Interactive Fly, Drosophila

The Rho1 gene is expressed throughout embryogenesis in a widespread manner. Rho1 mRNA is detectable at relatively uniform levels throughout the embryo (Hariharan, 1995).

Large-scale movements of epithelial sheets are necessary for most embryonic and regenerative morphogenetic events. The cellular processes associated with germ band retraction (GBR) have been characterized in the Drosophila embryo. During GBR, the caudal end of the embryo retracts to its final posterior position. Using time-lapse recordings, it has been shown that, in contrast to germ band extension, cells within the lateral germ band do not intercalate. In addition, the germ band and amnioserosa move as one coherent sheet, and the amnioserosa strongly shortens along its dorsal-ventral axis. Furthermore, during GBR, the amnioserosa adheres to and migrates over the caudal end of the germ band via lamellipodia. Expression of both dominant-negative and constitutively active RhoA in the amnioserosa disrupts GBR. Since RhoA acts on both actomyosin contractility and cell-matrix adhesion, it suggests a role for such processes in the amnioserosa during GBR. The results establish the cellular movements and shape changes occurring during GBR and provide the basis for an analysis of the forces acting during GBR (Schock, 2002).

GBR is completed during embryonic stage 12. This is a time of exceptional morphogenetic activity: the midgut fuses and encloses the yolk sac laterally; the tracheal pit extensions fuse to form the tracheal tree, and the segmental furrows form from anterior to posterior. At stage 12, the embryo consists of two major epithelia, the squamous extraembryonic amnioserosa and the ectodermal germ band epithelium, as well as a mesenchymal mass of mesodermal and central nervous system precursor cells. Also found in the embryo are the epithelia of the foregut, hindgut, and salivary and tracheal pit invaginations, which are of ectodermal origin, and the midgut epithelium, which forms at that stage by mesenchymal to epithelial transition. The syncytial yolk sac, which is enclosed by a yolk sac membrane, sits in the middle of the embryo at the beginning of GBR, but moves more dorsally, directly beneath the amnioserosa, by the end of GBR. Analysis was focused on the amnioserosa and the germ band, because they appear to be the most likely candidates of the above tissues to participate in GBR. Cells in the germ band do not intercalate. GBR is therefore not a reversal of germ band extension (Schock, 2002).

Rather, reciprocal cell shape changes within the amnioserosa and the germ band are associated with the changes in embryo morphology at this stage. That is, the amnioserosa shortens along the DV axis, while the germ band elongates along the DV axis. This is possible because the amnioserosa and germ band are tightly attached to each other via adherens junctions, i.e., both epithelia move as one coherent sheet (Schock, 2002).

The boundary between amnioserosa and germ band was investigated at high magnification to obtain an idea of whether the shape changes observed are of an active or a passive nature. It was assumed that contractile forces would be exerted along the plasma membranes, because the actin cytoskeleton is localized cortically in both germ band and amnioserosa cells. The row of leading edge germ band cells is pulled in where the amnioserosa membranes perpendicular to the leading edge are attached. This suggests that amnioserosa cells contract along their DV axis. Cells of the germ band would be expected to push into the larger amnioserosa cells in the case of an active DV extension of the germ band, thus resulting in a convex shape of the amnioserosa-germ band boundary (Schock, 2002).

The presence of protrusions is demonstrated; these are formed predominantly at the posterior edge of the amnioserosa projecting toward the posterior. These protrusions exhibit high levels of dynamic actinGFP at the migration front, indicating actin polymerization. These protrusions have been classified as lamellipodia, because their appearance, behavior, and dynamic actin content are identical to lamellipodia in other motile cells. These lamellipodia migrate over the germ band instead of being passively dragged by the retracting germ band. The lamellipodia may migrate on an apical extracellular matrix secreted by germ band cells as a precursor to the larval cuticle. These observations indicate that the overlap of the amnioserosa over the caudal end of the germ band during GBR is maintained by lamellipodia-mediated migration. Furthermore, both constitutively active and dominant-negative RhoA disrupt GBR, when expressed in the amnioserosa. This suggests that actomyosin contractility or cell migration within the amnioserosa contribute to GBR, since these processes are affected by expression of rhoA mutants in tissue culture (Schock, 2002).

The amnioserosa is required for GBR because embryos that lack this tissue fail to undergo GBR. It has been proposed that this requirement for the amnioserosa may involve signaling from the amnioserosa to the germ band. The cell shape changes and motility observed within the amnioserosa and the overexpression experiments suggest that the amnioserosa additionally contributes to GBR in other ways than signaling (Schock, 2002).

The processes observed in this study allow several mechanisms, which are not mutually exclusive, to participate in GBR. (1) Segment furrow formation within the germ band may facilitate GBR by causing AP shortening of the germ band. (2) Active DV shortening of the amnioserosa may contribute to GBR by pulling in the lateral sides of the anterior germ band, thereby resulting in retraction of the germ band behind the bend of the U-shaped germ band-extended embryo. (3) The pulling force that appears to be exerted by DV shortening of the amnioserosa may be assisted by active DV extension of the germ band cells. (4) The overlap of the amnioserosa over the germ band may allow proper deployment of forces occurring within the amnioserosa (Schock, 2002).

Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)^Pbl-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF^Pbl-dependent control themselves. Microscopy of Drosophila S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, a separate RhoGEFPbl-dependent pathway was uncovered that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF^Pbl-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. It is proposed that such interactions occur dynamically and transiently to promote furrow stability (Hickson, 2008).

Drosophila S2 cell lines expressing anillin-GFP were generatated. The anillin-GFP fusion rescued loss of endogenous anillin and its localization paralleled that of endogenous anillin. In interphase it was nuclear, at metaphase it was uniformly cortical, and in anaphase it accumulated at the equator while being lost from the poles. In some highly expressing cells, nuclear anillin-GFP formed filaments not ordinarily seen with anillin immunofluorescence, but these disassembled upon nuclear envelope breakdown and the overexpression had no appreciable effect on the progress or success of cytokinesis (Hickson, 2008).

Tests were performed to see whether RhoGEF^Pbl contributes to anillin localization during cytokinesis. After 3 d of RhoGEF^Pbl RNAi or Rho1 RNAi, anillin-GFP was found to be localized to the cortex in metaphase but does not relocalize to the equator during anaphase, indicating a requirement for RhoGEF^Pbl, consistent with prior analysis of fixed RhoGEF^Pbl mutant embryos. Because anillin can bind F-actin and phosphorylated myosin regulatory light chain (MRLC), RhoGEF^Pbl might regulate anillin indirectly through its control of F-actin and myosin II (Hickson, 2008).

Latrunculin A (LatA) was used to test whether F-actin was required for anillin-GFP localization. A 30-60-min incubation of 1 µg/ml LatA abolished cortical anillin-GFP localization in metaphase, indicating an F-actin requirement at this phase. However, when anillin normally relocalizes to the equator (~3-4 min after anaphase onset), anillin-GFP formed punctate structures that became progressively more filamentous over the next few minutes, reaching up to several micrometers in length and having a thickness of ~0.3 µm. These linear anillin-containing structures contained barely detectable levels of F-actin and formed specifically at the plasma membrane and preferentially at the equator, although subsequent lateral movement often led to a more random distribution. Thus, anillin responds to spatiotemporal cytokinetic cues even after major disruption of the F-actin cytoskeleton. A substantial (albeit incomplete) reacquisition of cortical phalloidin staining was observed in cells fixed after washing out the drug for a few minutes. In live cells, LatA washout immediately after formation of the anillin structures allowed the preformed structures to migrate from a broad to a compact equatorial zone as the cells attempted to complete cytokinesis. This movement indicates that an F-actin–dependent process can contribute to the equatorial focusing of anillin (Hickson, 2008).

The influence of RhoGEF^Pbl on anillin behavior was tested in LatA. After RNAi of RhoGEF^Pbl or Rho1, anillin-GFP remained cytoplasmic through anaphase. Thus RhoGEF^Pbl and Rho1 are required for anaphase anillin behavior, whether the cortex is intact or disrupted by LatA treatment (Hickson, 2008).

Tests were performed to see whether myosin II impacts anillin-GFP localization. Compared with controls, RNAi of the gene encoding MRLC spaghetti squash (MRLC^Sqh) inhibited cell elongation during anaphase, slowed furrow formation, and delayed and diminished the equatorial localization of anillin-GFP. However, unlike after RhoGEF^Pbl RNAi, equatorial accumulation of anillin-GFP was not altogether blocked. It was still recruited but in a broad zone. Furthermore, in the presence of LatA, MRLC^Sqh RNAi did not affect the formation of the anillin-GFP structures. It is concluded that myosin II contributes to the equatorial focusing of anillin when the F-actin cortex is unperturbed but that myosin II is dispensable for anillin behavior in LatA (Hickson, 2008).

Collectively, these data suggest that multiple RhoGEF^Pbl-dependent inputs control anillin localization. The slowed equatorial accumulation of anillin when myosin II function was impaired indicates a myosin II-dependent input. That reassembly of the cortical F-actin network (after washout of LatA) allowed preformed anillin structures to move toward the cell equator indicates an F-actin-dependent input. This is consistent with the concerted actions of myosin II and F-actin driving cortical flow, as observed in other cells, and is reminiscent of the coalescence of cortical nodes during contractile ring assembly in Schizosaccharomyces pombe. However, the F-actin- and myosin II-independent behavior of anillin in LatA indicates an additional RhoGEF^Pbl-dependent input. Thus, RhoGEF^Pbl can control anillin behavior in anaphase via a previously unrecognized route. Immunofluorescence analysis revealed extensive colocalization between endogenous Rho1 and anillin-GFP in LatA, indicating that Rho1 was itself a component of these structures. These findings are consistent with the idea that Rho1 and anillin directly interact (Hickson, 2008).

Myosin II localization was studied, since it can bind anillin and is controlled by RhoGEF^Pbl. MRLC^Sqh-GFP is able to localize to the equatorial membrane independently of F-actin, and in doing so forms filamentous structures resembling those observed with anillin-GFP. Indeed, anillin and MRLC^Sqh (detected as either MRLC^Sqh-GFP or with an antibody to serine 21-phosphorylated pMRLC^Sqh) colocalize (, although they were often offset as if labeling different regions of the same structures (Hickson, 2008).

The effects of anillin RNAi on MRLC^Sqh-GFP localization were tested. MRLC^Sqh-GFP recruitment and furrow initiation appeared normal, but within a few minutes of initiation, furrows became laterally unstable and oscillated back and forth across the cell cortex, parallel to the spindle axis, in repeated cycles, each lasting ~1-2 min and eventually subsiding to yield binucleate cells after ~20 min. The phenotype was very similar to that reported for anillin RNAi in HeLa cells and represents a requirement for anillin at an earlier stage than previously noted in Drosophila. Thus, a conserved function of anillin is to maintain furrow positioning during ingression (Hickson, 2008). <{>In LatA, anillin RNAi did not prevent equatorial MRLC^Sqh-GFP recruitment, but instead of appearing as persistent linear structures distorting the cell surface, a more reticular and dynamic structure lacking cell surface protrusions was observed. Thus myosin II can localize independently of both anillin and F-actin but the filamentous appearance of myosin II in the presence of LatA requires anillin, indicating that anillin can influence myosin II behavior in the absence of F-actin, whereas myosin II appeared capable of influencing anillin behavior only in the presence of F-actin (Hickson, 2008).

