Isozyme-specific stimulation of phospholipase C-gamma2 by Rac GTPases

The regulation of the two isoforms of phospholipase C-gamma, PLCgamma(1) and PLCgamma(2), by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCgamma isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCgamma may also be regulated by Rho GTPases. In this study, PLCgamma(1) and PLCgamma(2) were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCgamma activity. PLCgamma(2), but not PLCgamma(1), was markedly activated in intact cells by constitutively active Rac1(G12V), Rac2(G12V), and Rac3(G12V) but not by Cdc42(G12V) and RhoA(G14V). The mechanism of PLCgamma(2) activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCgamma(2)-activating protein-tyrosine kinases. Activation of PLCgamma(2) by Rac2(G12V) in intact cells coincided with a translocation of PLCgamma(2) from the soluble to the particulate fraction. PLCgamma isozyme-specific activation of PLCgamma(2) by Rac GTPases (Rac1 approximately Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5'-(3-O-thio)triphosphate caused a marked stimulation of PLCgamma(2) but had no effect on the activity of PLCgamma(1). PLCgamma(1) and PLCgamma(2) have previously been shown to be indiscriminately activated by PtdInsP(3) in vitro. Thus, the results suggest a novel mechanism of PLCgamma(2) activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP(3).     

Hematopoietic-specific Rho GTPases Rac2 and RhoH and human blood disorders

The small guanosine triphosphotases (GTPases) Rho proteins are members of the Ras-like superfamily. Similar to Ras, most Rho GTPases cycle between active GTP-bound, and inactive GDP-bound conformations and act as molecular switches that control multiple cellular functions. While most Rho GTPases are expressed widely, the expression of Rac2 and RhoH are restricted to hematopoietic cells. RhoH is an atypical GTPase that lacks GTPase activity and remains in the active conformation. The generation of mouse knock-out lines has led to new understanding of the functions of both of these proteins in blood cells. The phenotype of these mice also led to the identification of mutations in human RAC2 and RHOH genes and the role of these proteins in immunodeficiency diseases. This review outlines the basic biology of Rho GTPases, focusing on Rac and RhoH and summarizes human diseases associated with mutations of these genes.     

Differential regulation of cyclooxygenase 2 expression by small GTPases Ras, Rac1, and RhoA

Cyclooxygenase 2 (COX-2) is an immediate early gene induced by a variety of stimuli and its expression is stimulated by individual activation of Ras or Rho GTPases. Here we investigate the role of coordinate activation of Ras and Rho GTPases in the induction of COX-2. Individual expression of constitutively active Ras, RhoA, or Rac1 was capable of stimulating COX-2 expression in NIH3T3 cells, but co-expression of constitutively active RhoA with either constitutively active Ras or Rac1 was required for full stimulation of COX-2 expression. Serum growth factors differentially activated Ras, RhoA, and Rac1, which correlated with the activation of Raf-1, ERK, and c-Jun as well as with induction of COX-2. Inhibition of Ras significantly blocked the activation of Raf-1, ERK, and c-Jun and the stimulation of COX-2 expression in response to serum. In contrast, inhibition of Rho family GTPases partially blocked serum induction of ERK activation but had little effects on COX-2 expression. Both inhibitors of MEK (PD098059) and JNK (SP600125) inhibited serum induction of COX-2. PD98059 only inhibited constitutively active Ras-induced COX-2 expression, while SP600125 significantly inhibited both constitutively active Ras- and RhoA-induced COX-2 expression. Together, our data suggest that constitutively active oncogenic Ras and Rho coordinately stimulate COX-2 expression whereas transient activation of Ras but not RhoA or Rac1 mediates the induction of COX-2 in response to serum. Furthermore, ERK and JNK activation are both required for serum- and oncogenic Ras-mediated COX-2 expression whereas only JNK activation is required for oncogenic RhoA-mediated stimulation of COX-2 expression.     

Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

Structural basis for the signaling specificity of RhoG and Rac1 GTPases

RhoG is a new GTPase that has high sequence similarity with members of the Rac subfamily (Rac1, Rac2, and Rac3), including the regions involved in effector recognition and binding. To characterize its biological properties, we have compared the activity of RhoG and Rac1 in a number of experimental systems, including the study of their subcellular localization, oncogenic potential, activation of effectors, and effect on F-actin dynamics. Our study indicates that RhoG and Rac1 share overlapping, but not identical, signal transduction pathways. In contrast to previous results, we also provide evidence that RhoG works in parallel to Rac1 rather than as a Rac1 upstream activator. Using an extensive collection of Rho/Rac1 chimeras and point mutants, we demonstrate that the different biological properties of RhoG and Rac1 can be traced to specific amino acid variations in their switch I, beta2/beta3 hairpin, alpha5 helix, and C-terminal polybasic regions. Taken collectively, our results highlight the complexity of the signal transduction pathways activated by Rho/Rac GTPases and provide insight into the structural determinants that mediate the differential engagement of biological responses by GTPases of very similar structure.     

Small GTPases Rap1 and RhoA regulate superoxide formation by Rac1 GTPases activation during the phagocytosis of IgG-opsonized zymosans in macrophages

Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b(558). In addition, neutrophils from rap1A(-/-) mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22(phox). 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22(phox), and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22(phox) that activates NADPH oxidase and produces superoxide.     

Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2

Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian erythroblasts.     

RhoA and Rac1 GTPases mediate the dynamic rearrangement of actin in peripheral glia

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, we specifically labeled the peripheral glia of Drosophila melanogaster using an Actin-GFP marker and analyzed their development in the embryonic PNS. 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. We 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. We 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.     

Comparative functional analysis of the Rac GTPases

Small GTPases of the Rho family including Rac, Rho and Cdc42 regulate different cellular processes like reorganization of the actin cytoskeleton by acting as molecular switches. The three distinct mammalian Rac proteins share very high sequence identity but how their specificity is achieved is hitherto unknown. Here we show that Rac1 and Rac3 are very closely related concerning their biochemical properties, such as effector interaction, nucleotide binding and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis support the idea that altered dynamics of Rac2 at the switch I region may be responsible for different biochemical properties. These results provide insight into the individual functionalities of the Rac isoforms.     

TGFbeta1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA

TGFbeta is a potent regulator of cell differentiation in many cell types. On aortic endothelial cells, TGFbeta1 displays angiogenic properties in inducing capillary-like tube formation in collagen I gels, in vitro. We investigated cytoskeletal changes that precede tube formation and related these alterations to the effects of TGFbeta1 on the activation state of members of the RhoGTPase family. TGFbeta1 promotes cell elongation and stress fiber formation in aortic endothelial cells. Using cell lines with inducible expression of Rac1 mutants, we show that these events are mimicked by expression of dominant-negative Rac1 whereas the constitutively active mutant prevents the TGFbeta1-mediated change of phenotype. Although TGFbeta1 induces an initial rise in the Rac1-GTP content, this phase is followed by a prolonged loss of the active form. In contrast, RhoA activity increases progressively and reaches a plateau when Rac1-GTP is no longer detectable. Prolonged inhibition of Rac1 appears necessary and sufficient for the increase in RhoA-GTP. In situ examination of Rho activity in TGFbeta1-treated cells provides evidence that active RhoA relocalizes to the tips of elongated cells. Inhibiting the Rho effector ROCK abrogates tube formation. Thus, Rac1 and RhoA are regulated by TGFbeta1 in the process of endothelial tube formation in collagen I gels.     

Rac1 and RhoA GTPases have antagonistic functions during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts

Background information. N‐cadherin, a member of the Ca²⁺‐dependent cell—cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N‐cadherin‐dependent cell—cell contacts in myoblasts remain unexplored. Results. In the present study, we show that N‐cadherin‐dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N‐cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N‐cadherin‐dependent cell contacts, characterized by the immobilization of a pool of N‐cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F‐actin filaments. We also demonstrated that the formation of N‐cadherin‐dependent cell—cell contacts requires a co‐ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N‐cadherin‐dependent cell—cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N‐cadherin‐dependent cell contact formation, since, via ROCK (Rho‐associated kinase) signalling and myosin 2 activation, it allows the stabilization of N‐cadherin at the cell—cell contact sites. Conclusions. We have shown that Rac1 and RhoA have opposite effects on N‐cadherin‐dependent cell—cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.     

Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.     

