RhoGef2
DEVELOPMENTAL BIOLOGY

Embryonic

A Drosophila gene has been identifed that has substantial sequence homology to a distinct class of proto-oncogenes that includes DBL, VAV, Tiam-1, ost and ect-2. It has predicted Rho or Rac guanine exchange factor (Rho/RacGEF) and pleckstin homology (PH) domains with the PH immediately downstream of the Rho/RacGEF. Rho/RacGEFs catalyze the dissociation of GDP from the Rho/Rac subfamily of Ras-like GTPases, thus activating the target Rho/Rac. Members of the Rho/Rac subfamily regulate organization of the actin cytoskeleton, which controls the morphology, adhesion and motility of cells. Message from this gene is found throughout oogenesis and embryogenesis. Of particular interest, message is most abundant in furrows and folds of the embryo where cell shapes are changing and the cytoskeleton is likely to be undergoing reorganization (Werner, 1997).

Compartmentalisation of Rho regulators directs cell invagination during tissue morphogenesis

During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe, which recognizes the active, GTP-bound form of Rho1 and that can be expressed in vivo using the UAS/GAL4 system, thus facilitating the visualisation of RhoGTPase, evidence is presented that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination (Simoes, 2006).

Using a probe that allows the visualisation of Rho1 activity in the course of normal development, evidence is presented that this GTPase is active at the apical side during the process of cell invagination. In the spiracles Rho1 activity is essential to control this movement, similarly to that previously shown during Drosophila gastrulation, when mesodermal cells fail to invaginate after inhibition of Rho1 function. An apical enrichment is observed of Myosin II, a possible target of activated Rho1, analogous to that reported in other tissues of the fly embryo where this type of movement occurs, such as the mesoderm and the salivary glands. Inhibition of Rho1 activity results in a disorganised pattern of apical Myosin II and F-Actin in the spiracle cells. It is suggested that concentration of active Rho1 at the apical side organises the Actin cytoskeleton and promotes high Myosin II accumulation/activity in this region, leading to a contractile Actin-Myosin based force to produce a wedge-shaped cell (Simoes, 2006).

These data show that spatial restriction of Rho1 activity is achieved by distinct mechanisms. (1) Albeit ubiquitous, Rho1 protein is strongly enriched on the apical side of the invaginating spiracle cells. (2) To ensure that this GTPase is active exclusively on that side of the cell, opposing Rho regulators are differentially distributed in two distinct membrane domains: two Rho activators, RhoGEF64C and RhoGEF2, are apically localised, whereas a Rho inhibitor, the RhoGAP Cv-c, occupies the complementary, basolateral domain. Cell shape changes and inward cell movements driving invagination are impaired if Rho1 becomes activated in a spatially unrestricted manner. These observations stress the importance of finely tuning Rho1 localisation and activation during normal tissue morphogenesis (Simoes, 2006).

Several mechanisms might be at work to achieve the specific localisation of the Rho regulators that direct cell invagination. In the case of RhoGEF64C, its mRNA and protein are apically localised, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to activate Rho1 in a spatially restricted manner. Recent studies show that the mRNA of RhoA can also be transported and locally translated in the axons and growth cones of embryonic rat neurons, where RhoA controls growth cone collapse in response to Semaphorin 3A. This shows that intracellular mRNA transport of Rho GTPases and of their regulators may be an important mechanism to control spatial GTPase activation (Simoes, 2006).

Loss of function of the RhoGEFs involved in spiracle invagination leads to variable apical defects, which are compatible with a partial loss of Rho1 function: knocking out RhoGEF64C resulted in a mild disruption of cortical Actin without blocking invagination, while the absence of RhoGEF2 could result in a complete failure of the invagination process. These results suggest that several RhoGEFs are required to properly activate Rho1 during spiracle cell movement and organ shaping (Simoes, 2006).

One interesting observation from these studies is the fact that mutants for the RhoGAP Cv-c did not show ectopic activated Rho1 on the basolateral membrane where this RhoGAP was localised. Thus, several mechanisms must be at work to ensure that Rho1 activity is excluded from the basolateral domain during cell invagination: the presence of at least one RhoGAP on the basal membrane, the apical restriction of RhoGEFs and the existence of low levels of Rho1 protein on the basolateral side of the cells. In addition, it was also observed that spiracles from severe cv-c mutants showed lower levels of apical Rho1-GTP than their wild-type counterparts, correlating with the disruption of their apical Actin. Defects in apical Actin/Myosin II have also been reported during invagination of the tracheal pits in cv-c mutants. Taken together, these observations suggest that GTP hydrolysis is a necessary step in the regulation of Rho1 function during cell invagination and the RhoGAP Cv-c may help to maintain a steady state level of apical Rho1-GTP (Simoes, 2006).

Based on the differential distribution of Rho1 GEFs and GAPs, a model is proposed in which Rho1 must shuttle back and forth between two membrane compartments, being GTP-bound on the apical cell membrane and GDP-bound on the basolateral side. Thus, during tissue morphogenesis, epithelial cells can couple their apical-basal polarity to the spatial control of small RhoGTPase function (Simoes, 2006).

RhoGTPases act as dynamic switches in many developmental and cellular contexts. In order to understand how they orchestrate these dynamic processes, their activity states needs to be visualised over time. It is anticipated that this work and the tools described will provide a basis for studying Rho1 activity in vivo. It will be interesting to extend this analysis to other contexts in which Rho GTPases are known to act -- such a dorsal closure, neurulation, wound healing -- and to identify the Rho regulators involved in each case, relating their spatial/temporal distribution with the patterns of Rho GTPase activity (Simoes, 2006).

The Frizzled-dependent planar polarity pathway locally promotes E-cadherin turnover via recruitment of RhoGEF2

Polarised tissue elongation during morphogenesis involves cells within epithelial sheets or tubes making and breaking intercellular contacts in an oriented manner. Growing evidence suggests that cell adhesion can be modulated by endocytic trafficking of E-cadherin (E-cad), but how this process can be polarised within individual cells is poorly understood. The Frizzled (Fz)-dependent core planar polarity pathway is a major regulator of polarised cell rearrangements in processes such as gastrulation, and has also been implicated in regulation of cell adhesion through trafficking of E-cad; however, it is not known how these functions are integrated. This study reports a novel role for the core planar polarity pathway in promoting cell intercalation during tracheal tube morphogenesis in Drosophila embryogenesis, and evidence is presented that this is due to regulation of turnover and levels of junctional E-cad by the guanine exchange factor RhoGEF2. Furthermore, it was shown that core pathway activity leads to planar-polarised recruitment of RhoGEF2 and E-cad turnover in the epidermis of both the embryonic germband and the pupal wing. This study thus reveals a general mechanism by which the core planar polarity pathway can promote polarised cell rearrangements (Warrington, 2013).

Looking in three different tissues, this study found that core planar polarity pathway activity promotes E-cad turnover from junctions, most likely via local recruitment and regulation of RhoGEF2 and RhoA activity. In general terms, it is believed that local assembly or disassembly of adherens junctions through trafficking of E-cad is likely to be important for polarised tissue rearrangement; however, few specific contexts in which this occurs have been identified (Warrington, 2013).