Septins are multimeric filament-forming proteins that can bind anillin in vitro and function with anillin in vivo. Using an antibody to the septin Peanut, it was found that in nontransfected S2 cells, septin^Pnut localized to the cleavage furrow and midbody where it colocalized with anillin. Unexpectedly, the septin^Pnut antibody also strongly labeled bundles of cytoplasmic ordered cylindrical structures, each ~0.6 µm in diameter and of variable length (up to several micrometers). These staining patterns could be greatly reduced by septin^Pnut RNAi and were thus specific. The cylindrical structures did not appear to be cell cycle regulated, as they were apparent in interphase, mitotic, and postmitotic cells. They also did not colocalize with anillin, nor did their stability rely on anillin. Incubation with 1 µg/ml LatA before fixation inevitably led to disassembly of most of these large structures; however, the resulting distribution of septin^Pnut depended on the cell cycle phase. In LatA-treated interphase cells, when anillin is nuclear, septin^Pnut formed cytoplasmic rings, ~0.6 µm in diameter, which are similar to the Septin2 rings seen in interphase mammalian cells treated with F-actin drugs or in the cell body of unperturbed ruffling cells (Kinoshita, 2002; Schmidt, 2004). In LatA-treated mitotic cells, septin^Pnut was diffusely cytoplasmic (or barely detectable) in early mitosis, whereas in anaphase/telophase, it localized to the same plasma membrane-associated anillin-containing filamentous structures (Hickson, 2008).

Anillin behavior was analyzed after septin^Pnut RNAi. Although unable to fully deplete septin^Pnut, it was found that anillin could localize to the equatorial cortex in regions devoid of detectable septin^Pnut, which is consistent with findings in C. elegans (Maddox, 2005). Importantly, in septin^Pnut-depleted cells, anillin-GFP still localized to the plasma membrane in LatA but no longer appeared filamentous, indicating that septin^Pnut is essential for the filamentous nature of the structures and that Rho1 can promote the association of anillin with the plasma membrane independently of septin^Pnut. However, in this case the plasma membrane to which anillin-GFP localized subsequently exhibited unusual behavior. It was internalized in large vesicular structures, apparently in association with midzone MTs. Although this phenomenon is not understood, it may be related to events induced by point mutations in the septin-interacting region of anillin that give rise to abnormal vesicularized plasma membranes during Drosophila cellularization (Hickson, 2008).

The effects were tested of anillin RNAi on the localization of septin^Pnut. Using Dia as a furrow marker, 3 d of anillin RNAi prevented the furrow recruitment of septin^Pnut. In LatA-treated cells, anillin RNAi did not affect the formation of septin^Pnut rings in interphase cells, but it greatly reduced the formation of septin^Pnut-containing structures during anaphase/telophase. Thus, anillin is required for the furrow recruitment of septin^Pnut and for the formation of septin^Pnut-containing structures in 1 µg/ml LatA. In contrast, Dia could still localize to the equatorial plasma membrane after combined anillin RNAi and LatA treatment, indicating that it can localize independently of both anillin and F-actin. Thus, although Dia partially colocalized with anillin in LatA, this likely reflected independent targeting to the same location rather than an association between anillin and Dia (Hickson, 2008).

These data argue that Rho1, anillin, septins, and the plasma membrane participate independently of F-actin in the formation of a complex. However, anillin, septins and F-actin can also form a different complex in vitro, independently of Rho (Kinoshita, 2002). Perhaps two such complexes dynamically interchange in vivo (Hickson, 2008).

The involvement of MTs in anillin behavior was tested in LatA. Overnight incubation with 25 µM colchicine effectively depolymerized all MTs in mitotic cells and promoted mitotic arrest, as expected. Using Mad2 RNAi to bypass the arrest, anillin-GFP was observed during mitotic exit in the absence of MTs and in the presence of LatA. Under such conditions, anillin-GFP formed filamentous structures very similar to those formed when MTs were present, indicating that MTs were dispensable for their formation. However, the structures appeared uniformly around the plasma membrane rather than restricted to the equatorial region, which is consistent with the role MTs play in the spatial control of Rho activation (Hickson, 2008).

The LatA-induced anillin structures localize to the ends of nonoverlapping astral MTs directed toward the equator. Live imaging of cells coexpressing cherry-tubulin and anillin-GFP revealed bundles of MTs associating with the filamentous anillin-GFP structures as they formed. Colocalization between anillin-GFP structures and MT ends persisted over many minutes, even after considerable lateral movement at the membrane. Thus, although the anillin structures formed independently of MTs, they stably associated with MTs. These findings support prior biochemical evidence for interactions of MTs with both anillin and septins (Sisson, 2000) and reveal a potential positive-feedback loop in which MTs directed where Rho1-anillin-septin formed linear structures at the plasma membrane, whereas the structures in turn associated with the MT ends. An MT plus end-binding ability of anillin-septin could explain the furrow instability phenotype elicited by anillin RNAi. Accordingly, anillin may physically link Rho1 to MT plus ends during furrow ingression, thereby promoting the focusing and retention of active Rho1, thus stabilizing the furrow at the equator (Hickson, 2008).

These live-cell analyses highlight an unusual behavior of the Rho-dependent anillin-containing structures at the plasma membrane. Initially forming beneath and parallel to the plasma membrane, the structures then often lifted on one side to appear perpendicular to the cell surface while remaining anchored at their base by MTs. This reorientation is interpreted as reflecting avid binding to and subsequent envelopment by the plasma membrane. Although intrinsically stable, the structures exhibited dynamic movement within the plane of the plasma membrane and were capable of sticking to one another, via their ends, giving rise to branched structures that were also capable of breaking apart. Anillin has a pleckstrin homology domain within its septin-interacting region and a membrane-anchoring role of anillin has long been postulated (Field, 1995). These data support such a role and suggest that it is controlled by Rho (Hickson, 2008).

The data highlight the complexity of RhoGEF^Pbl signaling and lead to a model in which multiple Rho-dependent inputs synergize to control anillin behavior during cytokinesis (Hickson, 2008). At the appropriate time and location of normal cytokinesis, several proteins, including Rho1, MRLC^Sqh, Dia, anillin, and Septin^Pnut, localized to the equatorial membrane in the presence of LatA. Of these (and apart from Rho1 itself), anillin and Septin^Pnut were uniquely and specifically required for the formation of the linear filamentous structures describe in this study. The behaviors of these structures are consistent with prior studies of ordered assemblies of septins and anillin and of interaction between anillin and MTs. Although such structures are not normally seen in furrowing cells, anillin localizes to remarkably similar filamentous structures in the cleavage furrows of HeLa cells arrested with the myosin II inhibitor blebbistatin (Hickson, 2008).

It seems unlikely that LatA induces the described structures through nonspecific aggregation of proteins. Rather, it is proposed that LatA blocks a normally dynamic disassembly of Rho1-anillin-septin complexes (by blocking an F-actin-dependent process required for the event) and that continued assembly promotes formation of the linear structures. Because blebbistatin slows F-actin turnover, blebbistatin and LatA may have induced filamentous anillin-containing structures via a common mechanism. A dynamic assembly/disassembly cycle involving anillin could promote transient associations between the plasma membrane and elements of the contractile ring and MTs, properties that could contribute to furrow stability and plasticity. Finally, because local loss of F-actin accompanies and may indeed trigger midbody formation, LatA treatment may have stabilized events in a manner analogous to midbody biogenesis and could therefore be useful in understanding this enigmatic process (Hickson, 2008).

Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper testing of this hypothesis. Loss- and gain-of-function approaches were used to examine the role of Diaphanous in Drosophila morphogenesis. Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. Constitutively active Diaphanous was used to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. This hypothesis was tested by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis (Homem, 2008).

Previous analysis of Diaphanous-related formins (DRF) function in morphogenesis was hampered by mammalian DRF redundancy and the key role of DRFs in cytokinesis. The genetic tools available in Drosophila were used to partially circumvent these difficulties. Dia is known to regulate cellularization. The analysis revealed new roles during morphogenesis. In embryos with severely reduced Dia function, gastrulation is disrupted. Apical constriction of central furrow cells is delayed or blocked in a subset of cells, suggesting a possible role for Dia in regulated apical constriction. Previous work suggested that an unknown Rho effector regulates actin during this process; perhaps this is Dia. Adjacent cells, which are stretched during invagination, become massively multinucleate in dia⁵M mutants. This may reflect the fact that the cells of the blastoderm undergo an incomplete form of cytokinesis, remaining connected to the underlying yolk by actin-lined yolk canals. These canals normally close at the onset of gastrulation in ventral furrow cells; this may prevent cell membrane rupture initiated at the yolk canal under stress. Dia may regulate yolk canal closure; it localizes there at the end of cellularization and similar defects are seen in mutants lacking the septin Peanut, a yolk canal component. Alternately, Dia may stabilize cortical actomyosin or its connections to AJs, with its absence weakening cortical integrity under stress (Homem, 2008).

Dia also plays a key role during germband retraction. The most striking effect of reduced Dia/Rho1 function is altered amnioserosal cell protrusiveness. They normally make stable AJs and form a tight tissue boundary with the epidermis; this is destabilized in dia/Rho1 and dia⁵M/Z mutants. Altered protrusive activity was seen in other contexts: dia⁵M/Z ectoderm exhibited cortical blebbing, and dia/Rho1 and dia⁵M/Z amnioserosal cells had altered protrusiveness during dorsal closure. Reduced Dia function destabilized AJs, and the striking increase in cell protrusiveness in dia/Rho1 mutants was substantially rescued by increasing Myosin phosphorylation. These data support the hypothesis that Dia normally helps restrict actomyosin contractility to AJs, and in its absence, AJs are destabilized and myosin activity outside AJs stimulates protrusiveness. Interestingly, in cultured cells, fully assembled AJs inhibit Rac1 and lamellipodial activity while increasing Rho and contractility, and myosin is required for mouse Dia1-induced AJ strengthening (Homem, 2008).

Given these morphogenetic roles, the mechanisms of action of Dia was explored, initially hypothesizing that it would primarily affect actin. Dia^CA expression did elevate F-actin levels (reducing Dia did not dramatically decrease F-actin, but residual Dia may suffice). However, surprisingly, Dia activation also increased Myosin accumulation and cell contractility, and some effects of reduced Dia function were rescued by increasing Myosin activation. This suggests a surprising role for Dia in regulating Myosin levels/activity. The data also suggest that Dia stabilizes AJs, supporting earlier work in cultured cells. There are intimate connections between AJs and cortical actomyosin: AJs help organize the apicolateral actin ring while cortical actin stabilizes AJs. Interestingly, in cultured cells Myosin-mediated contraction of actin filaments is essential for cell-cell contact expansion, and mouse Dia1 AJ stabilization requires myosin activity, providing further evidence that contractility, AJ stability and Dia activity are mechanistically linked. The actomyosin network may also stabilize AJs by preventing endocytosis. Perhaps Dia, like formin 1, directly binds AJs. Linear actin filaments promoted by Dia at AJs might recruit α-catenin, which then might inhibit Arp2/3-induced actin branching, facilitating formation of a belt of unbranched actin. Thus, Dia is poised to act at the interface between AJs, cortical actin and contractility (Homem, 2008).

