Roles of p190 RhoGAP in mouse development. Development, 2000.

8/6/2019 Roles of p190 RhoGAP in mouse development. Development, 2000.

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INTRODUCTION

Members of the Rho family of small GTPases, which includesthe Rho, Rac and Cdc42 proteins, function as critical regulatorsof actin cytoskeleton organization. As such, these proteinsmediate a variety of cellular processes, including migration,adhesion and shape change (Hall, 1998; Van Aelst andDSouza-Schorey, 1997). These cellular functions of the RhoGTPases have recently been linked to several of themorphogenetic events associated with normal embryonicdevelopment in mammals and in other multicellular organisms(Settleman, 1999). Like the Ras GTPases, Rho proteins cyclebetween a GDP-bound inactive form and a GTP-bound activeform in a tightly regulated manner (Nobes and Hall, 1994).Regulation of the nucleotide state of the Rho GTPases isaccomplished by the action of three major classes of proteins:guanine nucleotide exchange factors, guanine nucleotidedissociation inhibitors and GTPase activating proteins (GAPs).

GAPs function by stimulating the relatively weak intrinsicGTP hydrolyzing activity of their substrate GTPases, thereby

inactivating them. Among the numerous RhoGAPs that havebeen described, p190 RhoGAP, together with a closely relatedprotein, p190-B, accounts for a substantial fraction of the totalRho inhibitory activity in cultured cells (Burbelo et al., 1995;Settleman et al., 1992a; Vincent and Settleman, 1999), andboth proteins are widely expressed in adult mammalian tissues(Burbelo et al., 1998; Settleman et al., 1992a). P190 RhoGAPwas first identified as the major binding partner of p120RasGAP in Src-transformed cells (Ellis et al., 1990). Thus,p190 RhoGAP may integrate signals transduced by the Ras andRho family GTPases. P190 RhoGAP contains an amino-terminal GTPase domain (Foster et al., 1994) and a carboxy-terminal RhoGAP domain that preferentially interacts with theRho GTPase (Settleman et al., 1992b). Although the preciserole of the GTPase domain of p190 RhoGAP has not beenestablished, it appears to regulate the ability of p190 RhoGAPto function as a Rho regulator in cultured cells (Tatsis et al.,1998).

Evidence for a role for p190 RhoGAP in regulating the actincytoskeleton comes from several studies with cultured

4891Development 127, 4891-4903 (2000)Printed in Great Britain The Company of Biologists Limited 2000

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Rho GTPases direct actin rearrangements in response to avariety of extracellular signals. P190 RhoGAP (GTPaseactivating protein) is a potent Rho regulator that mediatesintegrin-dependent adhesion signaling in cultured cells.We have determined that p190 RhoGAP is specificallyexpressed at high levels throughout the developing nervoussystem. Mice lacking functional p190 RhoGAP exhibitseveral defects in neural development that are reminiscentof those described in mice lacking certain mediators ofneural cell adhesion. The defects reflect aberrant tissuemorphogenesis and include abnormalities in forebrainhemisphere fusion, ventricle shape, optic cup formation,neural tube closure, and layering of the cerebral cortex.

In cells of the neural tube floor plate of p190 RhoGAPmutant mice, polymerized actin accumulates excessively,suggesting a role for p190 RhoGAP in the regulation ofRho-mediated actin assembly within the neuroepithelium.

Significantly, several of the observed tissue fusion defectsseen in the mutant mice are also found in mice lackingMARCKS, the major substrate of protein kinase C (PKC),and we have found that p190 RhoGAP is also a PKCsubstrate in vivo. Upon either direct activation of PKC orin response to integrin engagement, p190 RhoGAP israpidly translocated to regions of membrane ruffling,where it colocalizes with polymerized actin. Together, theseresults suggest that upon activation of neural adhesionmolecules, the action of PKC and p190 RhoGAP leads to amodulation of Rho GTPase activity to direct several actin-dependent morphogenetic processes required for normalneural development.

