Rho1


DEVELOPMENTAL BIOLOGY

Embryonic

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).

Rho-dependent control of anillin behavior during cytokinesis

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 RhoGEFPbl-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 RhoGEFPbl-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 RhoGEFPbl contributes to anillin localization during cytokinesis. After 3 d of RhoGEFPbl 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 RhoGEFPbl, consistent with prior analysis of fixed RhoGEFPbl mutant embryos. Because anillin can bind F-actin and phosphorylated myosin regulatory light chain (MRLC), RhoGEFPbl 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 RhoGEFPbl on anillin behavior was tested in LatA. After RNAi of RhoGEFPbl or Rho1, anillin-GFP remained cytoplasmic through anaphase. Thus RhoGEFPbl 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 (MRLCSqh) inhibited cell elongation during anaphase, slowed furrow formation, and delayed and diminished the equatorial localization of anillin-GFP. However, unlike after RhoGEFPbl 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, MRLCSqh 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 RhoGEFPbl-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 RhoGEFPbl-dependent input. Thus, RhoGEFPbl 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 RhoGEFPbl. MRLCSqh-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 MRLCSqh (detected as either MRLCSqh-GFP or with an antibody to serine 21-phosphorylated pMRLCSqh) colocalize (, although they were often offset as if labeling different regions of the same structures (Hickson, 2008).

The effects of anillin RNAi on MRLCSqh-GFP localization were tested. MRLCSqh-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 MRLCSqh-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, septinPnut localized to the cleavage furrow and midbody where it colocalized with anillin. Unexpectedly, the septinPnut 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 septinPnut 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 septinPnut depended on the cell cycle phase. In LatA-treated interphase cells, when anillin is nuclear, septinPnut 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, septinPnut 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 septinPnut RNAi. Although unable to fully deplete septinPnut, it was found that anillin could localize to the equatorial cortex in regions devoid of detectable septinPnut, which is consistent with findings in C. elegans (Maddox, 2005). Importantly, in septinPnut-depleted cells, anillin-GFP still localized to the plasma membrane in LatA but no longer appeared filamentous, indicating that septinPnut is essential for the filamentous nature of the structures and that Rho1 can promote the association of anillin with the plasma membrane independently of septinPnut. 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 septinPnut. Using Dia as a furrow marker, 3 d of anillin RNAi prevented the furrow recruitment of septinPnut. In LatA-treated cells, anillin RNAi did not affect the formation of septinPnut rings in interphase cells, but it greatly reduced the formation of septinPnut-containing structures during anaphase/telophase. Thus, anillin is required for the furrow recruitment of septinPnut and for the formation of septinPnut-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 RhoGEFPbl 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, MRLCSqh, Dia, anillin, and SeptinPnut, localized to the equatorial membrane in the presence of LatA. Of these (and apart from Rho1 itself), anillin and SeptinPnut 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).

Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis

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. DiaCA 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 DiaCA, suggesting that Myosin activation is an important part of the process. However, MLCK did not precisely mimic DiaCA, 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).

Coordination of Rho family GTPase activities to orchestrate cytoskeleton responses during cell wound repair

Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. This paper describes how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model. Rho, Rac, and Cdc42 were found to rapidly accumulate around the wound and segregate into dynamic, partially overlapping zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation, leading to its association with actin. Rho1, along with Cdc42, is necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization toward the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. Rho1 GTPase was found to use several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions. These results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single-cell wound repair in the Drosophila embryo. Wounding triggers the formation of arrays of Rho GTPases that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, is required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell's needs (Abreu-Blanco, 2013).

Actin and myosin II recruitment and organization at the wound edge are key and conserved elements of the single cell wound repair process in different organisms. In epithelial cells and Xenopus oocytes, wounding induces a strong cytoskeletal response dependent on calcium and Rho family GTPase signaling. In particular, the contractile actomyosin ring formed in Xenopus oocyte wounds is accompanied by the flow of cortical F-actin filaments toward the wound from neighboring regions. Myosin II also accumulates as foci at the wound edge, and its recruitment is independent of F-actin and cortical flow, although its subsequent assembly into a continuous ring does require an intact F-actin network. Interestingly, this study found that actin and myosin II are both actively mobilized toward the wound in the Drosophila syncytial embryo, in a process dependent on cortical flow (Abreu-Blanco, 2013).

This mobilization of myosin II is necessary for proper actomyosin ring assembly and function. In the context of Drosophila cell wound repair, Rac coordinates the actin and microtubule network that influences actin and myosin recruitment, consistent with its known functions in regulating the polymerization of both actin and microtubules. The sharp actomyosin ring formed at the leading edge of the wound, as well as the accompanying halo of dynamic actin, provides the contractile force driving rapid wound closure. This study found that Rho1 is necessary for myosin II activation, leading to its association with actin: actin fails to accumulate as a ring at the wound edge when myosin is disrupted. This is consistent with Rho's known role in increasing the phosphorylation of the myosin II regulatory light chain via its downstream effector Rok. Rho1, along with Cdc42, also functions upstream of the assembly and constriction of the actomyosin ring, where they likely trigger actin filament formation and subsequent actomyosin ring stabilization (Abreu-Blanco, 2013).

In addition to Rho family GTPases regulating actomyosin ring assembly, the actin and myosin II cytoskeleton reciprocally mediates GTPase function. For example, the assembly/ disassembly of the actin network in Xenopus oocyte wounds has been shown to disrupt the local activation and inactivation patterns of Rho and Cdc42, which are required for wound healing progression. In the Drosophila model, actin is required for the translocation, refinement, and maintenance of the Rho family GTPase arrays, while myosin II is required for their recruitment and organization. The actomyosin ring acts as scaffold for signaling molecules that, in turn, are responsible for the polymerization of actin and activation of myosin II. Stabilization of the actin network by Jasplakinolide treatment provides strong evidence that the actomyosin array and the underlying signals translocate together. A striking observation from this model is that Rho1, the first Rho family GTPase to be recruited, accumulates at the wound site even when the actin and myosin II cytoskeleton is severely disrupted, indicating that its recruitment is independent of cytoskeleton integrity. Cdc42 and Rac1, which accumulate after Rho1, are sensitive to disruption of actin and myosin II. Thus, Rho family GTPases are required in Drosophila syncytial embryos for proper wound healing through reciprocal regulation with the dynamic and integrated actin and myosin II networks (Abreu-Blanco, 2013).

Rho family GTPases localize at the wound edge with a precise and characteristic organization pattern. This study shows the recruitment of Rac GTPases to the wound: Rac1 and Rac2 accumulate as graded ring-like arrays at the wound edge. By analyzing the distribution of the endogenous Rho GTPases, it was found that Rho, Cdc42, and Rac (Rac1/2) form partially overlapping concentric arrays that correlate with their specific functions during wound repair. Another interesting observation is that Rho is recruited to the wound before the onset of actin and myosin II recruitment and followed by Cdc42 and Rac (Rac1/2) with a 90 s delay. The localization of active Rho GTPases was analyzed using a combination of activity biosensors and downstream effectors. In the Drosophila model, active Rho1 localizes as a discrete array internal to the actomyosin ring. This is in contrast to that observed in Xenopus oocyte wounds, where active Rho and Cdc42 arrays are formed as discrete concentric rings overlapping with myosin II and actin, respectively. Although the timing and configuration of the GTPase arrays is different in the two cell wound models, they both achieve the same end result: organization of a highly contractile actomyosin ring and a dynamic surrounding zone of actin and myosin assembly/ disassembly, thereby ensuring that a region of highly contractility enriched in myosin II is followed by one of low contractility where actin can assemble. Indeed, the organization of Rho GTPases as local arrays is not only restricted to contractile ring structures such as those observed in wound repair and cytokinesis, but is a common strategy in different biological processes. In the context of cell migration, coordinated zones of Rho family GTPases at the cell's leading edge regulate the waves of cellular protrusion and retraction necessary for migration. In this scenario, Rho activation occurs first, followed by Cdc42 and Rac1 with a 40 s delay. Rho accumulates at the front of the cell concomitant with protrusions and its levels are reduced during retraction, while Rac1 accumulates behind the Rho array with its levels remaining high during the retraction phase. These patterns of activation and organization correlate with their proposed activities: Rho modulates contraction and polymerization, whereas Cdc42 and Rac1 regulate adhesion dynamics (Abreu-Blanco, 2013).

Negative feedback among Rho GTPases is a conserved theme in multiple cellular and developmental processes. In the Xenopus wound model, Rho has been shown to negatively modulate the integrity of the Cdc42 array, while inhibition of Cdc42 activity strongly suppresses local Rho activation. Moreover, Abr, a protein with Rho/Cdc42 GEF and Cdc42 GAP activity has been shown to accumulate at the Rho zone where its GAP activity is required to locally suppress Cdc42 activity, allowing Rho and Cdc42 to segregate into their respective zones. It was not possible to specifically deplete Cdc42 in the Drosophila syncytial embryo wounding assays, so its effects on Rho1 and Rac were not specifically addressed in this model. Nonetheless, the results support the idea of GTPase crosstalk playing a role in the control of GTPase array organization, segregation, and levels. A notable example of crosstalk in this context comes from the expansion of the Rac1 wound-induced array in embryos where Rho1 levels were depleted, suggesting that high levels of Rho1 at the wound edge inhibit Rac1 accumulation at the interior of the actomyosin ring, thereby allowing Rac1 to control the dynamic actin halo surrounding the wound. These intricate and highly regulated interactions among Rho family GTPases allow them to efficiently execute their multiple functions in the cell (Abreu-Blanco, 2013).

A current challenge in the field is to determine how Rho GTPases are maintained locally at high levels that dynamically adjust during wound closure. Recent studies in the Xenopus model propose a mechanism of GTPase treadmilling wherein Rho and Cdc42 are subject to rapid local activation and inactivation, which is different for each GTPase: the Rho activity zone is shaped by trailing edge inactivation, whereas Cdc42 undergoes variable inactivation across the array. The development of biologically active photoactivatable Rho family GTPase reporters will be necessary to determine the relevance of this option in the Drosophila cell wound model. An alternate possibility is that Rho family GTPases and their regulators are anchored at the plasma membrane in an actin-dependent manner. Putative candidates for anchoring proteins are the ERM family proteins, which are known to interact and regulate Rho GTPases in different cellular process (Abreu-Blanco, 2013).

It was surprising to find that Rho1 utilizes multiple downstream effectors simultaneously during wound repair, begging the question of how this specificity is achieved, maintained, and tweaked dynamically. Considering the complexity of wound repair, cytoskeletal responses are likely regulated via several signaling pathways that converge on the contractile ring. These pathways require precise coordination to provide and integrate the different components required for the dynamic assembly and disassembly of contractile ring machinery as the wound is drawn closed. Rho family GTPases, rather than switching the behavior of the entire cell, would need to be capable of locally modulating the dynamics of the cytoskeleton from one part of the cell to another. This specificity associated with simultaneous recruitment and function of downstream effectors is likely to be a key regulatory feature for dynamic orchestration of cell wound repair. Future challenges include defining the molecular composition of these signaling modules and delineating the combinations and specific subcellular localizations of Rho GTPases, GEFs, GAPs, upstream regulators, and downstream effectors that are needed for the proper functioning of these pathways (Abreu-Blanco, 2013).

TGF-β signals regulate axonal development through distinct Smad-independent mechanisms

Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Transforming growth factor β (TGFβ) signals regulate axon growth. In most contexts, TGFβ signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFβ responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFβ receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFβ receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo (Ng, 2008).

Once growing axons reach the correct postsynaptic target, axon outgrowth terminates and synaptogenesis begins. These studies suggest that TGFβ signals play a role. When Babo is inactivated, MB axon growth does not terminate properly and overextends across the midline. Consistent with this, CA Babo expression results in precocious termination, forming axon truncations. How Babo is spatially and temporally regulated remains to be determined. Analogous to the Drosophila NMJ, MB axon growth might be terminated through retrograde signalling. Target-derived TGFβ ligands could signal to Babo (on MB axon growth cones) and stop axons growing further. In an alternative scenario, TGFβ ligands might act as a positional cue that prevents MB axons from crossing the midline. Recent data have shown that Babo acting through Smad2 restricts individual R7 photoreceptor axons to single termini. Loss of Babo, Smad2, or the nuclear import regulator Importin α3 (Karyopherin α3 - FlyBase), results in R7 mutant axons invading neighbouring R7 terminal zones. With the phenotype described in this study, Babo could similarly be restricting MB axons to appropriate termination zones, its loss resulting in inappropriate terminations on the contralateral side (Ng, 2008).

In contrast to MB neurons, Babo inactivation in AL and OL neurons resulted in axon extension and targeting defects. This might reflect cell-intrinsic differences in the response in different neurons to a common Babo signalling program. This may be the case for MB axon pruning and DC axon extension, which require Babo/Smad2 signals. Whether these differences derive from cell-intrinsic properties, or from Babo signal transduction, they underline the importance of Smad-independent signals in many aspects of axonal development (Ng, 2008).

The results suggest that Smad-independent signals involve Rho GTPases. One caveat in genetic interaction experiments is that the loss of any given gene might not be dosage-sensitive with a particular assay. Nevertheless, all the manipulations together suggest that Babo-regulated axon growth requires Rho1, Rac and LIMK1. How Babo signals involve Rho GTPases remains to be fully determined. In addition to LIMK1, which binds to Wit, one possibility, as demonstrated for many axon guidance receptors, is that the RhoGEFs, RhoGAPs and Rho proteins might be linked to the Babo receptor complex. Thus, ligand-mediated changes in receptor properties would lead to spatiotemporal changes in Rho GTPase and LIMK1 activities (Ng, 2008).

The data suggest that a RhoGEF2/Rho1/Rok/LIMK1 pathway mediates Babo responses. Whether Rac activators are required is unclear, as tested RacGEFs do not genetically interact with babo. In this respect, rather than through GEFs, Babo might regulate Rac through GAPs, by inhibiting Tumbleweed (Tum) activity (Ng, 2008).

Do mutations in Rho1 and Rac components phenocopy babo phenotypes? β lobe overextensions are observed in Rok, Rho1 and Rac mutant neurons. In MB neurons, Rac GTPases also control axon outgrowth, guidance and branching. Rho1 also has additional roles in MB neurons. Although Rho1 mutant neuroblasts have cell proliferation defects, single-cell αβ clones do show β lobe extensions. RhoGEF2 strong loss-of-function clones do not exhibit axon overextension. As there are 23 RhoGEFs in the Drosophila genome, there might well be redundancy in the way Rho1 is activated. LIMK1 inactivation in MB neurons was reported previously. Axon overextensions were not observed as LIMK1 loss results in axon outgrowth and misguidance phenotypes. This suggests that LIMK1 mediates multiple axon guidance signals, of which TGFβ is a subset in MB morphogenesis (Ng, 2008).

Although their phenotypes are similar, several lines of evidence indicate that CA Babo does not simply reflect LIMK1 misregulation in MB neurons. First, whereas LIMK1 genetically interacts with most Rho family members and many Rho regulators, CA babo is dosage-sensitive only to Rho1 and Rac and specific Rho regulators, suggesting that Babo regulates LIMK1 only through a subset of Rho signals (Ng, 2008).

Second, the LIMK1 misexpression phenotype is suppressed by expression of wild-type cofilin (Twinstar Tsr), S3A Tsr, or the cofilin phosphatase Slingshot (Ssh). By contrast, only wild-type Tsr, but not S3A Tsr or Ssh, suppresses CA Babo. The suppression by wild-type Tsr might reflect a restoration of the endogenous balance or spatial distribution of cofilin-on (unphosphorylated) and -off (phosphorylated) states within neurons. Indeed, optimal axon outgrowth requires cofilin to undergo cycles of phosphorylation and dephosphorylation. Since S3A forms of cofilin cannot be inactivated and recycled from actin-bound complexes, wild-type cofilin is more potent in actin cytoskeletal regulation (Ng, 2008).