The Yin-Yang relationship between adhesion and protrusiveness is a very interesting one, underlying epithelial-mesenchymal transitions. Stable AJs probably actively inhibit protrusiveness, and Dia may assist this, acting in a reinforcing loop that concentrates actomyosin activity at AJs. There are interesting parallels with the role of Dia in cytokinesis. Dia stabilizes Myosin at the midzone. In its absence Myosin is delocalized around the cortex, leading to abnormal protrusiveness. Future work will reveal how Dia fits into the regulatory network coordinating adhesion and cytoskeletal regulation (Homem, 2008).

From these data, a mechanistic model was developed for the regulation of actin, Myosin and AJs by Dia during cell shape change, in which Dia activation stabilizes actin and active Myosin at AJs. In radially symmetric amnioserosal cells, Dia promotes organization/activation of an apical actomyosin network linked to AJs, inducing precocious apical cell constriction. Activation of endogenous Dia may regulate normal amnioserosal constriction: this now needs to be tested. Dia activation in cells where AJs are planar polarized, such as epidermal cells, promotes actomyosin organization preferentially at cell borders where AJs are enriched. This leads to cell widening or helps cells resist elongation, generating groove-cell-like morphology. Once again, Dia may be normally activated specifically in groove cells to modulate their shape (Homem, 2008).

How does Dia activation activate Myosin? It does not occur primarily through SRF, which triggers Myosin accumulation but not cell shape changes. Myosin activation via MLCK partially mimicked Dia^CA, suggesting that Myosin activation is an important part of the process. However, MLCK did not precisely mimic Dia^CA, suggesting that Dia acts preferentially at specific sites such as AJs rather than globally activating Myosin. The dual effects on actin and Myosin suggests the speculative possibility that feedback mechanisms exist to coordinate the Rho-regulated actin and Myosin pathways. Recent work revealed that active Dia1 can activate RhoA by binding the Rho-GEF LARG, and strong genetic interactions were seen between dia and the LARG relative RhoGEF2, making this idea more plausible. Future work is needed to test this hypothesis (Homem, 2008).

Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis. Drosophila embryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellularization, a specialized form of cytokinesis. During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton throughout phylogeny and in Drosophila. To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme from Clostridium botulinum or with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate downstream effectors. Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors. Of the 10 agents microinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests. Time-lapse video records of embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a dose-dependent fashion. Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do not proceed to gastrulation. It is concluded that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning (Crawford, 1998).

In mammalian cells, Rho and Cdc42 effectors function antagonistically. In competition are two distinct small GTPase protein-driven processes: the formation of stress fibers drive by Rho and the formation of filopodia driven by Cdc42. In Drosophila, Rho and Cdc42 effectors function antagonistically, but in contrast to the mammalian case the two phenotypes are indistinguishable. By this hypothesis, Rho and Cdc42 effectors function in independent pathways: Rho effector function is required for cellularization and maintenance of the actinomysin hexagonal array, whereas Cdc42 effector function antagonizes this process. Cdc42 effectors may inhibit Rho effector function directly, thereby phenocopying the disruption of Rho function generated by the microinjection of C3 exoenzyme or N19Rho. An alternative hypothesis that explains these data requires a mechanism of Rho subfamily regulation of the actomyosin cytoskeleton in a single pathway that depends only on Rho effector function during Drosophila cellularization. By this hypothesis, Rho and Cdc42 share a common factor that is required for Rho function, but is sequestered by GTP-bound Cdc42 (Crawford, 1998).

What is the role of myosin in cellularization? Since myosin function is necessary for cytokinesis and cellularization, a mechanism whereby myosin is regulated through phosphorylation by a Rho effector provides an attractive model. Because it is likely that force production for cytokinesis continuously requires activated myosin, it would be predicted that inhibition of Rho activity would block further progression of the cellularization front. Thus, agents that interfere with Rho activity would be expected to prevent the activation of myosin function and the formation of myosin bipolar filaments. It is possible that Rho effects on myosin can also explain the observed changes in the organization of actin. Indeed, Rho activity in the bundling of preexisting actin filaments may be directly or indirectly dependent on myosin function. Bipolar myosin filament formation can stimulate the formation and organization of filamentous actin; therefore, inhibition of myosin function may also explain the observed disruption of actin distribution (Crawford, 1998).

Embryos with a homozygous mutation for the null alleles of the Rho1 gene exhibit an 'anterior open' phenotype, consistent with a defect in morphological movements or cell polarity in the embryonic epidermis. Homozygous mutant clones of cells carrying null alleles in imaginal tissue fail to proliferate, indicating a requirement for Rho1 in cell growth and/or viability. However, clones of hypomorphic alleles give rise to mutant tissue with specific patterning defects. Loss of photoreceptors is also occasionally observed, a phenotype that becomes more common with strong alleles. In the eye, ommatidia are incorrectly rotated relative to the equator, and sometimes exhibit inappropriate chiral forms. The misrotation of ommatidia mutant for Rho1 is evident early in the 3rd instar imaginal disc (during the time when tissue polarity genes are required), showing that it results from an early failure in polarity establishment. Clones of Rho1 hypomorphic alleles in the wing also show a characteristic tissue polarity phenotype: this is manifest both in abnormal wing hair polarity, and in a multiple wing hair phenotype. Such multiple wing hair phenotypes are typical of tissue polarity genes, such as multiple-wing-hair and inturned, whereas frizzled and dishevelled mutations largely show a wing hair polarity defect, with only a low incidence of multiple wing hairs (Strutt, 1997).

Since the multiple wing hair phenotype partially masks any underlying polarity defect, a weaker loss-of-function Rho1 state was generated by misexpressing a dominant-negative form of the Rho1 protein in part of the wing. This gives rise to a phenotype in which fewer cells show a multiple wing hair phenotype and a polarity defect more typical of frizzled and dishevelled is evident. It is concluded Rho1 is required for both wing hair polarity and determination of hair number in the wing. The tissue polarity-like phenotypes of Rho1 mutations in the eye and wing suggests that Rho1 is a common factor required for the generation of polarity in epidermal wing structures. Rho1 clones exhibit only an autonomous effect, with surrounding tissue appearing phenotypically normal. This differs from the frizzled phenotype, which also exhibits directional non-autonomy in both the eye and wing. dsh has been shown to function autonomously in the notum and wing. Similarly, clones of dsh in the eye show only an autonomous effect. Thus the dsh phenotype resembles Rho1 more closely than fz in this regard (Strutt, 1997).

The tissue polarity genes of Drosophila are required for correct establishment of planar polarity in epidermal structures, which in the eye is shown in the mirror-image symmetric arrangement of ommatidia, relative to the dorsoventral midline. Mutations in the genes frizzled, dishevelled and prickle-spiny-legs (pk-sple) result in the loss of this mirror-image symmetry. Little is known of the signaling pathway(s) involved other than that Dsh acts downstream of Fz. Mutations have been identified in Drosophila Rho1; by analysis of their phenotypes it has been shown that Rho1 is required for the generation of tissue polarity. Genetic interactions indicate a role for Rho1 in signaling mediated by Fz and Dsh. Overexpression of Fz in a subset of photoreceptor precursors gives ommatidial polarity defects, characterized by misrotations and incorrect chiralities. Consistent with this phenotype representing overactivation of the Fz signaling pathway, Rho1 phenotypes are dominantly suppressed by fz mutations. The phenotype due to overexpression of Fz is also dominantly suppressed by dsh. Overexpressed Dsh is dominantly suppressed by Rho1 mutations. Given the similarity of the mutant phenotypes of fz, dsh and Rho1, as well as these genetic interactions, it is proposed that Rho1, like Dsh, acts downstream of Fz in the generation of tissue polarity (Strutt, 1997).

Mutations in the gene basket, which encodes a JNK/SAPK homolog, suppress the Fz overexpression phenotype and the Dsh overexpression phenotype to a similar extent as does mutation of Rho1. The embryonic phenotypes of Rho1 and bsk mutants are similar, supporting a role for Bsk in Rho signaling. Eye clones have been generated for bsk hypomorphic mutations, and these do not show an ommatidial polarity defect (or any other significant eye phenotype). However, the phenotype of bsk null clones is not known. These data are consistent with a Fz/Rho1 signaling cascade analogous to the yeast pheromone signaling pathway. This pathway has been proposed as an activator of the serum response factor (SRF) in vertebrate cells (Strutt, 1997).

During patterning of the Drosophila eye, the Notch-mediated cell fate decision is a critical step that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. Evidence is presented that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. The planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. In the light of these findings, it is concluded that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. A new model is proposed for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled (Strutt, 2002).

A number of lines of evidence have previously suggested that Rho/Rac GTPases and the JNK cascade are required for ommatidial polarity decisions and, in particular, the R3/R4 fate decision. These include the following: overexpression of Fz or Dsh in the eye gives a polarity phenotype that is dominantly suppressed by RhoA, bsk, hep, and Djun; RhoA clones or expression of dominant-active/negative RhoA or Rac1 gives ommatidial polarity phenotypes; overexpression of dominant-active/negative JNK pathway components and human Jun elicits ommatidial polarity defects, and expression of a Dl enhancer trap is altered by overexpression of either fz or dsh or by activated human Jun, Hep, RhoA, or Rac1. These observations led to the hypothesis that higher levels of Fz/Dsh signaling in R3 result in higher activation of Dl transcription in R3 via a Rho GTPase/JNK cascade, biasing the N/Dl feedback loop to produce high N in R4 (Strutt, 2002).

However, the data do not support the hypothesis that activation of Dl transcription via Rho GTPases/JNK cascade is the primary mechanism for biasing N activity in R3/R4 during normal eye development. Reexamination of RhoA phenotypes indicates that these rarely affect R3/R4 fate and ommatidial chirality, and RhoA activity is not required for N repression in R3. No role is found for Rac in this process, a finding confirmed by the recent report that deletion of all three Rac homologs in the Drosophila genome has no effect on planar polarity. In addition, notwithstanding the observation that loss of Djun activity can result in ommatidial polarity defects in 2%–3% of ommatidia, mosaic ommatidia where the presumptive R3 lacks Djun activity have wild-type levels and polarity of Notch signaling, so no evidence is found that Djun is directly modulating N activity in R3/R4. It is also interesting to note that although a double mutant combination of the JNK pathway components hemipterous and puckered produces an ommatidial polarity phenotype, this apparently consists entirely of rotation and not chirality defects (Strutt, 2002).

