folded gastrulation


REGULATION

Transcriptional Regulation

In mutants of snail or twist, transcription of fog is normal in the posterior midgut primordium but almost completely eliminated on the ventral side. Maternal-effect ventralizing mutations that expand the expression of twist and snail also expand the domain of fog transcription. In embryos from torpedoQY mutant mothers, Twist protein expression extends farther laterally, but the ventral furrow is usually split into two narrow ventrolateral invaginations by an unknown patterning mechanism. In this case, fog is transcribed in two separate ventrolateral stripes (Costa, 1994).

In mutants of huckebein and tailless, genes known to specify, respectively, adjacent posterior and anterior domains of the posterior midgut invagination, fog transcription at the posterior pole does not extend as far anteriorly. The same lack of extention is evident in forkhead mutants. The double mutant huckebein tailless is the only double mutant combination of these three genes that completely eliminates the posterior midgut invagination; this combination also abolishes all expression of fog at the posterior pole. It is not clear how the anterior extent of fog transcription is delimited, since the domain of tailless expression and activity extends further to the anterior than the region of fog expression (Costa, 1994).

Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal (Dl) nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. What are the consequences of expressing a constitutively activated form of the Toll receptor, Toll(10b), in anterior regions of the early embryo? Using the bicoid 3' UTR, localized Toll(10b) products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic Dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo (Huang, 1997).

The Huang (1997) paper also clearly summarizes what is known about the regulation of genes involved in dorsal/ventral patterning. There are five distinct thresholds of gene activity in response to the Dorsal nuclear gradient in early embryos. The type I target gene folded gastrulation is activated only in response to peak levels of the Dl gradient, so that expression is restricted to a subdomain of the presumptive mesoderm. The PE enhancer from the twist promoter region exhibits a similar pattern of expression. This enhancer contains a cluster of low-affinity Dl binding sites that restrict expression to the ventral-most regions of early embryos. The type II target gene snail contains a series of low-affinity Dl-binding sites, as well as binding sites for the bHLH activator, Twist. The Dl and Twist proteins appear to make synergistic contact with the basal transcription complex, so that snail is activated throughout the presumptive mesoderm in response to both peak and high levels of the Dl gradient. The ventral midline arises from the mesoderm, which is derived from the ventral-most regions of the neuroectoderm. Mesectoderm differentiation is controlled by the bHLH-PAS gene, sim. Some of the E(spl) complex also exhibit early expression in the presumptive mesectoderm. A synthetic enhancer containing high-affinity Dl-binding sites and Twist binding sites exhibits expression in this region. The type IV target gene rhomboid is expressed in lateral stripes that encompass the ventral half of the presumptive neuroectoderm. These stripes are regulated by a 300-bp enhancer (NEE) that contains high-affinity Dl-binding sites, Twist-binding sites, and "generic" E-box sequences that appear to bind ubiquitously distributed bHLH activators (Daughterless and Scute), which are present in the unfertilized egg. The fifth and final threshold response is defined by the lowest levels of the Dl gradient. The zerknullt target gene is repressed by high and low levels of the gradient, so that expression is restricted to the presumptive dorsal ectoderm. The zen promoter region contains high-affinity Dl-binding sites and closely linked "corepressor" sites. Efficient occupancy of the Dl sites appears to depend on strong, cooperative DNA-binding interactions between Dl and the corepressors. The same low levels of Dl that repress zen also repress sog. The sim, E(spl), rho and sog expression patterns are restricted to the neurogenic ectoderm and excluded from the ventral mesoderm by Snail, which encodes a zinc finger repressor (Huang, 1997).

During early embryogenesis in Drosophila, Caudal mRNA is distributed as a gradient with its highest level at the posterior of the embryo. This suggests that the Caudal homeodomain transcription factor might play a role in establishing the posterior domains of the embryo, which undergo gastrulation and give rise to the posterior gut. By generating embryos lacking both the maternal and zygotic mRNA contribution, caudal has been shown to be essential for invagination of the hindgut primordium and for further specification and development of the hindgut. Mature embryos lacking cad activity (maternal and/or zygotic contributions) were examined to assess the requirement for cad in establishing the structures that arise from the posterior ~15% of the blastoderm embryo, namely the posterior midgut, Malpighian tubules and hindgut (Wu, 1998).

The stages of gastrulation can be observationally followed by using expression of brachyenteron byn as a marker for the hindgut primordium. In the wild-type embryo, byn is expressed in a ring at the circumference of the amnioproctodeal plate. The edges of this ring come together as the posterior midgut primordium invaginates during stages 6 and 7; the ring of the hindgut primordium then sinks inward during stage 8 and is completely internalized by the end of stage 9. The zygotically expressed cad stripe and the posterior wg stripe are also expressed in the bordering ring (i.e., the hindgut primordium) of the invaginating amnioproctodeal plate. Strikingly, in cad-deficient embryos, the byn-expressing ring of hindgut primordium draws together, but fails to invaginate, remaining on the outside of the embryo. Thus, although internalization of the Malpighian tubule and posterior midgut primordia is normal in cad-deficient embryos, the gastrulation movements necessary for internalization of the hindgut primordium do not occur in embryos lacking cad activity (Wu, 1998).