Redundant functions of Rac GTPases in inner ear morphogenesis

Development of the mammalian inner ear requires coordination of cell proliferation, cell fate determination and morphogenetic movements. While significant progress has been made in identifying developmental signals required for inner ear formation, less is known about how distinct signals are coordinated by their downstream mediators. Members of the Rac family of small GTPases are known regulators of cytoskeletal remodeling and numerous other cellular processes. However, the function of Rac GTPases in otic development is largely unexplored. Here, we show that Rac1 and Rac3 redundantly regulate many aspects of inner ear morphogenesis. While no morphological defects were observed in Rac3(-/-) mice, Rac1(CKO); Rac3(-/-) double mutants displayed enhanced vestibular and cochlear malformations compared to Rac1(CKO) single mutants. Moreover, in Rac1(CKO); Rac3(-/-) mutants, we observed compromised E-cadherin-mediated cell adhesion, reduced cell proliferation and increased cell death in the early developing otocyst, leading to a decreased size and malformation of the membranous labyrinth. Finally, cochlear extension was severely disrupted in Rac1(CKO); Rac3(-/-) mutants, accompanied by a loss of epithelial cohesion and formation of ectopic sensory patches underneath the cochlear duct. The compartmentalized expression of otic patterning genes within the Rac1(CKO); Rac3(-/-) mutant otocyst was largely normal, however, indicating that Rac proteins regulate inner ear morphogenesis without affecting cell fate specification. Taken together, our results reveal an essential role for Rac GTPases in coordinating cell adhesion, cell proliferation, cell death and cell movements during otic development.     

Pathogenicity island-dependent activation of Rho GTPases Rac1 and Cdc42 in Helicobacter pylori infection

Helicobacter pylori has been identified as the major aetiological agent in the development of chronic gastritis and duodenal ulcer, and it plays a role in the development of gastric carcinoma. Attachment of H. pylori to gastric epithelial cells leads to nuclear and cytoskeletal responses in host cells. Here, we show that Rho GTPases Rac1 and Cdc42 were activated during infection of gastric epithelial cells with either the wild-type H. pylori or the mutant strain cagA. In contrast, no activation of Rho GTPases was observed when H. pylori mutant strains (virB7 and PAI) were used that lack functional type IV secretion apparatus. We demonstrated that H. pylori-induced activation of Rac1 and Cdc42 led to the activation of p21-activated kinase 1 (PAK1) mediating nuclear responses, whereas the mutant strain PAI had no effect on PAK1 activity. Activation of Rac1, Cdc42 and PAK1 represented a very early event in colonization of gastric epithelial cells by H. pylori. Rac1 and Cdc42 were recruited to the sites of bacterial attachment and are therefore probably involved in the regulation of local and overall cytoskeleton rearrangement in host cells. Finally, actin rearrangement and epithelial cell motility in H. pylori infection depended on the presence of a functional type IV secretion system encoded by the cag pathogenicity island (PAI).     

The role of Rac1 and Rac2 in bacterial killing

The small Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 have distinct roles in regulating neutrophil chemotaxis; however, little is known about their possible unique roles in mediating bacterial killing. To elucidate the relative roles of Rac1 and Rac2 in regulating neutrophil-mediated bacterial killing, we utilized the previously described mice model in which mouse neutrophils are deficient in either Rac1, Rac2, or both isoforms. We demonstrate here that while both Rac isoforms are required for normal neutrophil chemotaxis and bacterial killing, they have non-overlapping roles in bacterial phagocytosis and NADPH oxidase function.     

Nitric oxide causes macrophage migration via the HIF-1-stimulated small GTPases Cdc42 and Rac1

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of tumor development. Recently, the tumor microenvironment, with the presence of tumor-associated macrophages (TAMs), has gained considerable interest. The mechanisms of macrophage/TAM migration as well as the role of HIF-1 in macrophages for tumor progression are still under debate. We present evidence that under normoxic conditions, nitric oxide (NO) promotes macrophage migration. The response was impaired in macrophages from leukocyte conditional HIF-1α^−/− mice. NO production and cell migration in response to cytokines were attenuated in macrophages from iNOS^−/− mice, suggesting that iNOS-derived NO transmits cytokine signaling toward cell migration. We further identified the small GTPases Cdc42 and Rac1 as effectors of the NO–HIF axis to drive macrophage migration by modulating the actin cytoskeleton. Our observations strengthen the role of HIF-1 in macrophages as a target of NO in facilitating functional responses such as migration.     

Cdc42Hs and Rac1 GTPases induce the collapse of the vimentin intermediate filament network

In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002, R(p)-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events.     

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.     

Rac1 and Rac3 GTPases regulate the development of hilar mossy cells by affecting the migration of their precursors to the hilus

We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus.