Lowe syndrome protein Ocrl1 is translocated to membrane ruffles upon Rac GTPase activation: a new perspective on Lowe syndrome pathophysiology

Oculocerebrorenal Lowe syndrome is a rare X-linked disorder characterized by bilateral cataract, mental retardation and renal Fanconi syndrome. The Lowe syndrome protein Ocrl1 is a PIP2 5-phosphatase, primarily localized to the trans-Golgi network (TGN), which 'loss of function' mutations result in PIP2 accumulation in patient's cells. Although PIP2 is involved in many cell functions including signalling, vesicle trafficking and actin polymerization, it has been difficult so far to decipher molecular/cellular mechanisms responsible for Lowe syndrome phenotype. We have recently shown that, through its C-terminal RhoGAP domain, Ocrl1 forms a stable complex with Rac GTPase within the cell. In line with this finding, we report here that upon epidermal growth factor induced Rac activation in COS-7 cells, a fraction of Ocrl1 translocates from TGN to plasma membrane and concentrates in membrane ruffles. In order to investigate the functionality of Ocrl1 in plasma membrane, we have analysed PIP2 distribution in human dermal fibroblasts (HDFs) from Lowe patients versus control HDFs. As revealed by both immunodetection and green fluorescent protein-PH binding, PIP2 was found strikingly to accumulate in PDGF induced ruffles in Lowe HDFs when compared with control. This suggests that Ocrl1 is active as a PIP2 5-phosphatase in Rac induced membrane ruffles. Cellular properties such as cell migration and establishment of cell-cell contacts, which depend on ruffling and lamellipodia formation, should be further investigated to understand the pathophysiology of Lowe syndrome.     

The effect of missense mutations in the RhoGAP-homology domain on ocrl1 function

Lowe syndrome is a rare X-linked disease characterized by congenital cataracts, defects in renal tubule cell function, and mental retardation. Mutations in the OCRL1 gene, which encodes ocrl1, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) 5-phosphatase, are the cause of Lowe syndrome. PtdIns(4,5)P(2), a substrate of ocrl1, is an important signaling molecule within the cell. OCRL1 is ubiquitously expressed and co-localizes with the trans-Golgi network (TGN) and endosomal proteins. The ocrl1 protein contains two recognizable domains, one a conserved Ptd(4,5)P(2) 5-phosphatase domain and the other with homology to Rho GTPase activating proteins (RhoGAPs). The objective of our study was to further characterize the ocrl1 RhoGAP-homology domain by analyzing the effect of two missense mutations in this domain, I751N and A780P, which were previously reported in Lowe syndrome patients. Both mutant proteins were expressed at levels similar to wild-type but their enzyme activity was reduced by 85-90%, indicating that the RhoGAP-homology domain is important for the enzymatic function of ocrl1. Study of a C-terminal region of wild-type ocrl1 containing this domain detected no GAP activity, eliminating the possibility of an effect by mutations in this domain on GTPase activation. Because members of the Arf family of small G-proteins are directly involved in (Ptd(4,5)P(2)) signaling and localize to the TGN like ocrl1, we analyzed by immunoprecipitation the interaction of ocrl1 with Arf1 and Arf6 via its RhoGAP-homology domain. Wild-type ocrl1, but not the I751N mutant protein, co-immunoprecipitated with these two Arf proteins. These results indicate that wild-type ocrl1 and Arf proteins can interact and that this interaction is disrupted by the mutation. It remains unknown whether a disrupted interaction between Arf and ocrl1 plays a role in the Lowe syndrome phenotype.     

OCRL localizes to the primary cilium: a new role for cilia in Lowe syndrome

Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of ocrl in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.     

A novel interstitial deletion in Xq25, identified by array-CGH in a patient with Lowe syndrome

The oculocerebrorenal syndrome of Lowe (OCRL) (MIM:309000) is an X-linked multisystemic disorder affecting the eyes, nervous system and kidneys due to mutations in OCRL1 gene. The gene contains 24 exons, and encodes a 105kDa phosphatydylinositol 4,5-biphosphate [PtdIns(4,5)P(2)] 5-phosphatase localized primarily in the trans-Golgi network and the lysosomes. The large majority of the OCRL1 mutations producing Lowe syndrome are either missense mutations localized mainly in the catalytic domain or non-sense/frameshift mutations resulting in truncated proteins. Rarely, in about 6% of the cases, the disease results from large gene deletions occurring in the 5' part of the gene. Here we report a new case of a patient with Lowe syndrome due to a deletion of about 4Mb, encompassing the OCRL1 gene, detected by PCR and CGH array. The mother was carrier of the same deletion.     