One process in which regulation of E-cad turnover is strongly linked to cell intercalation is elongation of branches in the Drosophila embryonic tracheal system. Loss of core pathway function and also reduction of RhoGEF2 and RhoA activity give a similar phenotype to blocking endocytosis in this tissue, resulting in increases in both overall levels of and the stable fraction of E-cad at junctions, and a delay in cell intercalation. Consistent with the increase in E-cad levels being the cause of the intercalation defect in core pathway backgrounds, this phenotype can be suppressed by lowering E-cad gene dosage. It is speculated that core planar polarity proteins might transiently show polarised distribution or activity in this context, thus selectively weakening junctions and allowing cells to slide over one another. However, consistent with previous studies, this study failed to detect such asymmetry. The possibility cannot therefore be ruled out that core pathway activity is uniform within cells in this tissue, and only plays a role in general modulation of E-cad trafficking (Warrington, 2013).

In the pupal wing, the core pathway has already been linked to regulation of E-cad trafficking, and evidence has been presented that this promotes junctional remodelling that gives rise to a regular hexagonal arrangement of the cells. The exact mechanism by which the core pathway modulates E-cad trafficking was not defined, although the observation that Sec5 is recruited to proximodistal junctions suggested that there might be a role for local exocytosis of E-cad. Looking at a stage shortly after junctional remodelling, when the core proteins are strongly asymmetrically distributed, planar-polarised localisation of RhoGEF2 to proximodistal junctions is observed, but also a decrease in overall levels and the stable fraction of E-cad in this position. This appears to rule out a role for increased E-cad exocytosis on proximodistal junctions. Interestingly, although Sec5 is best characterised as a component of the exocyst, it has also been implicated in endocytosis in the Drosophila oocyte and perhaps this is also true in the wing. It is not clear how planar-polarised E-cad trafficking would contribute to formation of a regular hexagonal array of cells, as removing E-cad from proximodistal distal junctions might be expected to cause shrinkage of these junctions at the expense of anteroposterior junctions. However, during the peak period of junctional rearrangement (from ~18 hours of pupal life), the planar-polarised asymmetric distribution of the core proteins is largely lost, and so it might be that during the crucial stage of morphogenesis the core pathway promotes relatively uniform endocytosis of E-cad (Warrington, 2013).

The observation of a role for the core pathway in modulating E-cad turnover in the epidermis of the embryonic germband is particularly intriguing, as loss of core pathway activity does not result in a defect in embryonic germband extension, even though a planar-polarised distribution of E-cad has been implicated as a key mechanism in promoting cell intercalation in this context. It is speculated that planar polarisation of E-cad might be only one of a number of mechanisms that operate redundantly during the crucial developmental event of germband extension. Among other mechanisms reported are localised actomyosin contraction at vertical junctions, inhibition of Bazooka localisation on vertical junctions by local Rho kinase (Rok) activity and alteration of Arm (β-catenin) dynamics on vertical junctions by localised activity of the Abl kinase. Interestingly, it was found that loss of core pathway activity also abolishes Zipper and Bazooka asymmetry, but not Arm asymmetry (Abl or Rok asymmetry was not examined in this study). Additionally, a planar-polarised distribution of activated Src kinase to horizontal junctions was observed. Although Src kinase is a known modulator of E-cad trafficking in the Drosophila embryo, the significance of its planar-polarised distribution is unclear, as loss of core pathway function did not affect the distribution of Src, but did block the planar-polarised distribution of E-cad (Warrington, 2013).

Another context in which the core pathway might modulate E-cad turnover is during ommatidial rotation in the developing Drosophila eye, in which possible involvement of both RhoA and the kinase Nemo have been reported (Warrington, 2013).

In summary, this study has presented evidence that the core planar polarity pathway acts to locally promote E-cad endocytosis via local recruitment of RhoGEF2 and activation of RhoA activity. This represents a mechanism by which the core pathway can promote planar-polarised cell rearrangements (Warrington, 2013).

Effects of Mutation or Deletion

Ras superfamily proteins function as regulated molecular switches that alternate between active GTP-bound and inactive GDP-bound states. The GTP/GDP balance of these small GTPases is dictated by the net rates of guanine nucleotide exchange and GTP hydrolysis; these rates are tightly controlled by three classes of regulatory molecules: (1) guanine nucleotide exchange factors (GEFs) act as positive regulators that promote the release of GDP and consequent formation of the active GTP-bound state; (2) GTPase activating proteins (GAPs) act as negative regulators that stimulate the intrinsic GTPase activity to cycle them back to the inactive GDP-bound form, and (3) guanine nucleotide dissociation inhibitors (GDIs), associate with GTPases to maintain the existing nucleotide-bound state. Rho, acting through other proteins, mediates actin rearrangements that are likely to be required for the numerous cell shape changes in a developing embryo.

Embryos lacking DRhoGEF2 fail to gastrulate due to a defect in cell shape changes required for tissue invagination. Expression of a dominant-negative Rho GTPase in early embryos results in similar defects. Of the DRhoGEF2 homozygotes, 100% die as late embryos or early larvae in the absence of obvious abnormalities. DRhoGEF2 mRNA is expressed uniformly at high levels in the syncitial blastoderm; levels decrease until, by the time gastrulation is initiated, no expression is detected. This apparent maternal contribution of DRhoGEF2 mRNA suggests a likely role for the encoded protein in early embryogenesis. Embryos lacking maternal DRhoGEF2 exhibit normal dorso-ventral and anterior-posterior patterning, as well as mesoderm. However, germband extension and posterior midgut invagination appear to be defective. In addition, the cells of the mesectoderm fail to intercalate at the ventral midline, indicating a defect in ventral furrow formation. In embryos lacking maternal DRhoGEF2, the process of gastrulation is highly disorganized and ventral furrow formation never occurs. In such embryos it appears that random cells within an approximately 20-cell width spanning the ventral midline undergo apical membrane constrictions. There is also a substantially reduced number of addition constrictions in neighboring cells, resulting in a pitted ventral surface. In addition to the defects in ventral furrow formation and invagination of the posterior midgut, DRhoGEF2 mutant embryos are defective in invagination of the anterior midgut, a closely related gastrulation event (Barrett, 1997 and Hacker, 1998).

In addition to the DRhoGEF2 defects, embryos expressing a dominant negative Rho1 exhibit obvious defects in gastrulation. While the furrow in dominant negative Rho1 embryos does form, it fails to extend at the posterior end, resembling the ventral furrow defects in folded gastrulation and concertina mutants. The T-shaped invagination of the anterior midgut does not form normally in these embryos and they also exhibit defects in posterior midgut invagination and germband extension. However, the cephalic furrow forms normally in both DRhoGEF2 and dominant negative Rho1 embryos (Barrett, 1997)

Evidence is also presented that DRhoGEF2 mediates these specific cell shape changes in response to the extracellular ligand, Folded gastrulation. fog was expressed ectopically from a huckebein promoter, which is normally active in a subset of cells at the anterior and posterior ends of the embryo. In all the hkb-fog expressing embryos a characteristic transient depression in the dorsal head region can be seen. The surfaces of cells in this depression exhibit membrane blebbing and constrictions closely resembling those normally seen in cells along the ventral furrow in wild-type embryos. In addition, the nuclei of these cells have migrated from an apical to a basal position. In the absence of DRhoGEF2, ectopic Fog expression fails to induce any detectable cell shape changes, despite equivalent levels of fog transgene expression. Together, these results establish a Rho-mediated signaling pathway that is essential for the major morphogenetic events in Drosophila gastrulation (Barrett, 1997).