The failure of the hindgut to become internalized in caudal-deficient embryos raises the question of whether cad might regulate a zygotically expressed gene required for the invagination of the amnioproctodeal plate. One gene known to be required for gastrulation is fog; fog mutant embryos lack not only the posterior midgut, but, as revealed by anti-Crb staining, the Malpighian tubules and hindgut as well. In the blastoderm stage embryo, fog expression is first activated in the region that will become the ventral furrow; shortly thereafter, expression is initiated in a posterior cap, in the region that will become the amnioproctodeal invagination. In cad-deficient embryos, fog expression in the prospective ventral furrow is normal, but is significantly reduced in the posterior cap. Thus, cad is required for the normal level of expression of fog in the prospective amnioproctodeal plate; decreased fog expression in cad-deficient embryos is likely responsible for the failure of the hindgut primordium to be internalized during gastrulation. Since fkh or wg mutant embryos do not display detectable defects in gastrulation, fog is the only gene presently known to mediate the effects of cad on gastrulation. In fog mutant embryos, none of the posterior gut primordia invaginate, while in cad-deficient embryos the posterior midgut and Malpighian tubule primordium do invaginate; thus, consistent with the in situ hybridization results, a low level of fog activity is present at the posterior of embryos lacking cad (Wu, 1998).

Signaling downstream of Fog: 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 (Grosshans, 2005; Padash Barmchi, 2005; 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).

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).


DEVELOPMENTAL BIOLOGY

Embryonic

fog is expressed in both ventral and posterior invagination primordia in a pattern that precisely precedes the pattern of apical constrictions. Zygotic fog transcription begins first in the ventral furrow primordium [Images] during cellularization, about 30 min before the start of constrictions. The anteroposterior extent of this expression is coincident with the domain of the ventral furrow. Transcription begins at the ventral midline and then rapidly spreads to more lateral regions, to encompass 12-14 cells. This process is complete before any of the cells begin to constrict their apices. fog is transcribed in only a subset of ventral furrow cells: the constricting cells that make up the initial invagination. This region corresponds approximately to the central domain cells and the results suggest that localization of fog expression may delimit the region of mesodermal precursors that undergo apical constriction to invaginate the ventral furrow. In the posterior midgut primordium, fog transcription begins about 10 min after the start of transcription on the ventral side. Transcripts are first detected in the cells immediately dorsal to the pole cell cluster. These cells do not require fog to constrict themselves, but the neighboring cells do. As the invagination closes, fog transcripts rapidly disappear. fog is expressed in the dorsoanterior region of cell cycle 14 embryos before the start of cellularization, but cells in this region do not constrict their apices and they remain on the surface of the embryo throughout gastrulation (Costa, 1994).

A Rho GTPase signaling pathway, in conjunction with concertina and folded gastrulation, is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation

A single Rho GTPase family member is capable of initiating several different processes, including cell cycle regulation, cytokinesis, cell migration, and transcriptional regulation. It is not clear, however, how the Rho protein selects which of these processes to initiate. Guanine nucleotide exchange factors (GEFs), proteins that activate Rho GTPases, could be important in making this selection. In vivo, DRhoGEF2, a GEF that is ubiquitously expressed and specific for Rho1, is reiteratively required for epithelial folding and invagination, but not for other processes regulated by Rho. The limitation of DRhoGEF2 function supports the hypothesis that the GEF selects the outcome of Rho activation. DRhoGEF2 exerts its effects in gastrulation through the regulation of Myosin II to orchestrate coordinated apical cell constriction. Apical myosin localization is also regulated by Concertina (Cta), a Galpha12/13 family member that is thought to activate DRhoGEF2 and is itself activated by a putative ligand, Folded gastrulation (Fog). Fog and Cta also play a role in the morphogenetic events requiring DRhoGEF2, suggesting the existence of a conserved signaling pathway in which Fog, Cta, and DRhoGEF2 locally activate Myosin for epithelial invagination and folding (Nikolaidou, 2004).

If the guanine nucleotide exchange factor (GEF) is important in selecting the outcome of activating Rho, then its function should be limited to a subset of those associated with the GTPase. To address this possibility, the in vivo function of DRhoGEF2 was investigated. Two hypomorphic alleles, DRhoGEF2PX6 and DRhoGEF2PX10, in combination with null alleles of DRhoGEF2, give adults that have crumpled and/or blistered wings. Earlier in development, the DRhoGEF24.1/DRhoGEF2PX6 wing discs appear buckled rather than conforming to the stereotypical folding pattern observed in the wild-type. This malformation is not a result of either improper patterning or loss of apico-basal polarity. It must therefore be caused by disruption of another mechanism -- for example, the propagation of a localized signal that brings about folding in specific places. To test this hypothesis, clones of DRhoGEF21.1 cells spanning a fold were generated (Nikolaidou, 2004).

