the role of rho family gtpases in development: lessons from drosophila melanogaster

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Molecular Cell Biology Research Communications 1, 87–94 (1999)

Article ID mcbr.1999.0119, available online at http://www.idealibrary.com on

INIREVIEW

he Role of Rho Family GTPases in Development:essons from Drosophila melanogaster

u Lu and Jeffrey Settlemanassachusetts General Hospital Cancer Center and Harvard Medical School, Building 149,

3th Street, Charlestown, Massachusetts 02129

eceived March 15, 1999

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The Rho family of Ras-related GTPases comprises anxpanding group of small GTP-binding proteins thategulate a variety of cellular functions (1). Genes en-oding Rho-related proteins have been identified ineveral organisms, including mammals, insects,lants, and yeast, indicating that they have beenighly conserved throughout evolution. Like their Rasounterparts, these GTPases function as molecularwitches, cycling between active GTP-bound and inac-ive GDP-bound states to mediate intracellular signalransduction pathways in response to extracellulartimuli. Functional studies of the three prototype Rhoamily GTPases, Rho, Rac, and Cdc42, have revealedhat these proteins perform essential functions in aariety of biological processes, including cell cycle pro-ression and gene transcription. However, their mostntensively studied function is that of cytoskeletal reg-lation (2).Several of the Rho family proteins can regulate actin

ytoskeleton remodeling in response to extracellularignals. When microinjected into fibroblasts, activatedho stimulates the formation of actin stress fibers and

ocal adhesions (3), Rac induces membrane rufflinglamellipodia formation) (4), and Cdc42 promotes theormation of filopodia (5). Moreover, inhibiting partic-lar Rho family GTPases in a variety of experimentalystems blocks certain cellular response to extracellu-ar stimuli, confirming their critical function as signalransduction intermediates. As potent regulators of thectin cytoskeleton, these proteins have also beeninked to cellular processes that determine cell shape,

otility, and adhesive properties (2). Thus, it has beenuggested that the Rho GTPases are likely to be im-ortant regulators of the numerous morphogeneticvents associated with the development of multicellu-ar organisms. Although it is generally appreciatedhat Rho GTPases are critical switching components

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heir precise mechanism of action and the organizationf their associated signal transduction pathways areust beginning to be elucidated.

iochemical Regulation of Rho GTPases

Much like their Ras counterparts, Rho GTPasesunction as molecular switches, and their ability toycle between active and inactive states is tightly reg-lated (1). While their intrinsic GTPase activities areelatively slow, the hydrolysis of bound GTP by acti-ated Rho proteins can be greatly accelerated by aamily of proteins called GTPase activating proteinsGAPs) (1). Thus, the GAPs promote GTPase inactiva-ion. Activation of the Rho GTPases is promoted by alass of proteins named guanine nucleotide exchangeactors (GNEFs). The GNEFs appear to function bytabilizing a nucleotide-free form of the GTPase, suchhat it can bind GTP (which is considerably more abun-ant in the cell than GDP), thereby becoming activated1). Together, these regulatory proteins provide strin-ent control over the nucleotide state of the GTPases,nd appear to link the activation of the Rho GTPases toell surface signals through mechanisms that are justeginning to be elucidated.

ho GTPase-Associated Signaling Pathways

Upon activation to the GTP-bound form, GTPasesndergo a conformational change that allows them to

nteract with so-called downstream effector targets,hich contribute to the cellular response to GTPasectivation. During the past few years, numerous pro-eins have been identified that bind specifically to thective GTP-bound Rho GTPases. Many of these pro-eins exhibit specific interactions with a particular RhoTPase, although a few of them appear to be sharedmong different Rho proteins (1). Thus, it has been