One factor not directly investigated is the STE20 kinase homolog encoded by msn. Loss of function analysis of msn does reveal some ommatidial chirality defects, albeit rarely. More compellingly, mosaic analysis suggests that if one cell of the R3/R4 pair is msn^-, this cell will generally (but not exclusively) take the R4 fate. This suggests a role for Msn in repression of N in R3, although the apparent rarity of chirality defects in ommatidia lacking msn function suggests this is a nonessential pathway (Strutt, 2002).

Taken together, the phenotypic evidence from loss-of-function studies does not support a primary role for Rho GTPases/JNK cascades in the R3/R4 fate decision. But the weight of genetic evidence does support a secondary role for some of the proposed pathway components, possibly in the augmentation of polarity decisions driven largely by asymmetric localization of polarity proteins and direct repression of N activity. In addition, the observation that RhoA mutations result largely in defects in ommatidial rotation supports the hypothesis that RhoA acts downstream of the planar polarity genes in regulating this aspect of ommatidial polarity (Strutt, 2002).

For some years, the standard view of planar polarity gene function has been that this is mediated by Rho GTPases and a JNK kinase cascade, most likely leading to a transcriptional response. In particular, it was thought that this pathway controlled R3/R4 photoreceptor fate in the developing eye, via transcriptional activation of Dl. Although Rho GTPases do regulate one aspect of planar polarity in the eye (ommatidial rotation), they do not appear to be the primary determinant of R3/R4 fate. Therefore, it is concluded that Rho GTPases/JNK cascades are not the major effectors of planar polarity activity in this context. Furthermore, it has been demonstrated that several planar polarity proteins adopt asymmetric subcellular localizations in the eye that correlate with N activation and R3/R4 fate. Therefore, an alternative model has been proposed for modulation of N activity via direct interactions with planar polarity proteins (and most probably Dsh) at the cell cortex (Strutt, 2002).

Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Genetic and overexpression assays have been used to show that Dishevelled activates JNK cascades. In contrast to the action of wingless-pathway components, mutations in rhoA, hemipterous, basket, and jun as well as deficiencies removing the Rac1 and Rac2 genes show a strong dominant suppression of a Dishevelled overexpression phenotype in the compound eye. In an in vitro assay, expression of Dsh has been shown to induce phosphorylation of Jun, indicating that Dsh is a potent activator of the JNK pathway. Whereas the PDZ domain of Dsh, known to be required in the transduction of the wingless signal, is dispensable for signal-independent induction of Jun phosphorylation, the C-terminal DEP domain of Dsh is found to be essential. The planar polarity-specific dsh1 allele is found to be mutated in the DEP domain. These results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions (Boutros, 1998).

How can Fz/Dsh signaling be linked to small GTPase and JNK/MAPK pathways? Recent studies provided evidence that links G protein-coupled receptors, which share structural features with Fz proteins, to MAPK signaling through heterotrimeric G proteins and PI-3 kinases. It is intriguing to speculate that a subset of Fz proteins might signal through a similar pathway. It was also shown recently that XWnt5A and rFz2, in a heterologous assay, increase intracellular calcium via G proteins and phosphoinositol signaling. A mutation in the beta-subunit of a heterotrimeric G protein in C. elegans prevents correct spindle orientation, a process that is believed to be dependent on a Wnt and a Fz receptor, but not on Arm. Further studies regarding a possible involvement of PI-3K and G proteins in planar polarity signaling may provide additional insight to the diversity of Fz-related signaling pathways (Boutros, 1998 and references).

Flies harbouring a Rho1 transgene, which is specifically expressed behind the morphogenetic furrow exhibit a dramatic dose dependent disruption of normal eye development. Flies bearing at least two copies of the transgene display a severe rough eye phenotype characterized by missing secondary and tertiary pigment cells, a substantial reduction in the number of photoreceptor cells and a grossly abnormal morphology of the rhabdomeres. The retina is markedly reduced in thickness. The lattice formed by the secondary and tertiary pigment cells is completely absent. Pigment cells are restricted to the apical regions of the retina and are likely to represent primary pigment cells. Photoreceptor cells of normal morphology are not evident, however, some rhabdomeres, often tortuous in morphology, are present below the layer of pigment cells. Lens facets and ommatidial bristles are still present. Cell fate determination in the imaginal disc occurs normally and abnormalities become manifest late in pupariation, coincident with the phase when the cells undergo major morphological changes. This phenotype is modified by mutations at several other loci that have been implicated in signal transduction, but not by mutations in ras pathway components. A chromosome bearing an allele of cdc37, allele e1E (also known as Enhancer of sevenless 3B), suppresses the Rho1 phenotype significantly. The function of cdc37 is unknown (Hariharan, 1995).

Rho GTPases play an important role in diverse biological processes, such as actin cytoskeleton organization, gene transcription, cell cycle progression and adhesion. They are required during early Drosophila development for proper execution of morphogenetic movements of individual cells and groups of cells important for the formation of the embryonic body plan. Loss-of-function mutations, isolated in the Drosophila Rho1 gene during a genetic screen for maternal-effect mutations, allow for an investigation of the specific roles Rho1 plays in the context of the developing organism. Rho1 is required for many early events: loss of Rho1 function results in both maternal and embryonic phenotypes. Embryos homozygous for the Rho1 mutation exhibit a characteristic zygotic phenotype, which includes severe defects in head involution and imperfect dorsal closure. Two phenotypes are associated with reduction of maternal Rho1 activity: the actin cytoskeleton is disrupted in egg chambers, especially in the ring canals; embryos display patterning defects as a result of improper maintenance of segmentation gene expression. Despite showing imperfect dorsal closure, Rho1 does not activate downstream genes or interact genetically with members of the JNK signaling pathway. The JNK pathway is used by Rho1 relatives Rac and Cdc42 for proper dorsal closure. Consistent with its roles in regulating actin cytoskeletal organization, Rho1 interacts genetically and physically with the Drosophila formin homolog, cappuccino. Rho1 interacts both genetically and physically with concertina, a Galpha protein involved in cell shape changes during gastrulation (Magie, 1999).

Rho1 is essential for zygotic function, since progeny that are homozygous for zygotic lethal alleles of Rho1 mutations die as embryos. Heterozygous Rho1 embryos are viable and have no embryonic cuticle defects, while homozygous Rho1 embryos die with holes in the dorsal anterior region of the cuticle and a disruption of the dorsal surface that stretches the ventral surface and causes the cuticles to bow slightly. To better identify the processes leading to such disruptions, scanning electron microscopy was used to visualize embryonic morphology throughout gastrulation. The most striking defects occur late in gastrulation when the embryos fail to undergo head involution, a process whereby the anterior structures of the embryo are internalized through dramatic cell shape changes and movements. The procephalon, which cannot secrete cuticle, remains on the exterior, leading to a characteristic dorsal anterior hole in the larval cuticle preparations. Additionally, the Rho1 mutant embryos display a puckered dorsal midline, suggestive of improper dorsal closure (Magie, 1999).

During dorsal closure in wild-type embryos, actin and myosin localize along the leading edge of the dorsal lateral epidermis as they extend dorsally. These proteins have been proposed to act as part of the driving force of the cell shape changes occurring at this time. Dorsal closure was examined in wild-type and Rho1 mutants using phalloidin to visualize actin structures at successive developmental time points. Actin is enriched in the cortices of individual cells, allowing for the documentation of cellular morphology during dorsal closure. As the two leading epithelial edges meet in wild-type embryos, they fuse from either end to form a straight, seamless dorsal midline. Embryos homozygous for the Rho1 mutation undergo dorsal closure, since the lateral epithelia do come together at the dorsal surface of the embryo; however, this process is disorganized, as compared to wild type. At higher magnification, cells along the dorsal midline in wild-type embryos are well ordered and columnar in shape. In Rho1 mutant embryos, cells along the dorsal midline are inappropriately shaped, pinched together in some regions and stretched out in others. This is especially clear in the later stages of dorsal closure, after the epithelia have come together (Magie, 1999).

Disruptions to the leading edge cytoskeletal components have been found using dominant negative and constitutively active transgenes of UAS-DRhoA or UAS-Dcdc42 expressed with Hs-GAL4. Segmentally reiterated splaying of cells and loss of myosin staining and phosphotyrosine nodes have been found in leading edge cells flanking the segment border. While aberrant cell shapes and inappropriate constriction of cells are seen along the leading edge, the segmental reiteration of these losses in the Rho1 loss-of-function mutation is not seen. In addition, nodes of phosphotyrosine expression are still visible in splayed out cells. To look further at cell shape along the leading edge, the expression of Fasciclin III (Fas III) was examined. While Fas III is present in the leading edge cells of wild-type embryos, it is not present in the dorsalmost edge of these cells until they are touching the cells of the opposing leading edge epithelia. In Rho1 mutant embryos, Fas III accumulates in the dorsalmost edge of the leading edge cells prematurely. When cells along the leading edge in Rho1 mutations constrict inappropriately, they appear to recognize their lateral neighbors as those on the opposing leading epithelial edge and elicit downstream events prematurely. Consistent with this possibility, the appearance of secondary 'midlines' perpendicular to the major midline, is sometimes observed (Magie, 1999).

Ectopic expression of dominant-negative and constitutively active forms of Drosophila Rho family members have identified a role for these proteins in regulation of actin structure in the ovary. The Rho1 mutation was initially identified in a genetic screen for maternal genes whose activity is essential for embryonic development. In this screen, a change-of-function mutation in an RNA polymerase II subunit, wimp, was used to reduce, but not eliminate, Rho1 maternal contribution. It is not possible to completely eliminate maternal Rho1 function: germline clones of Rho1 can not be generated due to the inviability of the clonal cells. In order to examine whether reduction of maternal Rho1 function has any effect on the actin cytoskeleton in ovaries, ovaries from mothers trans-heterozygous for Rho1 and wimp were stained with phalloidin. In wild-type egg chambers, actin structures are well organized. This includes the ring canals, portals consisting of two tightly bundled concentric rings of actin (resulting from incomplete cytokinesis) that allow for the exchange of cytoplasmic material between nurse cells and the developing oocyte. Egg chambers from females with reduced Rho1 function show a general disruption of the actin cytoskeleton, particularly in the outer ring canals and oocyte cortex. The inner ring canals appear relatively normal; oogenesis is able to proceed in these females leading to inviable embryos with patterning defects (Magie, 1999).

The maternal effect of the Rho1 mutation on embryos is distinct from its zygotic effects: embryos derived from mothers with reduced maternal Rho1 activity die with moderate segmentation defects as seen by the fusion of adjacent denticle bands in larval cuticles. In addition, approximately 10% of the dead embryos have cuticular holes. These cuticular holes are randomly placed and are not the same as holes on the dorsal surface resulting from failed dorsal closure. These holes could be the result of inappropriate cellularization or cell fate specification; cell death, or other morphogenetic processes whereby cuticle is not properly secreted. To begin distinguishing among these possibilities, embryos derived from mothers with reduced maternal Rho1 activity were stained with acridine orange, a marker for cell death. While the staining pattern is temporally and spatially dynamic, acridine orange staining patterns are similar in Rho1 mutants when compared to wild-type embryos. Embryos derived from mothers with reduced maternal Rho1 activity were stained with antibodies to phosphotyrosine to outline the cell shapes. Patches of irregularly shaped cells can be seen throughout the embryo, suggesting that Rho1 is likely to be required for proper cellularization and additional morphogenetic processes (Magie, 1999).

