of the mesoderm anlage. Forces generated by the cortical cytoskeletal network have a pivotal role in this cellular shape change. This study shows that the organisation of cortical actin is essential for stabilisation of the cellular surface against contraction. Mutation of genes related to heterotrimeric G protein (HGP) signaling, such as Gβ13F, Gγ1, and ric-8, results in formation of blebs on the ventral cellular surface. The formation of blebs is caused by perturbation of cortical actin and induced by local surface contraction. HGP signaling mediated by two Gα subunits, Concertina and G-iα65A, constitutively regulates actin organisation. It is proposed that the organisation of cortical actin by HGP is required to reinforce the cortex so that the cells can endure hydrostatic stress during tissue folding (Kanesaki, 2013).

The coordinated movement of cells is one of the foundations of tissue morphogenesis. The forces driving the cellular movements are generated by surface dynamics, such as rearrangements of cell adhesions and changes of the contractility of cortical acto-myosin networks. However, the surface mechanics resisting deformation forces and maintaining cortical integrity are not well understood (Kanesaki, 2013).

The shape of the cell surface can change dynamically. One notable surface feature is the bleb, a spherical protrusion of the plasma membrane observed in diverse cellular processes such as locomotion, division, and apoptosis. Formation of blebs is driven by hydrostatic pressure in the cytoplasm. According to the current model, blebbing starts with local compression of the cytoskeletal network and proceeds according to a subsequent increase of the pressure. The compression of the cytoskeleton is mediated by the contractile force of non-muscle myosin II (MyoII). Though it has been shown that various cells, such as germ line and cancer cells, utilise blebs for their motility, the role of blebs and the mechanism of blebbing in tissue morphogenesis are still largely unclear (Kanesaki, 2013).

Invagination of a cellular layer is one of the common events in tissue morphogenesis. In gastrulation in Drosophila, ventral cells of the blastoderm embryo invaginate and then differentiate to mesoderm. The process of mesoderm invagination can be grossly divided into two sequential steps: apical constriction and furrow internalisation. During apical constriction, ventral cells collectively contract their apices and consequently form a shallow furrow on the embryo. During furrow internalisation, the ventral furrow becomes deeper and the layer of cells becomes engulfed in the embryonic body. The molecular and cellular mechanisms underlying apical constriction have been studied extensively. The change of cellular shape is mediated by integrated functioning of the cortical acto-myosin network and cellular adherens junction complex. The force driving the constriction is generated by pulsed contractility of MyoII. The tensile force from individual cells is transmitted to epithelial tissue through the adherens junction, and the tissue generates feedback force that leads to anisotropic constriction of ventral cells (Kanesaki, 2013).

Heterotrimeric Gprotein (HGP) has an important role in apical constriction in Drosophila gastrulation. Signaling triggered by the extracellular ligand folded gastrulation (fog) promotes surface accumulation of MyoII in ventral cells, and the Fog signaling is mediated through an HGP α subunit encoded by concertina (cta). HGP belongs to the GTPase family, and its activity is regulated by multiple factors, including guanine nucleotide exchange factor (GEF). A previous study showed that ric-8 mutation results in a twisted germ-band due to abnormal mesoderm invagination. ric-8 was first identified as a gene responsible for synaptic transmission in Caenorhabditis elegans, and was shown to interact genetically with EGL-30 (C. elegans Gαq). Nematoda and vertebrate Ric-8 has GEF activity and positively regulates HGP signalingin vivo and in vitro. In Drosophila, Ric-8 is essential for targeting of HGPs toward the plasma membrane and participates in HGP-dependent processes such as asymmetric division of neuroblasts (Kanesaki, 2013 and references therein).

In this study, the precise role of ric-8 in mesoderm invagination was investigated. It was found that cortical stability of ventral cells is impaired in a ric-8 mutant. By a combination of genetic and pharmacological analyses, blebbing of ventral cells was found to be induced by either disruption of cortical actin or mutation of ric-8. It is suggested that HGP signaling constitutively organises cortical actin, thereby reinforcing the resistance of cells against deformation (Kanesaki, 2013).