CA Babo might not simply misregulate LIMK1 but also additional cofilin regulators. Recent data suggest that extracellular cues (including mammalian BMPs) can regulate cofilin through Ssh phosphatase and phospholipase Cγ activities. In different cell types, cofilin phosphorylation and phospholipid binding (which also inhibits cofilin activity) states vary and potently affect cell motility and cytoskeletal regulation. Whether a combination of LIMK1, Ssh and phospholipid regulation affects cofilin-dependent axon growth remains to be determined (Ng, 2008).

Third, by phalloidin staining, LIMK1, but not CA Babo, misexpression results in a dramatic increase in F-actin in MB neurons. Thus, CA Babo does not in itself lead to actin misregulation. Fourth, Babo also regulates axon growth independently of LIMK1 (Ng, 2008).

This study differs significantly from the canonical model of Smad signalling, in which type 1 receptors function downstream of the ligand-type 2 receptor complex. In this study, the gain- and loss-of-function results suggest that type 2 receptors act downstream of type 1 signals. Since ectopic only Wit and Put suppress the babo axon overextension phenotype, this implies that Smad-dependent and -independent signals have distinct type 1/type 2 receptor interactions. How these interactions propagate Smad-independent signals remains to be fully determined. Babo could act as a ligand-binding co-receptor with Wit and Put. In addition, Babo kinase activity could regulate type 2 receptor or Rho functions. The results suggest, however, that provided that Wit or Put signals are sufficiently high, Babo is not required. Whatever the mechanism(s), it is likely that Babo requires the Wit C-terminus-LIMK1 interaction to relay cofilin phosphoregulatory signals. How Put functions is unclear. Since the put135 allele (used in this study) carries a missense mutation within the kinase domain, this suggests that kinase activity is essential. put does not genetically interact with LIMK1. Since Put lacks the C-terminal extension of Wit that is necessary for LIMK1 binding, this suggests that Put acts independently of LIMK1. One potential effector is Rac, which, in the context of Babo signalling, also appears to be Pak1- and thus LIMK1-independent (Ng, 2008).

In MB neurons, Wit and Put can function interchangeably. In other in vivo paradigms, type 2 receptors are not interchangeable. However, since the Wit C-terminal tail is required to substitute for Put, this suggests that Wit axon growth signals are independent of its kinase activity. Together, this suggests that Smad-independent signals involve LIMK1-dependent and -independent mechanisms (Ng, 2008).

This study shows that Babo mediates two distinct responses in related MB populations. How do MB neurons choose between axon pruning and axon growth? The babo rescue studies suggest that whereas Baboa or Babob elicits Smad-independent responses, only Baboa mediates Smad-dependent responses. Since Babo isoforms differ only in the extracellular domain, differences in ligand binding could determine Smad2 or Rho GTPase activation. However, it is worth noting that in DC neurons, either isoform mediates axon extension through Smad2 and Medea. In addition, although expressed in all MB neurons, CA babo misexpression (which confers ligand-independent signals) perturbs only αβ axons. Thus, cell-intrinsic properties might also be essential in determining Babo responses (Ng, 2008).

Many TGFβ ligands signal through Babo. For example, Dawdle, an Activin-related ligand, patterns Drosophila motor axons, whereas Activin (Activin-β, FlyBase) is required for MB axon pruning. Whether these ligands regulate Babo MB, AL and OL axonal morphogenesis is unclear. Taken together, the evidence suggests that Babo signalling is varied in vivo and is involved in many aspects of neuronal development (Ng, 2008).

TGFβ signals are responsible for many aspects of development and disease and, throughout different models, Smad pathways are closely involved. Although Smad-independent pathways are known, their mechanisms and roles in vivo are unclear. TGFβ signals often drive cell shape changes in vivo. During epithelial-to-mesenchymal transition (EMT), cells lose their epithelial structure and adopt a fibroblast-like structure that is essential for cell migration during development and tumour invasion. TGFβ-mediated changes in the actin cytoskeleton and adherens junctions are necessary for EMT. Although Smads are crucial, TGFβ signals also involve the Cdc42-Par6 complex, resulting in cell de-adhesion and F-actin breakdown through Rho1 degradation. In other studies, however, TGFβ-mediated EMT has been shown to require Rho1, which can be regulated by Smad activity (Ng, 2008).

Many TGFβ-driven events in Drosophila are Smad-dependent. Whether Smad-independent roles exist beyond those identified in this study remains to be tested. This study therefore provides a framework to understand how non-Smad signals regulate cell morphogenesis during development (Ng, 2008).

Apical secretion in epithelial tubes of the Drosophila embryo is directed by the formin-family protein Diaphanous

Apical localization of filamentous actin (F-actin) is a common feature of epithelial tubes in multicellular organisms. However, its origins and function are not known. This study demonstrates that the Diaphanous (Dia)/Formin actin-nucleating factor is required for generation of apical F-actin in diverse types of epithelial tubes in the Drosophila embryo. Dia itself is apically localized both at the RNA and protein levels, and apical localization of its activators, including Rho1 and two guanine exchange factor proteins (Rho-GEFs), contributes to its activity. In the absence of apical actin polymerization, apical-basal polarity and microtubule organization of tubular epithelial cells remain intact; however, secretion through the apical surface to the lumen of tubular organs is blocked. Apical secretion also requires the Myosin V (MyoV) motor, implying that secretory vesicles are targeted to the apical membrane by MyoV-based transport, along polarized actin filaments nucleated by Dia. This mechanism allows efficient utilization of the entire apical membrane for secretion (Massarwa, 2009).

Apical localization of F-actin is a general feature of tubular epithelial structures. It has been observed in mammalian MDCK cells forming tubes in three-dimensional cell culture, in the cytoplasm underlying the apical membrane facing the lumen in mammalian secretory organs, such as the lacrimal gland, and in the different epithelial tubes of the Drosophila embryo. The lower level of gene duplication in Drosophila, and the ability to follow the consequences of targeted gene inactivation in the tubular structures, allowed identification of the mechanism responsible for nucleating the actin terminal web at the apical side of epithelial tube cells. This study has demonstrate that Dia, which is known to promote the formation of linear actin filaments, is responsible for producing this actin network in Drosophila embryonic tubular structures. Despite differences in the diameter and function of the different tubular organs, the polarized apical actin cables formed by Dia appear to have a common role in trafficking secretory vesicles to the apical tube surface (Massarwa, 2009).

While the role of Dia in promoting apical secretion spans the entire duration of tracheal morphogenesis, two other Formin-homology proteins act at very specific junctions of Drosophila tracheal morphogenesis. Formin 3 participates in the generation of a continuous dorsal trunk tube by promoting vesicular trafficking in the fusion cells of each metamer, perpendicular to the tube lumen. Another Formin domain protein, DAAM, promotes the organization of F-actin in rings around the circumference of the tracheal tube, at the final stages of tracheal morphogenesis (Massarwa, 2009).

It is likely that each of the three Formin domain proteins is regulated by distinct activators that are concentrated at different sites. The localized activation of Formin 3 may eventually lead to polarized vesicle movement, similar to Dia, but toward a different membrane. The activation of DAAM may be necessary for the localized synthesis of F-actin, which will modify the contours of the apical membrane, and thus define the shape of chitin layered on top. The function of Dia stands out, since it is required throughout tracheal development, and is also involved in morphogenesis of other tubular organs (Massarwa, 2009).

The mechanism of localized activation of Dia operates after apical-basal polarity of the cells has been established. Thus, no defects were observe in overall polarity in dia mutant embryos. It seems that the steps upstream to Dia activation utilize the existing polarity at multiple tiers in order to trigger Dia at a highly restricted position. The two Rho-GEF proteins, Gef2 and Gef64C, exhibit a tight apical localization in the cells forming the tubes. The single Rho1 protein, which is downstream to the Rho-GEFs, is again tightly localized to the apical surface in tubular structures. Binding of Rho1 to Dia leads to an opening of the autoinhibited form of Dia and to the formation of a Dia dimer representing the active form (Goode, 2007). Since GTP-bound Rho1 is the immediate activator of Dia, it is particularly important that Rho1 be embedded in the apical membrane, to ensure spatially restricted nucleation of actin polymerization. In C. elegans, a GEF and a Rho protein were shown to be essential for the development of the lumen of the excretory cell. It will be interesting to determine if a Dia-family protein is subsequently activated to promote secretion (Massarwa, 2009).

Dia is also apically localized, both at the mRNA and protein levels. Elimination of the dia 3'UTR demonstrated a persistence of apical protein localization, even when mRNA localization was lost, suggesting that there are two parallel and independent mechanisms for apical localization. The multiple tiers of apical localization assure that activated Dia will be highly restricted to the apical surface (Massarwa, 2009).

It is interesting to note that, while Gef2, Gef64C, Rho1, and Dia proteins are broadly expressed, partially due to maternal contribution of mRNA, they exhibit apical localization only in the tubular structures. This raises the possibility that genes that are specifically expressed in the tubular organs contribute to the apical localization. Alternatively, apical localization may rely directly on the specific phospholipid composition of the apical tube membranes. It will be interesting to determine if a common mechanism is responsible for the apical localization of the different proteins in the pathway, and if this mechanism relies on components that are restricted to the tubular organs (Massarwa, 2009).

The cellular machinery which dictates the apical localization of Rho-GEFs/Rho1/Dia appears to be in place early on. For example, expression of Dia-GFP in the trachea demonstrated apical localization of the protein already at the stage when the tracheal pits are formed. Yet, generation of the polarized actin cables by Dia, and their utilization for secretion, takes place at a later stage, and follows a stereotypic temporal order in the different tracheal branches. What triggers activation of Dia, following the apical localization of the different components? This study has demonstrated that both Gef2 and Gef64C are required to trigger Rho1, which activates Dia. While the activity of the two Rho-GEFs is similar, both have to accumulate to a critical level in order to activate the system. Thus, no secretion takes place when either of them is missing, or when each of them is present at half dose. The delay in activation of Dia and in secretion, may be explained by the time required to accumulate sufficient levels of Rho-GEF proteins. When the system was 'short circuited' by expression of activated Rho1, which was properly localized to the apical surface, Dia-dependent apical secretion was observed already at early stages of tracheal pit formation (Massarwa, 2009).

The results identify Drosophila MyoV (Didum) as a primary motor for apical trafficking of secretory vesicles along the polarized, Dia-nucleated actin cables in tubular organs. When the activity of MyoV was compromised in the tubular epithelia, apical secretion of cargos requiring Dia-generated actin cables was abolished. In contrast, since MyoV operates downstream to Dia, the actin cables themselves remained intact. An analogous role for MyoV has been recently demonstrated during trafficking of Rhodopsins to photoreceptor rhabdomers (Li, 2007). The functional link between the Dia pathway and MyoV was demonstrated by the ability of myoV RNAi to suppress constitutively activated Rho1 or Dia phenotypes. These results further support the direct link between Dia and apical secretion (Massarwa, 2009).

The polarized actin network formed via the nucleating activity of Dia can account for the final phase of secretory vesicle transport to the apical plasma membrane. Class V myosins, such as MyoV, are known to be involved in transfer of vesicles from microtubules to cortical actin networks, suggesting that polarized microtubule arrays may promote the long-range trafficking of the secretory vesicles from their sites of formation to the cell cortex. Consistent with this scenario, a polarized arrangement was demonstrated of microtubulesin tube epithelial cells, the minus ends of which are in close proximity to the apical membrane, which remains intact in the absence of Dia. The universality of this system is highlighted by similarities to polarized secretion in budding yeast, where Myo2p-mediated transport of secretory vesicles into the bud utilizes Dia-generated actin bundles as tracks, in order to deposit the compounds for polarized cell growth (Massarwa, 2009).

When early steps in the secretory pathway are compromised by reducing the activity of the COPII or COPI complexes, accumulation of cargo is observed within the cells and reduced amounts are detected in the lumen. Since these manipulations block an early and global process of secretion, all cargo vesicles are affected (Tsarouhas, 2007). However, after exit from the Golgi, it appears that distinct classes of vesicles are generated, each containing a different set of cargos, and trafficked by a distinct mechanism. One class of vesicles contains chitin-modifying enzymes (such as Verm or Serp), and is targeted to the septate junctions. When the structure of the septate junctions was compromised, these proteins failed to be secreted. Another class of vesicles may contain transmembrane proteins that are deposited in the apical membrane, such as Crb (Massarwa, 2009).

This study now uncovers a third class of cargo vesicles. Several distinct cargos that are secreted to the apical lumen rely on Dia for their secretion. These cargos include the 2A12 antigen, Pio, and the artificial rat ANF-GFP construct. In the absence of Dia, these proteins failed to be secreted to the lumen, but also did not accumulate within the epithelial tube cells. It is believed that when secretion is disrupted, the vesicles are efficiently targeted for lysosomal degradation, since a block of lysosomal targeting facilitated intracellular accumulation of vesicles that failed to be secreted. Inability to secrete Pio resulted in tracheal defects that were similar to pio mutant embryos. Additional defects of dia pathway mutant embryos, such as highly convoluted tracheal branches, may stem from the absence of additional, yet unknown, proteins in the lumen. The mechanisms underlying the incorporation of distinct cargos into different secretory vesicles, as well as the recognition of each vesicle type by different motors and trafficking scaffolds, remain unknown (Massarwa, 2009).

In conclusion, this work has uncovered a universal mechanism, which operates in very different types of tubular epithelial structures in Drosophila. The conserved feature of an apical F-actin network in tubular epithelia of diverse multicellular organisms, and the high degree of conservation of the different components generating and utilizing these actin structures, strongly suggests that this polarized secretion mechanism is broadly used across phyla. The ability to generate polarized actin cables that initiate at the apical membrane provides an efficient route for trafficking vesicles by MyoV, leading to their fusion with the apical membrane and secretion. It is likely that different pathological situations manifested in aberrant formation of epithelial structures, or their utilization for secretion once the tubular organ is formed, represent defects in different components of this pathway. For example, it was shown that mutations in MyoVa in humans disrupt actin-based melanosome transport in epidermal melanocytes (Massarwa, 2009).

Interaction between Drosophila bZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. This study implicates Drosophila Activating transcription factor 3 (Atf3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. These data thus identify Atf3 as a new functional partner of Drosophila Jun during development (Sekyrova, 2010).

Metamorphosis of Drosophila larvae into pupae and adult flies provides remarkable examples of morphogenetic changes that involve replacement of entire cell populations. Epithelia that had served larval function undergo programmed cell death while imaginal cells proliferate and differentiate to take their position. The Drosophila abdomen is an attractive system for studying the developmental replacement of one epithelial cell population with another. Unlike the adult head and thorax with appendages, all forming from pre-patterned imaginal discs, the adult abdomen derives from histoblasts that reside in each abdominal segment. Soon after the onset of metamorphosis, the diploid histoblasts undergo an initial phase of synchronized cell divisions; later the histoblasts expand while proliferating and replace the old polyploid larval epidermal cells (LECs) that cover the surface of the abdomen. To free space for the histoblasts, LECs are extruded from the epithelial monolayer. In order to maintain integrity of the epithelia, changes in cell adhesion and cell migration must be precisely orchestrated during this tissue remodeling (Sekyrova, 2010).

Rho kinase signaling, which stimulates constriction of the apical actomyosin cytoskeleton through myosin phosphorylation, is necessary for the extrusion and the ensuing apoptosis of LECs. Perturbed myosin phosphorylation leaves the process of the epithelial exchange incomplete, with residual LECs obstructing closure of the adult abdominal epidermis at the dorsal midline. A similar defect results from compromised function of the ecdysone receptor (EcR), which is required for both the initial phase of histoblast proliferation and for the removal of LECs. Other factors besides Rho signaling and EcR that regulate the epithelial cell replacement are unknown (Sekyrova, 2010).