The central domain of DRhoGEF2 contains a likely phorbol ester-response motif. The homologous domain in Protein kinase C mediates kinase activation in response to diacylglycerol, which is generate by phospholipase C (PLC). Thus is it possible that the GEF activity of DRhoGEF2 is responsive to diacylglycerol. Since PLC-mediated production of diacylglycerol can be promoted by both receptor tyrosine kinase activation and by activation of receptor-coupled heterotrimeric G proteins, it is possible that the nucleotide-exchange activity of DRhoGEF2 is stimulated by signals transduced by both of these types of receptors. The presence of a PDZ domain in DRhoGEF2, suggests that it may interact with additional signaling proteins. Therefore, it appears that the GEF activity of DRhoGEF2 may be regulated by multiple upstream signals (Barrett, 1997).

It is postulated that Fog acts via the G alpha protein Concertina (see G protein salpha 60A: Evolutionary homologs section for more information about Concertina) to activate DRhoGEF2, thereby promoting Rho1 activation and consequent actin rearrangements. Significantly, the Drosophila G alpha subunit, Concertina, exhibits the strongest sequence similarity to the mammalian Galpha12 and Galpha13 proteins, which mediate the activation of Rho by LPA. Thus, it appears likely that a Rho-mediated signaling pathway linked to heterotrimeric G proteins has been evolutionarily conserved (Barrett, 1997 and references).

The obsevation that DRhoGEF2 is required for the cell shape changes induced by ectopic Fog expression strongly supports the model that a signal from Folded gastrulation via Concertina activates DRhoGEF2 (Barrett, 1997). There is, however, one case of a significant difference between the mutant phenotypes of fog and cta and those of DRhoGEF2: in contrast to DRhoGEF2, fog and cta are not essential for ventral furrow formation. For this reason DRhoGEF2 appears to be activated (at least to some extent) independently of either Fog or Cta. In fact, because of the non-essential function of fog and cta in the mesoderm, a second pathway instructing cells to undergo shape changes has been postulated (Costa, 1994). It is proposed that DRhoGEF2 identifies this pathway as a G-protein-coupled signaling cascade involving the GTPase Rho1. Whether this pathway is also required to transduce additional signals besides that of Fog is presently unclear and this question will require further attention in the future (Hacker, 1998).

Genetic analysis demonstrates a direct link between Rho signaling and nonmuscle myosin function during Drosophila morphogenesis

A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. The regulation of myosin activity has been explored by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, a screen was performed for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. A single missense mutation in the zipperEbr allele gives rise to its sensitivity to second-site noncomplementation. The Rho signal transduction pathway has been identified as necessary for proper myosin function. A lethal P-element insertion interacts genetically with zipper. Subsequently this second-site noncomplementing mutation has been shown to disrupt the RhoGEF2 locus. Two EMS-induced mutations, previously shown to interact genetically with zipperEbr, disrupt the RhoA locus. Further, their molecular lesions have been identified and it has been determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, it has been shown that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. These studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan (Halsell, 2000).

To identify loci encoding gene products that collaborate with nonmuscle myosin during morphogenesis, second-site noncomplementation screens were performed for the malformed adult leg phenotype (mlf). Depletion of myosin during leg imaginal disc morphogenesis results in mlf. A collection of 268 single, lethal P-element insertional mutations on the second chromosome were screened for genetic interactions with the zipEbr allele. Fourteen insertions failed to complement zipEbr. The strength of the genetic interaction is arbitrarily defined on the basis of the percentage of flies of the appropriate genotype that exhibit the malformed phenotype: weak interactions show penetrance of 10%-25% while intermediate interactions are 25%-75% penetrant. Eleven of the lethal P-element insertions identified are weak interactors. Three of the insertions are intermediate interactors. Two of these intermediate interactors are not second-site noncomplementing loci but are new zipper alleles, exhibiting intraallelic complementation. The third intermediate interacting mutation, l(2)04291, causes mlf flies in trans to zipEbr with a penetrance of 38% (Halsell, 2000).

The P-element insertion of l(2)04291 disrupts the RhoGEF2 locus. Genomic DNA flanking the P-element insertion was recovered by plasmid rescue, and by sequencing flanking DNA it was discovered that the P element lies within an intron that interrupts the 5' UTR of the RhoGEF2 gene (Barrett, 1997; Hacker, 1998). To further confirm that the genetic interaction observed with zipEbr results from a mutation in RhoGEF2, two EMS-induced mutant RhoGEF2 alleles, 1.1 and 4.1, were tested in the malformed leg assay. Both alleles interact with zipEbr; the penetrance of the malformed phenotype in double heterozygous flies is 33% with the RhoGEF21.1 allele and 27% with the RhoGEF24.1 allele, comparable to that seen with the original P-insertional allele (Halsell, 2000).

In addition to the malformed legs observed in flies double heterozygous for mutant RhoGEF2 and zipEbr, malformed wings were observed at comparable frequencies. Between 80% and 97% of the flies exhibiting a malformed leg phenotype also exhibit malformed wings. In contrast, most other loci that interact with zipper do not exhibit significant wing defects. Malformed wings are rarely observed when the legs are wild type. Taken together, these data indicate a requirement for RhoGEF2 during myosin-driven leg and wing imaginal disc morphogenesis (Halsell, 2000).

RhoAE3.10 genetically behaves as a severe allele, yet molecularly results from a single amino acid change that converts a cysteine at position 189 to a tyrosine residue. This missense mutation causes severe effects because it alters the first residue, cysteine, in the CaaX box. The CaaX box is a common feature of members of the Ras-superfamily of small GTPases. Functionally, the cysteine residue is the site of a post-translational prenylation modification. Subsequent to this modification further lipid modifications may occur, and in most cases, the final three amino acids are removed. These modifications are required for proper association of the small GTPase and the membrane; without this association, the GTPase is nonfunctional. These functional relationships have been demonstrated for numerous Ras superfamily members, including Rho. Site-directed mutagenesis that changes the CaaX box cysteine to serine of the S. cerevisiae RhoA homolog, Rho1, results in the failure of the mutated Rho1 protein to repartition from the cytosolic compartment to the membrane. Further, these Rho1 mutant cells fail to grow. In mammalian tissue culture, CaaX box-mutated RhoB cannot be lipid modified, and these cells lose their ability to become transformed in sensitized backgrounds. Therefore, it is likely that the RhoAE3.10-encoded protein cannot be post-translationally modified, resulting in a complete loss of RhoA function. Similarly, the nonsense mutation at residue 180 in the J3.8 allele would remove the CaaX box and an additional nine amino acids and, therefore, would also behave as a severe RhoA allele (Halsell, 2000).

However, on the basis of the differences observed in their genetic interactions with Df(2R)Jp1 and their levels of reduced viability in trans to zipEbr, RhoAE3.10 appears to be a more severe allele than RhoAJ3.8. It is hypothesized that the protein encoded by RhoAE3.10 may have a partial dominant-negative effect because it does not repartition properly. On the other hand, the premature stop codon in RhoAJ3.8 may give rise to an unstable gene product. Since appropriate antibodies directed against Rho are not yet available, this alternative cannot be adequately evaluated (Halsell, 2000).