Ventral cells intrinsically exhibit a few small blebs during mesoderm invagination. This indicates that surface contraction during apical constriction induces blebbing even in normal invagination. This study found that Ric-8 and HGP signaling are required for suppression of abnormally large blebs, and for the stabilisation of the cortex in invaginating cells. The physical mechanism underlying blebbing has been studied extensively in cultured cells. The contractile force of the acto-myosin network causes an increase of hydrostatic pressure in the cytoplasm, which leads to detachment of the plasma membrane from the cortical actin layer. The dynamics of blebs observed in ric-8 ventral cells were similar to those reported in cultured cells in terms of time and size, suggesting that the mechanisms underlying blebbing in these two systems are conserved (Kanesaki, 2013).

The average size of blebs changes as development proceeds: blebs become larger during furrow internalisation. Immuno-fluorescence imaging revealed that MyoII is abnormally accumulated beneath enlarged blebs in the ric-8 mutant. This correlation suggests that MyoII acts to induce an increase of hydrostatic pressure. Although MyoII is an indispensable factor for apical constriction, its activity can also cause malformation of the cells. How MyoII accumulates abnormally in the ric-8 mutant remains unclear. It cannot be ruled out that other processes of mesoderm invagination, such as mechanical stress from surrounding cells, also contributes to the enlargement of blebs. During apical constriction, epithelial tissue generates tension along the anterior-posterior axis, and ventral cells undergo constriction in an anisotropic manner. Similar force may also be generated at the tissue level during furrow internalisation, causing the cells there to be squeezed, and consequently increasing the intracellular pressure. Blebbing in the ric-8 mutant may be a consequence of abnormal cytoskeletal networks and physical stress acting cell to cell. In normal situations, cells would resist such physical stress and maintain the surface integrity, thereby supporting correct morphogenetic movements (Kanesaki, 2013).

This study demonstrates that HGP signaling has two functions in mesoderm invagination: induction of the apical constriction via MyoII accumulation and maintenance of the cellular surface via organisation of cortical actin. Although Fog is required for apical constriction, F-actin is organised in a Fog-independent manner, suggesting that these two functions are regulated in different ways. cta mutants and G-iα65A mutants showed similar phenotypes regarding cortical actin, suggesting that these Gα paralogs have overlapping functions. Because the Drosophila genome encodes 6 Gα subunits and 5 of them are expressed in early embryos, the contribution of G α paralogs other than Cta and G-iα65A to the suppression of blebbing cannot be rule out. The finding that ric-8, Gβ13F, and Gγ1 mutants showed blebbing, a hallmark of severely disturbed cortical actin, supports the idea that multiple HGP pathways control cortical actin redundantly. However, currently it is not known whether those signaling pathways act on the same downstream effectors. Considering that most blastoderm cells showed a dispersed signal of GFP-Moesin in the mutants, HGPs appear to be rather constitutive regulators of cortical actin organisation. Nevertheless, the abnormality of the cortex does not affect the morphology of the 'standstill' cells that do not carry out the inward movement. Thus, HGPs are required to reinforce the cortex so that the cells can endure the stress generated during tissue folding (Kanesaki, 2013).

It was previously reported that ric-8 is required for Drosophila gastrulation. This study extensively investigated mesoderm invagination and found that apical constriction is indeed compromised in the ric-8 mutant. Based on the observation of Fog-dependent MyoII accumulation, it is concluded that ric-8 is required for Fog-Cta signaling. It is unlikely that this phenotype is a secondary consequence of the disorganised F-actin in the ric-8 mutant, because actin was organised normally in the fog mutant embryo and ectopic Fog expression induced cell flattening even in late B-treated embryos. These findings instead suggested that Fog-Cta signaling and actin organisation are separate pathways and Ric-8 is involved in both pathways (Kanesaki, 2013).