In large mutant clones that are less influenced by physical constraints, the folds fail to follow the line of the fold in wild-type tissue. Bifurcation of folds does not occur in wild-type discs, supporting the idea that the mutant tissue is unable to respond to a localized signal to fold. Although the clonal and DRhoGEF24.1/DRhoGEF2PX6 mutant tissues do appear folded, the irregularity of the folds indicates that this is probably a consequence of passive folding, as is seen in the gastrulation mutants and murine neurulation mutants that fail to invaginate tissue appropriately (Nikolaidou, 2004).

The possibility was investigated that other events involving epithelial invagination or folding might also require DRhoGEF2 activity. One such event is the invagination of a placode to form a salivary gland tube on both sides of the embryo. Combinations of dominant-negative alleles with a putative null allele of DRhoGEF2 showed that in 93% of embryos some or all of the salivary-gland cells fail to invaginate and instead remain on the outside. Because maternally provided DRhoGEF2 is vital for epithelial invagination in gastrulation, this and the above two phenotypes represent three examples of the requirement for DRhoGEF2 in epithelial-layer morphogenesis (Nikolaidou, 2004).

If DRhoGEF2 is participating in the selection of the cell's response to activated Rho, then its function should be limited. Rho is known to play a role in cytokinesis, cell cycle regulation and planar polarity. The large size of clones of DRhoGEF2, equivalent numbers of cells in twin wild-type and mutant clones, and normal polarity of mutant tissue indicate that unlike Rho, DRhoGEF2 is not required for any of these processes, nor is it required for apico-basal polarity. No significant defects were seen in the gross morphology of the nonepithelial tissues of muscles and neurons in late-stage DRhoGEF24.1/DRhoGEF26.5 and DRhoGEF24.1/DRhoGEF25.1 embryos. In addition, the normal cell cycle control shows that the convolution of DRhoGEF2 mutant wing discs is not a result of excessive proliferation (Nikolaidou, 2004).

Although the possibility exists that DRhoGEF2 has a function not addressed, it seems likely that its role is confined to the control of epithelial morphogenesis. This limit of DRhoGEF2 function suggests that it is important in selecting a role for Rho only in epithelial morphogenesis, whereas other GEFs would activate Rho in other processes; for example Pebble activates Rho primarily in cytokinesis, and Trio acts on Rac in neuronal outgrowth (Nikolaidou, 2004).

To study in more detail the mechanism by which DRhoGEF2 affects epithelial morphogenesis, the possible targets of DRhoGEF2 activation have been considered. One of these is myosin II. During gastrulation, Zipper (Zip), the heavy chain of myosin II, appears to accumulate on the apical side of the mesodermal precursors in the ventral furrow (VF). To address the possibility that apical myosin localization is required for other invagination events, salivary-gland formation was analyzed in embryos expressing the myosin light chain, Spaghetti squash (Sqh), as a fusion with green fluorescent protein (Sqh-GFP). Although Sqh-GFP is present at the cortex of all the cells, it is concentrated at the apical surface of salivary-gland precursors that are about to invaginate or are in the process of invaginating. Sqh-GFP does not accumulate apically until invagination, as demonstrated by the lack of apical localization in cells that are present more anteriorly in the placode but that will invaginate later (Nikolaidou, 2004).

It is not clear if this apical myosin accumulation is present in time to contribute to apical constriction. To resolve this question, the localization of Sqh-GFP was observed in the invaginating VF during gastrulation. In wild-type cells, Sqh-GFP is maintained at the tip of the growing membrane that forms between the nuclei during cellularization, the process immediately prior to gastrulation. At the end of cellularization, Sqh-GFP begins to decrease on the basal side and accumulate on the apical side of the ventral cells, i.e., only those that will constrict apically. This redistribution of myosin precedes apical cellular constriction, suggesting that it contributes to the process. Basally located Sqh-GFP is subsequently lost, and the apical levels increase (Nikolaidou, 2004).

In DRhoGEF2 germline clone-derived (GLC) embryos (i.e., those lacking maternal DRhoGEF2), Zip, the myosin heavy chain, is lost from the basal side of cells in the developing VF, but it accumulates at much lower levels on the apical side than it does in the wild-type. These results imply that a signal through DRhoGEF2 is needed in order for the ventral cells to induce apical Zip localization. In contrast, relocalization of β-heavy spectrin occurred normally in DRhoGEF2 GLC embryos, indicating that cell polarity is maintained in these cells and that at least some forms of protein relocalization, especially that of a protein that is found in close proximity to Zip (Nikolaidou, 2004).