Studies reveal that multiple processes require myosin function throughout Drosophila development, including oogenic cell migrations, larval cytokinesis, and imaginal disc morphogenesis. Strong or null alleles of zipper are embryonic lethal, fail during dorsal closure, and give rise to embryos with dorsal cuticular holes. Additionally, myosin immunolocalization studies suggest that myosin is required during stages not yet tested functionally, including embryonic cellularization and gastrulation. RhoGEF2 and RhoA also function at least during a subset of the morphogenetic processes that require myosin (Halsell, 2000 and references therein).

Mutations in the Drosophila RhoGEF2 gene have been identified by three distinct means: phenotypic suppression of ectopically expressed RhoA (Barrett, 1997); genetic screens for maternally encoded molecules required during early Drosophila embryogenesis (Hacker, 1998), and genetic screening for molecules required for myosin function. Maternal depletion of RhoGEF2 results in defects during gastrulation (Barrett, 1997; Hacker, 1998). Specifically, embryos lacking maternal RhoGEF2 fail during apical constriction of ventral furrow cells. Interestingly, myosin localizes to the apical ends of these ventral furrow cells. This observation coupled with the genetic interaction between RhoGEF2 and myosin during leg morphogenesis suggests that RhoGEF2 may exert some of its effect during gastrulation via the activity of myosin in these cells (Halsell, 2000 and references therein).

RhoA mutations are recessive embryonic lethals. Zygotic depletion of RhoA results in an anterior dorsal hole in the cuticle. This defect has been characterized as a dorsal closure phenotype. Dorsal closure is an embryonic morphogenetic event in which the lateral epidermis moves over the dorsal side of the embryo, ultimately fusing along the midline. If dorsal closure fails, then cuticular holes result. Typically, these holes are more posteriorly localized than those observed in RhoA mutants. However, certain zipper alleles give rise to cuticular holes that extend from the posterior one-third of the embryo to the anterior end. These extensive cuticular holes are consistent with the head involution defects observed in zipper mutants and may reflect combined defects in head morphogenesis and dorsal closure. Therefore RhoA loss-of-function mutations may more accurately represent a particular sensitivity in head morphogenesis to perturbation rather than being dorsal closure mutants per se (Halsell, 2000 and references therein).

Nonetheless, RhoA function during dorsal closure has been implicated by analysis of embryos expressing dominant negative RhoA transgenes. In wild-type embryos, the leading-edge cells and the adjacent lateral cells elongate during dorsal closure. When dominant-negative RhoA is driven in the leading edge by utilizing the GAL-4 UAS system, stretching of the leading cells initiates but is ultimately lost, and the lateral cells never elongate. The Jun-kinase signal transduction cascade acts during dorsal closure and induces expression of the TGFß gene, decapentaplegic (dpp), in the leading-edge cells. Leading-edge dpp expression is a prerequisite for elongation of the flanking lateral cells. In the dominant-negative RhoA embryos, dpp expression is wild type, therefore the authors suggest that RhoA acts upstream of a separate transcriptional pathway. Three observations suggest that RhoA may function directly upstream of myosin in the leading edge. (1) It has been shown that RhoA signaling is necessary for myosin-driven cell shape changes during leg imaginal disc morphogenesis. (2) zipper mutants lose myosin in the leading-edge cells, and, subsequently, the leading-edge cells fail to elongate. (3) Myosin is delocalized in leading-edge cells expressing dominant negative RhoA. Taken together, these results suggest that RhoA signaling may have a direct cellular output at the level of myosin activity in the leading-edge cells and may not exert its effect via a transcriptional pathway (Halsell, 2000 and references therein).

Numerous pharmacological, cell culture, and biochemical studies implicate the Rho subfamily of GTPases as signal transducers upstream of actin cytoskeleton rearrangements and myosin regulation. In Drosophila, injection of mutant forms of Rho or Cdc42 proteins induces gross malformations in the actomyosin cytoskeleton, disrupting a specialized embryonic cytokinesis known as cellularization. When dominant-negative Rac1 is expressed at later stages of embryogenesis, the actomyosin cytoskeleton is disrupted in the leading-edge cells during dorsal closure. In Swiss 3T3 cells, the Rho GTPase induces the formation of actin stress fibers. Further, it has been demonstrated that contractility of the actin cytoskeleton, presumably mediated by myosin, is required for stress fiber formation and that this contractility is downstream of Rho signal transduction (Halsell, 2000 and references therein).

In metazoans, nonmuscle myosin and smooth muscle-based contractility depend on the phosphorylation state of the noncovalently bound regulatory light chain. Molecularly, activated Rho may modulate the phosphorylation state of the regulatory light chain. Biochemical analysis reveals that activated Rho binds and activates a variety of effectors, including a group of serine/threonine kinases known as Rho kinase/ROK and p160ROCK/ROKß. In vitro biochemical assays reveal that Rho kinase can phosphorylate the regulatory light chain at its activating sites and induce myosin activity. Further, Rho kinases phosphorylate the myosin binding subunit of myosin phosphatase and thus repress its activity; the net result is a further increase in the phosphorylation state of the regulatory light chain (Halsell, 2000 and references therein).

Genetic screens for morphogenesis defects in C. elegans have also identified mutations in loci encoding Rho signal transduction components. Mutations in the C. elegans Rho kinase locus, let-502, disrupt embryonic elongation, while mutations in the regulatory subunit of the myosin phosphatase gene, mel-11, suppress the let-502 morphogenetic defect (Wissmann, 1997). These results suggest that Rho signal transduction is upstream of myosin-driven morphogenesis in C. elegans. This hypothesis cannot be tested directly because myosin mutations that affect cell sheet morphogenesis have not been identified in C. elegans. Nonmuscle myosin is encoded by more than one locus and functional redundancy of these loci may preclude the isolation of morphogenetic myosin mutations (Halsell, 2000 and references therein).

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).

RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation.

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitant with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. Both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. These results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane (Grosshans, 2005).

This morphological analysis of the mutant phenotypes reveals a new function of RhoGEF2 and dia in the formation of the furrow canal. This function is consistent with the co-localisation of both proteins with F-actin at the furrow canal and the reduced amounts of F-actin in RhoGEF2 and dia mutants. Biochemical analysis demonstrates actin polymerisation by Dia and thus supports the model that RhoGEF2 and Dia organise actin filaments to control the formation of the furrow canals. Furthermore, evidence is provided that the previously characterised genes nullo and sry- alpha act in a genetic pathway in parallel to RhoGEF2 and dia, suggesting that they control two distinct aspects of furrow canal formation. This conclusion is based on the assumption that amorphic situtations were used in these experiment. The possibility cannot be excluded that RhoGEF2 and dia stabilise the furrow canal rather than control its initial formation. A function in the formation is supported by the observation that the proportion of nuclei in multinuclear cells does not increase in the course of cellularisation (Grosshans, 2005).