This study implicates Atf3 (A3-3 -- FlyBase), the single Drosophila ortholog of the vertebrate Activating transcription factor 3 (ATF3) and Jun dimerization protein 2 (JDP2) in abdominal development. ATF3 and JDP2 belong among basic region-leucine zipper (bZIP) proteins, some of which play important roles in epithelial morphogenesis. Particularly the functions of Jun and Fos bZIP proteins in epithelial closure events during development are well understood owing to genetic studies in Drosophila. By contrast, no morphogenetic function has yet been reported for Atf3 in Drosophila (Sekyrova, 2010).

Mammalian ATF3 and JDP2 form homodimers but preferentially dimerize with members of the Jun subfamily (Aronheim, 1997; Hai, 1989; Hsu, 1991), functioning either as transcriptional activators (ATF3-Jun) or repressors (JDP2-Jun). Based mainly on cell-culture studies, multiple roles in cell proliferation, differentiation and apoptosis have been ascribed to ATF3 and JDP2. Atf3-/- mice are viable but suffer from altered glucose and immune homeostasis. Also Jdp2-/- mice survive but produce extra fat in their brown adipose tissue. In vivo significance of the interaction between the ATF3 or JDP2 proteins and Jun remains unclear (Sekyrova, 2010).

This study shows that Atf3 interacts biochemically and genetically with Jun in Drosophila. Temporal downregulation of atf3 transcription during metamorphosis is crucial, since sustained atf3 expression alters adhesive properties of LECs, thus preventing their extrusion and replacement by the adult epidermis. This effect of Atf3 requires the presence of Jun (Sekyrova, 2010).

Among Drosophila bZIP proteins, the predicted product of the CG11405 gene (also referred to as a3-3), located on the X chromosome, shows the closest similarity to the mammalian ATF3 and JDP2 proteins. The DNA-binding/dimerization bZIP domains of the human ATF3 and Drosophila Atf3 proteins are identical in 60% of their amino acids; there is 58% identity between Atf3 and JDP2 in this region (Sekyrova, 2010).

Dimerization between Atf3 and Jun in Drosophila has been theoretically predicted and confirmed by a yeast two-hybrid screen. To demonstrate direct binding, co-immunoprecipitation experiments were conducted. The endogenous Jun protein from Drosophila S2 cells co-precipitates with a transiently expressed Atf3 whereas Fos did not. A DNA mobility-shift assay with recombinant bZIP domains of Atf3, Jun and Fos was conducted to test for their DNA-binding properties. Atf3 specifically bound an ATF/CRE consensus element but not the AP-1 site, which was recognized by the Jun-Fos (AP-1) complex. Although Atf3 bound DNA by itself, presumably as a homodimer, the binding was enhanced in the presence of Jun. Fos did not synergize with Atf3 in DNA binding. Excess unlabeled DNA bearing the ATF/CRE binding site competed for the Atf3 bandshift activity whereas the AP-1 binding element did not. These results have shown that, like ATF3 or JDP2 in mammals, Atf3 in Drosophila selectively dimerizes with Jun, with which it cooperatively and specifically binds the ATF/CRE DNA element (Sekyrova, 2010).

To test whether Atf3 and Jun interact in vivo, experiments were conducted in the Drosophila compound eye, the precise structure of which sensitively reflects genetic interactions. Overexpression of atf3 under the GMR-Gal4 driver disrupted the ommatidial arrangement, resulting in smaller eyes with a glossy appearance. This atf3 misexpression phenotype could be completely suppressed by simultaneous RNAi-mediated knockdown of jun but not of fos. Conversely, the phenotype was exacerbated when jun was overexpressed in the eye together with atf3, suggesting that it is Atf3 in a complex with Jun that derails the normal eye development. Neither RNAi nor overexpression of jun alone had any effect on eye morphology. Interestingly, like depletion of Jun, co-expression of fos under the GMR-Gal4 driver completely averted the atf3 misexpression phenotype, restoring the normal appearance of the eye. Expression of fos or its mutant versions alone had no effect. These data can be explained by the ability of the surplus Fos to bind Jun and thus reduce its availability for interaction with the Atf3 protein. This interpretation is further supported by experiments showing that expression of the truncated bZIP domain of Fos is sufficient to suppress the Atf3 gain-of-function phenotype, whereas its transcription activation domain or phosphorylation sites are dispensable (Sekyrova, 2010).

Taken together, these results show that Atf3 cooperates with Jun, as Jun is specifically required for an effect caused by overexpression of Atf3 in the developing eye. Given the capacity of both Atf3 and Fos to bind Jun, and based on the ability of Jun to enhance and of Fos to suppress the Atf3 gain-of-function phenotype, it is suggested that Atf3 and Fos compete for their common partner Jun in vivo (Sekyrova, 2010).

To find out whether Atf3 is required for Drosophila development and whether its absence might resemble a phenotype caused by loss of its partner Jun, atf3 mutant flies were generated. The longest deletion (line atf376) obtained by imprecise excision of a P element, removed the entire bZIP domain of Atf3, and atf376 hemizygous (male) larvae lacked detectable atf3 mRNA. Thus, atf376 probably represents a null allele. Most atf376 larvae die soon after hatching and during all three larval stages. Only a few (approximately 2%) reach the third instar but die before metamorphosis as defective pseudopuparia. Expression of atf3 cDNA under the ubiquitous armadillo (arm-Gal4) driver rescued some atf376 hemizygotes to adults, confirming that loss of atf3 was the cause of the lethal phenotype. Interestingly, the moribund atf376 larvae abnormally enlarged lipid droplets in their fat body, thus displaying a phenotype reminiscent of that in mice lacking one of the Atf3 orthologs, JDP2. However, in contrast to viable Jdp2 or Atf3 knockout mice, atf3 is an essential gene in Drosophila (Sekyrova, 2010).

Fly embryos lacking the function of Jun or Fos die because of the failed dorsal closure. However, atf376 embryos develop normally, without the dorsal open defect, even when derived from atf3-deficient germline clones induced in atf376/ovoD1 mothers. Thus, unlike its partner Jun, Atf3 is not required for dorsal closure, suggesting that dorsal closure is regulated by Jun-Fos dimers and that the Atf3-Jun complex has another function later in development (Sekyrova, 2010).

Consistent with the vital requirement for Atf3 during larval stages, atf3 mRNA was expressed in embryos and larvae. Expression then sharply declines by the late-third larval instar, and no atf3 mRNA was detected by northern blot hybridization in wandering larvae and during metamorphosis from the time of puparium formation until the second day of pupal development. Detailed RT-PCR analysis showed that atf3 downregulation coincided with the cessation of feeding and the onset of metamorphosis [0 hours after puparium formation (APF)]. A pulse of expression occurred at 6 hours APF. RT-PCR from isolated fat body and abdominal integuments, together with in situ hybridization performed on puparia at this stage, showed that atf3 mRNA was primarily present in the larval epidermis (LECs) during the expression peak at 6 hours APF. From the time of head eversion (12 hours APF) the mRNA level remained low until the second day of pupal development, and then it grew steadily during morphogenesis of the adult. Quantitative RT-PCR revealed a 4.3-fold difference in atf3 mRNA abundance between 0 and 72 hours APF. In contrast to the tight regulation of atf3, the mRNAs of fos and jun fluctuated little during the examined period. Therefore, unlike Jun or Fos, Atf3 was dynamically regulated during metamorphosis at the level of transcription (Sekyrova, 2010).

The precise temporal control of atf3 expression suggested that the rise and subsequent fall of Atf3 during metamorphosis might be critical for the complex morphogenesis occurring in fly pupae. This possibility was tested by means of sustained expression of the full-length Atf3 protein using the UAS-Gal4 system with various drivers. A striking, fully penetrant metamorphic defect was observed with the pumpless (ppl) Gal4 driver. Although ppl>atf3 animals developed normally until the pupal stage, they failed to complete fusion of the adult abdominal epidermis. A dorsal cleft in the abdomen remained that could not be covered with the adult cuticle, and consequently 86% of the flies died inside the puparium. All of the ppl>atf3 adults that did eclose showed abdominal lesions filled with the old pupal cuticle lacking adult pigmentation and bristles, often with a clot covering a bleeding wound. Adults with the same abdominal cleft (but otherwise normal) also emerged when atf3 was moderately and ubiquitously misexpressed under the arm-Gal4 driver, suggesting that abdominal morphogenesis was the process most sensitive to ectopic Atf3 (Sekyrova, 2010).

The adult fly abdomen derives from histoblasts that proliferate, replace LECs and finally differentiate, giving rise to the adult cuticle. Therefore, the observed abdominal defect suggested a compromised function of the epidermis, either LECs, histoblasts or both cell types. To distinguish between these possibilities, expression of the ppl-Gal4 driver was first examined in the epidermis. It was found that ppl-Gal4 was active in LECs but not in histoblasts. Second, another driver, Eip71CD-Gal4, which was inactive in histoblasts but strongly expressed in LECs, was examined. Eip71CD-Gal4-driven misexpression of atf3 mostly produced lethal pupae lacking adult cuticle, but it occasionally yielded adults with a dorsal abdominal cleft. In addition to being active in LECs, both ppl-Gal4 and Eip71CD-Gal4 (data not shown) were also expressed in the fat body. However, no abdominal defects occurred when atf3 was misexpressed under either of three fat-body-specific Gal4 drivers, Lsp2, Cg or C7. Third, to rule out the possibility that ectopic Atf3 affected the imaginal epidermis, its expression was directed to histoblasts by using the escargot (esg) and T155 Gal4 drivers; in neither case the fusion of the adult abdominal epidermis was affected (Sekyrova, 2010).

To finally confirm that abdominal morphogenesis was disrupted by sustained atf3 activity in LECs, atf3 was induced by using the flp-out technique. Owing to the timing of heat-shock induction to the mid-third instar, this method triggers expression in the polyploid larval cells but not in the diploid histoblasts . Misexpression of atf3 under the actin promoter following the flp-out event invariantly led to an abdominal cleft. The lesions were often more severe than those observed in ppl>atf3 animals, affecting also lateral and ventral parts of the abdomen. Together, the above data demonstrate that the sustained expression of atf3 prevents fusion of the adult abdominal epidermis by acting upon LECs, suggesting that the replacement of these obsolete larval cells by adult histoblasts requires the developmental downregulation of atf3 expression (Sekyrova, 2010).

To understand the cellular events underlying the incomplete epithelial closure in ppl>atf3 animals, cell membranes were visualized by antibody staining of the septate junction component, Discs large 1 (Dlg1), or used a transgenic DE-cadherin::GFP fusion protein (shg::gfp). In wild-type animals 24 hours APF, LECs covering the surface of the abdomen gave way to the rapidly expanding nests of histoblasts that began to fuse laterally and ventrally. In ppl>atf3 pupae the histoblast nests also spread, and at least at 16 hours APF, before their fusion, they comprised normal numbers of histoblasts. By 48 hours APF a control abdomen was fully covered with adult epidermis consisting exclusively of histoblasts, now forming sensory bristles. Histoblasts in ppl>atf3 abdomens also differentiated the adult cuticle with sensory bristles, although polarity of the bristles near the dorsal cleft was altered. However, in contrast to the control, a large population of LECs remained in the dorsal abdomen of ppl>atf3 animals at 48 hours APF. The membranes of the persisting LECs accumulated the Dlg protein, and although these cells became severely deformed they survived throughout metamorphosis to the adult stage. When visualized in live ppl>atf3 pupae, the apical junctions of the remaining LECs displayed interdigitation and accumulation of DE-cadherin::GFP. Another adherens junction component, the Drosophila β-catenin Armadillo, was also enriched in atf3-expressing LECs (Sekyrova, 2010).

Cooperation between adherens junctions and the apical ring of actomyosin cytoskeleton is required for basal extrusion of LECs. The altered pattern of DE-cadherin and β-catenin therefore suggests that excessive Atf3 might prevent LEC extrusion through stabilization of the cell-cell adhesion complex. To examine the effect of Atf3 on LECs in further detail, the flp-out technique, which allows comparisons of atf3-misexpressing and control LECs within one tissue, was employed. Membrane interdigitation occurred between atf3-positive LECs already at 18 and 24 hours APF, even in areas where the LECs had no contact with histoblasts. At 48 hours APF only LECs expressing atf3 persisted, apparently being squeezed by the expanding histoblasts. The membrane-associated DE-cadherin::GFP signal was stronger in adjacent atf3-positive LECs compared with non-induced LECs, and quantitative analysis of confocal images acquired at 18 hours APF and at 24 hours APF both revealed a statistically significant 1.4-fold increase of the DE-cadherin::GFP signal intensity upon atf3 induction. Enrichment of DE-cadherin on apical membranes of atf3-expressing LECs was further confirmed on confocal cross sections (Sekyrova, 2010).

Although some atf3-positive LECs began the extrusion process, they could not detach from the apical surface even when entirely surrounded by histoblasts, possibly being tethered to it by the excessive adhesion protein. By contrast, control LECs did completely separate from the epithelium. In addition, LECs overexpressing atf3 displayed apical enrichment of moesin, an actin-binding protein of the ERM (ezrin, radixin, moesin) family, which links transmembrane proteins to cortical actin filaments. Interestingly, prominent accumulation of DE-cadherin was also observed in atf3-expressing clones of epithelial cells within the hinge region of wing discs that form the adult thorax, indicating that the effect of Atf3 on cell adhesion components may not be limited to larval epithelia (Sekyrova, 2010).

In summary, these results show that deregulation of atf3 expression causes marked changes of cell membranes, including interdigitation and accumulation of cell adhesion molecules, suggesting that LEC adhesiveness might be increased. Although some of the affected LECs initiate extrusion, this process stays incomplete. Consequently, the adhering LECs present a physical barrier for the migrating histoblasts (Sekyrova, 2010).

Rho kinase (Rok)-dependent phosphorylation of myosin regulatory light chain was shown to be required for LEC extrusion. To examine a possible relationship between the Rok-dependent cytoskeletal regulation and Atf3, the function of the GTPase Rho1 (also called RhoA), which acts immediately upstream of Rok, was disrupted. RNAi silencing of Rho1 using the ppl-Gal4 driver produced a phenocopy of atf3 misexpression, causing a dorsal abdominal cleft in 100% of ppl>Rho1(RNAi) adults, of which most died in the puparium and about 12% eclosed, similar to ppl>atf3 animals. However, when Rho1 RNAi and misexpression of atf3 in LECs were combined, the abdominal defect became more severe, not allowing any pharate adults to eclose. Conversely, co-expression of a dominantly active Rho1V14 protein suppressed the otherwise fully penetrant abdominal defect in some ppl-atf3 flies. Surprisingly, it was found that the endogenous Rho1 protein was mislocalized in atf3-misexpressing LECs, showing a diffuse cytoplasmic signal, compared with membrane localization in control LECs. These results suggest a genetic interaction between Rho signaling and atf3, and support the idea that excess Atf3 prevents extrusion of LECs by altering their cell adhesion properties (Sekyrova, 2010).

Disturbed function of the ecdysone receptor (EcR) has been shown to prevent extrusion of LECs, causing a dorsal abdominal cleft that closely resembles the Atf3 gain-of-function phenotype. Therefore whether stimulating EcR-dependent signaling by addition of the natural agonist 20E might overcome the defect caused by sustained atf3 expression was examined. Indeed, supplying third-instar ppl>atf3 larvae with dietary 20E increased the number of eclosing adults, the abdominal scars of which were in 22% of the cases partially or completely sealed with normal adult cuticle (Sekyrova, 2010).

Atf3 interacts with Jun to form a DNA-binding complex and genetically when overexpressed in the developing compound eye. To see if this interaction is biologically relevant during abdominal morphogenesis, whether Atf3 relies on the presence of Jun to cause the dorsal cleft phenotype was tested. First, it was confirmed that Jun is indeed expressed in LECs during metamorphosis. RNAi-mediated depletion of Jun in animals that misexpressed atf3 under the ppl-Gal4 driver restored viability of adults from 14% (atf3 alone) to 100%. Strikingly, 87% of the ppl>atf3, jun(RNAi) adults eclosed with a completely normal abdomen. By contrast, RNAi knockdown of Fos in ppl>atf3 background did not improve the abdominal defect. RNAi silencing of either jun or fos alone under the ppl-Gal4 driver had no effect on the abdomen. These results demonstrate that Atf3 requires its partner Jun but not Fos to disrupt abdominal morphogenesis. Similar to the situation in the compound eye, the effect of misexpressed atf3 can be neutralized by simultaneously expressing Fos or its truncated bZIP domain under the ppl-Gal4 driver. Therefore, the model in which Atf3 and Fos compete for their common partner Jun may be extended to the developing abdomen (Sekyrova, 2010).