Key words: Rho GTPase, Morphogenesis, Signal transduction, Actin,PKC, Cell adhesion, Mouse

SUMMARY

The adhesion signaling molecule p190 RhoGAP is required for

morphogenetic processes in neural development

Madeleine R. Brouns1, Stephen F. Matheson1, Kang-Quan Hu1, Ivana Delalle2, Verne S. Caviness, Jr2,Jerry Silver3, Roderick T. Bronson4 and Jeffrey Settleman1,*

1Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, MA 02129, USA2Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA3Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, USA4Department of Pathology, Tufts University Schools of Medicine and Veterinary Medicine, Boston, MA 02111, USA*Author for correspondence (e-mail: [email protected])

Accepted 30 August; published on WWW 24 October 2000


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fibroblasts. For example, overexpression of p190 RhoGAPcauses cells to become rounded with long, beaded extensions,and is associated with collapse of the actin cytoskeleton (Tatsiset al., 1998). In addition, lysophosphatidic acid (LPA)-inducedactin stress fiber formation, which is Rho-mediated, is inhibitedby microinjection of the RhoGAP catalytic domain of p190RhoGAP (Ridley et al., 1993). P190 RhoGAP has also been

observed to rapidly co-localize with polymerized actin upongrowth factor stimulation (Chang et al., 1995).

Several lines of evidence indicate that p190 RhoGAPmediates a signal transduction pathway downstream ofintegrins, the receptors for extracellular matrix proteins. Forexample, expression of the isolated amino-terminal regionof RasGAP, which is constitutively associated with p190RhoGAP, results in a reduction in focal contacts and animpaired ability to adhere to fibronectin (McGlade et al.,1993). Furthermore, p190 RhoGAP is rapidly recruited tothe plasma membrane upon antibody-induced integrinengagement, where it co-localizes with cortical actin (Burbeloet al., 1995). Finally, p190 RhoGAP is recruited to thecytoskeleton following adhesion of fibroblasts to fibronectin

(Sharma, 1998). These observations suggest that p190RhoGAP mediates an adhesion signaling pathway and that itacts through its ability to regulate the Rho GTPases, whichhave previously been shown to be required for integrin-dependent adhesion signaling (Clark et al., 1998; Hotchin andHall, 1995).

We have examined the in vivo function of p190 RhoGAP bya targeted gene disruption strategy in mice. We first determinedthat p190 RhoGAP is highly and specifically expressed in thedeveloping nervous system of the mouse, suggesting a role forthe protein in neural development. Mice lacking a functionalp190 RhoGAP exhibit several specific defects in neuraldevelopment that appear to reflect a role for the protein inadhesion signaling. We also find evidence of a role for PKC inregulating p190 RhoGAPs response to adhesion moleculeactivation. Taken together, our findings suggest that p190RhoGAP is critical for several aspects of tissue morphogenesisin the developing nervous system by regulating Rho GTPase-mediated actin organization in response to the engagement ofadhesion molecules.

MATERIALS AND METHODS

In situ hybridization

Embryos were fixed in 4% formaldehyde by immersion (at E12.5) ortranscardiac perfusion (at E15.5 and E18.5) and cryoprotected in

Dulbeccos PBS containing 20% sucrose. 20 m sections were cut bycryostat. [35S]UTP-labeled riboprobes were synthesized from apBluescript SK+/ phagemid containing p190 RhoGAP cDNA(bp 1199-1792). Hybridizations were carried out under standardconditions. Slides were dried and exposed to -Max film. Sectionswere immersed in Kodak NTB-2 autoradiography emulsion.

Whole-mount in situ hybridization was carried out essentially aspreviously described (Wilkinson, 1992). Wild-type E8.5 embryoswere fixed in 4% paraformaldehyde at 4C for 3-12 hours. Sampleswere rinsed in PBT (PBS + 0.1% Tween 20), dehydrated in methanoland stored at 20C. After rehydration, the embryos were hybridizedto riboprobes overnight at 70C in hybridization buffer (50%formamide, 5 SSC pH 4.5, 50 g/ml yeast RNA, 1% SDS, 50 g/mlheparin) containing 1 g/ml digoxigenin-labelled RNA probe.

pBluescript SK+/ plasmid containing p190 RhoGAP codingsequence (bp 1694-3610) served as template for synthesis of probes.Following extensive washing, embryos were incubated overnight at4C with alkaline phosphatase-conjugated anti-digoxigenin antibodythat had been preabsorbed with heat-inactivated mouse embryopowder. Alkaline phosphatase activity was visualized using nitroblue-tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate for 1-2 hours at room temperature.