The following arguments support the hypothesis that RhoGEF2 and dia act in the same genetic pathway that controls spatially restricted assembly of actin filaments. In both dia and RhoGEF2 mutants the morphology of the furrow canal is disrupted. The furrow canals are much larger than normal and filled with cytoplasmic blebs. Both proteins are localised at the furrow canal and both precede the appearance of the cellularisation front. The localisation of both proteins does not depend on F-actin. However, they are directly or indirectly involved in the assembly of F-actin since the amount of F-actin is reduced at the furrow canal of the mutant embryos. The strongest argument for a functional connection is that Dia localisation at the furrow canal depends on RhoGEF2 during the early phase of cellularisation. Rho1 may mediate this functional link by direct interactions with RhoGEF2 and Dia. However, the findings do not show that RhoGEF2 exclusively functions via dia. Other targets of Rho1-GTP, like citron kinase, protein kinase N or Rho kinase may be activated in parallel to Dia. Although a reduction of MyoII at the furrow canal was observed during the first half of cellularisation in embryos from RhoGEF2 germline clones, correspondingly lower MyoII levels are also observed in embryos from dia germline clones, which indicates that the reduction of MyoII may be a consequence of reduced F-actin levels. Consistent with the reduction of F-actin at the furrow canal, levels of MyoII were also reduced in the mutant embryos. In contrast to the reduction at the furrow canal, cortical F-actin appeared to be increased in some embryos from dia germline clones. This increase was variable and not observed in all of the experiments, however (Grosshans, 2005).

The difference in the RhoGEF2 and dia mutant phenotypes clearly shows that dia has additional functions and may be controlled by other not yet identified factors besides RhoGEF2. Whereas RhoGEF2 mutants pass through the cleavage cycles without obvious defects, dia is involved in formation of pole cells and pseudo cleavage furrows. As a possible consequence of these additional functions, dia mutants in contrast to RhoGEF2 mutants often have a more disrupted F-actin array, larger furrow canals and a more disturbed cellularisation than RhoGEF2 mutants. Furthermore in the early phase of cellularisation Dia localisation depends on RhoGEF2, whereas later, after the furrow has formed, Dia becomes enriched to a certain degree at the cellularisation front independently of RhoGEF2. One gene that may act in parallel to RhoGEF2 to control Dia localisation is Abl. Embryos from Abl germline clones have reduced amounts of Dia at the furrow canal and show a disrupted F-actin array similar to that observed in dia and RhoGEF2 mutants. However, the molecular link between Abl and Dia is elusive and no abnormalities in the morphology of the furrow canal in Abl mutants have been described. Thus Dia may be controlled and activated by multiple pathways including RhoGEF2 among others (Grosshans, 2005).

It is not known how the position of the invaginating plasma membrane is determined. RhoGEF2 and Dia are not likely to be part of a pattern formation process, but their localisation reflects an early readout of this pattern, since the nuclei and centrosomes are properly arranged in RhoGEF2 and dia mutants. RhoGEF2 and Dia proteins are early markers for these sites and precede furrow canal formation because specific staining was detected for both Dia and RhoGEF2 when the nuclei were still spherical and when the cellularisation front was not yet visible. Other factors beside RhoGEF2 and Dia are also involved in furrow canal formation, because many furrow canals still form in RhoGEF2 and dia mutants, which indicates that there is genetic redundancy (Grosshans, 2005).

At present it can only be speculated about which factors and mechanisms are responsible for RhoGEF2 localisation. Candidates may be among the group of genes involved in furrow canal formation. However, for all of these mutations no ultrastructural analysis has been reported that would allow the morphological defect to be defined and their function for furrow canal formation to be compared with the function of RhoGEF2 and dia. Among this group are Rab11 and nuf, which encode a GTPase of the recycling endosome and its putative effector. Considering the assumed biochemical activities, it is conceivable that vesicle targeting is important for transporting factors to the site of membrane invagination. This raises the possibility that RhoGEF2 is transported by such vesicles to the sites of membrane infolding. Analysis of RhoGEF2 protein distribution in nuf and Rab11 mutants and the phenotype of double mutants may address this hypothesis. Alternatively, RhoGEF2 may be transported to the site of the future furrow canal along microtubules that form open baskets around the nuclei, or other recruiting factors may precede at the site of membrane invagination (Grosshans, 2005).

Furthermore, slow as molasses (slam) is required for timed formation of the furrow and invagination of the membrane in the first half of cellularisation. Like Dia and RhoGEF2 Slam protein localises to the furrow canal and localisation precedes furrow canal formation. Slam may act by recruiting MyoII to the furrow canal, but the biochemical activities of Slam have not been defined. Although the membrane does not invaginate initially in slam mutants, a complete F-actin array is visible. Thus despite the overlapping localisation of Slam, RhoGEF2 and Dia, their functions are clearly distinguishable (Grosshans, 2005).

How do RhoGEF2 and Dia act in furrow canal formation? If the biochemical activity of Dia is considered to nucleate actin filaments and the enlarged and labile furrow canals in the dia mutants, it is conceivable that Dia organises and assembles a coat of F-actin at the site of membrane invagination and furrow canal formation. The coat of F-actin may be important for the compactness and stability of the furrow canal to prevent infoldings of the cytoplasm. Such a function may be related to the function of F-actin in endocytic events. The subset of actin filaments controlled by RhoGEF2 would not significantly contribute to pulling in the plasma membrane, since membrane invagination proceeds with normal speed in RhoGEF2 mutants. Alternatively, RhoGEF2 and Dia may perform their function independently of actin polymerisation. Although the amount of F-actin is reduced in the mutants, the possibility that the polymerisation activity of Dia is not required for all or part of its function cannot be excluded. Dia may also influence the organisation of microtubules, as interactions of mDia1 with microtubules and EB1, a microtubule-associated protein, have been described (Grosshans, 2005).

The differences in protein localisation and mutant phenotypes of RhoGEF2 and nullo suggest that they have distinct activities. In contrast to the frequently missing furrow canals in single mutants, their complete absence in embryos lacking both gene functions clearly implies, however, that their functions are redundant from a genetic point of view. These results show that RhoGEF2 and dia are required for the formation of a compact and stable furrow canal. If one of the two pathways is disturbed, the furrow canal can still form, albeit with a lower and variable efficiency that depends on the conditions. For example the nullo phenotype is strongly temperature sensitive. However, if both pathways are affected, furrow canals do not form at all. Future studies will resolve how the actin filaments are involved in bending the plasma membrane that leads to the furrow canal and will further demonstrate how RhoGEF2 protein is expressed in the hexagonal array to serve as a template for local actin polymerisation (Grosshans, 2005).

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help in the understanding of animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. This study reports the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, these data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction (Fox, 2007).

Regulation of apical constriction during Drosophila VF formation is a paradigm for how signal transduction directs morphogenesis. This study identified Abl as a novel regulator of this process. The results suggest that Abl acts in parallel to the known signaling pathway that promotes apical myosin activation by helping to organize a continuous apical actin network. Furthermore, the results help to explain the greater severity of the RhoGEF2-mutant phenotype relative to other VF mutants by suggesting that RhoGEF2 plays crucial roles in both myosin and actin regulation (Fox, 2007).

Previous work established myosin as a key output of RhoGEF2 signaling during mesoderm internalization. However, ambiguities remained regarding the circuitry of this pathway, since the RhoGEF2 phenotype is much more severe than that of cta or fog mutants, suggesting that a simple linear pathway is unlikely. The data suggest that RhoGEF2 plays dual roles in actin and myosin regulation, and thus its inactivation has more severe effects (Fox, 2007).