The possibility was considered that myosin localization is also regulated by other components of the DRhoGEF2 signaling pathway. By analogy to the mammalian and C. elegans orthologs and as a result of genetic studies , DRhoGEF2 is thought to participate in a signal transduction pathway, which is called here the DRhoGEF2 signaling pathway, initiated by Folded gastrulation (Fog) and propagated by Concertina (Cta). Mutations in both these genes result in gastrulation defects. In embryos derived from cta mutant mothers, a low level of Zip accumulates on the apical side only of apically constricting cells in the invaginating VF. This is also true in DRhoGEF2 GLC embryos. In contrast, there is no apical myosin apparent in the cells that do not constrict their apical surface. These data clearly link the presence of apical myosin with apical constriction and indicate that in gastrulation this is controlled by the DRhoGEF2 signaling pathway. The link between DRhoGEF2 and Myosin is also supported by the documented genetic interactions between DRhoGEF2 and zip in leg and wing development (Nikolaidou, 2004).

It is not clear how DRhoGEF2 influences the apical accumulation of myosin. It could act via the Rho effector Rho kinase. When activated by Rho in mammalian cells, Rho kinase is responsible for revealing the actin binding site on the regulatory light chain of myosin II. Thus, in DRhoGEF2 mutants, a possible failure in the activation of Rho1 and Rho kinase would result in the inability of myosin to bind actin (Nikolaidou, 2004).

If DRhoGEF2 is required reiteratively for epithelial morphogenesis, it is hypothesized that Fog and Cta might also be used reiteratively. mRNA for fog is expressed in invaginating tissue during gastrulation and salivary-gland formation, suggesting that Fog also participates in invagination of the salivary gland. At present there are conflicting reports regarding the role of fog in salivary gland formation. This study finds that some or all cells fail to invaginate in 90% of the embryos. Because invagination in gastrulation is cell autonomous, it is considered more likely that this phenotype results from a lack of fog in these cells rather than because of earlier developmental defects (Nikolaidou, 2004).

The possibility was addressed that the pathway is also important in wing development. Initial descriptions indicate that Fog and Cta play no role in this process. However, demonstrating a previously undisclosed role for Fog and Cta, combinations of mutations in fog or cta and DRhoGEF2 result in synergistic effects on wing development. Together, these results point to the reiterative use of the DRhoGEF2 signaling pathway in development to bring about epithelial folding or invagination (Nikolaidou, 2004).

Preliminary data indicate that the folds in the wing disc are brought about by apical cell constriction. It is therefore proposed, because both gastrulation and salivary gland invagination also involve apical cell constriction, that this is a major aspect of DRhoGEF2 function. The location of the folds in wing discs is highly stereotypical, which would suggest that specific signals are activated in these locations to initiate folding. One candidate for this signal is Fog, which is perhaps acting in conjunction with a second signal to bring about epithelial folding in the wings. In gastrulation, fog and cta are essential, but their phenotypes are not as strong as that observed after the removal of maternal DRhoGEF2, again indicating the requirement for additional signals that activate DRhoGEF2. The nature of this additional signal, or signals, remains elusive (Nikolaidou, 2004).

Analysis and reconstitution of the genetic cascade controlling early mesoderm morphogenesis in the Drosophila embryo

To understand how transcription factors direct developmental events, it is necessary to know their target or 'effector' genes whose products mediate the downstream cell biological events. Whereas loss of a single target may partially or fully recapitulate the phenotype of loss of the transcription factor, this does not mean that this target is the only direct mediator. For a complete understanding of the pathway it is necessary to identify the full set of targets that together are sufficient to carry out the programme initiated by the transcription factor, which has not yet been attempted for any pathway.In the case of the transcriptional activator Twist, which acts at the top of the mesodermal developmental cascade in Drosophila, two targets, Snail and Fog, are known to be necessary for the first morphogenetic event, the orderly invagination of the mesoderm. A system of reconstituting loss of Twist function by transgenes expressing Snail and Fog independently of Twist was used to analyse the sufficiency of these factors¡Va loss of function assay for additional gene functions to assess what further functions might be needed downstream of Twist. Confirming and extending previous studies, Snail was shown to play an essential role, allowing basic cell shape changes to take place. Fog and at least two other genes are needed to accelerate and coordinate shape changes. Furthermore, this study represents the first step in the systematic reconstruction of the morphogenetic programme downstream of Twist (Seher, 2007).

In addition to Twist, Snail and Fog, there are genes in four regions of the autosomal genome which upon deletion lead to abnormalities during ventral furrow formation. Is it likely that all zygotically active genes that participate in normal mesoderm invagination have been detected? Although the assay proved to be sufficiently sensitive to identify a number of mutants, it is conceivable that further genes with even less pronounced mutant phenotypes were missed. Further, genes with completely redundant functions, for example, because duplicates exist in distinct regions of the genome, might not give a loss-of-function phenotype. Such genes might be identifiable only via sophisticated genetic screens (modifier screens) or appropriate molecular approaches (Seher, 2007).