Given that HGPs constitutively regulate F-actin, the signaling seems to be active in most blastoderm cells. Some unknown extracellular ligand and its receptor thus appear to be expressed to activate HGPs. It is also possible that cytoplasmic HGP regulators such as Pins, Loco, or other RGS proteins are involved in the activation. In the formation of the blood-brain barrier in Drosophila, Pins and Loco positively regulate HGP signaling. Embryos mutant for Pins also show abnormal cellular movements during mesoderm invagination. It is also intriguing to hypothesise that Ric-8 participates in the activation of HGPs through its GEF activity, which has been characterised both in vivo and in vitro. This hypothesis suggests the possibility that HGPs are endogenously activated. Future analysis of the responsible cytoplasmic regulators may clarify the mechanism of HGP regulation, and may give new insights regarding the intricate network of HGP signaling in animal development (Kanesaki, 2013).

How might HGP be functionally linked to actin polymerisation? Since G α12/13 participates in the activation of Formin family proteins in mammalian fibroblasts and a human Formin inhibits the formation of blebs in a prostate cancer cell line, a candidate factor regulating actin filaments downstream of HGP could be Diaphanous (Dia), a Drosophila Formin. Although it has been shown that organisation of actin via Dia is required for ventral furrow invagination, it is unclear whether Dia is also required for cortical stability during morphogenesis. Considering that Dia is an actin nucleator, it is speculated that Dia might act in the assembly of the actin meshwork and thereby reinforce the cortex. Indeed, it was observed that the dia mutant embryos showed cellular deformation during gastrulation, suggesting the functional relevance of the actin nucleator in the suppression of blebs. Further analysis will be required to clarify the functions of Dia (Kanesaki, 2013).

Previous studies demonstrated that ventral cells form a particular type of F-actin meshwork that makes a basic frame for apical constriction. RhoA- and Abelson-mediated signaling is required for organisation of the apical F-actin meshwork, while the Fog-Cta pathway is not. Thus, it is surprising that the mutants for HGPs, including Cta, showed a defect of cortical actin. HGP signaling may organise only a moiety of F-actin which is distinct from the one specifically accumulated at apices. HGP signaling regulates the organisation of cortical actin and mediates the establishment of the blood-brain barrier in Drosophila , suggesting that this function of HGPs is rather common in fly embryogenesis (Kanesaki, 2013).

Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation

Gastrulation of Drosophila melanogaster proceeds through sequential cell movements: ventral mesodermal (VM) cells are induced by secreted Fog protein to constrict their apical surfaces to form the ventral furrow, and subsequently lateral mesodermal (LM) cells involute toward the furrow. How these cell movements are organized remains elusive. This study observed that LM cells extend apical protrusions and then undergo accelerated involution movement, confirming that VM and LM cells display distinct cell morphologies and movements. In a mutant for the GPCR kinase Gprk2, apical constriction expands to all mesodermal cells and the involution movement is abolished. In addition, the mesodermal cells halt apical constriction prematurely in accordance with the aberrant accumulation of Myosin II. Epistasis analyses revealed that the Gprk2 mutant phenotypes are dependent on the fog gene. Overexpression of Gprk2 suppresses the effects of excess Cta, a downstream component of Fog signaling. Based on these findings, it is proposed that Gprk2 attenuates and tunes Fog-Cta signaling to prevent apical constriction in LM cells and to support appropriate apical constriction in VM cells. Thus, the two distinct cell movements in mesoderm invagination are not predetermined, but rather are organized by the adjustment of cell signaling (Fuse, 2013).

In the Gprk2 mutant embryos, cell movements triggered by Fog signaling were compromised. fog is genetically epistatic to Gprk2, indicating that Gprk2 functions by acting on Fog signaling. LM cells undergo apical constriction in the Gprk2 mutant, suggesting that Gprk2 normally inhibits Fog signaling in LM cells. Premature termination of apical constriction and abnormal accumulation of Myosin were also observed in the Gprk2 mutant, suggesting that Gprk2 adjusts Fog signaling to an appropriate level in VM cells. Thus, Gprk2 regulates Fog signalingin a cell group-dependent manner. But what are the underlying molecular mechanisms (Fuse, 2013)?