Protein analysis

Analysis of p190 RhoGAP protein expression, tyrosinephosphorylation, and RasGAP association was carried out aspreviously described (Hu and Settleman, 1997). Subcellularfractionation was performed by tissue homogenization in ice-coldhypotonic buffer (20 mM Hepes pH 7.5, 2 mM MgCl2, 10 mM KCl)containing protease and phosphatase inhibitors, followed bycentrifugation in a microfuge (10,000 g for 4 minutes at 4C) to yieldthe crude extract. Ultracentrifugation (100,000 g for 20 minutes at4C) of the crude extract yielded the soluble fraction (supernatant),whereas lysis of the obtained high-speed pellet in hypotonic buffersupplemented with 0.5% NP-40 and 400 mM NaCl, followed bycentrifugation (25,000 g for 10 minutes at 4C), yielded the insolublefraction.

Targeting of the p190 RhoGAP gene

Genomic clones containing p190 RhoGAP coding sequence wereisolated from a 129/SvJ genomic library. The targeting strategy tookadvantage of the fact that the amino-terminal 80% of the p190RhoGAP coding sequence is contained in a single large exon. Thep190 RhoGAP targeting vector was constructed by inserting a 1.4 kb

BamHI-EcoRI 5 genomic fragment and a 6.5 kb XbaI 3 genomicfragment in the pPNT vector (Tybulewicz et al., 1991) in oppositetranscriptional orientation to the PGK-neoR cassette. This resulted ina deletion of 1.5 kb of the first exon, thereby eliminating thetranslation initiation site. NotI linearized targeting vector waselectroporated into D3 ES cells. Transfected ES cells were selectedfor growth in G418 and gancyclovir. Correctly targeted ES clones (5of 500 screened) were detected by Southern blot analysis ofHindIII

digested genomic DNA, using a 5 external probe. Two 3 externalprobes were used to confirm the absence of rearrangements 3 of thetargeted region. ES cells from each of the five identified p190RhoGAP heterozygous clones were injected into C57BL/6Jblastocysts to generate chimeric mice. Germline transmission wasdetermined by crosses between chimeras and C57BL/6J mice andconfirmed by Southern blot analysis of tail DNA. Subsequentgenotypic analysis was performed by PCR. ES cell culture andblastocyst injections were performed essentially as described byHogan et al. (1994).

Immunostaining of cultured cells

Mouse embryonic fibroblasts (MEFs) were prepared as previouslydescribed (Hogan et al., 1994) and maintained in DMEM/15% fetalbovine serum. Cos-7 cells, maintained in standard culture conditions,

were transfected using the DEAE-Dextran method with the epitope-tagged p190 expression vector, RcHAp190 (Hu and Settleman, 1997).For immunostaining with anti-p190 RhoGAP or anti-HA-tagantibodies, cells were plated for the indicated times on glasscoverslips that were coated with fibronectin (0.001% w/v in PBS),poly-L-lysine (0.01% w/v in H2O) or were untreated. Cells were fixedin 4% paraformaldehyde in PBS (20 minutes), rinsed in PBS,permeabilized in 0.1% Triton X-100/PBS (2 minutes) and blockedwith 1% normal goat serum, 0.1% BSA in PBS (1 hour). Primaryantibodies were diluted in 0.1% BSA/PBS and applied to coverslipsfor 1 hour (anti-p190 RhoGAP, Transduction Laboratories, 1:50; anti-HA-Tag 12CA5, 1:1). Coverslips were rinsed in PBS and incubatedin Cy3-conjugated secondary antibody (Jackson ImmunoResearch),diluted in PBS (1:300) in the presence of FITC-conjugated phalloidin

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(Sigma) (1:300) for 30 minutes. Coverslips were rinsed in PBS,mounted on glass slides and viewed with a Zeiss fluorescencemicroscope.