Role of the Rho GTPase-activating protein RICS in neurite outgrowth

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase-activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS(-/-) neurons was higher than that in wild-type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS(-/-) mice bore longer neurites than those from wild-type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.     

Unusual renal features of Lowe syndrome in a mildly affected boy

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by congenital cataracts, mental retardation, and renal tubular dysfunction. The gene responsible for OCRL was identified by positional cloning and encodes a lipid phosphatase, phosphatidylinositol 4,5, bisphosphate [PtdIns(4,5)P2]5-phosphatase, which localizes to the Golgi apparatus and is suspected to play a role in Golgi vesicular transport [Suchy et al., 1995]. In addition to the ocular and renal manifestations, most boys with OCRL have cognitive problems and maladaptive behaviors including tantrums and stereotypies. We report a boy with a history of congenital cataracts and mild developmental delay who was also found to have hematuria with proteinuria but minimal signs of renal tubular dysfunction. Subsequent renal biopsy was compatible with a diagnosis of a noncomplement fixating chronic glomerulonephritis. Despite the atypical renal findings, skin fibroblast analysis for PtdIns (4,5)P2 5-phosphatase was performed, and enzyme activity was low, consistent with the diagnosis of OCRL. Western blot analysis from cell lysates showed the ocrl protein was decreased in size and amount. Our report shows atypical renal features of OCRL in a mildly affected boy. The possibility of OCRL should be considered in boys with cataracts and glomerular disease, even in the absence of renal tubular defects and frank mental retardation usually associated with the syndrome. Am. J. Med. Genet. 95:461-466, 2000. Published Wiley-Liss, Inc.     

Oculo-cerebro-renal Lowe syndrome: clinical, biochemical and molecular studies in a Moroccan patient

The oculo-cerebro-renal syndrome of Lowe is a rare X-linked disorder, caused by the inositol biphosphate 5-phosphatase deficiency, localized to the Golgi complex. Several mutations were reported in patient's OCRL gene leading to enzyme deficiency. We report a Moroccan case of OCRL syndrome of Lowe with a neo mutation in exon 10. The patient aged of 19 months was referred to our medical centre because of a psychomotor retardation. He had a medical history of eye abnormalities including cataract and bilateral glaucoma, diagnosed when he was 5 weeks old. Cataract has been treated after chirurgical therapy but ocular hypertonia persisted. Physical examination revealed an axial hypotonia and walking difficulties. Laboratory tests revealed a moderate acidosis (20 mmol/L), a slight decrease of serum phosphate level (24 mg/L) and an increased serum phosphatase activity. Further studies showed mild proteinuria, urinary bicarbonates loosing and generalised hyperaminoaciduria. Based on both clinical and biological data, Lowe syndrome has been suggested. In this context, molecular investigation has been performed using dHPLC/sequencing techniques which allow identifying an original mutation c.776T>C (p.Phe259Ser), localized on the exon 10 of the OCRL gene. The mutation was not found in the probant's mother suggesting a neo mutation. Lowe syndrome is a rare hereditary X-linked disorder resulting from a variety of heterogeneous mutations of OCRL gene. Indeed, numerous mutations have been reported, variations were noted concerning their localization as well as their type. To our knowledge, this is the first report of the neo mutation c.776T>C of OCRL gene and the first published case report of the Lowe syndrome in a Moroccan patient.     