It is known that GPCR kinase phosphorylates the C-terminal region of GPCR, and regulates GPCR signaling by multiple mechanisms. The phosphorylated GPCR dissociates from the G protein and is internalized from the plasma membrane. This produces a negative-feedback loop for GPCR signaling. Theoretically, the negative-feedback loop stabilizes the signaling and generates biphasic output from fluctuating inputs: OFF for low inputs and ON for high inputs. It is speculated that Gprk2 might phosphorylate a GPCR and might generate biphasic output for Fog signaling in a spatial manner: OFF in LM cells and ON in VM cells. The Fog receptor is expected to be a GPCR, since a G protein (Cta) functions downstream of Fog. Identification of the Fog receptor would help to clarify the molecular functions of Gprk2 (Fuse, 2013).

The kinase activity of Gprk2 is essential for gastrulation. Although it is not yet known what substrates are phosphorylated by Gprk2 in this process, one might be Gprk2 itself because it was observed that Gprk2 protein was phosphorylated in S2 cultured cells and that the phosphorylation was abolished in the K338R mutant of Gprk2. Autophosphorylation of other GPCR kinases has been demonstrated previously and is thought to stimulate their binding to GPCR. Autophosphorylation of Gprk2 might play a similar role (Fuse, 2013).

In addition to its kinase activity, GPCR kinase has an RGS domain, which exhibits GAP (GTPase activating protein) activity and functions in recycling of the Gα protein. Therefore, whether Gprk2 exhibits GAP activity for Cta is an intriguing issue. Indeed, this possibility was supported by genetic data showing that Gprk2 suppresses the effect of Cta overexpression, but not that of Cta Q303L, the GTP-bound form of Cta protein. Cta Q303L might not be subject to the inhibitory effect (GAP activity) of Gprk2, although the alternative explanation has not been ruled out that the inhibition of Cta Q303L might require more Gprk2 protein than does the inhibition of wild-type Cta. Considering that GPCR kinase regulates GPCR signaling by multiple mechanisms, it is suggested that the repression of Cta activity might be one of several means by which Gprk2 regulates Fog signaling (Fuse, 2013).

Fog signaling stimulates the apical localization of Myosin, which generates a force to constrict the apical cell surface. In the wild-type embryo, Myosin protein appears and disappears at the apical surface in a dynamic pattern that they described as 'pulsed coalescence'. In the Gprk2 mutant, Myosin continued to accumulate on the entire apical surface of mesodermal cells. Similar phenomena were also observed in Cta-overexpressing ectodermal cells, and this phenotype was suppressed by simultaneous expression of Gprk2. It is suggested that Gprk2 normally attenuates Fog-Cta signaling to an appropriate level, and such refinement might contribute to controlling the dynamics of Myosin protein (Fuse, 2013).

Previous studies showed that Gprk2 acts in Hedgehog (Hh) signaling for imaginal disc patterning. In this process, Gprk2 phosphorylates a GPCR, Smoothened, and potentiates Hh signaling. Thus, Gprk2 plays roles in multiple signaling pathways in various contexts during development (Fuse, 2013).

The movements of LM cells were characterized, and were found to extended apical protrusions. Some examples have been documented of the extension of protrusions by epithelial cells, such as dorsal ectodermal cells of embryos and wing disc cells of larvae in Drosophila. However, the mechanisms that induce the protrusion and the roles of protrusion in directional cell movement are not understood. Since it was observed that apical protrusions in LM cells always pointed toward the ventral furrow and that cells close to the furrow extended longer protrusions than cells distant from it, it is speculated that the apical protrusion might be induced by the apically constricting neighbors. Indeed, in cta mutant embryos the apical protrusions did not always point toward mid-ventral, but rather frequently pointed toward the slight depressions that were formed at random positions by uncoordinated apical constriction. One possibility is that mechanical or chemical signals that emanate from apically constricting cells might induce apical protrusions in surrounding cells (Fuse, 2013).