1522-4724/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

difficult to establish the mechanisms by which signal-i

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Vol. 1, No. 2, 1999 MOLECULAR CELL BIOLOGY RESEARCH COMMUNICATIONS

ng specificity is achieved in vivo.Many of the putative GTPase effector targets are pro-

ein kinases. For example, the Rho GTPase associatespecifically with several identified protein kinases, in-luding the PKC-related PKN (6, 7) and PRK2 kinases (8,), the ROK (Rho Kinase) family of serine/threonine ki-ases (10), and the Citron kinase (11). The closely relatedac and Cdc42 GTPases associate with a distinct familyf kinases referred to as the PAK (p21-activated) kinases12). These kinases are all moderately activated uponinding to the GTP bound forms of the respectiveTPases, suggesting that upon GTPase activation, a sig-al is transduced to these protein kinases that leads tohe phosphorylation of substrates. However, for most ofhese kinases, the relevant substrate targets remain un-dentified. In addition to these protein kinases, severalipid kinases have also been identified as putative Rhoffector targets (13–15).Much less is known about the upstream signaling

athways that lead to activation of the Rho GTPases.ho proteins can be activated by various extracellulartimuli, such as LPA (lysophosphatidic acid), growthactors, bombesin and bradykinin (1). The LPA, brady-inin and bombesin receptors belong to the seven-ransmembrane-domain family and are linked to het-rotrimeric G proteins, which may be required forctivating Rho GTPases. The Rho/Rac GNEFs appearo be regulated by a variety of factors, including phos-horylation, lipid interactions, and membrane localiza-ion, indicating that diverse regulatory inputs may betilized to promote their ability to activate the Rhoamily GTPases (1). Despite these observations, theignaling pathways leading from cell surface receptorctivation to Rho/Rac/Cdc42 GTPase activation areargely unknown.

enetic Analysis of Rho GTPase Signalingin Drosophila

As described above, biochemical studies in mammalianystems have yielded substantial information regardinghe biochemical regulation of Rho GTPases, and the iden-ification of many of their downstream targets. However,he biological significance of these findings, in most cases,emains somewhat elusive. As an alternative approach,enetic analyses of the evolutionarily conserved RhoTPases in simpler organisms, such as Drosophila mela-ogaster, have begun to provide additional clues to theirunction. Drosophila, a model organism that is ideallyuited to genetic and developmental studies, has beenarticularly useful in deciphering the organization of theas-mediated signal transduction pathway (16). Re-

ently, this system has proved to be equally as powerful aool to study Rho-associated signaling pathways in an inivo context.

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onents of mammalian signaling pathways appear toe well conserved, provides a setting in which loss-of-unction and gain-of-function of particular genes in aariety of developmental processes can be directly ex-mined. The ability both to easily generate transgenicy lines and to perform genetic interaction studies, asell as mutational “modifier” screens, has provided

nvestigators with powerful tools for dissecting signal-ng pathways in vivo. In the following sections, recenttudies will be summarized in which the Drosophilaystem has been used to examine the regulation andunction of the Rho family GTPases in a variety ofevelopmental processes. As described below, thesetudies have revealed the power of this system in elu-idating the role of the Rho GTPases and their associ-ted signaling pathways in regulating the morphoge-etic events required for the normal development ofulticellular organisms.

he Drosophila Rho GTPases and Their Expressionin Embryogenesis

At least five Drosophila Rho GTPases have beendentified thus far (17–20). These include Rho1, Rac1,ac2, Cdc42, and RhoL, which are 70-90% identical inmino acid sequence to their mammalian counterparts.hese GTPases are expressed throughout embryogen-sis, and some are widely expressed in many tissuessuch as Rho1 and Rac2) while others exhibit moreestricted expression in the mesoderm, gut, and ner-ous system later in development (such as Rac1 anddc42) (17, 18). This is consistent with the notion thatho GTPases are important regulators of actin cy-

oskeletal changes required for the numerous morpho-enetic events in Drosophila development. In the nextections, studies addressing the role of Rho familyTPases in several of the well studies aspects of Dro-

ophila development are summarized.

ogenesis

Drosophila oogenesis takes place in an organ struc-ure referred to as the egg chamber. Each egg chamberonsists of 15 nurse cells and a single oocyte that areonnected through actin-rich structures called ring ca-als, and the 16-cell germ line is covered by a single

ayer of somatic cells known as follicle cells. In additiono the ring canals, the cortical regions of all germ cellsnd the adherent junction type connections betweenhe germ cells and follicle cells also contain high con-entrations of filamentous actin (21). During oogenesis,urse cells eventually “dump” all of their cytosolic con-ents to the oocyte through the ring canals using anctin-myosin contractile system, while the follicle cellsndergo characteristic shape changes and migrationshat may be cytoskeleton-mediated. Therefore, it wasredicted that Rho GTPases play a role in oogenesis.