Identification of two novel mutations in the OCRLI gene in Japanese families with Lowe syndrome

Kubota T, Sakurai A, Arakawa K, Shimazu M, Wakui K, Furihata K, Fukushima Y. Identification of two novel mutations in the OCRL1 gene in Japanese families with Lowe syndrome. Clin Genet 1998: 54: 199–202. 0 Munksgaard, 1998
The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked disorder with features of congenital cataracts. Fanconi syndrome of the renal tubule, and mental retardation. The OCRLI gene has been positionally cloned and shown to encode a phosphatidylinositol 4.5–biphos-phate-5–phosphatase. OCRL is thus thought to be an inborn error of inositol polyphosphate metabolism. We analyzed the gene in two Japanese OCRL patients and their families by DNA sequencing and mismatch polymerase chain reaction (PCR) followed by restriction digestion. A novel nonsense mutation (C1399T) replacing the glutamine of codon 391 (Gln 391 Stop) was identified in exon 12 in 1 patient and also in his mother. A novel missense mutation (C1743G) was identified in exon 15 in the second patient, his mother and maternal grandmother. The missense mutation predicts a substitution of serine for arginine (Ser 505 Arg) in a domain highly conserved among the inosi-tol-5–phosphatase family. Our observations expand the range of OCRLI mutations that cause Lowe syndrome. and will be useful for genetic counseling in these two Fdmilies.     

Interaction of alphaPIX (ARHGEF6) with beta-parvin (PARVB) suggests an involvement of alphaPIX in integrin-mediated signaling

Members of the Rho GTPase family are key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton. It is now well established that these small GTPases are also crucial for neuronal morphogenesis and connectivity. Moreover, mutations in ARHGEF6 (also known as alphaPIX or Cool-2 ), encoding a Rac1/Cdc42-specific guanine nucleotide exchange factor, have been implicated in X-linked mental retardation. In an attempt to get insight into the biological function of ARHGEF6 and the upstream signaling cascades leading to its activation, we used the full-length coding region of ARHGEF6 as bait in yeast-two hybrid screens and identified PARVB (beta-parvin or affixin) as a novel binding partner. The interaction was confirmed by co-immunoprecipitation and GST pull-down. We showed by immunofluorescence that ARHGEF6 and PARVB co-localize at the cell periphery to lamellipodia and ruffles in well-spread and actively spreading cells adhered to fibronectin. In addition, interaction of ARHGEF6 to ARHGEF7 (betaPIX or Cool-1), a close homolog of ARHGEF6, was confirmed. In in vivo assays, two ARHGEF6 mutations identified previously in patients with X-linked non-specific mental retardation, ARHGEF6 deltaaa56-83 and deltaaa396-776, abolished interaction of ARHGEF6 to PARVB. Binding between ARHGEF6 and ARHGEF7 was not affected by ARHGEF6 deltaaa56-83 but did not occur with ARHGEF6 deltaaa396-776. These data suggest that both the N-terminal calponin homology (CH) and C-terminal coiled-coil domains are necessary for the ARHGEF6-PARVB binding. In contrast, it seems that only the coiled-coil domain is required for the interaction and heterodimerization of ARHGEF6 and ARHGEF7. PARVB is known to interact with integrin-linked kinase (ILK) and is involved in the early stage of cell-substrate interaction through integrins. The identification of PARVB as an ARHGEF6 interacting partner together with the co-localization of ARHGEF6 and ILK in spreading cells suggest that ARHGEF6 is involved in integrin-mediated signaling leading to activation of the GTPases Rac1 and/or Cdc42.     

Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes

Motile lymphocytes adopt a polarized morphology with different adhesion molecules (ICAM, CD43 and CD44) and ERM actin-binding proteins concentrated on the uropod, a slender posterior appendage with important functions in cell-cell interactions and lymphocyte recruitment. We have studied the role of Rho family of GTPases (Rho, Rac and Cdc42) in the control of lymphocyte polarity and migration by analyzing the effects of exogenously introduced Rho GTPase mutants. Transfection of T cell lines that constitutively display a polarized motile morphology with activated mutants of RhoA, Rac1 and Cdc42 impaired cell polarization. A guanosine nucleotide exchange factor for Rac, Tiam-1, induced the same effect as activated Rac1. Conversely, dominant negative forms of the three GTP-binding proteins induced a polarized phenotype in constitutively round-shaped T cells with redistribution of ICAM-3 and moesin to the uropod in an integrin-dependent manner. On the other hand, overexpression of dominant negative Cdc42 and activated mutants of all three Rho GTPases significantly inhibited SDF-1alpha-induced T cell chemotaxis. Together, these data demonstrate that Rho GTPases regulate lymphocyte polarization and chemokine-induced migration, and underscore the key role of Cdc42 in lymphocyte directional migration.     