From these data, a mechanistic model was developed for the regulation of apical constriction during VF formation. The regulation of actin localization by Abl and RhoGEF2 promotes organization of the apical actin network in constricting cells. It is suggested that Abl regulates actin by actively downregulating cortical Ena in mesoderm, thus leading to polarized actin accumulation, similar to the role that it was shown to play in follicle cells. RhoGEF2 plays a distinct, Cta-independent role in the effective assembly of organized apical actin. While RhoGEF2 and Abl are modulating actin assembly, the mesodermal transcription machinery activates Fog-Cta signaling, apically stabilizing RhoGEF2. This allows the efficient activation of apical myosin. Coupling of these two cues -- an organized apical actin ring at AJs and stable apical myosin activation -- cooperate to ensure highly coordinated actomyosin constriction throughout the sheet of mesodermal cells in a short timeframe (Fox, 2007).

This model helps explain the mutant phenotypes observed in this and previous studies. In abl mutants, Fog-Cta allow RhoGEF2 stabilization and myosin contraction, but the lack of organized mesodermal actin in these mutants, which results from inappropriate Ena regulation, prevents the uniform assembly of actin-based contractile rings. cta mutants lack a stabilizing signal for RhoGEF2, preventing uniform apical myosin activation and uniform constriction. However, some cells can constrict without Fog-Cta, accumulating apical myosin levels comparable to those in wild type. In RhoGEF2 mutants, the combined failure to stabilize/activate myosin and a lack of organized apical actin severely compromises apical constriction. The similarity between RhoGEF2 and cta;abl mutants supports this model, as both processes should be compromised (Fox, 2007).

The model suggests that organized apical actin is an essential prerequisite for cell constriction. Although both Abl and RhoGEF2 regulate actin localization, the data argue that each acts independently. First, actin defects arise during cellularization, when Abl and RhoGEF2 have non-overlapping localizations. Second, whereas Abl clearly acts through Ena, loss of RhoGEF2 disrupts actin without altering Ena localization. Finally, Abl is not a Rho effector in S2 cells (Fox, 2007).

Several unanswered questions remain. With respect to abl, a major question is why do some cells apically constrict while others fail? This phenotype resembles the cellularization defects of abl mutants, in which only some cells fail to reorganize actin into furrows. However, all cells exhibit excess apical Ena and thus form abnormally long, apical microvilli. Perhaps, in some cells, furrow actin assembly drops below a crucial threshold and furrows fail. In the VF, the absence of Abl may have similar effects. VF defects could result from both competition for cellular actin and recruitment of other regulators (e.g. the formin Diaphanous) to ectopic locations, preventing their action in VF formation. This may reduce actin assembly into contractile rings. When constriction initiates, stochastic variations in ring strength may lead some rings to fail, leading to unconstricted cells. Future work is needed to identify the full set of actin regulators involved, and to assess how they work. Interestingly, recent work implicates Abl in epithelial-mesenchymal transitions. Whereas Abl disrupts VF formation, Twist is normally localized in abl mutants, suggesting that this major regulator of such transitions is not an Abl target in flies (Fox, 2007).

The data also reveal the importance of mesodermal Ena downregulation. This may result from increased mesodermal Abl activity, suggested by elevated levels of mesodermal Abl relative to non-mesoderm; however, this remains to be tested. It is also necessary to identify the mechanism by which Abl regulates Ena. In some places, such as the syncytial blastoderm, Abl localizes to sites where Ena is normally absent and, in the absence of Abl, ectopic Ena is found at these sites. This suggests that Abl actively antagonizes Ena localization. At other times and regions, however, such as the leading-edge during dorsal closure, Abl co-localizes with Ena, and thus may hold it in an inactive state. In VFs, Abl localizes to the apical-lateral cortex, and Ena localizes to this site in its absence. Further studies of Abl action will be needed to clarify the mechanisms by which it downregulates Ena (Fox, 2007).

Interestingly, manipulating mammalian Ena/VASP can affect cell contractility Thus, Ena-downregulation may permit proper VF cell contractility. Testing this hypothesis will be important (Fox, 2007).

The results also raise questions regarding RhoGEF2. The model suggests that RhoGEF2 acts via two mechanisms, only one of which is Cta-dependent. Perhaps another upstream cue acts on RhoGEF2 to promote actin organization. Because RhoGEF2 mutants have actin-organization defects in all cells, this regulator may act in all cells prior to gastrulation. However, the data do not rule out a second mesoderm-specific RhoGEF2 regulator acting in parallel to Cta. Although Rho-Kinase is a potential Rho effector with respect to myosin, another effector may regulate actin organization. Attractive candidates are the Formins, which reorganize actin in many processes (Fox, 2007).

The data strengthen the idea that different cytoskeletal regulators direct distinct morphogenetic processes. Both Abl and Fog regulate mesodermal apical constriction but are dispensable for germband cell-cell intercalation. Thus, although both processes require dynamic myosin reorganization, distinct regulators act in each (Fox, 2007).

The picture becomes more complex when considering other roles of Fog, Cta and RhoGEF2. All are required for internalization of the posterior midgut and salivary glands, but these cells internalize in abl mutants. Thus, different types of apical constriction may be regulated differently. It will be interesting to explore the roles of Fog, Cta and RhoGEF2 during dorsal closure, which requires Abl (Fox, 2007).

This work supports mechanistic connections between VF formation and neural tube closure. Both involve actin-based apical constriction to internalize a sheet of cells into a tube. Mice lacking Abl and Arg kinases have neural tube defects, and actin organization in neuroepithelial cells appears altered; interestingly, these cells have ectopic actin that is less polarized than normal, similar to what was observed in abl-mutant VFs. Furthermore, double-mutant analysis suggests that mammalian Ena plays a role in neural tube closure in conjunction with Profilin. Thus, Abl-Ena signaling may represent a conserved mechanism of actin regulation during apical constriction. New mechanistic insights can now be pursued in mammals (Fox, 2007).

Rho also regulates neural tube closure. Mice lacking p190RhoGAP have neural tube defects. Interestingly, p190RhoGAP is an Arg substrate in the brain, suggesting possible direct links between Abl and Rho in apical constriction. The role of Drosophila p190RhoGAP in the VF has yet to be examined, but RhoGAP68F is implicated in VF formation. Future work in both flies and mice will provide further mechanistic insights into conserved mechanisms of apical cell constriction (Fox, 2007).

DRhoGEF2 and diaphanous regulate contractile force during segmental groove morphogenesis in the Drosophila embryo

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. This study shows that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. The Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. It is proposed that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis (Mulinari, 2008).

Analysis of segmental groove morphogenesis revealed that during invagination groove founder cells undergo two distinct phases of shape change. Initial apical constriction is associated with accumulation of F-Actin, DRhoGEF2 and Dia at the site of groove initiation. Together with the loss of apical constriction in DRhoGEF2 or dia mutants and the observation that DRhoGEF2 or constituatively active diaCA-overexpressing grooves form earlier, are deeper, and persist longer than wild-type grooves, this indicates that contractile forces in epidermal cells provide a driving force for groove morphogenesis (Mulinari, 2008).

Subsequent to apical constriction, groove founder cells and their immediate neighbors elongate their apical-basal axis and extend their basal sides inward. Apical-basal elongation does not occur when DRhoGEF2 or diaCA are overexpressed, suggesting that activation of these factors may counteract this specific change in cell shape. Inward movement of the epithelial baseline is not affected, and it occurs to a similar depth as in wild type, suggesting that it is not resulting from a pushing force generated by cell elongation. Expression of DRhoGEF2 in underlying mesodermal cells does not affect groove formation. Thus, cell autonomous bending of the epithelium rather than a pulling force originating in underlying tissues may be the main contributor to groove invagination (Mulinari, 2008).