In one study, dominant-negative forms of Rac1,CcmmfdbcItct

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dc42 and RhoL were specifically expressed in a spe-ialized subset of follicle cells (border cells) which nor-ally migrate to the anterior tip of the egg chamberidway through oogenesis (19). While no requirement

or Cdc42 or RhoL was observed, expression ofominant-negative Rac1 nearly completely blockedorder cell migration without affecting other follicleells, suggesting a specific role of Rac1 in this process.n the same study, it was demonstrated that while allhree GTPases are required for transfer of nurse cellontents to oocytes, they each perform additional cellype specific functions during oogenesis.

astrulation

Drosophila gastrulation commences immediately fol-owing the cellularization of the syncitial blastoderm.his evolutionary conserved developmental process in-olves major morphogenetic events including ventralurrow formation and midgut invagination (22). Ven-ral furrow formation is largely dependent onytoskeleton-mediated cell shape changes within aroup of mesodermal precursors located along theength of the mid-ventral surface of the embryo, andesults in the internalization of the presumptive meso-erm. In slightly later developmental stages, similarell shape changes are responsible for the invagina-ions of anterior and posterior endodermal primordia,hich subsequently give rise to the midgut. These cell

hape changes involve actin cytoskeleton-mediatedonstriction of the apical membranes followed by aell-shortening event (23).

Recent studies revealed that a Rho GTPase signalransduction pathway controls the gastrulation process24, 25). Specifically, a Drosophila RhoGEF (dRhoGEF2)as identified that was found to be essential for cell

hape changes in gastrulation. Furthermore, expressionf a dominant-negative Rho1 mutant in early embryoslocks ventral furrow formation as well as midgut invag-nation, suggesting that activation of Rho1 by dRhoGEF2s essential for cell shape changes required for gastrula-ion (24). Two previously identified Drosophila gastrula-ion mutants, folded gastrulation (fog) (26) and concer-ina (cta) (27), exhibit very similar defects to that ofRhoGEF2. Interestingly, Fog is an extracellular ligandor an unidentified membrane receptor and Cta is a Gubunit of a heterotrimeric G protein that may be coupledo the Fog receptor. It is tempting to speculate that Fogctivates a receptor coupled G-protein containing Cta,hich in turn activates the down stream Rho1 GTPase

hrough dRhoGEF2 and ultimately leads to cell shapehanges in gastrulation. Indeed, ectopic fog expressionnduces cell shape changes that can be blocked in eitherhe cta (28) or dRhoGEF2 mutant background (24). Annalogous pathway has been described in mammalianells in which LPA activates the LPA receptor coupled

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-protein, leading to subsequent activation of the RhoTPase via a RhoGEF (29), indicating that this path-ay of Rho activation has been conserved evolutionarily

Fig. 1).

orsal Closure

Dorsal closure in Drosophila development occursidway through embryogenesis. It is a major morpho-

enetic event in which embryonic epidermal cellstretch along their dorsal-ventral axis. Consequently,he two lateral epidermal cell sheets eventually meett the dorsal midline, thereby closing the dorsal side ofhe embryo. The process involves neither cell divisionor migration, and is solely dependent on cytoskeleton-ediated cell shape changes within a subset of embry-

nic cells (30). Defects in dorsal closure result in a holen the dorsal side of the larval cuticle, and can easily beetected. Recently, the Rho family proteins and theirssociated pathways have been implicated in dorsallosure (31–33).The mutant phenotype of one of the Rho GTPases

as been described. Rho1 null allele mutants display aorsal hole in the larval cuticle (33) (Fig. 2). Similarefects are seen in embryos expressing dominant-egative Rac1 (31) or Cdc42 (32) using a heat-shockromoter, suggesting a role for all three of theseTPases. Interestingly, mutants of several kinaseathway genes that function downstream of the respec-ive Rho GTPases in mammalian system also exhibitorsal closure defects. For example, mutations in theNK cascade kinases, including hemipterous (the Jun-terminal Kinase Kinase) (34) and basket (JNK) (Fig.