Carrier assessment in families with lowe oculocerebrorenal syndrome: novel mutations in the OCRL1 gene and correlation of direct DNA diagnosis with ocular examination

Lowe oculocerebrorenal syndrome (OCRL) (MIM 309000) is a rare X-linked multisystem disorder characterized by congenital cataracts, muscular hypotonia, areflexia, mental retardation, maladaptive behavior, renal tubular dysfunction, vitamin-D-resistant rickets, and scoliosis. The underlying gene OCRL1 is located on chromosome Xq25-q26 and contains 24 exons. It encodes a 105-kDa phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) 5-phosphatase that is localized to the Golgi complex. To confirm the clinical diagnosis and to assess the carrier state of female relatives for genetic counseling we examined 6 independent patients and their families (a total of 23 individuals) using an improved mutation screening strategy for the OCRL1 gene by sequencing of large PCR amplicons. Four novel and two known mutations were identified: three premature terminations caused by either frameshift mutations (1899insT in exon 17 and 2104-2105delGT in exon 18) or a nonsense mutation (1399C > T in exon 12), two missense mutations (1676G > A and 1754C > T in exon 15), and a 6-bp deletion (1609-1614delAAGTAT in exon 14). An ophthalmological examination was performed in all patients and 14 female relatives. All genotypically proven carrier females showed characteristic lenticular opacities, while all proven noncarriers were lacking this phenotypic finding. The results confirm that ophthalmological evaluation is an apparently reliable first-line method to ascertain the carrier state in Lowe oculocerebrorenal syndrome. The high expressivity of lenticular symptoms in OCRL1 gene carriers is consistent with the hypothesis that (PtdIns[4,5]P(2)) 5-phosphatase activity has low functional reserve capacity for maintaining a balanced homeostasis of lenticular metabolism.     

In Vivo Rho GTPase-Activating Protein Activity of Pseudomonas aeruginosa Cytotoxin ExoS

ExoS is a bifunctional type III cytotoxin secreted by Pseudomonas aeruginosa, which comprises a C-terminal ADP ribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain. In vitro, ExoS is a Rho GAP for Rho, Rac, and Cdc42; however, the in vivo modulation of Rho GTPases has not been addressed. Using a transient transfection system and delivery by P. aeruginosa, interactions were examined between the Rho GAP domain of ExoS and Rho GTPases in CHO cells. Rho GTPases were expressed as green fluorescent protein (GFP) fusion proteins to facilitate quantitation. GFP fusions of wild-type and dominant active Rho, Rac, and Cdc42 localized to discrete regions of CHO cells and appeared functional based upon their modulation of the actin cytoskeleton. Coexpression of the Rho GAP domain of ExoS changed the intracellular distribution of GFP-Rac and GFP-Cdc42 from a predominately membrane location to a cytosolic location. Coexpression of the Rho GAP domain of ExoS did not change the distribution of GFP-Rho, which was primarily in the cytosol. Coexpression of dominant active Rac (DARac) and DACdc42 inhibited actin reorganization by the Rho GAP domain but did not maintain the formation of actin stress fibers, which indicated that Rho had been inactivated. Similar results were observed when ExoS was delivered into CHO cells by P. aeruginosa. These data indicate that in vivo the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibition of Rho.     

Dynamic expression patterns of RhoV/Chp and RhoU/Wrch during chicken embryonic development.

Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch-1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development.     

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Background : We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin‐mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. Results : During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenin δ1 mRNAs with a splice‐site morpholino caused lateral widening and anterior‐posterior shortening of the presomitic mesoderm and somites and a shortened anterior‐posterior axis. These phenotypes indicate a cell‐migration effect. Co‐injection of low amounts of wild‐type Cdc42 or wild‐type Rac1 or dominant‐negative RhoA mRNAs, but not constitutively‐active Cdc42 mRNA, rescued these p120 catenin δ1‐depleted embryos. Conclusions : These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development. Developmental Dynamics 241:1545–1561, 2012. © 2012 Wiley Periodicals Inc.     

Impaired neural development in a zebrafish model for Lowe syndrome

Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.     