Cell adhesion assays

A quantitative cell adhesion assay was carried out in 96-wellmicrotitre plates, essentially as previously described (McGlade et al.,1993). Briefly, wells were coated overnight at 4C with fibronectin(0.1-10 g/ml) diluted in PBS (25 l/well). Wells were washed severaltimes with PBS and blocked with PBS containing 2.5 mg/ml BSA for2 hours at 37C. Fibroblasts were trypsinized, centrifuged in DMEM-15% FCS (5 minutes, 1500 rpm at room temperature) and washedthree times in serum-free DMEM. Cells were then resuspended inDMEM containing 2.5 mg/ml BSA, to a concentration of 3105

cells/ml, and plated in triplicate on fibronectin-coated wells (100l/well). Cells were allowed to adhere at 37C, after which wells wereaspirated and washed once with PBS. Attached cells were fixed in3.7% paraformaldehyde for 30 minutes and stained overnight with0.5% Toluidine Blue in 3.7% paraformaldehyde at room temperature.Wells were rinsed with PBS and cell adhesion was quantitated bymeasuring absorbance at OD 540 using an automated microtiter wellplate reader.

To analyze reorganization of the actin cytoskeleton during adhesion

and spreading of MEFs on fibronectin, glass coverslips were coatedwith 10 g/ml fibronectin in PBS for 30 minutes at 37C in 12-wellplates. Wild-type and mutant MEFs were trypsinized and plated at lowdensity (104 /well) on the fibronectin-coated coverslips for the timesindicated, washed once with PBS, fixed with 4% formaldehyde in PBSfor 15 minutes at room temperature and stained for actin as describedabove.

Histological analysis

Newborn mice were sacrificed by CO2 asphyxiation and fixed byimmersion in Bouins fixative. Coronal paraffin sections were stainedwith Haematoxylin and Eosin. E15-E17 embryos were fixed bytranscardiac perfusion with 4% paraformaldehyde. 1 m plasticsections were taken through selected slabs at 50 m intervals, so thatthe entire region containing the forebrain commissures, optic nerves

and optic chiasm were sampled. Sections were heat dried ontomicroscope slides, stained with Toluidine Blue, coverslipped withPro-Texx mounting medium and photographed. For Nissl staining,brains were fixed by immersion (4% paraformaldehyde) followed bycryoprotection in PBS/20% sucrose. Coronal cryostat sections werethen stained with Cresyl Violet acetate (0.5% in 1% acetic acid) for10 minutes.

Immunohistochemistry

Whole embryos (E10.5-E14.5) or heads (E16.5) were fixed in 4%paraformaldehyde at 4C for 2-3 hours or overnight, respectively.Following fixation, whole embryos, dissected brains or whole headswere washed with PBS, cryoprotected in PBS-20% sucrose overnight,frozen in OCT and cut on a cryostat. For immunofluorescence,sections (10-14 m) were air-dried for 30 minutes, washed with PBS,

permeabilized in PBS-0.1% Triton X-100 for 10 minutes, blockedwith PBS-10% normal goat serum (NGS) containing goat anti-mouseF(ab)2 fragments (10 g/ml) for 30 minutes, washed briefly with PBSand then incubated with primary antibody diluted in PBS-1% NGSfor 2 hours. Subsequently, sections were washed with PBS, followedby incubation with Cy3-conjugated secondary antibody for 1 hour.After PBS washes, sections were coverslipped using polyvinylalcohol-based aqueous mounting medium. For F-actin staining,sections were treated as above, omitting the F(ab)2 fragments in theblock solution. Sections were incubated with fluorescent phalloidinfor 1 hour followed by PBS washes and mounting.

The cortactin primary antibody (4F11, Upstate Biotechnology, Inc.)was used at 1:200. Goat anti-mouse F(ab)2 fragments (JacksonImmunoResearch) were used as indicated. The Rat-401 antibody

(Developmental Studies Hybridoma Bank) was used at 1:1 dilution.The p190 RhoGAP monoclonal antibody (Transduction Laboratories)was used at 1:50. Cy3-conjugated goat anti-mouse secondaryantibody (Jackson ImmunoResearch) was diluted 1:500. FITC-conjugated phalloidin (Sigma) was diluted 1:200, rhodamine-conjugated phalloidin (Sigma) was diluted 1:500.