The loss of the genes uncovered by the deficiencies results only in a delay of furrow formation, and not in the complete failure of invagination. If these genes are Twist targets, there are different possible explanations for the mutants showing such weak phenotypes. The genes might control an essential process parallel to that controlled by Snail, but act in a redundant manner in the pathway, such that disruption of only one of their functions does not lead to the disruption of furrow formation. Alternatively, only the pathway controlled by Snail may be essential, with other genes acting in parallel affecting only the speed and efficiency of furrow formation. The severe phenotype seen in the double mutants of Df(3R)TlP and fog as well as the enhancement of the fog phenotype by the loss of one copy of Snail argue for the latter scenario, i.e. two parallel pathways, both of which are essential (Seher, 2007).

Embryos were created in which the function of the mesodermal transcription factor twist was replaced by two of its downstream targets, snail and fog. The analysis of these embryos concentrated on the first phase of mesoderm morphogenesis, during which cell shape changes internalize the prospective mesoderm. The subsequent epithelial–mesenchymal transition, cell division and cell migration depend on other Twist targets, such as string, htl and dof. Since these are not expressed in the twist,PE::fog;2xPE::sna (twist driven fog and snail) embryos, this aspect of morphogenesis cannot occur in the reconstituted twist embryos (Seher, 2007).

The fog and snail transgenes had distinguishable effects in twist embryos. The PE::fog transgene induces the earliest event of the typical cell shape changes, apical flattening, and enhanced apical constrictions of ventral cells. By contrast, nuclear movement away from the apical cell surface was not significantly improved, nor was cell shortening observed. While the 2xPE::snail transgene also led to some improvement in apical flattening, it had additional effects. A larger number of cells showed distinctive nuclear movement, and a higher frequency of deep invaginations were scored, suggesting a role for Snail in releasing nuclei. With the combination of both transgenes nearly normal cell shape changes occurred which resulted in the formation of proper furrows in a substantial number of embryos. Specifically, many cells assumed the typical wedge-shape of ventral furrow cells, showing that snail and fog are sufficient to induce this shape in the absence of further Twist targets (Seher, 2007).

However, it appears that snail and fog cannot be the only targets downstream of twist to control mesoderm invagination. If they were, they should be able to replace twist function completely and fully restore furrow formation. It is therefore concluded that besides snail and fog other twist target genes must exist which are necessary to orchestrate the formation of the ventral furrow in the accurate, fast and stable fashion in wildtype embryos (Seher, 2007).

The as yet unknown targets must be involved in those events which were not restored in twist,PE::fog;2xPE::sna embryos: the speed of the process, adhesion between apical surfaces, and cell shortening during invagination. The latter process occurs efficiently in snail mutants, confirming that it is not Snail-dependent. Since fog does not contribute to this process it is likely that one or more other twist targets are involved in cell shortening (Seher, 2007).

In summary, the zygotic control of ventral furrow formation branches into separable functions downstream of Twist, the induction of the basic cell shape changes of ventral cells, and the control of the speed, accuracy and coordination of the shape changes. One of the known targets of Twist, the repressor Snail, is necessary to allow the shape changes to occur, whereas Fog and probably other Twist targets are responsible for accuracy and efficiency. Together, they ensure the rapid and regular formation of the ventral furrow. Ventral furrow formation may be an adaptation to the rapid early embryogenesis of long germ insects, serving to position mesodermal cells at a site where they can efficiently begin their FGF-dependent spreading on the inner surface of the ectoderm. The experimental system used in this study may be extended to test the function of the whole set of Twist targets, once they have been identified, for their ability to re-establish mesoderm invagination in the absence of Twist, and thereby reconstruct fully the pathway from a 'selector' gene to the cell biological processes it controls (Seher, 2007).

Effects of mutation or deletion

In embryos mutant for folded gastrulation (fog), cell shape changes that take place during gastrulation occur but the timing and synchrony of the constrictions are abnormal. A similar phenotype is seen in a maternal effect mutant, concertina (cta). fog encodes a putative secreted protein whereas cta encodes an a-subunit of a heterotrimeric G protein. It has been proposed that localized expression of the fog signaling protein induces apical constriction by interacting with a receptor whose downstream cellular effects are mediated by the Cta Galpha protein. In order to test this model, fog has been ectopically expressed at the blastoderm stage using a heat shock inducible promoter. In addition, the constitutive activation of Cta protein has been examined by blocking GTP hydrolysis using both in vitro synthesized mutant alleles and cholera toxin treatment. Activation of the fog/cta pathway by any of these procedures results in ectopic cell shape changes in the gastrula (Morize, 1998).

The most immediate response of this activation is an apical flattening of all cells in the early gastrula, such that these cells appear tightly cohesive and lack their typical dome-shaped apical surfaces. Flattening occurs very soon after heat shocked embryos have cellularized: it is always observed by the time heat-shocked embryos have begun gastrulation, but never in embryos fixed during the cellularization process itself. Simultaneous with apical flattening, ectopic expression of fog disrupts the ordered hexagonal arrangement of cells on the surface of the embryo. The cells appear to be stretched or pulled in an isotropic fashion. Their overall appearance suggests that the forces that cause apical flattening also pull on adjacent cells. These forces appear to be uniform over the surface, such that no cells at this time are able to fully constrict. Constitutively active alleles of cta induce all the features seen with ubiquitous expression of fog (overall flattening of the surface of the embryo, disruption of the regular array of cells, interference with the cephalic furrow formation and non responsiveness of mitotic cells), although the frequencies are low and somewhat variable (Morize, 1998).