FIG. 1. Evolutionary conserved Rho-mediated signal transduc-ion pathways for cell shape changes required for Drosophila gastru-ation (left panel) and neuronal cell shape changes in mammalsright panel).

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arget gene puckered (JNK phosphatase) (42), all resultn dorsal closure defects.

Recent studies have established a dorsal closureodel in which Rac and/or Cdc42 signals through the

NK cascade to induce expression in the leading edgeells (the dorsal-most role of epidermal cells known tonitiate the dorsal-ventral stretching of the two epider-

al cell sheets) of Dpp, a secreted TGF-b-like ligand. Itppears that secreted Dpp then signals the stretchingf the more lateral epidermal cells, resulting in theovement of the two cell sheets toward the midline

36–39). Thus far, the precise role of Rho1 in thisrocess has not been determined.The identity of signaling components that initiate

he dorsal closure process is still unclear. In particular,he pathways by which the Rho and Rac GTPases arectivated to initiate the stretching of epidermal cells,hich is required for dorsal closure, have not yet beenlucidated. However, it is likely that GNEFs will benvolved, as well as membrane receptors and/or adapt-rs. One such candidate is mbc (myoblast city), a genehich is also required for dorsal closure and appears to

unction as a Rac-specific upstream regulator of theorsal closure process (43, 44). Mbc was identified in autational modifier screen designed to identify specific

omponents of the Rac signaling pathway, and theuman Mbc homologue, DOCK180, was subsequentlyound to function as an upstream regulator of Racctivity (44). Thus, the genetic approach was able toeveal the function of a human protein whose cellularole was previously unknown.

uscle Development

As in vertebrates, Drosophila muscle developmentnvolves the fusion of myoblasts to form mature synci-

FIG. 2. Cuticle preparations of (A) wild type, (B) Rho172O (a nullllele of Drosophila Rho1), and (C) bsk1 (a strong loss-of-functionllele of Drosophila JNK), showing dorsal closure defects in basketbsk) and Rho1 mutant embryos.

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ssembly of actin filaments (46), suggesting that RhoTPases might be involved. Indeed, one study using

ransgenic flies expressing mutant forms of RhoTPases has revealed a role for Rac in myoblast fusion

18). Expression of dominant-negative Rac1 initiallyelays the fusion process, followed by excessive fusionf myoblasts, while constitutively active Rac1 com-letely blocks fusion, indicating that precise control ofhe Rac activity is essential for normal myoblast fu-ion. Despite a high degree of structural similarityetween Rac and Cdc42, similar experiments usingdc42 transgenic flies did not reveal a role of Cdc42 inyoblast fusion, indicating that this is a Rac specific

unction and that closely related Rho GTPases performifferent biological functions.The signaling pathway involved in the Rac-mediatedyoblast fusion is unknown. However, it was found

hat the Rac regulator, mbc, is also required for myo-last fusion (43). Although it is unclear from the Mbcrotein sequence how Rac might be activated by Mbc, its known that the mammalian Mbc homologue,OCK180, associates with the adapter protein Crk

47), which binds the RacGEF, Vav (48). This raises theossibility that a multi-protein complex containingbc might function to recruit Rac to its GNEF, thereby

ctivating the Rac mediated signaling pathway(s) re-uired for both myoblast fusion and dorsal closure.