OCRL1 mutation analysis in French Lowe syndrome patients: implications for molecular diagnosis strategy and genetic counseling

The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked recessively inherited disease characterized by a severe pleiotropic phenotype including mental retardation, bilateral congenital cataract, and renal Fanconi syndrome. The gene responsible for OCRL encodes an inositol polyphosphate-5-phosphatase. We performed mutation analysis in 36 families and characterized 27 new mutations with two of them being recurrent mutations. The panel of mutations consisted of 27 truncating mutations (frameshift, nonsense, splice site mutations, and large genomic deletions), one in-frame deletion, and six missense mutations. The four large genomic deletions occurred in the first half of the gene, whereas all the remaining mutations took place in the second part of the gene and were concentrated in a few exons. This distribution may be of interest in terms of screening strategy when looking for unknown mutations. Haplotyping of the families was performed to analyze segregation of the mutated loci, and revealed a somatic mosaicism in one family. This is the second case of mosaicism we characterized in a total panel of 44 unrelated families affected by Lowe's syndrome. Considering the low number of families investigated, it appeared that somatic and germinal mosaicisms are quite common in this disease and must be taken into account for genetic counseling.     

Regulation of cell function by rho family GTPases

Rho GTPases act as molecular switches to control many basic cellular activities that are also critical to the specialized functions of phagocytic leukocytes. Our laboratory has studied the regulation of Rho GTPase function, how these GTPases interact with specific effectors to modulate cell function, and how these events are coordinated in the stimulated cell. Areas of major interest include NADPH oxidase regulation by Rac2, Rac- and Cdc42-mediated control of the actin-myosin cytoskeleton via p21-activated kinase (PAK), and modulation of the apoptotic program by Rho GTPases and PAK.     

Identification of a novel deletion of the entire OCRL1 gene detected by FISH analysis in a family with Lowe syndrome

The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked multisystem disorder affecting the lens, kidney and brain. The gene involved (OCRL1) has been identified and is known to encode a phosphatidylinositol 4,5-bisphosphate 5-phosphatase. Mutations in OCRL1 have been shown to be causative of OCRL. To date, most of the mutations identified have consisted of simple or point mutations and there is one report of a 1.4-kb deletion. We investigated the OCRL1 gene in a male patient with OCRL by the polymerase chain reaction and found that the entire OCRL1 gene was deleted. Fluorescence in situ hybridisation analysis (FISH), with cosmid probes that span the entire OCRL1 gene, was used to confirm this deletion and subsequently identify it in the proband's mother. This is the first report of a whole gene deletion of OCRL1 and thus expands the range of mutations that give rise to OCRL. The use of the FISH technique facilitated carrier and prenatal testing for the deletion in the family.     

A GAL4‐inducible transgenic tool kit for the in vivo modulation of Rho GTPase activity in zebrafish

Background: Rho GTPases are small monomeric G‐proteins that play key roles in many cellular processes. Due to Rho GTPases' widespread expression and broad functions, analyses of their function during late development require tissue‐specific modulation of activity. The GAL4/UAS system provides an excellent tool for investigating the function of Rho GTPases in vivo. With this in mind, we created a transgenic tool kit enabling spatial and temporal modulation of Rho GTPase activity in zebrafish. Results: Transgenic constructs were assembled driving dominant‐negative, constitutively active, and wild‐type versions of Cdc42, RhoA, and Rac1 under 10XUAS control. The self‐cleaving viral peptide F2A was utilized to allow bicistronic expression of a fluorescent reporter and Rho GTPase. Global heat shock of hsp70l:gal4⁺ transgenic embryos confirmed GAL4‐specific construct expression. Western blot analysis indicated myc‐tagged Rho GTPases were expressed only in the presence of GAL4. Construct expression was confined to proper cells when combined with pou4f3:gal4 or ptf1a:gal4. Finally, transgene expression resulted in reproducible defects in lens formation, indicating that the transgenes are functional in vivo. Conclusions: We generated and validated 10 transgenic lines, creating a versatile tool kit for the temporal‐spatial modulation of Cdc42, RhoA, and Rac1 activity in vivo. These lines will enable systematic analysis of Rho GTPase function in any tissue of interest. Developmental Dynamics 245:844–853, 2016. © 2016 Wiley Periodicals, Inc.