The Rho1 loss-of-function mutation does not exhibit all the phenotypes expected if it is the primary target of genes such as DRhoGEF2: whereas DRhoGEF2 mutations and ectopic expression of dominant negative Rho1 block gastrulation, this phenotype is not observed with the Rho1 loss-of-function zygotic mutation. To determine if maternal Rho1 activity is masking this phenotype, the Rho1 zygotic phenotypes were examined when maternal Rho1 activity was reduced. Homozygous Rho1 mutant embryos derived from mothers with reduced maternal Rho1 activity exhibit both the maternal segmentation phenotype and the zygotic morphogenetic phenotypes. New phenotypes are not uncovered and the zygotic Rho1 phenotype is not enhanced in this background (Magie, 1999).

The segmentation phenotype in embryos with reduced maternal Rho1 activity indicates a maternal role for Rho1 in patterning events that establish the embryonic body plan. To identify the developmental stage at which Rho1 is necessary, embryos derived from mothers with reduced maternal Rho1 activity were stained with a collection of antibodies recognizing segmentation gene products, including Bcd (maternal), Hb and Kr (gap), Eve and Ftz (pair rule), and En (segment polarity). All of the antibodies tested show that segmentation products are set up properly in Rho1 maternal embryos. However, while the En segment polarity protein is initially expressed properly, it fails to maintain its proper expression and shows severe aberration in pattern by stage 9. Since Wingless (Wg) signaling is necessary for the maintenance of En expression, Wg expression was examined in these embryos. Like En, Wg expression is initiated correctly, but fails to be maintained properly. No genetic interactions were detected betweeen Rho1 and hemipterous and basket (members of the JNK signaling pathway), chickadee (cytoskeletal protein), puckered (dorsal closure mutant), wingless and armadillo (Wingless signaling), anterior open (ras signaling), or Egfr (EGF receptor). No genetic interaction with DRhoGEF2 could be detected; however, only trans-heterozygous interaction was examined since DRhoGEF2 and Rho1 map next to each other and no recombinant double mutant chromosomes have been recovered (Magie, 1999).

The lack of genetic interactions of Rho1 with Wg and JNK signaling components during oogenesis and early embryonic development was unexpected in light of Rho1's requirement for these factors during eye development and the classical dorsal closure defects described for dominant negative and constitutively active Rho1 transgenes. Activation of the JNK signaling pathway by the Rac and Cdc42 GTPases results in the induction of dpp and puckered (puc) expression in the leading edge cells. Since inability to detect genetic interactions does not rule out a role for Rho1 in JNK signaling, dpp and puc expression were examined in embryos homozygous mutant for Rho1. Consistent with the lack of genetic interactions, dpp and puc expression in Rho1 homozygous mutant embryos are indistinguishable from wild type. These results suggest that Rho1 mediates a pathway for epidermal cell shape changes that is independent of the previously reported Rac-mediated JNK cascade required for dorsal closure and does not share all the properties attributed to it based on ectopic expression of its dominant negative and constitutively active versions (Magie, 1999).

Genetic interactions of Rho1 were detected with two mutations: cappuccino (capu; formin homolog) and concertina (cta; Ga protein). Egg chambers from mothers heterozygous for Rho1 (Rho1/+) exhibit normal actin morphology. Egg chambers from mothers trans-heterozygous for Rho1 and capu (Rho1;capu/+) or cta (Rho1:cta/+) exhibit disruptions of the ovarian actin cytoskeleton, similar to those in females with reduced maternal Rho1 activity. While capu has been shown to affect actin integrity during oogenesis, similar studies have not been reported for cta. Phallodin staining was examined in egg chambers from homozygous cta mothers and similar, albeit weaker, defects are found in the actin cytoskeleton. The interaction of Rho1 with capu is more severe than with cta: the subsequent embryos from the Rho1/cta interaction survive, whereas the embryos from Rho1/capu are inviable and exhibit severe patterning defects. An in vitro binding assay was used to examine the interaction specificity between the Rho1 and Cta or Capu proteins. Rho1 fused to glutathione S-transferase (GST-Rho1) was expressed in bacteria and immobilized on glutathione-Sepharose beads. Rho1 was then tested for its ability to bind 35 S-labeled full-length Cta or Capu proteins. Consistent with the observed genetic interactions, Rho1 specifically pulls down full-length Cta or Capu. Interestingly, Capu preferentially interacts with GTP-bound Rho1, whereas Cta interacts equally with GTP- or GDP-bound Rho1 (Magie, 1999).

The numerous seemingly distinct biological responses of the Rho GTPase suggests that its activation must be both temporally and spatially regulated. Part of this regulation is likely to come from interaction of Rho with different GEFs. The mechanisms that lead to activation of Rho family proteins by extracellular signals are thought to be similar to those of Ras: they are mediated by GEFs linked to heterotrimeric G protein coupled membrane receptors. A large family of RhoGEFs have been identified in mammalian systems, some of which are specific for a particular family member (e.g., Lbc for Rho; Tiam1 for Rac), while other GEFs act on all members. Three RhoGEFs have been identified in Drosophila, but little is known about their specificity. No mutations corresponding to DRhoGEF1 have been reported (Werner, 1997). DRhoGEF2 has been shown to affect many of the morphogenetic movements associated with gastrulation and suppress genetic phenotypes associated with overexpression of wild type or constitutively active Rho (Barrett, 1997 and Häcker, 1998). However, while both Rho1 and DRhoGEF2 loss-of-function mutations affect gastrulation, their phenotypes are very different: no trans-heterozygous genetic interactions between DRhoGEF2 and loss-of-function Rho1 mutations have been detected. The third Drosophila RhoGEF, Pebble, does interact genetically with Rho1 loss-of-function mutations. Since the identified RhoGEFs do not have completely overlapping phenotypes with Rho1 loss-of-function mutations, it is likely that additional RhoGEFs exist. Similarly, since phenotypes associated with loss-of-function mutations in Rac, Cdc42 and RhoL have not yet been reported, the specificity of the existing RhoGEFs is not yet known (Magie, 1999 and references therein).

Work in fibroblasts suggests a role for subunits of the heterotrimeric Galpha proteins (G12 and G13) in Rho-mediated signaling. While the exact link between the G proteins and Rho family proteins has not been described, a physical interaction between specific RhoGEFs and Galpha proteins was recently reported. GEFs are thought to act immediately upstream of Rho family proteins. concertina is a Galpha-like G protein that is important in transducing signals necessary to appropriately organize cell shape changes during Drosophila gastrulation. Ectopic expression studies utilizing dominant-negative Rho1 have led to the implication of concertina in the cell shape changes leading to proper ventral furrow formation; this is consistent with studies showing disruption of Drosophila cellularization after microinjection of the botulinum C3 exoenzyme Rho-specific inhibitor. While Rho1 loss-of-function mutations do not show the same severe cellularization or gastrulation phenotypes of DRhoGEF2, Rho1 does interact both genetically and physically with cta, suggesting that Rho1 is likely to be a downstream effector of the Cta Galpha protein in the ovary. Interestingly, Cta interacts equally with the GTP- and GDP-bound forms of Rho1 and may form a complex including GEFs. Proper oogenesis and morphogenesis in Drosophila are dependent on Rho1 activity. Because these are complicated developmental processes involving multiple cellular events, it is expected that a large number of genes are involved in regulating and executing them. To understand the biochemical mechanisms through which Rho family proteins regulate the organization of the actin cytoskeleton, gene transcription, and their other associated activities, identification of regulatory factors and cellular targets is essential. Drosophila offers a genetically amenable system in which to systematically identify components of the Rho pathway required for the proper execution of these events. Future genetic screens with loss-of-function Rho1 mutations should also help in identification of regulators and effectors, an important step in describing the pathways through which Rho acts in the organism (Magie, 1999 and references therein).

To investigate the possibility that mutations in Rho1 cause defects during cell division, Rho1 embryos were stained with antibodies against lamin and alpha-spectrin. Embryos homozygous for null alleles of Rho1 (Rho1^72O and Rho1^72R) show many binucleate cells in the head region. Occasionally, a few polyploid cells are also observed in thoracic or abdominal segments. Although the latter phenotype is observed with low penetrance, binucleate ectodermal cells could never be found in wild-type embryos. The relatively weak phenotype caused by Rho1 mutations (unlike that of pebble) is most likely due to maternally provided Rho1 protein. Thus, it appears that Rho1 is required for cytokinesis, and that decreased levels of Rho1 may account for the observed phenotype in cytokinesis (Prokopenko, 1999).

To further demonstrate the role of Rho1 in cytokinesis, the effects of expression of a dominant-negative form of Rho1, Rho1^N19 were analyzed. A dominant-negative form of H-Ras, H-Ras^N17, cannot interact with downstream target proteins. In addition, H-Ras^N17 competes with normal H-Ras for binding to rasGEF, which results in formation of inactive H-Ras^N17-rasGEF complexes and depletion of the pool of the endogenous rasGEF leading to a dominant-negative effect. Therefore, a dominant-negative Rho1 may produce a phenotype similar to or stronger than loss-of-function alleles of Rho1. Ectopic expression of Rho1^N19 creates a phenotype that is much worse than loss of zygotic Rho1. Expression of Rho1^N19 in ectodermal stripes leads to a complete block of cytokinesis leading to polyploidy of almost every cell within the affected segment. In contrast, expression of wild-type Rho1 or dominant-negative Rac1 (Rac1^N17 or Rac1^L89) does not affect cytokinesis or completion of mitosis. Interestingly, coexpression of Rho1^N19 and Pbl (unlike Pbl alone) results in the mislocalization of Pbl to the cell cortex in interphase and late mitotic cells, further indicating that the two proteins interact during cytokinesis. These results, together with other data, suggest that activation of the Rho1 GTPase by Pbl, its putative exchange factor, is required for the initiation of cytokinesis, because mutations in either protein or overexpression of an inactive protein result in accumulation of multinucleate cells (Prokopenko, 1999).

Previously, Drosophila Rho1 has been shown to be required for cellularization, gastrulation, dorsal closure, and generation of tissue polarity, but not for cytokinesis. The data suggest that the molecular basis of the genetic interaction between pbl and Rho1 is a physical interaction between Pbl and Rho1 proteins, because the two proteins interact in a yeast two-hybrid assay. Both genetic data and two-hybrid assay results indicate that this interaction is specific for Rho1, but not Rac1 or Cdc42. This interaction presumably results in the activation of the Rho1 GTPase by Pbl and induction of the signaling cascade that initiates the assembly of the contractile ring (Prokopenko, 1999 and references therein).