eural Development

The neuronal growth cone is a highly dynamic actin-ich structure located at the tip of a growing axon. Therganized rearrangement of axonal actin structures iseminiscent of the Rac induced lamellipodia and Cdc42nduced filopodia seen in fibroblasts; moreover, highxpression levels of both Rac and Cdc42 in the Dro-ophila nervous system have been observed during latetages of embryogenesis (18). Therefore, a role for Racr Cdc42 in axon outgrowth and guidance during de-elopment is expected. Expression of dominant-egative Rac1 in neuroblasts or in the mature centralnd peripheral nervous system caused embryonic le-hality associated with severe loss of axons (but notendrites), suggesting a specific role of Rac in axonutgrowth initiation (18). Similar but more severe ax-nal losses were observed when activated Rac wasxpressed. In addition, by expressing activated Rac atlightly later stages of neuronal development so thatxon outgrowth is initiated, it was determined thatroper Rac activation is required for axon elongation.xpression of mutant forms of Cdc42 was found toause qualitatively different neuronal defects (18). No-ably, activated Cdc42 inhibited both axon and den-rite outgrowth. Moreover, affects on neuronal positionn addition to axon outgrowth were observed, suggest-

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ng an additional role of Cdc42 in neuronal migration.urther studies are required to examine the defectsssociated with the loss-of-function mutants of bothTPases before firm conclusions regarding the normal

oles of Rac and Cdc42 in neuronal development can berawn.The organization of Rac and Cdc42 mediated signal

ransduction pathways in Drosophila neuronal devel-pment remain largely unknown. However, a few stud-es suggest that some of the same types of componentssed in other Rho signaling pathways are involved. Forxample, a Drosophila RhoGEF, still life, was recentlydentified in a genetic screen searching for motorctivity-defective mutants (49). Still life expression inate embryogenesis is restricted in neurons, predomi-

FIG. 3. Scanning electron microscopy images of Drosophila eGMR-Cdc42 transgenic flies, showing the “rough-eye” phenotypes inyes, respectively.

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antly in the synaptic regions, suggesting a role inynaptic function, and still life mutant flies exhibiteduced locomotion. Transgenic flies expressing a pu-ative activated form of the Still life protein in neuronsisplay defects in motor axon elongation and formationf synaptic arbors on target muscles. In another report,xpression of a dominant-negative form of Rac1 in neu-ons caused axons associated with the intersegmentalerve b to bypass and extend beyond their normalynaptic targets (50). This “bypass” phenotype is en-anced by the absence of the receptor-tyrosine phos-hatase, DLAR, which has also been implicated inotor axon guidance (51). Since the mammalian homo-

ogue of DLAR interacts directly with Trio, a proteinhat contains two Rac/Rho GEF-like domains (52), it is

from (A) wild type, (B) pGMR-Rho1, (C) pGMR-Rac1, and (D)nsgenic flies that overexpress Rho1, Rac1, and Cdc42 in Drosophila

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ossible that DLAR activates Rac1 through a Trio-likeEF to mediate an axon guidance signal.

ye Development

Excessive activity of the Rho GTPases perturbs theormal development of the eye (17). When overex-ressed in transgenic lines, wild type forms of Rho1,ac1, Rac2, and Cdc42 can each disrupt the normalmmatidial structure of the eye and result in an exter-ally “rough” phenotype (Fig. 3). For example, expres-ion of a Rho1 transgene causes a rough-eye phenotypessociated with disruption of the morphology of thehotoreceptors (17). In addition, the lattice formed byecondary and tertiary pigment cells as well as theeneral ommatidial architecture is completely dis-upted. Furthermore, disruption of the normal appear-nce of the polymerized actin in pupal eye discs fromhese transgenics suggests that the ability of Rho1 toffect actin organization plays an important role in eyeorphogenesis. Expression of Rac1, Rac2, and Cdc42TPases in the eye similarly disrupts normal eye de-elopment, although the observed defects are differentn each case, indicating that this system is useful foristinguishing the activities of closely related RhoTPases in vivo (44).

issue Polarity

Polarized epithelial cells perform a variety of special-zed functions and epithelial polarity plays an impor-ant role in Drosophila morphogenesis. Drosophilapithelial cells are derived from the invaginated

Summary of Rho GTPase Signaling Components Involvedin Various Drosophila Developmental Processes