Cell collapse assay

NIH3T3 cells were transfected by calcium phosphate with eitherRCHAp190 (wild type p190 RhoGAP) or the R6 truncation mutantcorresponding to the amino-terminal deletion of the GTPase domain.The R6 truncation mutant was made by deleting the amino-terminalregion of p190 RhoGAP at anXbaI site that is 1.1 kb from the startcodon. 24 hours post-transfection, cells were re-plated onto glasscoverslips, and after an additonal 24 hours, cells were fixed andimmunostained with either 12CA5 antibody (RCHAp190) or p190RhoGAP monoclonal antibody A5D12. Cells that exhibited proteinexpression were scored, using fluorescence microscopy, as eitherbeing morphologically unaffected, partially collapsed, or completelycollapsed.

Scanning electron microscopy

Embryos were washed in PBS, then fixed in Karnovskys fixative (pH

7.5) at room temperature for 30 minutes. Subsequently, embryos wererinsed in 0.125 M sodium cacodylate buffer (pH 7.3), 310 mOsm,postfixed in 2% osmium tetroxide for 45 minutes, rinsed in 0.125 Msodium cacodylate buffer (pH 7.3) and dehydrated in graded alcoholsto 100% ethanol. Following critical point drying and sputter coatingwith gold/palladium, samples were viewed on an Amray 1000scanning electron microscope.

PKC phosphorylation assay

In vitro PKC assays were performed in a solution containing 20 mMMOPS buffer, pH 7.2, 25 mM -glycerophosphate, 1 mM sodiumorthovanadate, 1 mM DTT, 1 mM CaCl2, 0.1 M ATP and 10 Ci [-32P]ATP. Where indicated, the reaction was supplemented with 20 ngpurified brain PKC (Upstate Biotechnology, Inc.), 0.1 mg/mlphosphatidylserine and diglycerides and/or 100 ng of purified,

baculovius-produced p190 RhoGAP protein. Following incubation at30C for 10 minutes, samples were analyzed by 7.5% SDS-PAGE,followed by autoradiography. For in vivo PKC phosphorylationassays, early passage MEFs were serum-starved (DMEM-0.1% FCS)overnight, preincubated for 2 hours in phosphate-free DMEMcontaining 0.1% phosphate-free, dialyzed FCS, then incubated in thesame medium containing 32P (200 Ci/ml) for 3 hours. Cells werethen stimulated with TPA (100 ng/ml) for the indicated times, washedtwice with ice-cold PBS, scraped in 1 ml of ice-cold PBS andcentrifuged for 1 minute at 4000 rpm at room temperature. Cell pelletswere lysed in 50 mM Hepes pH 7.4, 150 mM NaCl, 1.5 mM MgCl2,5 mM EGTA, 10% glycerol, 1% Triton X-100 with protease andphosphatase inhibitors. Debris was removed by centrifugation andlysates were precleared by incubation with protein A beads. Proteinswere immunoprecipitated with anti-p190 RhoGAP and protein A-

Sepharose for 2 hours at 4C. Immune complexes were washed fourtimes in 20 mM Hepes pH 7.5, 150 mM NaCl, 10% glycerol, 0.1%Triton X-100, boiled in gel loading buffer and separated by 7.5%SDS-PAGE, which was followed by autoradiography.

In vivo PKC activation

Cos-7 cells transfected with RcHAp190 RhoGAP were plated on glasscoverslips in 12-well plates. 48 hours after transfection a subset ofwells was treated for 30 minutes with the phorbol ester TPA (100ng/ml) by direct addition to the medium. Mouse 3T3 fibroblasts werecultured overnight on glass coverslips and similarly treated with TPA.Cells were then washed once with PBS, fixed with 4% formaldehydein PBS for 15 minutes at room temperature and stained for p190RhoGAP and actin as described above.


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RESULTS

Neural expression of p190 RhoGAP during mouseembryogenesis

We previously reported that the p190 RhoGAP gene isexpressed ubiquitously in adult rodents (Settleman et al.,1992a). To determine whether the expression of p190 RhoGAPis developmentally regulated we performed in situhybridization analysis on tissue sections of mouse embryos atseveral gestational stages. We found that p190 RhoGAPmRNA is specifically expressed at high levels in the brain,spinal cord and eyes of developing mouse embryos (Fig. 1D-F). In brain, expression appears to be widespread, with someenrichment in the cortical plate (Fig. 1A-C). The p190RhoGAP protein product and its binding partner RasGAP arealso expressed uniformly throughout neural development (E12-P5) (Fig. 1G). Thus, during embryogenesis, the expression ofp190 RhoGAP is largely restricted to the nervous system. Inaddition, p190 RhoGAP protein is expressed in all regions ofthe mature nervous system of adult animals (Fig. 1H). We havealso found that p190 RhoGAP is expressed in a variety ofneuronal and glial cell lines, suggesting that it is not restrictedto a particular cell type in the nervous system (data not shown).

Generation of p190 RhoGAPmutant mice

To identify a potential role for p190 RhoGAP in neuraldevelopment, we disrupted the gene in mice by targetedhomologous recombination in embryonic stem cells (Fig. 2A-C).The targeting strategy involved deleting 1.5 kb of amino-terminalcoding sequence from a single large exon (3.8 kb) that encodesmost of the p190 RhoGAP protein. Mice heterozygous for thetargeted allele develop normally and display no abnormalities,and intercrossing these animals yieldsp190 RhoGAP/animalsat the expected frequency at birth (25%). The majority ofp190RhoGAP/mice are grossly indistinguishable from their normallittermates throughout gestation (Fig. 2F), although 95% diewithin the first 2 days after birth (Fig. 2G). The remaining mutantanimals are runted and die within 3 weeks.

Immunoblots of embryo brain lysates using an amino-terminal p190 RhoGAP antibody indicated that the full-lengthprotein is not expressed in homozygous mutant animals (Fig.2C). However, with other p190 RhoGAP-specific antibodies,we observed that an altered form of the protein is expressedfrom the targeted allele by an internal translational initiation(Fig. 2D). This truncated form of p190 RhoGAP specificallylacks the amino-terminal GTPase domain (Fig. 2E), and isexpressed at levels comparable to the wild-type protein.


Fig. 1. P190 RhoGAP gene expression during mouse embryogenesis. Coronal (A-C) and sagittal (D-F) tissue sections were hybridized toantisense (A-F) and sense (D) p190 RhoGAP [35S]UTP-labelled ribonucleotide probes. Expression is largely restricted to the central nervoussystem (CNS). No detectable hybridization is present with the sense probe. (A-C) Analysis at E12.5 (A), E15.5 (B) and E18.5 (C)demonstrating p190 RhoGAP expression throughout the brain with an enrichment in the developing cortical plate. (D,E) At E12.5, the highestlevel of p190 RhoGAP expression is detected in the spinal cord and a slightly lower level of expression is seen in the developing brain. (F) AtE15.5, strong expression of p190 RhoGAP mRNA can be detected throughout the CNS. (G) Immunoblot of p190 RhoGAP and its bindingpartner RasGAP in brain lysates from mice at the indicated stages of development. (H) Immunoblot of p190 RhoGAP in protein lysates fromthe dissected nervous system of a wild-type adult mouse. br, brainstem; cer, cerebellum; crt, cortex; cp, cortical plate; cw, cerebral wall; hip,

hippocampus; hyp, hypothalamus; mid, midbrain; olf, olfactory bulb; sc, spinal cord; str, striatum.


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The p190 RhoGAP mutation is a loss-of-functionalleleSeveral lines of evidence indicate that the truncated form ofp190 RhoGAP expressed in the mutant mice represents eithera severely defective loss-of-function allele or a null allele.First, we have not observed any defects in more than 100heterozygous mutant animals that have been examined. Thus,by genetic criteria, the p190 RhoGAP mutation is a loss-of-

function allele and is not functioning in a dominant-interferingmanner. Second, we have found that each of the knownfunctional domains of p190 RhoGAP is defective in the mutantprotein. Thus, the GTPase function is lost by the deletion ofthe entire catalytic region. In addition, unlike the wild-typeprotein, the mutant protein fails to undergo tyrosinephosphorylation or RasGAP association in brain (Fig. 3A). TheGTPase domain has recently been shown to be required for