The small GTPases Rac and Rho act as cellular switches in many important biological processes. In the fruit fly Drosophila, RhoA participates in the establishment of planar polarity, a process mediated by the receptor Frizzled (Fz). Thus far, analysis of Rac in this process has not been possible because of the absence of mutant Rac alleles. The roles of Rac and Rho in establishing the polarity of ommatidia in the Drosophila eye were investigated. By expressing a dominant negative or a constitutively activated form of Rac1, Rac signaling was interfered with specifically and ommatidial polarity was disrupted. The resulting defects are similar to the loss/gain-of-function phenotypes typical of tissue-polarity genes. Through genetic interaction and rescue experiments involving a polarity-specific, loss-of-function dishevelled (dsh) allele, Rac1 was found to act downstream of Dsh in the Fz signaling pathway, but upstream of, or in parallel to, RhoA. Rac signals to the nucleus through the Jun N-terminal kinase (JNK) cascade in this process. By generating point mutations in the effector loop of RhoA, it was found that RhoA also signals to the nucleus during the establishment of ommatidial polarity. Nevertheless, Rac and RhoA activate transcription of distinct target genes. Thus Rac is specifically required downstream of Dsh in the Fz pathway. It functions upstream or in parallel to RhoA and both signal to the nucleus, through distinct effectors, to establish planar polarity in the Drosophila eye (Fanto, 2000).

Random mutagenesis of activated mammalian Rho^V14 has led to the identification of mutations in the effector loop (a portion of the GTPase responsible for interaction with several effectors) that block either its action on cytoskeletal dynamics or on transcriptional activation of SRF. The F39V mutation impedes the formation of actin stress fibers but does not interfere with the activation of SRF-mediated transcription, separating the two effects of Rho^V14. The mutation E40L interferes with both SRF activation and the formation of stress fibers (Fanto, 2000).

The relevant mutations were recapitulated in the activated Drosophila Rho^V14 protein and they were expressed under the control of sev-gal4 in the eye disc (sev;Rho^V14 F39V and sev;Rho^V14 E40L). The sev;Rho^V14 F39V flies display a phenotype that is indistinguishable from that of sev;rho^V14 alone, with loss of photoreceptors and misorientation of otherwise wild-type clusters. This was evident even when the transgene was expressed at lower levels. Increasing the expression levels of sev;Rho^V14 F39V (two copies) led to an enhancement of both the polarity and the photoreceptor recruitment phenotypes. In contrast, sev;Rho^V14 E40L flies never displayed polarity defects, both when the transgene was expressed at low and at very high levels. Nevertheless, this mutant maintained the ability of sev;Rho^V14 to cause photoreceptor loss: although a large number of ommatidia had lost several photoreceptors, all the remaining ommatidia with wild-type complement had the correct polarity. This indicates that removing the function required for nuclear signaling (equivalent to SRF activation in cell culture) eliminated the ability of sev;Rho^V14 to induce polarity defects, suggesting that nuclear signaling by RhoA is critical for ommatidial polarity determination (Fanto, 2000).

To better characterize nuclear signaling by Rac and RhoA, the expression of puckered (puc) and Delta (Dl) were studied. Dl is the only known transcriptional target of Fz signaling in R3, and puc-lacZ expression serves as a measure of JNK activity in vivo. The puc gene is a transcriptional target of JNK signaling in Drosophila, and encodes a dual specificity protein phosphatase that acts as a negative regulator of JNK itself in a feedback loop. In the wild type, very weak beta-galactosidase expression from the puc enhancer trap line is detectable in all photoreceptor precursors. Expression of sev;rac^V12 lead to strong upregulation of puc-lacz in one or, more frequently, two cells of the cluster, identified as R3/R4 precursor cells, consistent with the expression pattern of sev;Rac^V12. These data resemble the upregulation of puc-lacz when the JNK pathway has been activated in the same cells (Fanto, 2000).

In contrast, Rho^V14 affects puc-lacz expression differently. Although in sev;Rho^V14 eye discs puc-lacz expression is upregulated in some cells at a later stage, these were not identifiable as the R3/R4 pair, but were often found in the position of the R2/R5/R8 precursors (where sev is not expressed). This suggests that the effect seen is not a direct consequence of Rho activation, but more likely a secondary effect (RhoA^V14 E40L fails to induce significant puc-lacz expression). Thus, the direct transcriptional activation of puc-lacz in R3/R4 correlates with the genetic interactions with the JNK module, suggesting a difference in the action of Rac and RhoA (Fanto, 2000).

An important aspect of R3/R4 cell fate and ommatidial polarity determination is the upregulation of Dl expression in the R3 precursor by Fz. Dl then signals to Notch on the R4 precursor, resulting in the choice of the R4 cell fate. In addition to Fz, other components of the Fz/planar-polarity pathway have also been found to upregulate Dl transcription. Thus, whether Rac and RhoA also regulate Dl transcription was investigated by monitoring Dl-lacZ expression in sev;Rac^V12 and sev;Rho^V14 eye discs (Fanto, 2000).

In the wild type, Dl is expressed dynamically in photoreceptor precursors behind the furrow. Within the R3/R4 pair, it is expressed in R3 from rows 4 to 8, whereas it remains at lower levels in R4. In contrast to the difference in puc expression, both sev;Rac^V12 and sev;Rho^V14 upregulated Dl-lacz expression in both R3/R4 precursors. The RhoA^V14 E40L isoform that is impaired in nuclear signaling does not affect Dl expression, confirming the importance of nuclear signaling by RhoA. These effects are very similar to those of sev;Fz, supporting the idea that Rac and RhoA act downstream of Fz in the regulation of the R3/R4 cell fate. Their different effects on puc-lacz indicate that their downstream effectors in nuclear signaling are distinct (Fanto, 2000).

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. The sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. These data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, these results show that there is negative feedback between ERM activation and activity of the Rho pathway (Speck, 2003).

Rho-induced phosphorylation of a C-terminal threonine has been shown to relieve the inhibitory intramolecular association and generate the active conformation of the ERM molecule. This residue is conserved in Drosophila Moesin at amino acid position 559. To test the role of Thr 559 phosphorylation in Moesin activation, two mutant cDNA constructs were generated: Moe(T559D), a phosphomimetic form, and Moe(T559A), a form that cannot be phosphorylated at this residue. Moe^G0323 hemizygous males are rescued to viability by transgenes expressing Moe(T559D), but not by Moe(T559A), implying that Moe(T559D) is an active form whereas Moe(T559A) is inactive (Speck, 2003).

The phosphomimetic form of ERM proteins has constitutively active properties because it may be locked in the open conformation (Gautreau, 2000). To examine the effects of ERM activation, Moe(T559D) was overexpressed under region-specific Gal4 drivers in wild-type imaginal discs. A marked upregulation of cortical F-actin was observed in cells that express Moe(T559D). By contrast, little or no effect was seen with Moe⁺ and Moe(T559A) overexpression. The accumulation of cortical actin induced by the gain-of-function mutant is complementary to the reduction of apical actin seen in the Moesin loss-of-function background, indicating that Moesin controls assembly of cortical actin (Speck, 2003).

The pleiotropic nature of the Moe^G0323 phenotype suggests an involvement in a signalling pathway that controls cell adhesion and motility. Given the crucial function carried out by the Rho family of small GTPases in regulating these processes and the evidence for Rho regulation of ERM function the relationship between Moesin and Rho signalling in vivo was examined by manipulating the genetic dose of Rho1, a Drosophila RhoA homolog, in a Moe^- background. The null Rho172R allele was used to reduce by half maternal and zygotic Rho function. Halving the dose of Rho1 not only ameliorates the Moe^G0323 disc epithelium organization and actin localization phenotypes, but also strongly suppresses Moe^G0323 lethality. A similar suppression of lethality was observed with other Rho1 alleles and when the dose of Drok, a Rho effector kinase, was reduced, and a weaker effect was observed with another downstream effector of Rho1 signalling, the non-muscle myosin-II heavy chain gene zipper). The observed suppression was not due to enhanced Moesin protein production or stability. Taken together, these results suggest that Moesin functions antagonistically to activity of the Rho pathway in regulating epithelial polarity and integrity (Speck, 2003).

The ultrastructure was examined of wild-type and Moe^G0323 epithelial cells as well as that of Moe^G0323 cells that are also heterozygous for Rho172R. In transmission electron micrographs, wild-type epithelial cells have apical finger-like microvilli that tend to congregate near the areas of contact between adjacent cells. In the Moesin mutant, the microvilli are replaced by large apical protrusions that are often of irregular shape. Reduction of Rho activity partially suppresses this Moe^- phenotype, and results in cells that lack the protrusions but still display abnormally large microvillus-like structures. These results suggest that Moesin may have a Rho-independent role in maintaining apical integrity that is not essential for viability (Speck, 2003).

If Moesin normally functions as an antagonist of Rho pathway activity, then the effects of Rho pathway hyperactivation should be similar to the Moe loss-of-function phenotype. To test this hypothesis, whether Rho1 overexpression phenotypes in wing imaginal discs mimic those seen in Moe loss-of-function mutants was investigated. Cells overexpressing Rho1 mislocalize F-actin and lose epithelial characteristics. As with Moe^G0323 cells, Rho1-expressing cells in the blade region of the wing imaginal disc lose junctional markers and drop basally. These cells also assume a mesenchymal character, become motile and invade between wild-type epithelial cells. Similar, but more disruptive phenotypes were observed when a constitutively active form of Rho1, Rho1V14, was overexpressed in wild-type imaginal discs. Therefore, Rho1 overexpression strongly resembles Moe loss of function, suggesting that Moesin and Rho1 exert opposite effects on the epithelium (Speck, 2003).

To test further the relationship of Moesin with the Rho pathway, whether a reduction in Moe function would suppress a phenotype associated with downregulation of Rho1 was investigated. Rho1 has been shown to function downstream of dishevelled (dsh) in the planar cell polarity (PCP) pathway that regulates the polarity and number of hairs generated by each wing blade cell. Each wild-type wing cell produces a single hair; however, multiple wing hairs result when Rho pathway function is impaired. In flies that are mutant for the dsh1 allele (which inactivates the PCP but not the wingless-signalling function of dishevelled), double-hair-producing cells occurred at a frequency of 6.3%. Removal of a single dose of Moe suppresses the number of cells with double hairs in dsh¹ mutants to 1.2%, suggesting that Rho pathway function is upregulated in response to reduction in Moesin activity (Speck, 2003).

These genetic results in Drosophila suggest that Moesin functions antagonistically to Rho pathway activity. To distinguish between an effect on Rho itself and effects on a downstream component of the pathway, and to extend these results from Drosophila to mammalian cells, the effect of reducing ERM function in mammalian LLC-PK1 epithelial cells was examined. Use was made of an ERM truncation, ezrin^Act, that has dominant-negative properties in flies and reduces function of all three ERM proteins in these cells (as assayed by phospho-ERM staining, a marker for ERM activation. Expression of ezrin^Act resulted in increased levels of Rho activity, whereas expression of wild-type ezrin caused either no effect, or slightly decreased the level of Rho activity. These results indicate that ERM proteins negatively regulate Rho pathway function by altering the activation state of Rho itself, rather than a downstream component of the pathway (Speck, 2003).

Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference has identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. RhoGAP inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by RhoGAP. Local regulation of RhoGAP could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of RhoGAP by integrin and Src, both implicated in neural plasticity (Billuart, 2001).

Microinjection of the GAP domain of p190 into fibroblasts results in actin stress fiber disassembly, suggesting that it primarily acts on RhoA (Ridley, 1993). Consistent with this observation, overexpression of RhoA in MB neurons significantly enhances the p190 phenotype. If p190 acts on RhoA, one would further predict that activation of the RhoA pathway would mimic p190 loss-of-function phenotypes. To explore this possibility, active RhoA (RhoA V14) was expressed constitutively in MB neurons. OK107-driven RhoA V14 expression results in adult lethality. When reared at 18°C, a few escapers were recovered that exhibited complex MB axon defects. It was difficult to determine if the escapers shared qualitatively similar phenotypes to those caused by p190 RNAi. However, in pupae, a selective dorsal lobe reduction similar to the p190 RNAi phenotype was often observed (Billuart, 2001).

RhoA transduces signals to both the nucleus and the cytoskeleton. To address which downstream signaling pathway mediates axon retraction, activated RhoA mutants (RhoA V14) were used with additional 'effector domain' mutations. The F39V mutation blocks RhoA's function in inducing stress fiber formation without affecting nuclear signaling, whereas the E40L mutation allows both nuclear and cytoskeletal pathways weakly. When RhoA V14(E40L) was expressed in MB neurons, a dorsal lobe phenotype similar to the p190 phenotype was found. However, RhoA V14(F39V) expression had almost no phenotype, suggesting that a cytoskeletal pathway is responsible for RhoA's effect on axon branch retraction (Billuart, 2001).

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. These data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho (Fritz, 2002).

Thus, when expressed alone, only ctDrac and ctDcdc42 cause midline crossing errors. However, the mutant GTPases interact genetically with mutations in robo, Sos, and chic and with overexpression of ctMLCK. The interactions are surprisingly specific. Midline crossing errors caused by expression of ctDrac or ctDcdc42 are suppressed by heterozygous loss of Profilin and enhanced by expression of ctMLCK. These results indicate that Drac1 and Dcdc42 encourage axons to cross the midline by regulating actin polymerization and/or myosin activity. CtRho and dnRho interact strongly with expression of ctMLCK or heterozygous loss of Robo, which suggests that regulation of myosin activity by Rho is crucial for midline repulsion. This work demonstrates that Rho, Drac1, and Dcdc42 are involved in dictating which axon may cross the midline, presumably by aiding in the transduction of attractive and/or repulsive cues operating at the midline. By using mutations in signaling molecules known to prevent pCC/MP2 axons from crossing the midline, this analysis concentrates on how Rho, Drac1, and Dcdc42 may regulate cytoskeletal dynamics in response to midline repulsive cues (Fritz, 2002).

The Rho family of GTPases was first studied in fibroblasts where activation of Cdc42, Rac, or Rho results in production of filopodia, lamellapodia, and stress fibers, respectively. In wound-healing assays, Rac appears to control actin polymerization to provide the protrusive force needed for movement, while Cdc42 determines cell polarity to localize Rac activity to the leading edge of the cell. Rho seems to play a role in adhesion and spreading during cell migration. These same processes are involved in growth cone motility, which makes the Rho GTPases candidates for regulation of cytoskeletal dynamics during axon guidance. Experiments in neurons, both in vitro and in vivo, indicate that activation of Rac and/or Cdc42 increases axon outgrowth and this is opposed by activation of Rho, which leads to growth cone collapse or retraction. This is consistent with findings that expression of ctDcdc42 or ctDrac allow axons to ignore repulsive signals at the midline and continue extending across the midline (Fritz, 2002).

The role of Rho in midline repulsion is more difficult to determine since both dnRho and ctRho enhance the midline crossing phenotype of heterozygous robo mutants. This is consistent with the data in which both dnRho and ctRho enhance the ctMLCK phenotype. Similar complexities are seen in the literature; expression of a Rho GEF, which is expected to increase Rho activity, leads to increased attraction to the midline, even though activation of Rho usually leads to growth cone collapse or retraction. The complexity of the Rho interactions is understandable when the dual role of myosin activity during axon guidance is considered. The most documented connection between myosin activity and Rho is through the effector Rho Kinase (RhoK). RhoK phosphorylates MLC and also inactivates myosin phosphatase by phosphorylating its myosin binding subunit, leading to increased phosphorylation of MLC and therefore increased myosin activity. Myosin activation is needed both for the retrograde flow of actin that retracts filopodia and for the force that propels the growth cone forward. Repulsive guidance signals are expected to increase retrograde flow while preventing forward movement (Fritz, 2002).

Expression of dnRho may specifically interfere with retraction of filopodia in response to repulsive cues, leading to increased midline crossing errors. A global increase in myosin activity caused by expression of either ctRho or ctMLCK, or even a Rho GEF, may cause axon guidance errors by increasing the forward movement of the growth cone. Midline attractive activity (e.g., Netrins) probably also influences how much myosin activity is available to move a growth cone over the midline. The literature and these experiments are most consistent with a model in which Rho is activated by repulsive guidance signals. Activation of ephrinA5 receptors causes an increase in Rho activity resulting in a growth cone collapse. Plexin B, the receptor for repulsive semaphorins, binds to and seems to activate Rho. Activation of Robo by Slit recruits srGAP1, which appears to prevent it from binding to and inactivating Rho. The genetic interactions seen between Sos^e49 mutations and expression of ctRho or dnRho are consistent with Sos acting as a GEF for Rho in pCC/MP2 neurons. DnRho strongly enhances the midline crossing errors caused by loss of Sos, while ctRho almost completely suppresses them. Since Sos-dependent signaling pathways are required for response to midline repulsive cues, this is further evidence that Rho is activated downstream of repulsive guidance signals, although a role downstream of selected attractants cannot be ruled out (Fritz, 2002).

Clearly, regulation of Rho family GTPase activity is necessary to prevent axons from crossing the midline inappropriately. Midline repulsive signaling involves regulation of all three GTPases; Drac1 and Dcdc42 are likely downregulated, while Rho seems to be activated downstream of repulsive signals. The Rho family GTPases influence actin polymerization and/or myosin force generation to regulate the processes of growth cone motility that are required for proper response to axon guidance signals (Fritz, 2002).

The pleiotropic functions of small GTPase Rho present a challenge to its genetic analysis in multicellular organisms. The MARCM (mosaic analysis with a repressible cell marker) system has been used to analyze the function of RhoA in the developing Drosophila brain. Clones of cells homozygous for null RhoA mutations were specifically labeled in the mushroom body (MB) neurons of mosaic brains. RhoA is found to be required for neuroblast (Nb) proliferation but not for neuronal survival. Surprisingly, RhoA is not required for MB neurons to establish normal axon projections. However, neurons lacking RhoA overextend their dendrites, and expression of activated RhoA causes a reduction of dendritic complexity. Thus, RhoA is an important regulator of dendritic morphogenesis, while distinct mechanisms are used for axonal morphogenesis (Lee, 2000).

The MARCM system was used to label only homozygous mutant cells in mosaic organisms. In this system, the repressor of the GAL4 transcriptional activator, GAL80, is made from a ubiquitously expressed tubP-GAL80 transgene on the chromosome in trans to the homologous chromosome containing a RhoA null mutation. Both RhoA and tubP-GAL80 are distal to a pair of homologous FRT sites. Mitotic recombination at the FRT sites gives rise to a daughter cell that is homozygous for RhoA and lacks tubP-GAL80. The absence of GAL80 allows GAL4 to drive the expression of a UAS cell marker in precisely the cells that lack the RhoA gene. Using this system, mitotic recombination results in the generation and visualization of three types of clones in the Drosophila CNS: Nb, two cell, and single cell clones (Lee, 2000).

This study focuses on the mushroom body (MB) neurons of the Drosophila brain. A wild-type MB Nb clone generated in a newly hatched larva and examined at the wandering third instar stage (about 100 hr later) always contains over 150 neurons. These neurons project their dendrites into a region called the calyx and project their axons through the peduncle, into the medial and vertical lobes. MB Nb clones homozygous for RhoA null mutations consistently contain about 10-12 cells (hereafter referred to as RhoA cells or neurons); within each clone, two of these nuclei are much larger than the rest of the nuclei. When examined in adulthood, wild-type Nb clones contain over 500 neurons that project axons to five lobes, whereas RhoA Nb clones still contain about 10-12 cells with axons projecting to only one medial lobe. Expression of a UAS-RhoA(wt) transgene specifically in RhoA mutant Nb clones partially rescues the phenotype, indicating that the observed defect is a result of loss of RhoA function. The rescue is not complete, perhaps due to the late onset of GAL4-induced transgene expression resulting from the perdurance of the GAL80 repressor protein, weak expression of GAL4-C155 in MB Nbs, or both (Lee, 2000).

Rho is known to regulate cytokinesis and G1/S transition, both of which are essential for normal cell proliferation. To test whether RhoA Nb clones arrest proliferation at G1/S transition, the birth time of RhoA mutant cells was mapped more precisely. RhoA Nb clones were readily identifiable, using nuclear lamin staining, based on the presence of two adjacent large nuclei, and the MB region has been identified based on its unique BrdU incorporation pattern. In the majority of the Nb clones, the last mitosis, which gives rise to the two large nuclei, has occurred by 18 hr after heat shock. Yet, BrdU incorporation still persisted at 18-24 hr after heat shock in 100% of the samples tested, indicating that there is at least one additional round of DNA replication after the last mitosis in the majority of RhoA Nb clones. This experiment argues against a G1/S block as the main cause of cell cycle arrest in RhoA Nb clones. RhoA Nb clones are also unlikely to arrest at the G2 stage, since no RhoA mutant cells contain detectable cyclin B staining, a marker for G2. To test whether there is a cytokinesis defect in RhoA Nb clones, the number of cell bodies and nuclei in RhoA Nb clones was determined. Two large RhoA mutant nuclei, the products of the last mitosis in RhoA Nb clones according to the BrdU labeling time course, are always present in the same cell body. In addition, one or two cell bodies have been found to contain two small nuclei in each RhoA Nb clone. According to the cell proliferation pattern during neurogenesis, two big nuclei in one cell body are probably due to a cytokinesis defect in a Nb, and two small nuclei in one cell body are likely derived from a ganglion mother cell with a cytokinesis defect. Therefore, this observation supports a cytokinesis defect as the main cause of cell division arrest in RhoA Nb clones. This result is consistent with the recent finding that the Drosophila Rho1/RhoA, as well as a putative exchange factor, Pebble, is essential for cytokinesis during embryogenesis (Lee, 2000).