Although expression was induced at the blastoderm stage, the surface of heat-shockfog embryos remains flattened until midgastrulation when groups of cells on the dorso-anterior side of the embryo acquire a round shape. These groups of cells appear in the same sequence and spatial pattern as the mitotic domains described by Foe (1989). In all the regions where two domains of dividing cells are separate but close to each other, the intervening cells constrict their apices and form shallow grooves with a morphology reminiscent of the VF. It is proposed that the increased apical surface area of the dividing cells as they round up releases the tension in the surface of the embryo, such that the cells located next to these mitotically active domains are able to fully constrict their apices. The furrows that are formed by these constrictions are transient and persist only until the cells forming them begin to divide (Morize, 1998).

Uniform fog expression rescues the gastrulation defects of fog null embryos but not cta mutant embryos, arguing that cta functions downstream of fog expression. The normal location of the ventral furrow in embryos with uniformly expressed fog suggests the existence of a fog-independent pathway determining mesoderm-specific cell behaviors and invagination. Epistasis experiments indicate that this pathway requires snail but not twist expression (Morize, 1998).

The ability of Fog misexpression to produce apical flattening is somewhat surprising, given that cells of the ventral furrow flatten normally in fog mutants. The most obvious phenotype reported previously for fog is reflective of its effects on the subsequent apical constriction of the flattened cells. That fog affects both apical flattening and constriction suggests that the two processes may both involve the same fundamental mechanism, namely a contraction of the cortical actin-myosin network. In the favored model, the relevant actin cytoskeletal components for both flattening and constriction are initially organized under the dome-shaped apical surface of the cell at the completion of cellularization. As this dome is induced to constrict by the fog signaling pathway, the first morphological consequence is apical flattening, and the dome-like network resolves into a more disc-like shape. Further movement of myosin molecules leads to a progressive reduction of the diameter of the network resulting in apical constriction. Although both flattening and constriction would thus involve contraction of the same filamentous network, apical constriction is slower because it involves a greater displacement of the underlying cytoplasm, and because the forces exerted by neighboring cells on the individual cell make it harder to achieve than apical flattening (Morize, 1998).

This model is consistent with the results obtained with the ubiquitous activation of the fog/cta pathway. In order for a cell in an epithelial sheet to constrict its apex, it must exert force on its neighbors. If all cells in the sheet exert equal forces, a stable equilibrium will be established that should prevent any further changes in shape. Consistent with this view, ubiquitous fog expression initially produces no reduction in apical diameter. The irregular cellular outlines in such embryos, and the distortion of the cephalic furrow and dorsal transverse folds, suggest that the surfaces of the embryonic cells are under tension. In such embryos, every single cell is able to flatten its dome-shaped cortex, but none is able to reduce its apical diameter. Later, some cells are able to fully constrict their apices because they are located near a dividing mitotic domain and thus subject to less resistance from their neighbors (Morize, 1998).

In this model, fog expression in the VF primordium may contribute to apical flattening of the VF cells. The fact that fog activity is not required for this flattening suggests that some other partially redundant pathway also contributes to this cell shape change. Both the fog pathway and the alternate pathway may be essential for efficient apical constriction, but either may be sufficient for the initial flattening. Genes involved in the alternate pathway have not yet been identified but it is suspected that, like fog, at least some of these genes may be regulated in ventral cells by mesodermal programming (Morize, 1998).

The folded gastrulation gene is required during gastrulation for two morphogenetic movements: formation of the ventral furrow and invagination of the posterior midgut primordium. The mutant defects in invagination are more severe in the posterior midgut invagination than in ventral invagination. The primary defect appears to be in the spatial and temporal organization of the constriction. In wild-type embryos, the first cells to initiate constriction usually lie immediately dorsal to the pole cell cluster. In the next few minutes, the constrictions spread farther dorsally and onto the lateral sides and then finally reach the ventral side of the posterior pole such that the constriction zone encompasses about ten cells dorsal and ventral to the pole cells and about five cells on each lateral side. In embryos mutant for fog the cells immediately dorsal to the pole cell cluster initiate apical constriction normally, but constrictions fail to spread onto the lateral and ventral sides. Consequently, the posterior midgut primordium does not invaginate. The fog mutant phenotype therefore suggests that there are at least two mechanisms for initiating apical constriction in the posterior midgut primordium. One functions in cells located in the dorsoposterior region of the embryo and generates the early constrictions; this mechanism does not require fog activity. The second mechanism operates in cells located in the more peripheral regions of the invagination primordium and does require fog activity. It is suggested that fog coordinates cell shape changes during the second stage of invagination by inducing apical constriction of cells in spatially and temporally defined manners (Costa, 1994).