Fig. 2. Targeted disruption of the p190 RhoGAP gene by homologous recombination. (A) The 5 region of the wild-type p190 RhoGAP gene isshown on the first line, the gene targeting vector on the second line and the disrupted locus following homologous recombination on the thirdline. The thickened bar represents the large (3.9 kb) first exon, including the translation initiation site; the open boxes show the neomycinresistance gene (NEO) and the thymidine kinase gene (TK) cloned in the opposite transcriptional orientation relative to the p190 RhoGAPgene; the sequence that is deleted after homologous recombination is depicted as ; the indicatedHindIII fragments represent the wild-type (3.8kb) and the mutated (4.8 kb) alleles that hybridize to the 5 external probe shown under the third line. B,BamHI; H,HindIII; E,EcoRI; X,XbaI;N,NotI. (B) Genotyping of ES clones by Southern blot analysis of genomic DNA using the 5 external probe, depicted in A. An example of onecorrectly targeted clone (+/) is shown. (C) p190 RhoGAP protein expression in E15 brain lysates by immunoblotting with the p190 RhoGAPmonoclonal antibody D2D6 (amino-terminal epitope). RasGAP expression is indicated as an internal control for protein loading. Genotypes arerepresented as wild-type (+/+), heterozygous (+/) and homozygousp190 RhoGAP mutant (/). (D) Immunoblot of p190 RhoGAP protein inlysates from wild-type (+/+), heterozygous (+/), and homozygous (/)p190 RhoGAP mutant embryos using the p190 RhoGAP monoclonalantibody A5D12 (central region epitope). (E) Schematic illustration of the altered form of p190 RhoGAP expressed by internal translationalinitiation. (F) External appearance of E18.5 wild-type (+/+), heterozygous (+/) and homozygous (/)p190 RhoGAP mutant mice.(G) Survival percentages of wild-type (+/+), heterozygous (+/) and homozygous (/)p190 RhoGAP mutant mice at postnatal days (P) 0, 1, 2and 28.


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normal RhoGAP activity in vivo (Tatsis et al., 1998) and wehave similarly observed a substantial reduction in the ability ofthe GTPase-deficient form of the protein to disrupt the actincytoskeleton in transfected cells, despite retention of afunctional RhoGAP catalytic domain (Table 1). In addition, wehave determined that the mutant protein fails to localize

properly within cells (Fig. 3B,C). Approximately one-third(34%) of the wild-type protein is present in a detergent-insoluble fraction, whereas only a small fraction of the mutantprotein (7%) is present in the same insoluble fraction of brainlysates from p190 RhoGAP+/ animals (Fig. 3B). Differentialsubcellular localization is also indicated by the distinctimmunofluorescent staining patterns seen in wild-type and p190 RhoGAP mutant mouse embryonic fibroblasts (MEFs)(Fig. 3C). Taken together, these results indicate that thetruncated p190 RhoGAP is a severely defective loss-of-function protein, but do not rule out the possibility that acomplete deletion of the gene might result in a more substantialphenotype.

Fibroblasts derived from p190 RhoGAPmutantsappear normal

To identify potential defects in cells lacking a functional p190RhoGAP, we first examined MEFs. Homozygous mutant cells

in culture were compared to wild-type and heterozygousmutant cells in several assays of growth and morphology.Overall, no significant differences were observed between thecell populations in comparisons of proliferation rates,morphology, actin organization, migration, spreading, oradhesion to extracellular matrix (Fig. 4 and data not shown).In addition, all cell lines were readily established as stable,

immortalized 3T3 lines that also exhibit similar properties(data not shown). Since we have previously observed that theclosely related p190-B protein is also expressed in fibroblasts,and actually accounts for most of the RhoGAP activity in thosecells (Vincent and Settleman, 1999), it is likely that a redundantfunction of p190-B masks the absence of a functional p190RhoGAP in these cells.