This study has identified Atf3 as a new partner of Jun in Drosophila. Previously, Jun has only been known to dimerize with itself and with the Drosophila homolog of Fos. Functional analysis of Atf3 has not yet been reported. These biochemical data show that, similar to mammalian ATF3 and JDP2, the Atf3 protein selectively binds Jun but not Fos. Also consistent with the properties of ATF3 and JDP2 is the ability of Atf3 to bind the ATF/CRE response element alone or synergistically with Jun. In contrast to its mammalian counterparts, however, neither Atf3 alone nor in complex with Jun bound to the AP-1 element under the same conditions. The selective interactions of Atf3 point to distinct biological roles for the Atf3-Jun and the Fos-Jun dimers, respectively (Sekyrova, 2010).

This study has shown a genetic interaction between Atf3 and Jun. The evidence is based on the ability of ectopic Atf3 to disturb morphogenesis of the adult abdomen and the compound eye, which strictly depends on the availability of Jun. Importantly, none of the Atf3 gain-of-function phenotypes could be induced by misexpression of the truncated bZIP domain of Atf3, suggesting that the functional Atf3 protein in complex with Jun is required. Based on the selectivity of Atf3 in a DNA-binding assay, it is predicted that the Atf3-Jun complex regulates specific target genes distinct from those targeted by Fos-Jun dimers (Sekyrova, 2010).

The data also reflect a relationship between the AP-1 and Atf3-Jun complexes. Although Fos does not dimerize or bind DNA with Atf3, its ability to suppress the Atf3 misexpression phenotype in the eye suggests that Fos and Atf3 compete in vivo for their common partner Jun. The fact that the same suppression can be achieved by overexpressing either the truncated Fos bZIP domain or Fos lacking phosphorylation sites indicates that the suppression does not rely on a transcriptional function of Fos but probably occurs through sequestering of Jun, even by a transcriptionally inactive Fos protein. Early in vitro studies have proposed a competition model for the AP-1 and Atf3 proteins to explain a temporal regulation of gene expression in the regenerating liver. However, to date such a relationship among Fos, Jun and Atf3 has not been supported with direct genetic evidence (Sekyrova, 2010).

Removal of LECs is normally complete by 36 hours APF, at which time the sheets of histoblasts reach the dorsal midline. The data strongly support the argument that the temporal downregulation of atf3 expression during abdominal morphogenesis is necessary for LECs to be replaced by the adult epidermis. When experimentally sustained, atf3 activity in LECs interfered with this exchange by blocking extrusion and death of the LECs. This was evident as the atf3-expressing LECs survived within the epithelial layer for days after their scheduled destruction (Sekyrova, 2010).

Interdigitation of cell membranes and accumulation of adherens junction proteins in LECs suggested that ectopic Atf3 caused adjacent LECs to reinforce their mutual contacts. This probably resulted from altered distribution of the proteins, as levels of the shg (DE-cadherin) mRNA remained unchanged in LECs of ppl>atf3 animals. By contrast, junctions between atf3-expressing LECs and their normal neighbors or histoblasts were smooth and presumably less rigid. DE-cadherin was similarly enriched in clones of imaginal disc cells. These observations suggested that differential adhesion of atf3-expressing cells might have led to their sorting out from the surrounding epithelium. Even modest differences in cadherin levels have been shown to cause segregation of cells within a population by altering their adhesiveness (Sekyrova, 2010).

Recent live imaging data have revealed that migrating histoblasts push the LECs ahead of themselves towards the dorsal midline, where histoblasts fuse last. The atf3-expressing LECs that adhered to each other were probably moved and pressed by the expanding histoblasts to the dorsal side, whereas non-induced LECs were eliminated. This explains why the abdominal lesions primarily occurred at the dorsal midline, although flp-out experiments showed that atf3 misexpression could affect LECs in other areas as well. Strengthened contacts among persisting LECs probably blocked invasion of histoblasts in between them and inhibited LEC extrusion, eventually causing gaps in the adult epidermis (Sekyrova, 2010).

In accord with the notion that extrusion from the epithelium is a prerequisite for LECs to undergo apoptosis, it is assumed that sustained presence of Atf3 primarily enhanced adhesiveness of LECs, which only consequently prevented their death. This view is supported by the observation that membranes of atf3-expressing LECs interdigitated and accumulated DE-cadherin as early as 18-24 hours APF, even in areas of the larval epidermis that were far from histoblasts and where control LECs did not yet extrude. In addition, the Atf3 gain-of-function phenotype was stronger than abdominal closure defects caused by caspase mutation or inhibition. When the anti-apoptotic proteins p35 or DIAP1 (Thread — FlyBase) was misexpressed under the ppl-Gal4 driver, the resulting dorsal lesions were not lethal and were clearly milder than the broad, mostly fatal scars in ppl>atf3 animals. Compared with the large contiguous populations of persisting LECs in ppl>atf3 pupae, inhibiting apoptosis with p35 only allowed small islands of LECs to survive (Sekyrova, 2010).

Ecdysone signaling promotes replacement of the abdominal epithelia by stimulating both the early histoblast proliferation and the extrusion of LECs. As atf3 misexpression affected LECs but did not impair early histoblast proliferation, the latter possibility remains, that added 20E counteracted the effect of ectopic Atf3 by facilitating the extrusion process. Since normal 20E titers was detected in ppl>atf3 larvae or prepupae, the failure of LEC extrusion was not a result of steroid deficiency. Also, 20E had no effect on atf3 mRNA levels, at least in Drosophila S2 cells or third-instar larvae. Atf3 and ecdysone signaling therefore probably influence LEC extrusion by acting independently (Sekyrova, 2010).

Although the mechanism through which ecdysone contributes to LEC removal is unknown, one attractive possibility is that it might cooperate with Rho signaling, which is required for LEC extrusion as well. It has been demonstrated that genetic interaction between the 20E-response gene broad and components of the Rho pathway including RhoGEF2, Rho1 and myosin II is important for ecdysone-dependent epithelial cell elongation during Drosophila leg morphogenesis. The current data show that Rho1 becomes mislocalized in LECs upon atf3 misexpression and that Rho1 silencing enhances the abdominal gain-of-function phenotype of atf3. The exact relationship between Atf3, Rho1 and ecdysone remains to be determined. However, Atf3 clearly represents a new intrinsic regulator of epithelial cell replacement during Drosophila metamorphosis (Sekyrova, 2010).

Regulation of Drosophila mesoderm migration by phosphoinositides and the PH domain of the Rho GTP exchange factor Pebble

The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants, the phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B was identified. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, PtdInsP-binding of Pbl was examined, along with the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pbl(DeltaPH) mutants. Taken together, these results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration (Murray, 2012).

Effects of Mutation or Deletion

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 (Rho172O and Rho172R) 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, Rho1N19 were analyzed. A dominant-negative form of H-Ras, H-RasN17, cannot interact with downstream target proteins. In addition, H-RasN17 competes with normal H-Ras for binding to rasGEF, which results in formation of inactive H-RasN17-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 Rho1N19 creates a phenotype that is much worse than loss of zygotic Rho1. Expression of Rho1N19 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 (Rac1N17 or Rac1L89) does not affect cytokinesis or completion of mitosis. Interestingly, coexpression of Rho1N19 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 RhoV14 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 RhoV14. 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 RhoV14 protein and they were expressed under the control of sev-gal4 in the eye disc (sev;RhoV14 F39V and sev;RhoV14 E40L). The sev;RhoV14 F39V flies display a phenotype that is indistinguishable from that of sev;rhoV14 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;RhoV14 F39V (two copies) led to an enhancement of both the polarity and the photoreceptor recruitment phenotypes. In contrast, sev;RhoV14 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;RhoV14 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;RhoV14 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;racV12 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;RacV12. These data resemble the upregulation of puc-lacz when the JNK pathway has been activated in the same cells (Fanto, 2000).

In contrast, RhoV14 affects puc-lacz expression differently. Although in sev;RhoV14 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 (RhoAV14 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;RacV12 and sev;RhoV14 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;RacV12 and sev;RhoV14 upregulated Dl-lacz expression in both R3/R4 precursors. The RhoAV14 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).

Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity

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. MoeG0323 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 MoeG0323 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 MoeG0323 disc epithelium organization and actin localization phenotypes, but also strongly suppresses MoeG0323 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 MoeG0323 epithelial cells as well as that of MoeG0323 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 MoeG0323 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, ezrinAct, 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 ezrinAct 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).

Rho and axon guidance

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 Sose49 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).

RhoA regulates neuroblast proliferation and dendritic morphogenesis

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 zipperEbr allele gives rise to its sensitivity to second-site noncomplementation. The Rho signal transduction pathway has been identified as necessary for proper myosin function. A lethal P-element insertion interacts genetically with zipper. Subsequently this second-site noncomplementing mutation has been shown to disrupt the RhoGEF2 locus. Two EMS-induced mutations, previously shown to interact genetically with zipperEbr, disrupt the RhoA locus. Further, their molecular lesions have been identified and it has been determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, it has been shown that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. These studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan (Halsell, 2000).

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

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

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

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

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

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

Mutations in the Drosophila RhoGEF2 gene have been identified by three distinct means: phenotypic suppression of ectopically expressed RhoA (Barrett, 1997); genetic screens for maternally encoded molecules required during early Drosophila embryogenesis (Hacker, 1998), and genetic screening for molecules required for myosin function (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 ßPS 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).

The myogenic function of nonmuscle myosin-II has been analyzed by using Drosophila genetics to manipulate the levels of nonmuscle myosin-II heavy chain, PS2 integrin, and RhoA GTPase in vivo in the developing larval muscles. Both nonmuscle myosin-II and PS2 colocalize at muscle termini. However, in contrast to models based on cultured fibroblasts, there is no evidence for either nonmuscle myosin-II or RhoA function in PS2 clustering. Instead, the maintenance of nonmuscle myosin-II localization at muscle termini is dependent on the presence of PS2 integrin and the cytoplasmic tail of the ßPS integrin subunit is sufficient for this. Further, nonmuscle myosin-II maintenance at the muscle termini is compromised in ifSEF, a ßPS2 integrin subunit mutant that specifically disrupts myofibril formation. Through the analysis of actin distribution in the musculature of living wild-type and mutant embryos, it has been demonstrated that RhoA-independent nonmuscle myosin-II function is required for the proper sarcomeric organization of the muscle cytoskeleton. Finally, since nonmuscle myosin-II localizes to the Z-line in late larval muscle, it has been suggested that nonmuscle myosin-II functions at both the muscle termini and the Z-line to maintain the structural integrity of the sarcomere (Bloor, 2001).

Rho function in glial migration and nerve ensheathement

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).

Rho and dorsal closure

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: establishment and/or maintenance of the leading edge cytoskeleton versus its down regulation. It is proposed that Dcdc42 is the target of Dpp, released from cells flanking segment borders. Dpp targets Dcdc42 in the adjacent segmental border cells through Dpp's receptors Thick veins and Punt (Ricos, 1999). Down regulation of the leading edge cytoskeleton may be controlled by the serine/threonine kinase PAK (see Drosophila PAK-kinase), a potential Dcdc42 effector. Whereas dominant negative Dcdc42 leads to a loss of Pak, constitutive active Dcdc42 induces an increase in Pak levels. Paradoxically, constitutively active Dcdc42 can also lead to a loss of leading edge cytoskeleton and leading edge Pak in some embryos. These two seemingly opposite effects of constitutively active Dcdc42 can occur in the same embryo. It is possible that Pak, activated by Dcdc42 in segmental border cells, is localized within the leading edge cytoskeleton and contributes to cytoskeletal down regulation. This breakdown is first executed transiently in the segment border cells and then permanently along the leading edge at the end of DC. Thus constitutively active Dcdc42 may be capable of elevating Pak sufficiently along the leading edge to cause premature down regulation of Pak and the cytoskeleton as a whole (Harden, 1999).

RhoA is required for the integrity of the leading edge cytoskeleton specifically in the cells flanking segment borders. Dominant negative RhoA expression leads to loss of leading edge components and a loss of anterior-posterior contraction in several cells flanking each segment border. One possible explanation for the effects of dominant negative RhoA is that RhoA is required for the assembly of the leading edge cytoskeleton in the segment border cells, perhaps downstream of Drac1. An alternative interpretation is that RhoA is functioning as a negative regulator of the leading edge cytoskeletal losses that occur in wild-type embryos, and that when Rho A function is absent, these losses become permanent instead of transient. The interactions of the various small GTPases in regulating dorsal closure reveals that there is no evidence for the hierarchy of Rho subfamily activity described in some mammalian cell types. Rather, the results suggest that while each of the p21s are required for dorsal closure, they act largely in parallel (Harden, 1999).

A model is given of the control of DC by the Drac1/JNK and Dcdc42/Dpp pathways. Drac1/JNK signaling, initiated by an as yet unknown factor, assembles cytoskeletal components (F-actin, myosin and focal complexes) and other proteins (Dpp, Puckered and Pak) in the leading edge cells and initiates the cellular migration that characterizes DC. Dpp-activated signaling controls the dynamics of epidermal migration, via Dcdc42 and the Dpp pathway, through the serine/threonine kinase Pak, which transiently downregulates the leading edge cytoskeleton at the segmental borders. Transient downregulation of the actin cytoskeleton and focal contacts near the segment border cells is likely to cause local relaxation of the anterior-posterior tension along the LE. Such transient relief of tension may then limit excessive migration of leading edge cells toward each other and prevent the bunching and shearing of epidermal segments that occurs following impairment of Dpp/Dcdc42 signaling. Segment borders cells are potential regions of highest Dpp signaling, because they are adjacent to the highest local concentrations of Dpp protein, and they have high levels of Pak protein and transcripts for the Tkv receptor. Segmental border cells are the only places where transient downregulation of the leading edge cytoskeleton is ever seen in wild-type embryos during DC. As such, it is proposed that the role of Dcdc42/Dpp signaling is the induction of Pak to downregulate the leading edge cytoskeleton at the segment borders, introducing a degree of flexibility to the leading edge during the dorsal closure process (Ricos, 1999).

Throughout development, a series of epithelial movements and fusions occur that collectively shape the embryo. They are dependent on coordinated reorganizations and contractions of the actin cytoskeleton within defined populations of epithelial cells. One paradigm morphogenetic movement, dorsal closure in the Drosophila embryo, involves closure of a dorsal epithelial hole by sweeping of epithelium from the two sides of the embryo over the exposed extraembryonic amnioserosa to form a seam where the two epithelial edges fuse together. The front row cells exhibit a thick actin cable at their leading edge. The function of this cable has been tested by live analysis of GFP-actin-expressing embryos in which the cable is disrupted by modulating Rho1 signaling or by loss of non-muscle myosin (Zipper) function. The cable serves a dual role during dorsal closure. It is contractile and thus can operate as a 'purse string,' but it also restricts forward movement of the leading edge and excess activity of filopodia/lamellipodia. Stripes of epithelium in which cable assembly is disrupted gain a migrational advantage over their wild-type neighbors, suggesting that the cable acts to restrain front row cells, thus maintaining a taut, free edge for efficient zippering together of the epithelial sheets (Jacinto, 2002).