Apical protrusions became apparent when LM cells started to accelerate toward the ventral furrow. From this observation, it is supposed that the directional protrusion might contribute to the movement of LM cells. Given that the apical protrusion of LM cells is analogous to the pseudopod of cultured cells, the apical protrusion might act as a scaffold for pulling the cell body into the furrow. The fact that the apical protrusion was also observed in some ectodermal cells, which never undergo involution movement, suggests that the apical protrusion is not sufficient to induce involution movement and that other mechanisms might regulate the cell movement in parallel (Fuse, 2013).

Drosophila mesoderm invagination is driven by sequential movements of different cells. The apical constriction of VM cells is one of the essential movements in this process. It is expected that the involution movement of LM cells might be another of the cell movements driving mesoderm invagination. The movements of different cells would probably influence each other in a complex manner. Observations of LM movements might be explained by such a coordination of cell movements. For example, the apical constriction of VM cells might stretch LM cells and thereby prevent LM cell apical constriction, as previously suggested. VM cells might then continue to move inward and pull LM cells toward the ventral furrow. In addition, ectodermal cells might generate a force to push mesodermal cells inward. These possibilities are not mutually exclusive. Further analyses are required to clarify the role of each cell movement and the effect of coordinated movements in mesoderm invagination (Fuse, 2013).

In the Gprk2 mutant, LM cells underwent apical constriction instead of involution movement. Given the inhibition of Fog signaling in the wild-type LM cells, involution movement might be a default state of mesodermal cells without Fog signaling. As noted above, in the fog and cta mutant, apical constriction occurs in some VM cells, and involution-like movement operates in an uncoordinated manner. These uncoordinated cell movements finally result in disorganized, but nearly complete, mesoderm invagination. Thus, apical constriction and involution movements seem to be alternative choices for mesodermal cells, and robust mesoderm invagination might progress via either type of cell movement. In normal Drosophila embryos, cell movements are spatially and temporally organized, and such organization might ensure the correct shape of gastrulae (Fuse, 2013).

Cell movements in gastrulation show diversity among insects. For example, mosquito embryos undergo only apical constriction and no apparent involution process. Locust embryos undergo neither apical constriction nor involution, but instead utilize the delamination of individual mesodermal cells. Compared with gastrulation in these insects, Drosophila gastrulation is a more complex process and is completed within a shorter time (15 minutes compared with hours). The highly organized cell movements in Drosophila might enable this rapid completion of gastrulation. The molecular mechanisms underlying the evolution of insect gastrulation are an intriguing issue for future studies (Fuse, 2013).

Passive mechanical forces control cell-shape change during Drosophila ventral furrow formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. This work develops a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. Novel data is introduced that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. Maintenance of this constant volume is shown to be sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. The specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, this model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that is observed in the actual embryo, with no active force generation required other than apical constriction (Polyakov, 2014).

Embryo-scale tissue mechanics during Drosophila gastrulation movements
Morphogenesis of an organism requires the development of its parts to be coordinated in time and space. While past studies concentrated on defined cell populations, a synthetic view of the coordination of these events in a whole organism is needed for a full understanding. Drosophila gastrulation begins with the embryo forming a ventral furrow, which is eventually internalized. It is not understood how the rest of the embryo participates in this process. This study used multiview selective plane illumination microscopy coupled with infrared laser manipulation and mutant analysis to dissect embryo-scale cell interactions during early gastrulation. Lateral cells have a denser medial-apical actomyosin network and shift ventrally as a compact cohort, whereas dorsal cells become stretched. The behaviour of these cells affects furrow internalization. A computational model predicts different mechanical properties associated with tissue behaviour: lateral cells are stiff, whereas dorsal cells are soft. Experimental analysis confirms these properties in vivo.

Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis

Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. This study reports the function of a common pathway comprising the heterotrimeric G proteins Gα12/13 (Concertina), Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog (Poor gastrulation, Pog & CG31660) was identified as being required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. It is proposed that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators (Kerridge, 2016).

During tissue morphogenesis, cells rearrange their contacts to invaginate, intercalate, delaminate or divide. During Drosophila gastrulation, invagination of the presumptive mesoderm in the ventral region of the embryo and of the posterior midgut requires apical cell constriction, a geometric deformation that occurs in different organisms. Elongation of the ventral–lateral ectoderm requires cell intercalation, a general topological deformation associated with junction remodelling. In the ectoderm, the so-called ‘vertical junctions’, oriented along the dorsal–ventral axis, shrink, followed by extension of new ‘horizontal’ junctions along the anterior–posterior axis. Despite differences in the cell deformations associated with intercalation and apical constriction, recent studies revealed that both processes require myosin II (MyoII) contractility. Cell shape changes rely on the pulsatile activity of MyoII in the apical–medial cortex, whereby MyoII undergoes cycles of assembly and disassembly allowing stepwise deformation1. Moreover, each step of deformation is stabilized and thereby retained, contributing to the irreversibility of tissue morphogenesis. In the mesoderm, each phase of apical area constriction mediated by MyoII pulses is followed by a phase of shape stabilization involving persistence of medial MyoII. In the ectoderm, medial–apical MyoII pulses flow anisotropically towards vertical junctions resulting in steps of shrinkage that are stabilized by a planar-polarized pool of junctional MyoII. This ratchet-like behaviour of MyoII is regulated by the Rho1–Rok pathway and requires quantitative control over MyoII activation. Low Rho1/Rok activity fails to form actomyosin networks, intermediate activation establishes MyoII pulsatility and high activation confers stability. The signalling mechanisms that cause stepwise activation of MyoII by Rho1 remain unknown. It is also unclear whether different pathways for Rho1 activation operate in the mesoderm and in the ectoderm as indeed Rho1 can be activated by numerous signalling mechanisms or whether a common pathway might exist (Kerridge, 2016).

Tissue-specific factors can result in polarized shape changes by signalling through cell surface receptors. For instance, in Drosophila ectoderm, pair rule genes encoding transcription factors control planar-polarized enrichment of MyoII through the combinatorial expression of the surface proteins Toll2, Toll6 and Toll8 in stripes. Likewise, in the mesoderm, Twist and Snail induce expression of Fog, a secreted ligand, and a G-protein-coupled receptor (GPCR) Mist (methuselah-like 1), which is reported to transduce Fog. The downstream G protein Gα12/13 (known as Concertina (Cta) in Drosophila) is required for RhoGEF2 and thereby MyoII apical recruitment. As RhoGEF2 is a known GEF for Rho1, the requirement of Gα12/13 for RhoGEF2 apical recruitment suggests that GPCRs and G-protein signalling mediate MyoII activation through the Rho1 pathway. These considerations prompted asking whether G-protein signalling directly controls the different regimes of MyoII dynamics (pulsatility and/or stability) in the mesoderm and planar polarized activation of Rho1 and MyoII in the ectoderm (Kerridge, 2016).

This study reports the function of the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and regulating MyoII dynamics both in the mesoderm and in the ectoderm. Receptor activation, through the GEF activity of the GPCR, converts Gα from an inactive GDP-bound state, in a complex with Gβγ, to an active GTP-bound state. This results in dissociation of Gβγ, enabling binding of both Gα–GTP and Gβγ to their respective effectors for signalling. This study found that Gα12/13 and the Gβ13F/Gγ1 complex constitute distinct signalling modules, which regulate MyoII dynamics medial–apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog, was found to be required for cell shape changes associated with both mesoderm invagination and ectoderm elongation. During these morphogenetic events, Smog functions with other GPCRs, Mist in the mesoderm and an as yet unknown GPCR in the ectoderm, to activate the Rho1–Rok pathway. This results in stepwise activation of Rho1 and MyoII, ensuring irreversible cell shape changes (Kerridge, 2016).