In the ventral furrow, two populations of cells are also distinguised by differential timing of apical constriction and requirements for fog activity. During the earliest stages of invagination, only those cells close to the middle of the invagination primordia are likely to constrict, while more marginal cells remain unconstricted. Later constrictions spread out from the ventral midline to include an approximately 12 cell-wide domain. In fog mutants, the first phase is fairly normal while cells in more lateral regions show variable defects. Thus cells in the lateral regions of the ventral furrow are more dependent on fog activity to initiate constriction. In contrast to the complete absence of posterior midgut invagination in fog mutants, the invagination of the ventral furrow does take place in fog mutants, albeit in a delayed fashion. Overexpression of fog induces ectopic cell shape changes, specifically in cells of the dorsoanterior region, showing that cells outside the normal invagination primordia possess all the components necessary for apical constriction except adequate levels of fog activity (Costa, 1994).

Use of genetic mosaics allows an estimate of the range of Fog action. From the spatial correspondence between the boundary of fog mutant clones and the assesment of regions displaying morphological defects, it is concluded that fog must act locally with respect to the site of its expression. It is estimated that fog can induce apical constriction two to three cells away from its site of expression. Overexpression of fog in the dorsoanterior region of the embryo induces ectopic constrictions, indicating that localization of FOG transcripts may define domains of cell shape changes (Costa, 1994).

The ventral furrow and posterior midgut invaginations bring mesodermal and endodermal precursor cells into the interior of the Drosophila embryo during gastrulation. Both invaginations proceed through a similar sequence of rapid cell shape changes, which include apical flattening, constriction of the apical diameter, cell elongation and subsequent shortening. Based on the time course of apical constriction in the ventral furrow and posterior midgut, two phases in this process have been identified: (1) a slow stochastic phase in which some individual cells begin to constrict and (2), a rapid phase in which the remaining unconstricted cells constrict. Mutations in the concertina or folded gastrulation genes appear to block the transition to the second phase in both the ventral furrow and the posterior midgut invaginations (Sweeton, 1991).

Mutations at the folded gastrulation locus interfere with early morphogenetic movements in Drosophila melanogaster. fog embryos do not form a normal posterior midgut and although their germbands do elongate, they do not extend dorsally. As a result, when normal embryos have fully extended germbands, the germbands in mutant embryos are folded into the interior on the ventral side of the embryo. fog embryos continue to develop, but form disorganized first instar larvae. fog is a zygotically active gene expressed at least by 10 and 20 min after the onset of gastrulation. Mutations are viable in homozygous germ cells and the wild-type gene need not be expressed during oogenesis for survival of heterozygous progeny. Elimination of fog+ gene product from maternal germ cells does, however, affect the extent of folding observed during gastrulation in viable heterozygotes. Analysis of fog adult and larval gynandromorphs indicates that normal folded gastrulation gene function is only required at the posterior region of the embryo, most probably in the cells giving rise to the posterior midgut or proctodeum. The relative survival of fog mosaics suggests that embryos with mosaic "lethal foci" also die during embryogenesis, although the typical fog phenotype is only produced when the entire focus is mutant (Zusman, 1985).

The secreted cell signal Folded Gastrulation regulates glial morphogenesis and axon guidance in Drosophila

During gastrulation in Drosophila, ventral cells change shape, undergoing synchronous apical constriction, to create the ventral furrow (VF). This process is affected in mutant embryos lacking zygotic function of the folded gastrulation (fog) gene, which encodes a putative secreted protein. Fog is an essential autocrine signal that induces cytoskeletal changes in invaginating VF cells. This study shows that Fog is also required for nervous system development. Fog is expressed by longitudinal glia in the central nervous system (CNS), and reducing its expression in glia causes defects in process extension and axon ensheathment. Glial Fog overexpression produces a disorganized glial lattice. Fog has a distinct set of functions in CNS neurons. The data show that reduction or overexpression of Fog in these neurons produces axon guidance phenotypes. Interestingly, these phenotypes closely resemble those seen in embryos with altered expression of the receptor tyrosine phosphatase PTP52F. Epistasis experiments were conducted to define the genetic relationships between Fog and PTP52F, and the results suggest that PTP52F is a downstream component of the Fog signaling pathway in CNS neurons. Ptp52F mutants were found to have early VF phenotypes like those seen in fog mutants (Ratnaparkhi, 2007).

Reduction of Fog in neurons produces subtle axon guidance phenotypes affecting both motor neurons and CNS interneurons. Overexpression of Fog in neurons produces strong CNS phenotypes in which longitudinal axons abnormally cross the midline. The same phenotypes can be produced by overexpressing Fog in CNS longitudinal glia, which are in apposition to the axons. This results suggests that glial Fog causes cytoskeletal changes that alter axon guidance in neurons, implicating Fog as an exocrine as well as an autocrine signal during nervous system development. (Ratnaparkhi, 2007).