To investigate the function of the actin cable during dorsal closure, advantage was taken of rho1 and also zip alleles that produce phenotypes in which the actomyosin cable disassembles part way through the dorsal closure process but in which the overall tissue movement typically does not fail. These mutants offered the opportunity to analyze the effects of cable loss at the cellular level without the complete disruption of tissue architecture. Also, they allowed the use of live GFP-actin embryos to analyze the dynamic cell behavioral response and determine how these behavioral changes influence the capacity of epithelial cells to participate in a coherent forward-sweeping movement (Jacinto, 2002).

Scanning electron micrographs of rho1 and zip embryos that complete dorsal closure reveal significant similarities. In both cases, combinations of amorphic or strong mutations, zip1/zipIIX62 or zip1/zip1 in the case of zip, and rho172O/rho172R in the case of rho1, were used. Both zip and rho1 mutant embryos exhibit the same dramatic defects in head involution, a tissue movement distinct from dorsal closure, but a clear, posterior dorsal hole is observed in none of the rho1 mutants, and in only 59% of the zip/zipIIX62 or 8% of the zip1/zip1 embryos. The remaining zip mutants appear to complete dorsal closure successfully; although, frequently, like their rho1 counterparts, these embryos show puckering or segment misalignments along the closed midline seam. Interestingly, puckering has previously been observed in mutants in which epithelial movement is not properly downregulated at the midline (e.g., puckered), suggesting that the actin cable may be regulating cell spreading during some periods of dorsal closure. It is presumed that the phenotypic variation in zip embryos is due to differences in the maternal contribution to the myosin II RNA and protein pool, which must run out around the stage of dorsal closure (Jacinto, 2002).

Phenotypic similarities between zip embryos that complete closure of the dorsal hole and rho1 mutants are not surprising since rho1 and zip have previously been shown to interact genetically in Drosophila. Moreover, a signaling link has also been revealed in mammalian cell culture studies, with Rho-associated kinase (ROCK), a Rho effector, shown to regulate Myosin function, and thus the assembly and maintenance of cable-like stress fibers, by repression of Myosin Light Chain phosphatase and direct activation of the Myosin Light Chain. It is also likely that Rho1 regulates the cytoskeleton via alternative effectors. The kinase Protein kinase related to protein kinase N (see Rho1 Protein Interactions) has been shown to function downstream of Rho1 during Drosophila dorsal closure, and mDia, the murine homolog of Drosophila Diaphanous, has been shown to bind active Rho1 and contribute to the formation of stress fibers in mammalian cells by promoting actin polymerization. However, whether or not these signaling pathways regulate actomyosin contractility during dorsal closure has yet to be demonstrated. Interestingly, Rho1 also interacts with p120ctn and regulates cadherin-based adherens junctions in the Drosophila embryo, suggesting that the leading edge disorganization seen in rho1 mutants may be not only a consequence of actin cable misregulation but also a result of defective adherens junctions (Jacinto, 2002).

Further similarities between the rho1 and zip embryos were revealed when higher-resolution SEM was used to look at the leading edge cells during the final stages of dorsal closure. In both mutants, the normally straight, and apparently taut, epithelial leading edge now appears to be relatively disorganized and to have lost tension, with front row cells extending increased levels of filopodial and lamellipodial protrusions compared to their wild-type counterparts (Jacinto, 2002).

To observe the dynamic behavior of these cells, time-lapse confocal microscopy was used to analyze the final stages of dorsal closure in living, wild-type embryos versus rho1 and zip embryos, expressing GFP-actin fusion proteins using the GAL4-UAS system. The fusion protein was driven in the epidermis by the epithelial driver e22cGAL4, and only embryos that subsequently closed the dorsal hole are described. As expected from SEM observations, the cytoskeletal architecture of the leading edge that normally characterizes wild-type dorsal closure is lost in both rho1 and zip mutants. The actin cable, which is clearly apparent in the wild-type leading edge from stage 13 onward, fails to assemble or disassembles during the dorsal closure process in rho1 and zip mutants. The disassembly of the actin cable is temporally coincident with a transition from an organized to disorganized leading edge. In both mutants, loss of cable is also coincident with more exuberant filopodial extensions than in wild-type leading edge cells, and these filopodia frequently coalesce to form lamellipodia. In wild-type embryos, lamellipodia are generally more a feature of the final stages of dorsal closure, as opposing epithelial fronts make contact with one another (Jacinto, 2002).

The coalescing of filopodia to form lamellipodia in mutant embryos leads to a broader extent of protrusion per unit length of leading edge cells and an increase of up to 300% in the total protrusion area extending from these cells. It is suggested that the increased level of actin-based protrusions seen in both rho1 and zip embryos at a time when the leading edge cable is disassembling may reflect that the cable serves some function in repressing excessive protrusive activity in these front row cells. The link could simply be due to a greater availability of free actin monomers, but it might also be a consequence of changes in membrane and cortical actin stiffness at the free edge in these cells. In this regard, myosin II has been shown to play a role in maintaining the integrity and stiffness of the cortical cytoskeleton during Dictyostelium morphogenesis. It is not clear from this data whether downregulation of Rho in any way feeds back on Cdc42 activity, but no increase in thickness or apparent contractility of the actin cable is observed in cells expressing dominant-negative versions of Cdc42 (Jacinto, 2002).

These observations indicate that maintenance of a fully formed and functioning actin cable is not an absolute requisite for closure of the dorsal hole. In order to test further how a failure to activate Rho1 and assemble an actin cable might influence a cell's capacity to participate in dorsal closure, the enGAL4 driver was used to express both GFP-actin and either dominant-negative Rho1 (RhoN19) or constitutively active Rho1 (RhoV14) in 4- to 5-cell-wide epithelial stripes. These embryos were then imaged live or, alternatively, fixed and costained with Alexa594-phalloidin to reveal the actin machinery of all the cells, including the intervening wild-type stripes that do not express GAL4. Initial live analysis of these embryos, from the earliest stages of dorsal closure, revealed differences between leading edge cells that are blocked in Rho1 signaling and their wild-type neighbors. Rho1N19-expressing cells fail to assemble an actin cable but do express broad filopodia and lamellipodia. Without a cable, they do not constrict at their leading edge in the way that their wild-type neighbors clearly do. Subsequently, these mutant Rho1N19 stripes of cells sweep forward, apparently released from their usual constraints, overspilling and displacing their wild-type neighbors. Frequently, the dorsal edges of intervening wild-type stripes are enveloped by adjacent Rho1N19-expressing stripes, and this wild-type tissue is consequently trapped back from the leading edge. By the time that the dorsal hole is closed, most of the midline seam epithelium is taken up by Rho1N19-expressing cells that clearly have a migration advantage over their wild-type, cable-assembling neighbors. In the converse experiment in which GFP-actin was expressed and a constitutively active Rho1 construct (Rho1V14) was expressed using the enGAL4 driver, precisely the opposite effect is seen. Now, Rho1V14-expressing cells are more constricted than their wild-type neighbors at early stages, and they are subsequently outcompeted during dorsal closure, such that wild-type stripes tend to dominate the leading edge during dorsal closure. Thus, when dorsal closure is complete, the midline seam epithelium is largely wild-type (Jacinto, 2002).

These results suggest that the actomyosin cable has a dual role to play during dorsal closure. It is a driver of leading edge cell contractility from the earliest stages of dorsal closure, but it is also required for restraining the leading edge epithelial cells and maintaining a coherent and taut epithelial margin during the later phases of dorsal closure, particularly as the epithelial faces are being zippered together. It is proposed that, in addition to the previously described 'purse string' function, actomyosin cables may have a more subtle, but equally important, support role during morphogenetic episodes such as dorsal closure. These cytoskeletal structures, regulated by Rho activity, function to maintain epithelial coherence during coordinated epithelial movements, particularly at free epithelial edges where a taut front enables efficient zippering together and alignment of cells at a zipper seam, in a process that is perhaps analogous to zippering closed a very full luggage bag (Jacinto, 2002).

In a sense, the restraining function of the cable during dorsal closure is analogous to that which operates for the band of actin at the periphery of a tissue culture clone of epithelial cells. In this case, the growth factor, scatter-factor, can trigger disruption of the restraining actin band as well as dissolution of cell:cell junctions, and cells at the periphery are consequently now able to break free of their neighbors (Jacinto, 2002).

The dual role for the actomyosin cable during dorsal closure, operating both as a 'purse string' and as a cell restrainer, is likely to be a finely balanced operation. Modulation of normal Rho1 activity, as in the experiments reported above, results in either overcontractility of cells or their release from leading edge constraints. These data support previous evidence suggesting that multiple cytoskeletal events drive dorsal closure but demonstrate that these events must be finely balanced to ultimately produce the precisely organized zippering required for perfect midline fusion of the two epithelial faces (Jacinto, 2002).

Rho and tracheal tube fusion

Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short stop (shot). In these fusion cells, the apical surface determinant Discs lost (Dlt: now redefined as Drosophila Patj) is subsequently deposited and new lumen forms along the track. In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led to an investigation of whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. It is therefore proposed that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization (Lee, 2002).

The tracheal lumen is initially closed at branch tips. Concurrent with branching morphogenesis, specialized cells at branch tips, known as fusion cells, join branches into a continuous tubular network. This process of anastomosis requires each fusion cell to recognize its partner in the adjacent hemisegment and to form a lumen that connects the two branches. Shotgun, the Drosophila homolog of the cell adhesion molecule E-cadherin is integral to the initial fusion cell contact. Mutations in shotgun affect tracheal branch extension and lumen formation at anastomosis sites, as do mutations in armadillo, the Drosophila homolog of its effector ß-catenin. E-cadherin and ß-catenin control cell polarity and tube extension in culture, suggesting an evolutionarily conserved role for cadherin-mediated cell adhesion in apical surface regulation (Lee, 2002).

Tracheal cells face the lumen with their apical surfaces, suggesting that apically localized molecules play a role in lumen formation. Mutations in discs lost (dlt), which encodes an apically localized PDZ domain family protein, disrupt epithelial cell polarity, as do mutations in crumbs, which encodes a Dlt-associated, EGF repeat family transmembrane protein. crumbs mutant embryos are defective in forming mature zonula adherens (ZAs), structures at apical/lateral contacts between cells that contain E-cadherin (Lee, 2002).

Several studies suggest that these apical surface determinants and E-cadherin regulate the cytoskeleton and therefore control lumen formation and morphology. For example, Crumbs may attach ßH-spectrin, an F-actin cross-linker, to the apical membrane in Drosophila and mutations in a C. elegans ßH-spectrin moderately enlarge the lumen of the excretory canal. E-cadherin interacts with F-actin via multiple mechanisms. These include binding to p120 catenin (p120ctn), a negative regulator of the RhoA GTPase. RhoA controls the formation of F-actin-containing focal adhesions and stress fibers in cultured cells. These interactions with RhoA also potentially regulate apical membrane protein targeting, a process important for lumen development in culture (Lee, 2002).

Little is known about the cytoskeletal structures required for lumen formation and how apical surface determinants are localized. This study identifies an F-actin-rich track that is associated with E-cadherin-dependent contacts between fusion cells that appears to guide deposition of apical surface determinants and lumen formation. During anastomosis, Short stop accumulates at these contacts and transiently along the track. Mutations in shot and constitutively active alleles of the Drosophila RhoA (RhoA) GTPase specifically disrupt this contact and the associated track. Remarkably, the interactions of Shot with F-actin and its binding to microtubules are functionally redundant in organizing the track, suggesting that Shot acts with other pathways to organize F-actin and microtubules, rather than as an F-actin/microtubule cross-linker. It is proposed that in fusion cells, RhoA antagonizes Shot to regulate E-cadherin-associated cytoskeletal structures required for apical surface determinant localization and lumen formation (Lee, 2002).

The results presented here provide further insights into how the cytoskeleton and associated proteins support contact formation and subsequent apical surface remodeling. The F-actin- and microtubule-binding domains of Shot are required to maintain and remodel E-cadherin contacts and to assemble a track of F-actin and Shot in fusion cells. This track initiates at the E-cadherin contact and extends outwards from it to connect with the existing apical assemblies of F-actin and Shot. It is proposed that the track guides new apical surface formation. Apical surface determinants and membrane appear to accumulate along the track, possibly by spreading from existing apical concentrations. This track may also enable the fusion cells to contract and to draw the existing lumenal surfaces closer, as fusion cells appear notably less compact in shot mutant embryos (Lee, 2002).

Loss-of-function shot and gain-of-function RhoA alleles have similar phenotypes in fusion cells, and RhoA disrupts Shot localization. It is therefore proposed that RhoA negatively regulates track assembly and E-cadherin contact remodeling by Shot. Apically organized F-actin and adherens junctions in other tracheal cells appear to develop normally in shot mutant and RhoAV14 embryos, suggesting specific requirements for shot and RhoA during new apical surface formation in fusion cells. It is proposed that Shot and RhoA regulate E-cadherin-dependent cell adhesion in selected developmental contexts (Lee, 2002).

The analysis also indicates that RhoA is required for lumen formation, most probably by regulating the apical cytoskeleton or by affecting the transport of lumenal antigens. Similarities between RhoAV14 and shot mutant phenotypes suggest that RhoA could work either to antagonize Shot activity, or through parallel pathways acting on F-actin and microtubules. RhoA has many effectors that control F-actin distribution. RhoAV14 has been reported to stabilize subsets of microtubules in fibroblasts in culture via an F-actin-independent pathway. Shot localizes apically via its interactions with the cytoskeleton, and either these interactions or the cytoskeletal structures themselves may be RhoA-regulated (Lee, 2002).

In cells throughout the trachea, reduced RhoA activity disrupts lumen formation and partially disrupts Dlt localization. Tracheal expression of RhoAN19 does not appreciably affect E-cadherin localization. In cultured epithelial cells, E-cadherin localization is also resistant to RhoAN19. These findings are consistent with RhoA functioning downstream of or parallel to E-cadherin. E-cadherin-associated p120ctn negatively regulates RhoA, but whether a similar pathway operates in Drosophila is unknown (Lee, 2002).

In fusion cells, RhoA can also function upstream of E-cadherin, as constitutively active RhoAV14 affects E-cadherin localization selectively in these cells. E-cadherin distribution is more dynamic in fusion cells than in other tracheal cells, and may therefore be more sensitive to RhoAV14. RhoAV14 also affects new E-cadherin contacts in culture. Further experiments will reveal whether Shot, RhoA and E-cadherin function in a common, evolutionarily conserved pathway to regulate apical surface remodeling in fusion cells (Lee, 2002).

Specification of leading and trailing cell features during collective migration in the Drosophila trachea

The role of tip and rear cells in collective migration is still a matter of debate and their differences at the cytoskeletal level are poorly understood. This study analysed these issues in the Drosophila trachea, an organ that develops from the collective migration of clusters of cells that respond to Branchless (Bnl), a FGF homologue expressed in surrounding tissues. Individual cells in the migratory cluster were tracked and their features were characterized; two prototypical types of cytoskeletal organization were unveiled that account for tip and rear cells respectively. Indeed, once the former are specified, they remain as such throughout migration. Furthermore, it was shown that FGF signalling in a single tip cell can trigger the migration of the cells in the branch. Finally, specific Rac activation was found at the tip cells, and how FGF-independent cell features such as adhesion and motility act on coupling the behaviour of trailing and tip cells was analyzed. Thus, the combined effect of FGF promoting leading cell behaviour and the modulation of cell properties in a cluster can account for the wide range of migratory events driven by FGF (Lebreton, 2013).

Among the tracheal branches from each placode, two grow towards the ventral side of the embryo, one in the anterior and the other in the posterior region of the segment, the lateral trunk anterior (LTa) and the lateral trunk posterior (LTp) respectively. By a combination of migration, intercalation and elongation, the tip cell of the LTp migrates towards the central nervous system (CNS), and the resulting ganglionic branch (GB) connects the CNS to the main tracheal tube. Another cell from the LTp migrates towards the LTa of the adjacent posterior metamere and makes a fusion branch that connects the two LT branches. This study focused on this branch (LTp/GB) because its complex morphology and pattern of migration make it particularly appropriate for analysing the morphology and behaviour of the tip and trailing cell during tracheal collective migration (Lebreton, 2013).