It has been suggested that germband retraction that occurs in parallel to segmental groove formation may contribute to the forces that cause groove founder cells to invaginate. In DRhoGEF2 mutants, the posterior midgut primordium remains at the posterior pole due to failure of the endoderm to invaginate. Consequently, the germband is thrown into deep folds that illustrate the pressure exerted on the epithelial sheet. Yet, segmental grooves do not form in these embryos. Furthermore, local expression of DRhoGEF2 causes the formation of deep grooves in expressing cells but not in neighboring tissues. These observations do not exclude that global tissue rearrangements support segmental groove invagination, but they suggest that the major contributing force to groove formation is due to autonomous action of epidermal cells (Mulinari, 2008).

At the onset of stage 13 when germband retraction has completed, DRhoGEF2, F-actin, and myosin II accumulate apically in four to five cells posterior to the grooves. These cells subsequently constrict apically, which may bend the epithelium in an outward direction. Thus, groove regression may be actively driven by actomyosin-based constriction of epidermal cells. This is supported by previous findings showing that in zipper mutants, which partially lack nonmuscle myosin, grooves occasionally do not regress (Mulinari, 2008).

Groove regression coincides with the formation of denticles in ventral epidermal cells posterior to the groove. Denticle forming cells undergo cell shape changes that are followed by polarized accumulation of F-actin, myosin II, and Dia into condensations at the posterior cell boundary. DRhoGEF2 in contrast to Dia and myosin II, does not polarize to the F-Actin condensations and DRhoGEF2 mutants are able to form denticles. DRhoGEF2 is therefore unlikely to play a role in denticle formation. It is proposed that the narrowing of cells along the anterior-posterior axis that occurs throughout the ventral and lateral epidermis before hair formation may be due to DRhoGEF2-controlled cell contraction and may contribute to the regression of segmental grooves (Mulinari, 2008).

In conclusion, the data suggest that groove invagination and regression are active processes driven by acto-myosin-based contractile filaments in epidermal cells that are regulated by DRhoGEF2 and Dia in a concentration-dependent manner (Mulinari, 2008).

The small GTPase Rho1 regulates acto-myosin constriction through a pathway including Rho-kinase, the regulatory subunit of myosin light chain phosphatase and myosin II. DRhoGEF2 regulates cell contraction in different developmental contexts and DRhoGEF2-mediated recruitment of myosin II to the apical cortex and cell contraction require Rho-kinase, suggesting that DRhoGEF2 controls acto-myosin contraction through the Rho-kinase pathway (Mulinari, 2008).

Rho1 also regulates F-actin nucleation and polymerization through a pathway including Dia and the actin-binding protein Profilin. In cell culture systems, mammalian Dia1 has been shown to act as a positive feedback regulator of Rho-activity through direct interaction with the C terminus of the mammalian DRhoGEF2 homologue LARG. However, it is not clear whether Dia can act as an effector of DRhoGEF2 during morphogenesis in vivo. Drosophila Dia is required in processes such as metaphase furrow invagination, pole cell formation and cellularization that are also defective in DRhoGEF2 mutants consistent with the idea that both proteins may function in concert (Mulinari, 2008).

The data suggest that DRhoGEF2 and Dia are required for segmental groove morphogenesis. Interestingly, morphologic changes elicited by DiaCA at the epithelial and cellular level were distinct from those observed with DRhoGEF2. Whereas DRhoGEF2-overexpression caused cells to contract and take on a rounded shape, thereby reducing contact with each other, diaCA-expressing cells remained tightly packed and columnar. In addition, cells showed increased levels of the junctional proteins β-catenin and E-cadherin, suggesting a strengthening of cell-cell contacts. Consistent with this, mammalian Dia1 has been implicated in maintenance and stabilization of adherens junctions. In contrast, Rho-kinase-mediated generation of contractile force leads to the physical disruption of cell-cell contacts and cell rounding reminiscent of DRhoGEF2 overexpression. Another distinguishing feature between DRhoGEF2 and dia is the accumulation of F-actin in response to diaCA expression that is consistent with the role of Dia in nucleation and elongation of F-actin filaments. F-actin accumulation was not observed in response to DRhoGEF2-overexpression consistent with the view that DRhoGEF2 regulates F-actin contraction but not polymerization. This study found that DRhoGEF2 and Dia have qualitatively different effects on F-actin remodeling, supporting the view that they may be connected to the actin cytoskeleton by distinct Rho-effector pathways. Despite the differences, overexpression of either DRhoGEF2 or diaCA in some instances seems to result in increased contractility. In DRhoGEF2, this is likely due to direct regulation of acto-myosin contractility through the Rho-kinase pathway. In DiaCA, the increased levels of actin elicited by Dia activation might indirectly promote the formation of acto-myosin filaments. Alternatively, DiaCA could affect contractility through its effect on myosin (Mulinari, 2008).

Expression of either diaCA or DRhoGEF2 led to increased myosin II levels. In Schneider cells, DRok activity and myosin phosphorylation were found to be required for myosin II recruitment to contractile acto-myosin fibers, whereas Dia was required for maintenance of myosin at contractile rings. Similar to Rho-kinase, DRhoGEF2 is required for apical recruitment of myosin II in the ventral furrow during gastrulation. A role of Dia in this process has not been reported but it is possible that DRhoGEF2 and Dia may regulate different aspects of myosin function also in the Drosophila embryo (Mulinari, 2008).

In summary, DRhoGEF2 and Dia were found to be required for segmental groove formation, and they may act in concert to regulate a series of specific cell shape changes that lead to groove invagination. However, clear differences in the response of cells to activation of DRhoGEF2 or Dia at the morphologic and molecular level suggest that both genes may regulate the actin cytoskeleton through distinct effector pathways (Mulinari, 2008).

Genetic evidence for antagonism between Pak protein kinase and Rho1 small GTPase signaling in regulation of the actin cytoskeleton during Drosophila oogenesis

During Drosophila oogenesis, basally localized F-actin bundles in the follicle cells covering the egg chamber drive its elongation along the anterior-posterior axis. The basal F-actin of the follicle cell is an attractive system for the genetic analysis of the regulation of the actin cytoskeleton, and results obtained in this system are likely to be broadly applicable in understanding tissue remodeling. Mutations in a number of genes, including that encoding the p21-activated kinase Pak, have been shown to disrupt organization of the basal F-actin and in turn affect egg chamber elongation. pak mutant egg chambers have disorganized F-actin distribution and remain spherical due to a failure to elongate. In a genetic screen to identify modifiers of the pak rounded egg chamber phenotype several second chromosome deficiencies were identified as suppressors. One suppressing deficiency removes the rho1 locus, and using several rho1 alleles it was determined that removal of a single copy of rho1 can suppress the pak phenotype. Reduction of any component of the Rho1-activated actomyosin contractility pathway suppresses pak oogenesis defects, suggesting that Pak counteracts Rho1 signaling. There is ectopic myosin light chain phosphorylation in pak mutant follicle cell clones in elongating egg chambers, probably due at least in part to mislocalization of RhoGEF2, an activator of the Rho1 pathway. In early egg chambers, pak mutant follicle cells have reduced levels of myosin phosphorylation and it is concluded that Pak both promotes and restricts myosin light chain phosphorylation in a temporally distinct manner during oogenesis (Vlachos, 2011).