Studies of Fog signaling during gastrulation have indicated that the cytoskeletal changes produced by autocrine Fog involve maternal Cta and RhoGEF2, and nonmuscle myosin II. This study tested whether these components participate in Fog signaling in the nervous system by examining the zygotic phenotypes of cta and RhoGEF2 mutants (germline clones do not develop to this stage). Cta may also be involved in Fog signaling during nervous system development, because it was found that cta zygotic mutant embryos display the same defects in the CNS and neuromuscular system as do fog embryos. RhoGEF2 mutants, however, have no visible nervous system defects. Myosin II (zipper) mutant embryos have a variety of generalized defects that preclude analysis of specific axon guidance phenotypes. (Ratnaparkhi, 2007).

Most of the cells in the CNS of late embryos that express fog mRNA at high levels are Repo-positive longitudinal glia. These glia are required for normal morphogenesis of the CNS axon tracts; but no CNS axon phenotypes were observed when Fog expression was reduced in glia. To evaluate Fog's functions in glia, glial morphology was examined directly using a membrane-associated GFP marker. When Fog expression is reduced in glia, glial processes fail to extend normally and ensheath CNS axons. There are gaps in the regular array of glia, glial surface areas are smaller than in wild-type, and the glia have a rounded appearance. These changes in cell shape could involve nonmuscle myosin. (Ratnaparkhi, 2007).

Overexpression of Fog in glia confers lethality during early larval phases. Glial morphogenesis is affected by overexpression, but the phenotypes are different from those seen when Fog is reduced. Glia appear to have normal shapes, but the glial lattice is quite disorganized. In wild-type embryos, lines of glia define the positions of the longitudinal tracts, commissural tracts, and peripheral nerves; these regular arrays are not observed in Fog glial overexpression embryos. Thus, both reduction and elevation of glial Fog produces a disorganized glial lattice, suggesting that a precise level of the Fog signal is necessary for normal glial development (Ratnaparkhi, 2007).

The Fog receptor has not been identified, although it is speculated to be a GPCR because of the requirement of the G protein alpha subunit Cta for Fog signaling. However, existing genetic data do not show that Fog directly activates a GPCR; they are also consistent with models in which Fog regulates signaling through a GPCR-Cta pathway in an indirect manner by interacting with a non-GPCR receptor (Ratnaparkhi, 2007).

PTP52F, like most RPTPs, is an 'orphan receptor'. The motivation to conduct the experiments described in this paper arose from observations that fog and Ptp52F embryos display similar VF phenotypes, and that PTP52F is expressed in ventral furrow cells during the gastrulation phase (Schindelholz, 2001). Based on these results, it was asked whether PTP52F could be the elusive Fog receptor (Ratnaparkhi, 2007).

PTP52F is required for axon guidance in the embryonic CNS and neuromuscular system. Thus, to examine whether Fog and PTP52F might be part of the same signaling pathway, fog axon guidance phenotypes were examined, and genetic interactions between the two molecules were studied. The data show that fog and Ptp52F have similar LOF and GOF phenotypes in the CNS. In the neuromuscular system, LOF mutations in both genes cause SNa bifurcation phenotypes. The definition of a fog GOF CNS phenotype allowed performance an epistasis experiment, and this showed that PTP52F is required for signaling downstream of Fog, at least in the context of this phenotype (Ratnaparkhi, 2007).

The genetic results that were obtained indicate that PTP52F is involved in reception of the Fog signal by neurons, but do not prove that PTP52F is a Fog receptor. The results could also be explained if PTP52F positively regulates signaling through a Fog-GPCR-Cta pathway. For example, GPCRs are phosphorylated (on serine or threonine residues) and internalized; the activities of the relevant kinases and/or the proteins involved in internalization could be modulated by tyrosine phosphorylation. Tyrosine phosphorylation could also regulate effectors downstream of the G protein Cta. (Ratnaparkhi, 2007).

Direct biochemical interaction tests between the PTP52F extracellular domain and several versions of the Fog protein have not yielded positive results. However, Fog might be processed in vivo to create a functional ligand, and this processing does not occur in heterologous systems. In these experiments, it was found that Fog tagged at its N-terminus is secreted from insect cells when expressed using the baculovirus system, but the protein is degraded to produce a ladder of bands ranging in size from ~100 kD (the predicted size of glycosylated full-length Fog) to <20 kD. Fog tagged at its C terminus cannot be detected at all. Fog fused near its C terminus to human placental alkaline phosphatase (Fog-AP) can be expressed as a mixture of apparently full-length and degraded forms, but none of these proteins bound detectably to the tagged PTP52F extracellular domain. Taken together, these data suggest that the C terminal region of Fog is subject to degradation, and that full-length Fog is unstable. There are several dibasic sequences in Fog which could represent proteolytic cleavage sites, and it has been proposed that Fog could be processed in vivo to generate an active fragment that binds to the receptor. In the CNS, such a fragment might derive from the middle region of Fog, because it was observed that antisera against full-length Fog stain late embryos, while antisera against the first 300 amino acids of Fog do not (Ratnaparkhi, 2007).

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).


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folded gastrulation: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 12 December 2016 

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