The FGF signalling pathway is involved in many morphogenetic events requiring collective migration of cell clusters. However, it is not entirely clear whether in these events FGF signalling is directly involved in triggering cell migration, or alternatively if it is required for other processes such as cell determination which only affect cell migration indirectly. Moreover, while FGF might be required it is not clear either whether all the cells or just a subset of those need to directly receive the signal to sustain the migration of the entire cluster. One well-studied case is the role of FGF in the development of the zebra fish lateral line. In that case, FGF appears to be produced by the leading cells which signal to the trailing cells, the cells where FGF signalling is active. Restriction of FGF signalling is thereafter required for the asymmetric expression of the receptors for the chemokines that guide migration (Lebreton, 2013).

A very different scenario applies in the case of Drosophila tracheal migration. On the one hand, FGF is expressed in groups of cells outside the migrating cluster. On the other hand the results in the LTp/GB indicate that FGF signalling is required and sufficient in the leading cells, and not in the trailing cells, for the migration of the whole cell cluster. Therefore, in spite of its widespread involvement, the mechanisms triggered by FGF signalling in collective migration appear to be quite different (Lebreton, 2013).

Rho inactivation produced breaks and detachment in the LTp/GB cluster while its constitutive activation led these cells to hold together impairing migration. Likewise, upon Cdc42 inactivation LTp/GB cells were associated by thin extensions associated in some cases with breaks, while upon its constitutive activation, the LTp/GB transient pyramidal organisation did not evolve, or evolved much more slowly, towards branch elongation. However, the phenotypes from each RhoGTPase mutants don't look alike and the detailed analysis suggests that Rho impinges primarily on cell adhesion while Cdc42 does so on cell motility (Lebreton, 2013).

These results are consistent with previous findings that show a role for Rho in regulating adherens junctions stability and for Cdc42 as the main mediator of filopodia formation. It is noted, however, that Cdc42 was found to exert in the LTp/GB an opposite effect to the one identified in other systems, as Cdc42DN mutants showed more protrusions and were more actin-enriched basally than wild-type cells and Cdc42ACT mutants showed a reduced the motility of LTp/GB (Lebreton, 2013).

There is an increasing amount of data pointing to the different effect of RhoGTPases in vitro versus in vivo models and also among various cell types. A unidirectional assignment between a specific cellular process in vivo and a single RhoGTPase is probably an oversimplification and this was not the aim of the current study. Rather the study relied on mutant forms of the RhoGTPases to modulate cell features, either individually or collectively, to assess their role in the overall behaviour of the cell cluster. In doing so, the results point to a critical role for a balance between cell adhesion and cell motility for the collective migration of a cell cluster (Lebreton, 2013).

The results support the following model for the specification, features and behaviour of leading cells in the migration of the LTp/GB branch. Upon signalling from the FGF pathway, tip cells reorganise their cytoskeleton features (actin enrichment at the basal membrane, small apical surface and an apicobasal polarity along the proximo-distal axis), thereby enabling them to acquire leading behaviour. Indeed, FGF can induce migratory capacity to the whole cluster by signalling only the tip cells, where a dynamic transition between states of Rac activity is needed to acquire a leading role. How the behaviour of tip cells leads collective migration thereafter depends on the features of the cells in the cluster, which are determined by various regulators (among these, the RhoGTPases) which act, at least in part, in an FGF-independent manner. Ultimately, the balance between individual cell properties such as cell adhesion, motility and apicobasal polarity will (1) determine the net movement of the overall cell bodies or alternatively changes in cell shape in terms of elongation, (2) control the migratory speed and (3) define whether cells will migrate individually or in clusters and whether clusters will bifurcate in different paths. The combined effect of the changes promoting leading cell behaviour and modulation of cell features is likely to be a widely exploited mechanism in collective migration. In particular, the actual balance between these cell features may dictate the specifics of each migratory process and, consequently, the final shape of the tissues and organs they contribute to generate (Lebreton, 2013).

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells

Double-stranded RNA-mediated interference (RNAi) was used to study Drosophila cytokinesis. Double-stranded RNAs for anillin, RacGAP50C, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. The phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and led to a proposal that the central spindle and the contractile ring are interdependent structures. Finally, these results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis (Somma, 2002).

The finding that chicadee, four wheel drive (fwd), and Kinesin-like protein at 3A (klp3A) are not required for cytokinesis in S2 cells is not surprising, because previous studies pointed toward a specific involvement of these genes in meiotic cytokinesis of males. Null mutations in klp3A, a gene encoding a kinesin-like protein expressed both in testes and somatic tissues, disrupt meiotic cytokinesis but have no effect on larval neuroblast division. Similarly, flies homozygous for null mutations in fwd, which encodes a phosphatidyl-inositol kinase, are viable but male sterile, and are specifically defective in male meiotic cytokinesis. In contrast with fwd and klp3A that are not required for viability, chic is an essential gene that specifies a Drosophila homolog of profilin. However, both male sterile chic mutants and heteroallelic chic combinations resulting in lethality, display severe disruptions in meiotic cytokinesis but have no defects in neuroblast cytokinesis (Somma, 2002).

It was initially surprising to find that RNAi depletion of the Pnut protein, which shares homology with the yeast septins, does not markedly affect cytokinesis in S2 cells. This protein concentrates in the cleavage furrow of several Drosophila cell types; null pnut mutants die at the larval/pupal boundary and exhibit polyploid cells in their brains, consistent with a defect in cytokinesis. It is possible that the lack of an effect in pnut (RNAi) cells reflects a small amount of residual Pnut protein in these cells. However, it is instead believed that Pnut's role in cytokinesis is not fundamental to the process. The larval brains of null pnut mutants were reexamined and the presence of polyploid cells was confirmed. However, polyploid cells represent only 10.5% of the mitotic figures, indicating that most neuroblasts can undergo cytokinesis even in the absence of Pnut. In addition, Pnut is not required for cytokinesis during either male meiosis or the cystoblast divisions in the female germline. Taken together, these findings indicate that the Pnut function is either partially or totally dispensable for cytokinesis in Drosophila (Somma, 2002).

The phenotypical analyses of RNAi-induced mutants in the RacGAP50C, rho1, and sqh genes provide the first description of the cytological defects that lead to cytokinesis failures when the function of these genes is ablated. Previous studies have shown that mutations in rho1 and sqh disrupt mitotic cytokinesis but have not defined the cytological phenotypes elicited by these mutations. In addition, pav and pbl (RNAi) cells have been characterized; the phenotypes of these (RNAi) cells are consistent with those previously observed in animals homozygous for mutations in these genes (Somma, 2002 and references therein).

Cells in which the RacGAP50C, pav, pbl, rho1, and sqh genes are ablated by RNAi normally undergo anaphase A, but they then fail to elongate and to undergo anaphase B. After anaphase A, mutant cells proceed toward telophase and decondense their chromosomes, forming typical telophase nuclei. However, these cells fail to develop a central spindle, to assemble an actomyosin contractile ring and to concentrate anillin in the cleavage furrow. This results in the formation of short, aberrant telophases that are unable to undergo cytokinesis and will thus give rise to binucleated cells (Somma, 2002).

The functional ablation of genes influencing either the actin or the microtubule cytoskeleton have similar effects on cytokinesis. The genes pbl, rho1, and sqh likely play primary roles in controlling the actin cytoskeleton. The sqh gene encodes a regulatory light chain of myosin II. Rho1 is a member of the Rho family GTPases that cycle from an inactive GDP-bound state to an active GTP-bound state under the regulation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs enhance the exchange of bound GDP for GTP, whereas GAPs increase the GTPase activity of Rho. Rho proteins and Rho GEFs, such as Drosophila Pbl and human ECT2, localize to the cleavage furrow and are required for contractile ring assembly. In contrast, the activities of RacGAP50C and pav are likely to primarily influence the function of the central spindle. The Pav kinesin-like protein, a homolog of the C. elegans ZEN-4, is localized in the central spindle, and is thought to mediate microtubule cross-linking at the central spindle midzone. The RacGAP50C gene encodes a Rho GAP, and it is orthologous to the cyk-4 gene of C. elegans. CYK-4 interacts with ZEN-4, and the two proteins are mutually dependent for their localization to the central spindle. The complete absence of Pav immunostaining in RacGAP50C (RNAi) telophases suggests a similar interaction between RacGAP50C and Pav, pointing to a role of RacGAP50C in central spindle assembly. In summary, the cytological phenotypes of pbl, rho1, and sqh (RNAi) cells indicate that a primary defect in acto-myosin ring formation results in a secondary defect in central spindle assembly. The phenotypes of RacGAP50C- and Pav-depleted cells suggest the converse: that a primary defect in the central spindle can secondarily disrupt contractile ring formation. Thus, taken together, these data indicate that the central spindle and the actomyosin ring are interrelated structures. Although the molecular mechanisms underlying the cross talk between these structures is not conpletely understood, two possibilities can be envisioned. The formation and maintenance of both the central spindle and the actomyosin ring could be mediated by physical interactions between interzonal microtubules and components of the contractile ring. Alternatively, the central spindle and the contractile ring could be coupled by a checkpoint-like regulatory mechanism, which would inhibit the formation of either of these structures when the other is not properly assembled (Somma, 2002).

Although RacGAP50C, pav, pbl, rho1, and sqh (RNAi) cells display similar terminal phenotypes, the aberrant telophases observed in these cultures differ in both actin and anillin distribution. In rho1 telophases these proteins are excluded from the cell equator, in pbl they are uniformly distributed, and in RacGAP50C, pav, and sqh they concentrate in a wide equatorial band. This suggests that rho1 and pbl are required for actin and anillin accumulation in the equatorial region of the dividing cell. In contrast RacGAP50C, pav, and sqh seem to be required for the assembly of the contractile machinery from proteins already concentrated at the cell equator. In sqh (RNAi) cells the failure to assemble an actomyosin ring is likely to be a direct consequence of the depletion of an essential component of the ring. In RacGAP50C and pav cells this failure is instead likely to be a secondary effect of problems in central spindle assembly (Somma, 2002).

An interplay between the central spindle and the contractile ring has been suggested by studies on Drosophila male meiosis. Mutant spermatocytes in the chic, and dia loci, which encode products thought to be involved in contractile ring formation, and mutants in the kinesin-encoding gene klp3A, all display severe defects in both structures. Although all the extant results on Drosophila cells strongly suggest an interdependence of the central spindle and the contractile ring, it is currently unclear whether this is true in all animal cells. Studies on mammalian cells have shown that central spindle plays an essential role during cytokinesis. However, these experiments have provided limited information on whether perturbations in the actomyosin ring assembly disrupt the central spindle. The best evidence of an interplay between the central spindle and the contractile ring has been in rat kidney cells. By puncturing these cells with a blunt needle a physical barrier is created between the central spindle and the equatorial cortex. This barrier not only abrogates actomyosin ring assembly on the side of perforation facing the cortex, but also disrupts the organization of central spindle microtubules on the opposite side (Somma, 2002 and references therein).

In contrast, studies on C. elegans embryos indicate that, at least in the early stages of cytokinesis, the actomyosin ring and the central spindle can assemble independently. Why do Drosophila, and possibly mammalian cells, differ from C. elegans in the interactions between the central spindle and the contractile ring? It is believed that the answer to this question reflects differences in the distance between the central spindle and the equatorial cortex. In Drosophila and mammalian cells during central spindle assembly the equatorial cortex is very close to the interzonal microtubules. In contrast, in C. elegans embryos the central spindle assembles in the center of the cell when the cleavage furrow has just began to ingress, so that during their assembly the actomyosin ring and the central spindle lie a considerable distance apart. Only later in cell division, after substantial furrow ingression, can the actomyosin ring and the central spindle come into contact. It is thus hypothesized that in embryonic cells of C. elegans the cytokinetic process consists of two steps: an early step, where the central spindle and the contractile ring assemble independently in distant cellular regions, and a late step that begins when the central spindle and the contractile ring have come into contact. The early stage might be mediated by interactions between astral (rather than central spindle) microtubules and the contractile ring. The late step of C. elegans cytokinesis may then require that the contractile ring and the central spindle interact cooperatively to complete cytokinesis successfully. This two-step hypothesis also applies to other large cells, such as echinoderm eggs, where the central spindle and the cortex are separated by large masses of cytoplasmic material and seem to assemble independently (Somma, 2002 and references therein).

The syx1A gene, which encodes a t-SNARE, plays an essential role in embryonic cellularization, but its direct role in cytokinesis has not been demonstrated. In syx1A (RNAi) cells approximately half of the telophases are shorter that those of control cells and display severe defects in both the central spindle and the contractile ring. These findings are rather surprising, because there is abundant evidence that syntaxins are specifically involved in membrane fusion processes. Thus, the observations on syx1A (RNAi) cells raise the question of how a defect in membrane formation can affect both the central spindle and contractile ring assembly. Studies of C. elegans embryos depleted of the cytokinesis-specific Syntaxin-4 protein by RNAi have shown that in some of these embryos there is a complete failure of cleavage furrow ingression, suggesting an underlying defect in the contractile ring machinery. It has been thus proposed that formation of new membrane may positively regulate contractile ring assembly. In agreement with this hypothesis, it is suggested that RNAi-induced Syx1A depletion in S2 cells disrupts membrane formation at the site of cleavage furrow, causing a secondary defect in contractile ring formation and thus also in central spindle assembly (Somma, 2002).

Rho1 regulates signaling events required for proper Drosophila embryonic development

The Rho small GTPase has been implicated in many cellular processes, including actin cytoskeletal regulation and transcriptional activation. The molecular mechanisms underlying Rho function in many of these processes are not yet clear. In Drosophila, reduction of maternal Rho1 compromises signaling pathways consistent with defects in membrane trafficking events. These mutants fail to maintain expression of the segment polarity genes engrailed (en), wingless (wg), and hedgehog (hh), contributing to a segmentation phenotype. Formation of the Wg protein gradient involves the internalization of Wg into vesicles. The number of these Wg-containing vesicles is reduced in maternal Rho1 mutants, suggesting a defect in endocytosis. Consistent with this, stripes of cytoplasmic β-catenin that accumulate in response to Wg signaling are narrower in these mutants relative to wild type. Additionally, the amount of extracellular Wg protein is reduced in maternal Rho1 mutants, indicating a defect in secretion. Signaling pathways downregulated by endocytosis, such as the epidermal growth factor receptor (EGFR) and Torso pathways, are hyperactivated in maternal Rho1 mutants, consistent with a general role for Rho1 in regulating signaling events governing proper patterning during Drosophila development (Magie, 2005).

The data indicate that a number of signaling pathways important during early development in Drosophila are compromised in maternal Rho1 mutants. The observation that secretion of Wg protein is aberrant in these mutants together with the endocytosis defects observed in S2R+ cells treated with Rho1 dsRNA and in maternal Rho1 embryos indicates that Rho1 plays a general role in membrane trafficking processes in the early embryo. The biochemical mechanisms through which Rho proteins affect membrane trafficking are currently unclear. One possibility is that the function of Rho1 in this process is a byproduct of its regulation of the actin cytoskeleton. In yeast, there is evidence that the actin cytoskeleton is important in endocytosis, as mutations in actin and some actin-binding proteins inhibit endocytosis. In addition, yeast Rho1 has been shown to be involved in endocytosis of the α-receptor. In mammalian cells, treatment with pharmacological agents that perturb actin structure can affect endocytosis in a cell-type-specific way. In polarized epithelial cells, for example, treatment with the actin-depolymerizing drug cytochalasin D inhibits endocytosis specifically at the apical, but not the basolateral surface. RhoA has also been implicated in endocytosis in polarized epithelial cells. In Drosophila, Rho1 has clear roles in actin cytoskeletal regulation during oogenesis and embryogenesis, consistent with the notion that Rho1 may be acting primarily through its effects on the actin cytoskeleton (Magie, 2005).