First, this study reports that Gα12/13 and Gβ13F/Gγ1 function as distinct signalling modules that control Rho1 and MyoII in different domains. Gα12/13 activates medial–apical MyoII through its effector RhoGEF2 both in the ectoderm and the mesoderm. In mammals, p115–RhoGEF interacts directly with Gα12 suggesting that this may be a conserved signalling module. In contrast, Gβ13F/Gγ1 activates MyoII both at cell junctions and in the medial–apical domain. This modularity may provide distinct regulatory mechanisms for the activation of MyoII in different subcellular compartments owing to the existence of different molecular effectors of Gα–GTP and Gβγ. Second, stepwise activation of Rho1 by multiple GPCRs and their ligands determines the emergence of a pulsatile regime medial–apically, or stable activation. In the mesoderm, Smog and Mist GPCRs, together with high expression of their ligand Fog, ensure stabilization and rapid (<5 min) accumulation of MyoII ensuring apical constriction. In the ectoderm, low Fog expression and thus lower activation of Gα12/13 and RhoGEF2 is responsible for intermediate medial–apical activation of MyoII and pulsatility. Indeed, Fog, constitutively active Gα12/13QL and RhoGEF2 overexpression all lead to stable accumulation of MyoII instead of pulsation, similar to constitutively active RhoV14 (Kerridge, 2016).

Interestingly, the same receptor Smog controls MyoII activation in different subcellular domains during intercalation and apical constriction begging the question of how activation of Gα12/13 and Gβγ is differentially achieved in the ectoderm and the mesoderm. The polarization of Smog activation is to some extent imparted by the ligand. Fog/Smog regulates medial–apical accumulation of MyoII in the two tissues: Fog induces medial Rho1 and Rok activation in the mesoderm and ectoderm and, when ectopically expressed in the ectoderm, it can increase Rho1 and Rok in the medial cortex. This argues that another mechanism results in junction-specific activation of Smog, Gβ13F/Gγ1, Rho1 and Rok in the ectoderm (Kerridge, 2016).

It is possible that an unknown ectoderm-specific ligand activates Smog specifically at junctions. Junctional localization of the Rho1 pathway by Smog may also be imparted by subcellular processing of Smog signalling, such as localization/activation of downstream effectors of Gα12/13 and Gβγ. The recently identified Toll receptors required for MyoII planar-polarized activation may bias Smog signalling although the molecular mechanisms remain unclear. This could be through localization of RhoGEFs. In the mesoderm, the transmembrane protein T48 localizes RhoGEF2 apically through binding to its PDZ domain, and is required for apical MyoII activation in parallel with Smog, Gα12/13 and Gβγ. Similarly, other GEFs may be required for junctional Rho1 activation by Smog (Kerridge, 2016).

What might be the advantage of having multiple GPCRs? Gastrulation sets the foundation for all other future processes in development and hence requires robustness. GPCRs with similar functions yet subtle differences such as ligand specificity may offer advantages compared with single ligand–receptor pairs. For instance, high cortical tension associated with mesoderm invagination may require multiple GPCRs activating parallel pathways to attain efficiency of the process. Moreover, multiple GPCRs may concede tissue-specific regulation of the common G-protein subcellular pathways. Finally, multiple GPCRs can allow stepwise activation of MyoII. Although activation by one GPCR is sufficient to induce pulsatility, more GPCRs are required to shift the actomyosin networks to more stable regimes (Kerridge, 2016).

The discovery that Smog and heterotrimeric G protein activate Rho1 and MyoII in two different morphogenetic processes provides a potentially general molecular framework for tissue mechanics. It is proposed that different developmental inputs tune a common GPCR/G-protein signalling pathway to direct specific patterns and levels of Rho1 activation. Quantitative control specifies the regime of MyoII activation through Rho1, namely pulsatility or stability of MyoII. Modular control defines the subcellular domains where MyoII accumulates (medial–apical or junctions) depending on molecular effectors. How developmental signals tune GPCR signalling will be important to decipher (Kerridge, 2016).

Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids

Many models of morphogenesis are forced to assume specific mechanical properties of cells, because the actual mechanical properties of living tissues are largely unknown. This study measured the rheology of epithelial cells in the cellularizing Drosophila embryo by injecting magnetic particles and studying their response to external actuation. It was established that, on timescales relevant to epithelial morphogenesis, the cytoplasm is predominantly viscous, whereas the cellular cortex is elastic. The timescale of elastic stress relaxation has a lower bound of 4 min, which is comparable to the time required for internalization of the ventral furrow during gastrulation. The cytoplasm was measured to be approximately 103-fold as viscous as water. Elasticity was shown to depend on the actin cytoskeleton, and these results are discussed as to how they relate to existing mechanical models of morphogenesis (Doubrovinski, 2017).

Actomyosin meshwork mechanosensing enables tissue shape to orient cell force

Sculpting organism shape requires that cells produce forces with proper directionality. Thus, it is critical to understand how cells orient the cytoskeleton to produce forces that deform tissues. During Drosophila gastrulation, actomyosin contraction in ventral cells generates a long, narrow epithelial furrow, termed the ventral furrow, in which actomyosin fibres and tension are directed along the length of the furrow. Using a combination of genetic and mechanical perturbations that alter tissue shape, this study demonstrated that geometrical and mechanical constraints act as cues to orient the cytoskeleton and tension during ventral furrow formation. An in silico model of two-dimensional actomyosin meshwork contraction was developed, demonstrating that actomyosin meshworks exhibit an inherent force orienting mechanism in response to mechanical constraints. Together, these in vivo and in silico data provide a framework for understanding how cells orient force generation, establishing a role for geometrical and mechanical patterning of force production in tissues (Chanet, 2017).

Dynamic control of dNTP synthesis in early embryos

Exponential increase of cell numbers in early embryos requires large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about how embryos satisfy this demand. This study examined dNTP metabolism in the early Drosophila embryo, in which gastrulation is preceded by 13 sequential nuclear cleavages within only 2 hr of fertilization. Surprisingly, despite the breakneck speed at which Drosophila embryos synthesize DNA, maternally deposited dNTPs can generate less than half of the genomes needed to reach gastrulation. The rest of the dNTPs are synthesized 'on the go.' The rate-limiting enzyme of dNTP synthesis, ribonucleotide reductase, is inhibited by endogenous levels of deoxyATP (dATP) present at fertilization and is activated as dATP is depleted via DNA polymerization. This feedback inhibition renders the concentration of dNTPs at gastrulation robust, with respect to large variations in maternal supplies, and is essential for normal progression of embryogenesis (Song, 2017).


Chanet, S., Miller, C. J., Vaishnav, E. D., Ermentrout, B., Davidson, L. A. and Martin, A. C. (2017). Actomyosin meshwork mechanosensing enables tissue shape to orient cell force. Nat Commun 8: 15014. PubMed ID: 28504247

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Kerridge, S., Munjal, A., Philippe, J. M., Jha, A., de Las Bayonas, A. G., Saurin, A. J. and Lecuit, T. (2016). Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis. Nat Cell Biol 18(3): 261-70. PubMed ID: 26780298

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Rauzi, M., Krzic, U., Saunders, T. E., Krajnc, M., Ziherl, P., Hufnagel, L. and Leptin, M. (2015). Embryo-scale tissue mechanics during Drosophila gastrulation movements. Nat Commun 6: 8677. PubMed ID: 26497898

Song, Y., Marmion, R. A., Park, J. O., Biswas, D., Rabinowitz, J. D. and Shvartsman, S. Y. (2017). Dynamic control of dNTP synthesis in early embryos. Dev Cell 42(3): 301-308.e303. PubMed ID: 28735680

Genes involved in tissue development

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