RhoA neurons show a striking overextension of their dendrites. The 10-12 RhoA mutant neurons in Nb clones project extensive dendrites in wandering third instar larvae that appear to occupy the entire calyx region. Close examination reveals that RhoA neurons often projected their processes beyond the calyx region. To determine whether the overextended processes from the calyx were dendrites, the distribution of a fusion protein composed of the microtubule motor Nod and beta-galactosidase (Nod-beta-gal) was tested. Nod-beta-gal concentrates at the tips of dendrites and is largely absent from the axons of embryonic sensory neurons. In wild-type MB neurons, Nod-beta-gal is highly concentrated in the calyx and largely absent from peduncles and axon lobes in MB neurons. In RhoA neurons, Nod-beta-gal is also concentrated at the tips of all overextended, thus identifying these processes as dendritic in nature. Occasionally, staining for Nod-beta-gal distribution also reveals short overextension of dendrites in wild-type Nb clones. However, in RhoA clones, the length, frequency, and number of overextended dendrites were drastically increased, as compared with wild type (Lee, 2000).

Previous cell culture studies suggest that RhoA mediates cell rounding and neurite retraction in response to extracellular agonists. For instance, inhibition of RhoA via C3 exoenzyme ribosylation inhibits agonist-induced cell rounding and process retraction, whereas expression of activated RhoA(V14) results in agonist-independent process retraction and growth cone collapse. RhoA inhibition blocks the generation of actomyosin-based contractile forces, which are likely to be responsible for process retraction. Indeed, one specific effector pathway, the regulation of myosin light chain phosphorylation via the activation of Rho-associated kinase (ROCK), mediates RhoA-induced neurite retraction. In cultured neuronal-like cells, however, there is no morphological differentiation of axons and dendrites. Here it is demonstrated that RhoA is cell autonomously required to limit dendritic growth of CNS neurons in vivo. This also strongly suggests that RhoA is not essential for growth, guidance, degeneration, and regeneration of the axons of MB neurons. These findings are further supported by the fact that constitutively active RhoA expression results in a reduction of dendritic volume and density without detectable effects on axons. This studies demonstrate an evolutionarily conserved role for the GTPase RhoA in negatively regulating dendritic growth, probably via the regulation of actomyosin-based contractility (Lee, 2000).

What are the likely explanations for the preferential requirement of RhoA in dendritic but not axonal morphogenesis? One possibility is that in the clonal analyses described in this study, axons from RhoA neurons are not pioneer axons. RhoA axons may adhere to and follow the path of wild-type MB axons derived from neurons either generated from the same Nb before the clone is generated or from three other wild-type MB Nbs. In contrast to axons, lack of fasciculation of dendritic processes and their highly diverse branching patterns suggest that dendritic growth and elaboration from individual neurons may be more independent of one another. It remains possible that RhoA is required for the growth and guidance of pioneering axons, which may have different molecular requirements. However, the growth and guidance of such nonpioneering axons are disrupted when short stop is mutated. Another possible explanation is that the 'stop signals' for dendritic growth are sent from the termini of incoming axons, and RhoA is an essential component of such a signal transduction pathway. The signal transduction pathway at the axonal growth cone for stopping growth may be distinct from those within the dendritic equivalent. The differential requirement of RhoA in the morphogenesis of axons and dendrites may also reflect the inherent differences in these two neuronal compartments in their cytoskeletal polarity and process growth rate. For instance, a recent study of cultured hippocampal neurons reveals differential stability of the actin cytoskeleton in the growth cones of axons and dendrites (Lee, 2000).

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 zipper^Ebr 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 zipper^Ebr, 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 zip^Ebr allele. Fourteen insertions failed to complement zip^Ebr. 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 zip^Ebr 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 zip^Ebr 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 zip^Ebr; the penetrance of the malformed phenotype in double heterozygous flies is 33% with the RhoGEF2^1.1 allele and 27% with the RhoGEF2^4.1 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 zip^Ebr, 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).

RhoA^E3.10 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 RhoA^E3.10-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 zip^Ebr, RhoA^E3.10 appears to be a more severe allele than RhoA^J3.8. It is hypothesized that the protein encoded by RhoA^E3.10 may have a partial dominant-negative effect because it does not repartition properly. On the other hand, the premature stop codon in RhoA^J3.8 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 (this study). 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).

Nonmuscle myosin-II is a key motor protein that drives cell shape change and cell movement. The function of nonmuscle myosin-II has been analyzed during Drosophila embryonic myogenesis. Nonmuscle myosin-II and the adhesion molecule, PS2 integrin (Myospheroid), colocalize at the developing muscle termini. In the paradigm emerging from cultured fibroblasts, nonmuscle actomyosin-II contractility, mediated by the small GTPase Rho, is required to cluster integrins at focal adhesions. In direct opposition to this model, it has been found that neither nonmuscle myosin-II nor RhoA appear to function in PS2 clustering. Instead, PS2 integrin is required for the maintenance of nonmuscle myosin-II localization and the cytoplasmic tail of the ß_PS integrin subunit is capable of mediating this PS2 integrin function. Embryos that lack zygotic expression of nonmuscle myosin-II fail to form striated myofibrils. In keeping with this, a PS2 mutant that specifically disrupts myofibril formation is unable to mediate proper localization of nonmuscle myosin-II at the muscle termini. In contrast, embryos that lack RhoA function do generate striated muscles. Finally, nonmuscle myosin-II localizes to the Z-line in mature larval muscle. It is suggested that nonmuscle myosin-II functions at the muscle termini and the Z-line as an actin crosslinker and acts to maintain the structural integrity of the sarcomere (Bloor, 2001).

Peripheral glial cells in both vertebrates and insects are born centrally and travel large distances to ensheathe axons in the periphery. There is very little known about how this migration is carried out. In other cells, it is known that rearrangement of the Actin cytoskeleton is an integral part of cell motility, yet the distribution of Actin in peripheral glial cell migration in vivo has not been previously characterized. To gain an understanding of how glia migrate, the peripheral glia of Drosophila were labelled using an Actin-GFP marker and their development in the embryonic PNS was analyzed. It was found that Actin cytoskeleton is dynamically rearranged during glial cell migration. The peripheral glia were observed to migrate as a continuous chain of cells, with the leading glial cells appearing to participate to the greatest extent in exploring the extracellular surroundings with filopodia-like Actin containing projections. It is hypothesized that the small GTPases Rho, Rac and Cdc42 are involved in Actin cytoskeletal rearrangements that underlie peripheral glial migration and nerve ensheathement. To test this, transgenic forms of the GTPases were ectopically expressed specifically in the peripheral glia during their migration and wrapping phases. The effects on glial Actin-GFP distribution and the overall effects on glial cell migration and morphological development were assessed. It as found that RhoA and Rac1 have distinct roles in peripheral glial cell migration and nerve ensheathement; however, Cdc42 does not have a significant role in peripheral glial development. RhoA and Rac1 gain-of-function and loss-of-function mutants had both disruption of glial cell development and secondary effects on sensory axon fasciculation. Together, Actin cytoskeletal dynamics is an integral part of peripheral glial migration and nerve ensheathement, and is mediated by RhoA and Rac1 (Sepp, 2003).

The data suggest that RhoA and Rac1 are both involved in peripheral glial cell migration and nerve ensheathement, and have distinct effects on Actin rearrangement. For example, constitutively active Rac1 (V12) and RhoA (V14) expression results in halted migration of cell bodies as well as disrupted cytoplasmic process extension. The phenotypes of the two mutants are very different from one another. Rac1 (V12) mutants show ball-shaped collapsed glia, while RhoA (V14) mutants have very long, spike-shaped actin processes emanating from the cell bodies. The distinct and extreme phenotypes from these mutants suggest that there is a balance of RhoA and Rac1 activity in wild-type peripheral glia to generate normal migration and cytoplasmic process extension. The concept of a balance of GTPase function being necessary for glial cell migration is also supported by observations that glial cell migration is stalled in both the gain-of-function and loss-of-function mutations. These observations are interpreted as suggesting that there is a balance of GTPase activities that is necessary for glial cell migration. In other words, anything that affects this balance either through a loss of function or gain of function, affects the ability of glial cells to migrate (Sepp, 2003).

The well-characterized cultured fibroblast model has shown that Rac is involved in lamellipodia formation, while Rho mediates stress fiber polymerization and Cdc42 is involved in the extension of filopodia. It is possible that Rac1 and RhoA mediate the assembly of similar structures in peripheral glia. The long, straight actin fibers seen in constitutively active RhoA (V14) mutants could represent overextended stress fibers. Furthermore, the massive glial lamellar-like structures that are stimulated by Rac1L89 expression appear very similar to the lamellipodia of cultured fibroblasts. The biochemical activity of the Rac1L89 mutation is not known, and can act as either a dominant-negative or constitutively active form, depending on the cell type. The Rac1L89 phenotype in peripheral glia is most similar to overexpression of wild-type Rac1, suggesting that the ectopic lamellar structures are a result of moderate increase in Rac1 activity. Thus, it is possible that the Rac1L89 mutation causes Rac1 to be overactive but not as much as in the Rac1V12 mutation (Sepp, 2003).

It was interesting to note that the ectopic actin-containing projections of RhoAV14 and Rac1L89 mutants did not always reach over axon tracts, which are the normal peripheral glial migrational substrates in the wild type. For the steering of a migrating cell, large amounts of actin polymerization occur at the contact between the leading edge of the cell and the attractive migrational substrate. Perhaps the hyperactivity of the mutant GTPases enable the peripheral glia to extend processes out on less adhesive substrates compared with axons. It was also interesting to note that ectopic projections of peripheral glia (in the RhoAV14 and Rac1L89 mutants) do not interfere with axon pathfinding in the periphery. The ectopic glial projections could be a result of failed glial pathfinding instead. Interestingly, axons are capable of correctly migrating in the absence of glial sheaths (in the RhoAV14 and Rac1V12 mutants). Peripheral glia are know to be able to mediate sensory axon guidance to the CNS. Thus, peripheral glia most probably mediate sensory axon migration to the CNS using secreted cues (Sepp, 2003).

The Rho subfamily of Ras-related small GTPases participates in a variety of cellular events including organization of the actin cytoskeleton and signaling by c-Jun N-terminal kinase and p38 kinase cascades. These functions of the Rho subfamily are likely to be required in many developmental events. A study has been performed of the participation of the Rho subfamily in dorsal closure (DC) of the Drosophila embryo, a process involving morphogenesis of the epidermis. In this study (Harding, 1999), and one published subsequently on the same problem (Ricos, 1999), a distinction is made between two types of cells at the leading edge: one type is termed 'cells flanking segment borders' and the second type is termed 'segment border cells'. The two cell types alternate along the anterior to posterior axis. Drac1, a Rho subfamily protein, is required for the presence of an actomyosin contractile apparatus believed to be driving the cell shape changes essential to DC. Expression of a dominant negative Drac1 transgene causes a loss of this contractile apparatus from the leading edge of the advancing epidermis and dorsal closure fails. It is postulated that Drac1 triggers the JNK cascade within cells flanking segment borders. Dpp is the target of the JNK cascade in the cells flanking segment borders, and Dpp is released by these flanking cells, targeting the adjacent segment border cells (Harden, 1999).

Two other Rho subfamily proteins, Dcdc42 and RhoA, as well as Ras1 are also required for dorsal closure. Dcdc42 appears to have conflicting roles during dorsal closure: e