The observation that the segmentation phenotype in maternal Rho1 mutants is the result of general defects in membrane trafficking processes (both secretion and endocytosis) and not a primary effect on transcriptional activation has important implications for the interpretation of data linking Rho to disparate cellular processes. While current data cannot exclude the possibility that Rho directly acts in transcriptional activation or through many disparate mechanistic pathways, data are accumulating that suggest Rho may act primarily as a regulator of the actin cytoskeleton and other functions it has been linked to are indirect effects. For instance, the ability of Rho to influence transcriptional activation through the serum response factor (SRF), as well as affect cell cycle progression, is due to its direct effects on actin cytoskeletal regulation. Identifying the molecular mechanisms underlying each of Rho's activities will be crucial to determining whether Rho1 has direct effects on a number of pathways or has a small number of primary functions that indirectly affect other functions. Investigations of Rho GTPase function in genetically amenable model organisms are providing a diversity of developmental contexts in which to examine all aspects of Rho biology, and the ability to examine specific, loss-of-function phenotypes will continue to aid identification of the mechanisms underlying Rho function (Magie, 2005).

Rho1 regulates Drosophila adherens junctions independently of p120ctn

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies (Magie, 2002) linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. This study examined the roles of these candidate AJ regulators during Drosophila development. It was found that although p120 is not essential for development, it contributes to morphogenesis efficiency, clarifying its role as a redundant AJ regulator. Rho has a dynamic localization pattern throughout ovarian and embryonic development. It preferentially accumulates basally or basolaterally in several tissues, but does not preferentially accumulate in AJs. Further, Rho1 localization is not obviously altered by loss of p120 or by reduction of core AJ proteins. Genetic and cell biological tests suggest that p120 is not a major dose-sensitive regulator of Rho1. However, Rho1 itself appears to be a regulator of AJs. Loss of Rho1 results in ectopic accumulation of cytoplasmic DE-cadherin, but ectopic cadherin does not accumulate with its partner Armadillo. These data suggest Rho1 regulates AJs during morphogenesis, but this regulation is p120 independent (Fox, 2005).

In mammalian cells, p120 is a key regulator of cadherin-based adhesion. However, the universality of this role was called into question by the viability of p120 mutant flies (Myster, 2003). One caveat remained, however. Magie (2002) reported that rapidly depleting fly embryos of p120 by RNAi led to defects in morphogenesis and Rho1 localization. To resolve this discrepancy, p120 RNAi was carried out. The data confirm that knockdown of p120 does not result in lethality. Thus, Drosophila and C. elegans p120 are dispensable for development. By contrast, p120 knockdown in Xenopus or Mus musculus is lethal, suggesting differences in the importance of p120 in vertebrates versus invertebrates (Fox, 2005).

Since Drosophila has a single p120 family member, simple redundancy does not explain the difference between vertebrates and invertebrates. Two possible explanations exist. p120 proteins may play fundamentally different roles in the two groups of animals. Alternatively, the role of p120 in both may be similar, but the relative importance of p120 and unrelated, partially redundant regulators of cadherin and/or Rho may differ. The latter possibility is favored, because p120 binds to and promotes the function of AJs in vertebrates and invertebrates, and p120 has a conserved role in regulating morphogenesis, contributing to dorsal closure efficiency and regulating dendrite morphology in Drosophila and regulating gastrulation and craniofacial morphogenesis in Xenopus. One role of p120 is to inhibit cadherin endocytosis. Perhaps in invertebrates other regulators of cadherin trafficking compensate for its absence (Fox, 2005).

The second postulated role for p120 is as a Rho regulator. The viability of p120 mutants suggested that Drosophila p120 is not an essential Rho1 regulator. However, this did not rule out with overlapping functions. For example, Magie (2002) suggested overlapping roles for p120 and alpha-catenin, with p120 regulating Rho1 localization during dorsal closure. Thus dose-sensitive genetic interactions were sought between p120 and Rho1. Loss of p120 did not substantially affect Rho1 function, as assessed by cuticle phenotype. Further, loss of p120 did not enhance or suppress the effect of Rho1 on F-actin or DE-Cad localization during dorsal closure. p120 overexpression had only a slight effect on the Rho1 phenotype, a result that may reflect variation in genetic background. Thus, although Drosophila p120 preferentially binds inactive Rho1 (Magie, 2002), it is not a major dose-sensitive regulator of Rho1 (Fox, 2005).

The hypothesis that p120 regulates Rho1 localization was tested by examining several places in which Rho1 exhibits striking subcellular localization, and examining the place where Rho1 exhibits its zygotic phenotype: the dorsal closure front. No change was seen in Rho localization in p120 mutants. Therefore, if p120 regulates Rho1 localization, it must do so redundantly with other putative Rho1 regulators, such as alpha-catenin (Magie, 2002). These data do not rule out the possibility that p120 recruits a pool of active Rho1, which may be only a small fraction of total cellular Rho1 (Fox, 2005).

p120 appears to regulate RhoA during Xenopus development. Perhaps redundant Rho regulators act in parallel to p120 in flies. Alternatively, the role of p120 as a Rho regulator may not be conserved: the N-terminal domain of p120, which is implicated in regulating transitions between its adhesive and cytoplasmic roles, is not well conserved between mammalian and fly p120. Since p120 (Myster, 2003) and Rho1 mutations modify shg mutant phenotypes differently, p120 and Rho1 may act in separate pathways to regulate AJs in Drosophila (Fox, 2005).

Previous analyses of Rho1 localization (Magie, 2002; Padash-Barmchi, 2005) are extended by this study. It is dynamic, with Rho1 accumulating at different subcellular sites in distinct cell types, some consistent with proposed Rho functions. For example, mammalian RhoA regulates integrin-based cell-matrix junctions. The basal localization of Rho1 raises the possibility that it may regulate integrins in Drosophila. Rho1 accumulation in mesodermal cells is consistent with its role in regulating cell shape during mesoderm spreading. Relative levels of cortical Rho1 decrease through development. Perhaps at later stages Rho1 is activated by localized RhoGEFs. Consistent with this, RhoGEF2 is more cortically enriched during dorsal closure than Rho1. Thus, future studies will need to examine the localization of Rho1 regulators and effectors. Recent advances also allow visualization of active Rho GTPases. As much of the Rho1 pool may be inactive, application of this approach to flies will advance understanding of Rho1 function (Fox, 2005).

It was previously proposed that Rho1 is enriched at Drosophila AJs and that this is regulated by core AJ proteins (Magie, 2002). The current study examined this in follicle cells and embryos. In follicle cells, Rho1 localized to lateral and apical membranes in early egg chambers, and to lateral membranes later. In neither case was enrichment observed in AJs, although Rho1 is not excluded from them. In embryonic epithelia, Rho1 sometimes localizes uniformly to the basolateral membrane, while in other places it is enriched basally. During dorsal closure, when Rho1 exhibits its zygotic phenotype, Rho1 accumulates basal to AJs. The lack of preferential Rho1 localization at AJs does not rule out accumulation of a pool of active Rho1 at AJs: this will require reagents to measure Rho1 activation in vivo. The hypothesis that AJs regulate Rho1 localization was tested. In follicle cells mutant for DE-Cad and embryos mutant for arm or DE-Cad during dorsal closure, Rho1 localization is not obviously disturbed (Fox, 2005).

In cultured cells, Rho and AJs have a complex relationship. Rho regulates AJ stability, and conversely AJs regulate Rho activity. Further, different Rho effectors can promote or decrease AJ stability in cultured mammalian cells. This complex relationship was examined during morphogenesis, using genetic and cell biological assays. The data support the hypothesis that Rho1 is an important regulator of cadherin-based adhesion during embryonic development (Fox, 2005).

Loss of Rho1 leads to DE-Cad mislocalization (Magie, 2002), while dominant-negative Rho1 reduces DE-Cad in AJs, implicating Rho1 in regulating DE-Cad localization. The current results support this hypothesis. Cytoplasmic DE-Cad accumulation is consistent with a role for Rho1 in regulating either DE-Cad transport to or recycling from AJs. It was observed that the ectopic DE-Cad in Rho1 mutants accumulates independently of its binding partner Arm. In mammalian cells, newly-synthesized E-cadherin must bind ß-catenin before it can be transported to AJs, while endocytosed E-cadherin accumulates with either no or reduced amounts of ß-catenin. Thus the data are more consistent with ectopic DE-Cad accumulating after endocytosis. Consistent with this, mammalian RhoA regulates clathrin-mediated endocytosis, and Drosophila Rho1 regulates endocytosis of the ligand Wingless (Magie, 2005). Further, constitutively active Rac1, which can inhibit RhoA, triggers E-cadherin recruitment to intracellular vesicles in keratinocytes. Since high levels of Rho1 do not accumulate at AJs, either a small pool of active Rho1 at AJs is sufficient to inhibit cadherin endocytosis or the effect is more indirect, with Rho1 acting on the actin cytoskeleton or regulators of endocytic trafficking. The mechanism by which Rho1 regulates DE-Cad trafficking is an interesting question for future studies (Fox, 2005).

Mammalian p120 also regulates cadherin endocytosis. The viability of fly p120 mutants suggests that in flies this role is not rate limiting, although the enhancement of mutants with reduced DE-Cad by p120 is consistent with p120 playing a similar role (Myster, 2003). p120 and Rho could regulate DE-cad trafficking via the same or distinct pathways. The effect on DE-Cad trafficking in zygotic Rho1 mutants, which should have limiting levels of maternal Rho1, is not enhanced by removing p120. This is more consistent with a model in which the two proteins work in different pathways, and in which p120 acts partially redundantly with another unknown regulator (Fox, 2005).

Analysis of Rho1 and Rho1 shg mutants is consistent with the hypothesis that Rho1 regulates AJs, but suggests that their interactions are complex. A weak shg allele was enhanced, but stronger alleles were suppressed. There are several possible explanations for these contrasting results. Weak alleles (e.g. shgG119) make protein with reduced but residual function. If Rho1 negatively regulates cadherin endocytosis, more mutant DE-Cad protein might be endocytosed in Rho1's absence, further reducing functional DE-Cad and enhancing the phenotype. However, null or very strong shg alleles accumulate no functional DE-Cad at AJs, rendering regulation of cadherin endocytosis a moot point. The slight suppression by Rho1 of strong shg alleles may result from a reduction of morphogenetic movements, reducing cuticle disruption. Alternatively, some mutant DE-Cad proteins may be capable of coupling to Rho1 while others are not. Rho1 can bind alpha-catenin (Magie, 2002), and active Rho1 may be recruited to AJs by that interaction. shgG119 has a wild-type cytoplasmic domain and could presumably couple to Rho1; reducing Rho1 might further impair its function. By contrast, the shg2 mutation may impair Arm and/or alpha-catenin binding and thus Rho1 recruitment; if so this mutant protein would not be further impaired by Rho1 removal. Finally, the complex genetic interactions might reflect different requirements for Rho1 during neuroblast delamination and head involution, which are affected by strong or weak reduction in DE-Cad function, respectively. Future studies of Rho regulation of and by AJs will help distinguish between these possibilities (Fox, 2005).

Antagonistic roles of Rac and Rho in organizing the germ cell microenvironment

The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments. However, how microenvironments are shaped is poorly understood. In testes of Drosophila, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells. Germ cells signal via the Egf class ligand Spitz (Spi), and evidence is provided that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppresses the germ cell enclosure defects of the conditional spi allele. It is proposed that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. These novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage (Sarkar, 2007).

In the male gonad of Drosophila, germ cells are surrounded by somatic cells that define their cellular microenvironmen. Germline stem cells (GSCs) are attached to a cluster of nondividing cells at the apical tip, called hub cells, and associated with cyst progenitor cells (CPCs) that act as stem cells for the somatic support cell lineage. Two CPCs extend their cytoplasm around one GSC, toward the hub, and toward each other such that each GSC appears to be completely enclosed in its cellular microenvironment. GSCc and CPCs generate differentiating daughters, called gonialblasts and cyst cells, respectively. The gonialblasts undergo transit amplification divisions to produce 16 spermatogonia, which become spermatocytes, grow in size, undergo the meiotic divisions, and differentiate into sperm. Two cyst cells grow cytoplasmic extensions around one gonialblast to form the germ cell cellular microenvironment that controls various aspects of germ cell differentiation (Sarkar, 2007).

Germ cells associated with somatic cells mutant for the Map-Kinase Raf fail to differentiate and accumulated as early-stage germ cells instead. A similar accumulation of early-stage germ cells was observed in Egfrts mutant testes shifted to nonpermissive temperature, and in testes from animals mutant for Stem cell tumor (Stet; Rhomboid2), a protease that cleaves Egfr ligands. However, stet mutant germ cells in addition fail to associate with somatic support cells, suggesting that the Egfr pathway is required for setting up the critical cellular microenvironment (Sarkar, 2007).

Loss of spi results in a failure of germ cells to differentiate, similar to the effects of loss of stet or the Egfr. Wild-type testes are long (~2 mm) tubular structures that contain germ cells in a spatio-temporal order along the apical-to-basal axis. Early germ cells (GSCs, gonialblasts, and spermatogonia) are small and have small, densely packed nuclei in DAPI-stained preparations. Spermatocytes are located basal to the spermatogonia, and differentiating spermatids fill the distal part of the testis (Sarkar, 2007).

Animals carrying a temperature-sensitive allele of spi, spi77-20, die when raised at 29°C. However, spi77-20 animals raised at a slightly permissive temperature (27°C) survive and have tiny testes. Most of these testes (40 of 50) contain only small cells, as seen at the tip of wild-type testes, and do not have spermatocytes or differentiating spermatids. Staining with molecular markers revealed that the testes contains increased numbers of GSCs, gonialblasts, and spermatogonia compared to wild-type. The remaining testes (10 of 50) have high numbers of early germ cells and a few spermatocytes, but no differentiating spermatids (Sarkar, 2007).

Testes from spi77-20 animals raised at an intermediate permissive temperature (25°C) are longer than testes from animals raised at 27°C, but significantly shorter (500 μm-1.5 mm) than wild-type testes. A substantial part of the testes is occupied by tumor-like aggregates of early-stage germ cells. However, spermatocytes and differentiating spermatids are also present (Sarkar, 2007).

spi activity is both sufficient and required within the germ cells. Expression of a cleaved version of Spi (sSpi) in germ cells but not in somatic support cells of spi77-20 testes restores the wild-type phenotype, and germ cell clones mutant for spi accumulate at early stages based on phase-contrast microscopy and DAPI-stained preparations) (Sarkar, 2007).

spi was also required for somatic support cells to associate with and enclose the germ cells. Germ cell clones mutant for the conditional spi77-20 allele from animals raised at 27°C either do not associate with somatic support cells or associated with only one somatic support cell (4 of 20 clones), based on staining with soma-specific antibodies, such as the transcription factor Traffic Jam (Tj). Tj labels the nuclei of somatic support cells that are normally associated with early-stage germ cells (Sarkar, 2007).

Germ cell enclosure can be investigated by labeling testes with molecular markers such as antibodies against the membrane-bound β-catenin Armadillo (Arm) that labels the cell membranes of somatic support cells as they surround the germ cells. In wild-type testes, each GSC, gonialblast, and cluster of developing germ cells is associated with and surrounded by two somatic support cells. In testes from spi77-20 animals raised at 27°C, Tj-positive cells did not form cytoplasmic extensions around the germ cells. Similar results were obtained with other markers, including a cytoplasmic UAS-Green Fluorescent Protein (UAS-GFP) expressed in somatic support cells under control of a soma-specific Gal4-driver. In control testes, GFP is detected in the cell bodies of the somatic cells surrounding the germ cells. In contrast, in spi77-20 testes from animals grown at 27°C, GFP is detected in balls, most likely small round cell bodies of somatic support cells. Occasionally, cytoplasmic extensions emerged from somatic support cells, but they remained short and did not enclose the germ cells (Sarkar, 2007).