This study establishes the basal F-actin of the follicular epithelium as an attractive system for the genetic analysis of the signaling pathways regulating the formation of stress fiber-like structures. The actin bundles in the follicle cells appear to be similar to the ventral stress fibers of nonmotile cultured cells, for which one model of stress fiber formation is that it is driven by bundling of actin filaments by actomyosin contractility. Consistent with this model, the results indicate that the major cause of basal F-actin disruption in pak mutant cells is misregulated actomyosin contractility that can be suppressed by reduction of the Rho1 pathway. It was found that Pak regulates pMLC distribution during oogenesis, at first being required for pMLC and later restricting where it is present. Such conflicting roles for Pak have been reported in isolation in mammalian cell culture studies, but these results are the first to show that they can be temporally separated during development of an epithelial cell. Paks from diverse species can function as MLCKs, and such an activity for Pak is indicated in early stage egg chambers, where Pak's MLCK function is opposed by the Flap wing (Flw) MLC phosphatase. Later in oogenesis, around the time of egg chamber elongation, Pak restricts the distribution of MLC phosphorylation and comes into conflict with the Rho1/Rok pathway (Vlachos, 2011).

There are a number of ways that Pak could impinge on the Rok pathway, with one being at the level of RhoGEF2 at the top of the pathway. Pak is required for the basal localization of RhoGEF2, and the mislocalized RhoGEF2 seen in pak mutant clones could at least in part be responsible for the ectopic pMLC seen in older egg chambers. A protein similar to RhoGEF2 in mammals, P115-RhoGEF, appears to be negatively regulated by Pak binding to its DH-PH domain, but no similar physical interaction was found between Pak and the RhoGEF2 DH-PH, nor has an effect of Pak on Rho1-GTP levels been detected, although it is possible that there could be an effect not detectable by the assays that were used. A recent study showed that the PDZ domain of RhoGEF2 is required for its localization at the furrow canal during cellularization. Furthermore, the novel protein Slam, which complexes with the RhoGEF2 PDZ domain, is required for RhoGEF2 localization during cellularization, and it will be of interest to determine if Pak regulation of RhoGEF2 localization in the follicular epithelium involves the PDZ domain and/or Slam. Another possibility is that Pak regulates RhoGEF2 through a trimeric G-protein interaction. RhoGEF2 is a member of the RGS-containing family of GEFs that interact with the activated Gα subunits of trimeric G proteins through their RGS domain and members of the Pak family bind the Gβγ subunit complex through a motif conserved in Drosophila Pak (Vlachos, 2011).

Another route by which Pak could be restricting pMLC distribution is through regulation of a MLCK cooperating with Rok. Work on mammalian Pak has demonstrated that Pak can negatively regulate the activity of MLCK, thus reducing the level of MLC phosphorylation, and three potential MLCKs were considered as candidate Pak targets. Alleles of these genes did not suppress pak oogenesis defects, suggesting either that they are not regulated by pak during oogenesis or that more than one is being regulated by Pak. Another possibility is that Pak is directly regulating Rok in some manner to restrict the output of this pathway. Interestingly, in the columnar epithelial cells over the occyte in late egg chambers, Pak does not regulate MLC phosphorylation and this may be to allow the extensive actomyosin contractility likely to be required to shape these cells (Vlachos, 2011).

Finally, the possibility that Pak could be regulating pMLC levels simply by controlling the overall amount of MLC has not been eliminated, but this seems unlikely given the considerable evidence that vertebrate Pak regulates MLC phosphorylation (Vlachos, 2011).

The finding that RhoGEF2 is a basally localized regulator of actomyosin contractility in the follicular epithelium is consistent with numerous previous studies indicating that RhoGEF2 is the major activator of Rho1 during epithelial morphogenesis. Two other RhoGEFs known to regulate actin, Pebble and RhoGEF64C, did not affect the pak mutant egg chamber phenotype. Deficiencies and/or alleles disrupting 20 other predicted RhoGEFs were tested for the ability to suppress the dpak mutant egg chamber phenotype and found that none were effective. Similarly, a recent study tested predicted RhoGEFs as Rho1 regulators in driving epithelial morphogenesis during imaginal disc morphogenesis and concluded that RhoGEF2 is a key regulator. Many of the RhoGEFs have not been characterized functionally, although some have been shown to be GEFs for GTPases other than Rho1 and to function in nonepithelial cells such as neurons (Vlachos, 2011).

RhoGEF2 is enriched at the basal end of the follicle cells throughout oogenesis including the points of basal membrane separation between follicle cells that occurs during follicle cell flattening in late stage egg chambers. Recently, it was shown that the Rho1 actomyosin contractility pathway is required for this separation between follicle cells at the basal membrane and presumably this signaling is activated by RhoGEF2 (Vlachos, 2011).

RhoGEF2 alleles are much more effective than alleles of other Rho1 pathway components at extending the life span of pak mutant females, implying that RhoGEF2 may have roles independent of the Rho1 actomyosin contractility pathway that could be regulated by Pak. There is evidence that RhoGEFs have functions distinct from small GTPase activation; for example, Pebble has a Rho1-independent role in mesoderm migration (Vlachos, 2011).

In addition to the Rho pathway, an antagonistic relationship between Pak and the Dpp pathway in the regulation of egg chamber elongation was uncovered. A recent study of the Drosophila wing disc demonstrated that Dpp signaling regulates the subcellular distribution of Rho1 activity and MLC phosphorylation in epithelial cells. If this link between pathways also occurs in the follicular epithelium, it may be that loss of Dpp is suppressing the pak mutant phenotype through disruption of Rho1 signaling. Another possibility is that Dpp regulation of the actin filament cross-linking protein α-actinin in the follicular epithelium is relevant (Vlachos, 2011).

The ability of wun and wun2 alleles to suppress the pak egg chamber elongation defect might also be due to downregulation of the Rho1 pathway, as wun was picked up in an overexpression screen for suppressors of impaired Rho1 signaling. Wun and Wun2 belong to a family of lipid phosphate phosphatases that regulate the levels of lipids involved in signaling including lysophosphatidic acid, which is an important activator of the RhoA pathway (Vlachos, 2011).

Identification of novel Ras-cooperating oncogenes in Drosophila melanogaster: a RhoGEF/Rho-family/JNK pathway is a central driver of tumorigenesis

Nutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (RasACT) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with RasACT in tumorigenesis, a genome-wide screen was carried out for genes that when overexpressed throughout the developing Drosophila eye enhance RasACT-driven hyperplasia. RasACT-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1ACT), RhoGEF2, and pbl cooperated with RasACT, resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or >Rac1 with RasACT upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with RasACT, while in the clonal setting, JNK upregulation was sufficient for RasACT-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of RasV12-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2+ human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with RasACT in the whole-tissue context. Collectively this analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with RasACT (Brumby, 2011).


REFERENCES

Search PubMed for articles about Drosophila RhoGEF2

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Levayer, R., Pelissier-Monier, A. and Lecuit, T. (2011). Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis. Nat. Cell Biol. 13(5): 529-40. PubMed Citation: 21516109

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RhoGef2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 June 2014

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