Developmentalprocesses

Genes/Pathwaysinvolved References

ogenesis: border cellmigration

Rac1 (19)

ogenesis: transfer ofnurse cell contentsto oocytes

Rac1, Cdc42, RhoL (19)

astrulation Fog, cta, DRhoGEF2,Rho1

(24–27)

orsal closure Rho1, Rac1, Cdc42, JNKpathway genes, Dpppathway genes, andmbc

(31–44)

uscle development Rac1, mbc (18, 43)eural development Rac1, Cdc42, still life (18, 49, 50)ye development Rho1, Rac1, Rac2,

Cdc42(17, 44)

issue polarity: wingdevelopment

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issue polarity: eyedevelopment

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(33)

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uring larval development. These discs eventually giveise to all adult tissues. Aside from the commonly ob-erved apical-basal polarity in epithelial tissue cultureells, epithelial cells can also develop a planar polarityelative to the body axis (53). This is reflected in theppearance of distally pointed hairs on the Drosophilaing blades and the regular arrays of trapezoidal

haped rhabdomeres in the Drosophila compound eye.To study the role of Rho GTPases in establishing

issue polarity in the fly wing disc, mutant forms ofac1 and Cdc42 were expressed specifically in wingiscs (54). From those experiments, it was found thatac1 activity was essential for the proper assembly ofell adherens junctions as well as for the establishmentf planar polarity, while Cdc42 was found to be re-uired for epithelial cell shape changes, but is notequired for actin assembly at adherens junctions. Nei-her of these GTPases was required to maintain polar-ty once it was established. In addition, detailed exam-nation revealed distinct functions for Rac and Cdc42n regulating different aspects of wing hair formation55). Specifically, expression of dominant-negativedc42 stunts wing hair growth, leading to a failure ofing hair cells to accumulate actin in their distal re-ions. This suggests that Cdc42 might be responsibleor polarized membrane outgrowth which is analogouso the Cdc42 stimulated filopodia formation in fibro-lasts. On the other hand, dominant-negative Rac1romotes the outgrowth of multiple hairs from a singleell, which also exhibit gaps in junctional actin and aisorganized apical microtubule web, indicating thatac1 is responsible for both actin polymerization andicrotubule organization.The existing Rho1 mutant further confirmed the role

f Rho GTPases in establishing tissue polarity in wingevelopment (33). Wing clones containing hypomor-hic Rho1 alleles exhibit abnormal wing hair polarity.imilarly, in Rho1 eye clones, a large number of om-atidia were incorrectly oriented while the relative

osition of photoreceptors and accessory cells remainnaltered. This is the same phenotype seen in tworeviously identified tissue polarity mutants: frizzlednd disheveled. Frizzled appears to be a G-protein cou-led receptor, and disheveled is a cytoplasmic signal-ng molecule. Genetic experiments established thatho1 functions downstream of the frizzled-disheveledediated tissue polarity pathway (33), which is consis-

ent with the general theme linking G-protein coupledeceptors to the Rho signaling pathway.

ummary

It has become increasingly clear in the last few yearshat the Rho family GTPases regulate cytoskeletonearrangements that are essential for a variety of mor-hogenetic events associated with the development of

multicellular organisms. In particular, Drosophila hasptwmufiitTmiiiiifiiac

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rovided an excellent in vivo system for decipheringhe signaling pathways mediated by Rho GTPases, asell as establishing the role of these pathways in nu-erous developmental processes (Table I). Continuedse of this system will undoubtedly lead to the identi-cation of additional Rho signaling components and

nformation regarding the function and organization ofhe Rho signaling pathways in tissue morphogenesis.he striking similarity between Drosophila and mam-alian Rho signaling components identified thus far

ndicates that the Rho pathways are highly conservedn evolution. Therefore, the findings from the Drosoph-la system can be extrapolated to higher organisms,ncluding humans. Combined with the rapid progressn the human and Drosophila genome projects, thesendings should contribute greatly to our understand-

ng of mammalian Rho GTPase signaling pathwaysnd their roles in normal development and pathologi-al conditions.

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the role of rho family gtpases in development: lessons from drosophila melanogaster

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