The lack of cytoplasmic extensions from Tj-positive cells in spi77-20 mutant testes is similar to the phenotype observed in stet mutants. This suggests that the Egf class ligand Spi, expressed in germ cells and processed by Stet, stimulates the Egfr on somatic support cells, inducing them to send out cytoplasmic extensions to enclose the neighboring germ cells (Sarkar, 2007).

Association of germ cells with somatic support cells is sensitive to the level of Spi. Germ cell clones from spi77-20 animals raised at 25°C and germ cell clones from animals mutant for the spi2 allele often associated with more than two somatic support cells (Sarkar, 2007).

The growth of cytoplasmic extensions around the germ cells is also sensitive to the level of Spi. When spi77-20 animals are raised at 25°C, many Tj-positive cells form cytoplasmic extensions directed toward and/or around the germ cells. However, not every germ cell cluster appear to be associated with and/or surrounded by somatic support cells. Furthermore, many of the Tj-positive cells forme cytoplasmic extensions toward each other, suggesting that multiple somatic support cells may surround one tumor-like aggregate of germ cells. Similar abnormal associations of somatic support cells with germ cells are also seen in Egfrts mutants shifted to nonpermissive temperature. One possible explanation for the different phenotypes of loss compared to reduction of Egfr signaling is that different levels of Egfr stimulation may affect different cellular properties of somatic support cells, such as cell adhesiveness and/or growth (Sarkar, 2007).

To identify novel players in germ cell enclosure, the sensitized background of the spi77-20 allele was used to search for genetic modifiers. It was found that impaired activity of the small monomeric GTPase (small GTPase) Rac1 enhances the spi77-20 testes phenotype. Activity of Rac1 was impaired by two strategies-either by removing one copy of the rac1 gene or by expressing a dominant-negative version of Rac1 (dnRac1) in somatic support cells of testes from spi77-20 animals raised at 25°C. In either case, the enhanced testes are shorter than testes from spi77-20 animals raised at 25°C. In 12 of 20 enhanced testes, Tj-positive cells do not enclose the germ cells, and early-stage germ cells accumulate (Sarkar, 2007).

Reducing activity of Vav, a guanine nucleotide exchange factor for Rac-type small GTPases, from somatic support cells by antisense expression also enhances the spi77-20 testes phenotype from animals raised at 25°C. 11 of 20 enhanced testes were tiny and contained mostly early-stage germ cells that were not surrounded by somatic support cells. The enhanced phenotypes caused by impairing Rac or Vav raises the possibility that Rac1 and Vav act downstream of the Egfr in somatic support cells and that Vav plays a role in regulating somatic support cell-shape changes associated with germ cell enclosure (Sarkar, 2007).

In mammalian cells, autophosphorylation of specific Vav-binding motifs within the cytoplamic tail of the Egfr allows for binding and phosphorylation of mammalian Vav2. Phosphorylation of Drosophila Vav has been shown to depend on Egfr stimulation in both mammalian and Drosophila cultured cells, and Drosophila Vav bound to mammalian Egfr (Sarkar, 2007 and references therein).

Consistent with a role for Drosophila Vav in Egfr signaling in testes, Vav protein immunoprecipitates from testis extracts with an antibody against the Egfr. Vav does not immunoprecipitate from testis extracts that had been pretreated with phosphatase, suggesting that the interaction between Vav and the Egfr is phosphorylation dependent. The immunoprecipitated Vav band comigrates with a band detected by antibodies against phospho-tyrosine, suggesting that Vav is phosphorylated when in a complex with the Egfr (Sarkar, 2007).

In the classical view of the Drosophila Egfr pathway, only one docking protein-Downstream receptor kinase (Drk)-binds to the stimulated Egfr and activates a MAP-Kinase cascade for transcription of target genes. However, genetic and biochemical data suggest that the Egfr pathway is branched at the level of docking proteins and that the adaptor protein Vav binds to the Egfr to activate the small GTPase Rac1. These data suggest that Rac regulates cell-shape changes associated with germ cell enclosure, and studies on Raf suggested that it regulates the transcription of target genes. However, the possibility of crosstalk between the two branches cannot be excluded: Vav may contribute to transcriptional regulation and Map-Kinases may contribute to germ cell enclosure. A possible crosstalk is consistent with findings that in cultured Drosophila cells (Hornstein, 2003), Vav can contribute to Erk phosphorylation (Sarkar, 2007).

Surprisingly, impairing activity of the Rho-type small GTPase Rho1 has the opposite effect to impairing Rac1. Testes from spi77-20 animals raised at 27°C that express dominant-negative Rho1 (dnRho1) in somatic support cells are long and appear almost wild-type. In contrast to somatic support cells in spi77-20 testes from animals raised at 27°C without dnRho1 expression, the somatic support cells expressing dnRho1 enclose the germ cells. The same dominant suppression is observed in spi77-20, rho1/+ testes, indicating that expression of dnRho1 reflects loss of Rho1 activity (Sarkar, 2007).

These data raise the possibility that Rac and Rho have antagonistic effects on germ cell enclosure. Rac appears to be required for somatic support cells to grow cytoplasmic extensions around the germ cells, and Rho appears to suppress this growth. Antagonistic roles for Rac and Rho have also been reported in cultured mammalian cells, where Rac and Rho regulate cell-shape changes and growth via different effects on the actin cytoskeleton. Prominent readouts for small GTPase activities on the actin cytoskeleton are the appearances of ruffles and lamellipodia in the cell membranes (Sarkar, 2007).

To address a potential role of Rac and Rho in shape changes of somatic support cells, dominant-negative Rac or Rho were expressed in somatic support cells of otherwise wild-type testes, and transmission electron microscopy (TEM) was used to investigate changes in the membranes of somatic support cells surrounding single germ cells and spermatogonia at the apical tip of the testes. Germ cells and somatic support cells can be identified based on their different shapes and density of staining in TEM. In wild-type, the somatic support cells surrounding single germ cells and spermatogonia exhibit wavy plasma membranes, possibly analogous to membrane ruffles accompanying cellular growth and rearrangements of the actin cytoskeleton in cultured cells (Sarkar, 2007).

Somatic support cells expressing dnRac1 have much smoother plasma membranes than do wild-type somatic support cells. Conversely, somatic support cells expressing dnRho1 have lamellipodia-like extensions in their membranes. Lamellipodia-like extensions were not detected in somatic support cell membranes in serial sections of wild-type testes or in testes expressing dnRac1. In mammalian cells, formation of lamellipodia depends on Rac-type small GTPases. The presence of lamellipodia-like extensions in somatic support cells expressing dnRho1 suggests that Rac may become hyperactive in the absence of Rho. Based on these TEM data, it is hypothesized that, just as their mammalian counterparts do in cultured cells, Drosophila small GTPases may act on the cytoskeleton of somatic support cells to mediate cell-shape changes and growth of cellular extensions and that the effects of Rac and Rho are antagonistic (Sarkar, 2007).

This model predicts that expression of a constitutively active Egfr ligand in somatic support cells might compromise the differential in smGTPase activities. Indeed, forced expression of cleaved ligand in somatic support cells, but not in germ cells, closely mimics the effect of dnRho expression: the somatic support cells formed lamellipodia-like structures in their membranes (Sarkar, 2007).

This research on the Drosophila gonad provides a striking example how one cell type in tissue communicates with another cell type to induce and direct the formation of a proper cellular microenvironment: a signal from one cell induces subcellular changes throughout the body of another cells. This mechanism underlying the formation of a cellular microenvironment may be conserved across species (Sarkar, 2007).

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

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

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

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

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

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

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

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

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

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

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

Rho GTPase controls Drosophila salivary gland lumen size through regulation of the actin cytoskeleton and Moesin

Generation and maintenance of proper lumen size is important for tubular organ function. This study reports on a novel role for the Drosophila Rho1 GTPase in control of salivary gland lumen size through regulation of cell rearrangement, apical domain elongation and cell shape change. Rho1 controls cell rearrangement and apical domain elongation by promoting actin polymerization and regulating F-actin distribution at the apical and basolateral membranes through Rho kinase. Loss of Rho1 results in reduction of F-actin at the basolateral membrane and enrichment of apical F-actin, the latter accompanied by enrichment of apical phosphorylated Moesin. Reducing cofilin levels in Rho1 mutant salivary gland cells restores proper distribution of F-actin and phosphorylated Moesin and rescues the cell rearrangement and apical domain elongation defects of Rho1 mutant glands. In support of a role for Rho1-dependent actin polymerization in regulation of gland lumen size, loss of profilin (Chickadee) phenocopies the Rho1 lumen size defects to a large extent. Ribbon, a BTB domain-containing transcription factor, functions with Rho1 in limiting apical phosphorylated Moesin for apical domain elongation. These studies reveal a novel mechanism for controlling salivary gland lumen size, namely through Rho1-dependent actin polymerization and distribution and downregulation of apical phosphorylated Moesin (Xu, 2011).

Rho1 acts both in salivary gland cells and in the surrounding mesoderm to maintain apical polarity during gland invagination and to mediate cell shape change during gland migration. This study demonstrates a novel role for Rho1 in controlling salivary gland lumen size through regulation of actin polymerization and distribution and regulation of Moesin activity. By analyzing Rho1 alleles for which salivary gland cells invaginated and formed a gland, it was shown that zygotic loss of function of Rho1 results in shortening and widening of the gland lumen, which is accompanied by defects in cell shape change and cell rearrangement and failure of apical domains to elongate along the Pr-Di axis of the gland. These effects of Rho1 are mediated through Rok; inhibition of Rok completely phenocopies loss of Rho1 in these cellular events. Based on these studies, a model is proposed for Rho1 control of salivary gland lumen size, in particular lumen width, which is determined by cell rearrangement and apical domain elongation. Rho1 and Rok, through inhibition of cofilin, regulate cell rearrangement and apical domain elongation by promoting actin polymerization to localize F-actin at the basolateral membrane and by limiting the apical accumulation of F-actin. In parallel to its role in actin polymerization and distribution, Rho1 acts independently of Rok to limit apical p-Moe with Rib by an unknown mechanism and this function of Rho1 is specific for apical domain elongation. The data on cofilin (Twinstar) are consistent with those in cultured HeLa cells that showed that mammalian ROCK can inhibit cofilin activity indirectly through LIMK-mediated phosphorylation of cofilin (Xu, 2011).

Although manipulating Moe activity through gland-specific expression of MoeT559D was sufficient to completely phenocopy the Rho1 lumen defects, including cell rearrangement, it did so without disrupting actin polymerization or distribution. This is likely to be due to activated Moe strengthening the link between the actin cytoskeleton and the apical plasma membrane (without affecting levels of apical F-actin), which would increase apical membrane stiffness and remove the ability of gland cells to rearrange. Indeed, Moesin has been shown to control cortical rigidity during mitosis of cultured Drosophila S2R+ cells. Thus, Rho1 regulates cell rearrangement and apical domain elongation by controlling the actin cytoskeleton and Moesin activity through distinct mechanisms (Xu, 2011).

The observation that chic mutant glands phenocopy Rho1 mutant glands to a large extent, suggests that Rho1 control of salivary gland lumen size is mainly dependent on a requirement for Rho1 in actin polymerization. However, as the chic and Rho1 gland lumen phenotypes are not identical, with chic mutant glands lacking the apical accumulation of F-actin and p-Moe observed in Rho1 mutant glands, Rho1 probably has an additional function in limiting accumulation of F-actin and p-Moe at the apical membrane. This function of Rho1, at least for limiting apical F-actin, might partly involve Rab5- or Shi-mediated endosome trafficking, because inhibition of Rab5 alone or Shi alone led to accumulation of F-actin at the apical membrane. Although Rab5DN- or ShiDN-expressing salivary gland cells were enriched with apical F-actin, lumen size was not affected. This could be due to Rab5DN and ShiDN affecting a pool of apical F-actin distinct from that affected by Rho1 and/or because Rab5DN-expressing gland cells retain basolateral F-actin and the ratio of apical to basolateral F-actin is not altered sufficiently to cause lumen size defects. In Rho11B mutant gland cells, some early endosomes were not coated with F-actin. Actin is known to contribute to multiple steps of the endocytic pathway, including movement of endocytic vesicles through the cytoplasm and their transport to late endosomes and lysosomes. One possible mechanism by which Rho1 normally limits apical accumulation of F-actin is by promoting its removal from the apical membrane and accumulation on endocytic vesicles (Xu, 2011).

Currently, it is not know how Rho1 limits accumulation of apical p-Moe. Membrane localization and activity of Moesin can be regulated via a number of mechanisms, such as its phosphorylation on a conserved Threonine residue, binding to phosphatidylinositol-(4,5)bisphosphate [PtdIns(4,5)P2] and association with components of the sub-membrane cytoskeleton, such as Crb. Studies in cultured mammalian cells have demonstrated that Rho signaling activates Moe either through phosphorylation of Moe by ROCK or through ROCK-mediated inhibition of myosin phosphatase, which is known to dephosphorylate p-Moe. Although it is possible that Drosophila Rho1 positively regulates Moe activity by one or more of these mechanisms, this study shows that in the developing salivary glands Rho1 in fact negatively regulates Moe activity. In rib mutant embryos, in which p-Moe is enriched apically, salivary gland and tracheal cells showed decreased staining for Rab11 GTPase, which localizes to the apical recycling endosomes and to secretory vesicles destined for the apical membrane. Thus, Rho1, like Rib might limit apical p-Moe through its membrane transport (Xu, 2011).

In Drosophila imaginal disc epithelia, Moe negatively regulates Rho1 activity to maintain epithelial integrity and to promote cell survival. These studies demonstrating that in the developing salivary gland Rho1 antagonizes Moe activity by limiting its localization at the apical membrane, shed novel insight into the functional relationship between Rho1 and Moe. It is possible that in a dynamic epithelium, such as the developing salivary gland, Rho1 contributes to the precise spatial and temporal regulation of Moe activity to fine-tune selective changes in apical domain shape. By contrast, in the imaginal disc epithelium, Rho1 regulation of Moe might not be necessary and, instead, Moe regulation of Rho1 activity is required to maintain epithelial integrity and cell survival. Thus, Rho and Moe can antagonize each other's activities depending on the type of epithelia or cellular event (Xu, 2011).

Rescue studies with Rho1WT demonstrate that Rho1 functions predominantly in the salivary gland cells to control apical domain elongation and cell rearrangement. Interestingly, expression of Rho1WT in the mesoderm with twi-GAL4 has no effect on cell rearrangement and has little effect on apical domain elongation and lumen size, whereas it has been shown that Rho1WT expression in the mesoderm significantly rescues the gland migration defect of Rho11B mutant embryos. This suggests that gland migration and lumen size control are regulated by distinct mechanisms. In support of this conclusion, embryos mutant for multiple edematous wings, encoding the ?PS1 integrin subunit, which have defects in gland migration, show no defects in gland lumen width. Identifying the distinct and overlapping mechanisms by which salivary gland lumen width and length are controlled will help to elucidate the mechanisms by which lumen size is controlled in tubular organs (Xu, 2011).

Galpha73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in Drosophila haematopoiesis

JAK/STAT signalling regulates multiple essential developmental processes including cell proliferation and haematopoiesis while its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. These studies show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, while Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. This study shows that Galpha73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, it was also shown that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. These findings link JAK/STAT transcriptional outputs, Galpha73B activity and Rho1-dependent cytoskeletal rearrangements/cell motility and therefore connect a pathway associated with cancer with a marker indicative of invasiveness. As such, this study suggests a mechanism via which JAK/STAT pathway signalling may promote metastasis (Bausek, 2013).

Brumby, A. M., Goulding, K. R., Schlosser, T., Loi, S., Galea, R., Khoo, P., Bolden, J. E., Aigaki, T., Humbert, P. O. and Richardson, H. E. (2011). Identification of novel Ras-cooperating oncogenes in Drosophila melanogaster: a RhoGEF/Rho-family/JNK pathway is a central driver of tumorigenesis. Genetics 188: 105-125. PubMed ID: 21368274

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

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


Rho1: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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