Abnormal eye development in p190 RhoGAPmutantmice

A detailed histological analysis of developing embryosrevealed that defects inp190 RhoGAP/animals appear to belargely restricted to the developing nervous system. A smallpercentage of homozygous mutant animals (less than 2%) also

exhibit omphalocele, which is a failed fusion of the abdominalcavity (not shown). No abnormalities in any other organs wereobserved grossly or microscopically. Moreover, none ofnumerousp190 RhoGAP heterozygous mutant animals (morethan 100 examined) was found to exhibit any of the defectsdescribed below. Consistent with the observed enrichedexpression of p190 RhoGAP in embryonic eyes, we found thatall of thep190 RhoGAP/animals exhibit abnormalities in eyedevelopment (more than 50 examined). Eyes from p190RhoGAP/ animals display an apparent hyperplasia of theretinal pigmented epithelium (RPE) (Fig. 5B,D). Upondissection, we determined that eyes from p190 RhoGAPmutant embryos are significantly smaller than normal andexhibit a defect in closure of the optic fissure, leading tocoloboma (Fig. 5A-D). Most likely, the observed RPEhyperplasia is a secondary consequence of the neural retinadefect, as has been previously described in adult eye tissue(Campochiaro et al., 1994).


Table 1. The GTPase-deleted form of p190 RhoGAP isdefective for induction of cell collapse

Complete PartialNumber of collapse collapse No effect

Construct cells (%) (%) (%)

RcHAp190 385 67 15 18R6 439 13 13 74

NIH 3T3 cells were transfected with either wild-type p190 RhoGAP or anamino-terminally truncated form (R6) designed to mimic the form that isexpressed by internal translational initiation in mutant mice. After 48 hours,cells were immunostained with a p190 RhoGAP antibody, and expressingcells were scored for effect on the cytoskeleton: complete collapse, partialcollapse, or no effect.

Fig. 3. Thep190 RhoGAP mutation is a loss-of-function allele. (A) p190 RhoGAP immunoprecipitations of brain lysates from wild-type (+/+),p190 RhoGAP heterozygous (+/), and p190 RhoGAP homozygous mutant (/) animals, followed by immunoblotting with anti-p190RhoGAP (top), anti-pTyr (middle), or anti-RasGAP (lower) antibodies. (B) Fractionation of a heterozygous adult brain into detergent-solubleand -insoluble fractions, followed by immunoblotting with p190 RhoGAP antibody. (C) Subcellular localization of wild-type p190 RhoGAP(left) and the p190 RhoGAP mutant (right) demonstrated by p190 RhoGAP immunostaining of embryo-derived 3T3 fibroblasts.


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Closure of the optic fissure generally occurs between E11.5and E14.5 and marks the end of the morphogenetic phase inthe formation of the eye (Hero, 1990). To determine the cellularbasis for the observed eye defect, we examined retinal sectionsfrom wild-type and mutant embryos at several relevantdevelopmental stages. To identify potential abnormalities inactin cytoskeleton organization, we compared phalloidin-stained eye sections (Fig. 5E-J). This analysis revealedabnormal folding of the retina in the mutants, and although noobvious differences in the distribution of polymerized actinwere seen, the persistent invagination of the actin-rich basalmembrane in the mutants potentially reflects a subtle defect inactin organization in the absence of functional p190 RhoGAP.Moreover, the progression of the mutant phenotype throughthese stages of development clearly indicates that the observeddefect occurs during the normal course of eye development,and does not arise subsequent to the developmental phase.

Despite the defect in closure of the optic fissure, the retinallayers appear to be normal with regard to cell number and cell

type composition (data not shown). Examination of thelocalization of the cellular markers cortactin, a cortical actin-binding protein (Fig. 5K-O) and cadherin (not shown)indicated no defects in the polarization of retinal epithelialcells, but revealed an abnormal morphology of cells lining theoptic fissure in the mutants. Thus, the eye defects in p190RhoGAP mutant mice appear to be associated exclusively withdefective morphogenesis of the neural retinal tissue, and areassociated with aberrant epithelial cell shapes.

Forebrain defects in p190 RhoGAPmutant mice

The majority of brains dissected from p190 RhoGAP/

newborn animals are normal in size, weight, and externalappearance (see next section). However, they always exhibita substantial cleft between the cerebral hemispheres.Histological analysis revealed that these mice completely lacka corpus callosum, the major bundle of midline-crossingcortical axons in the forebrain (50 out of 50 examined),whereas in wild-type and heterozygous mutant littermates this

Fig. 4. Fibroblasts derived fromp190 RhoGAP mutants appear normal. (A-J) MEFs lacking functional p190 RhoGAP exhibit normal actincytoskeleton rearrangements during adhesion and spreading on fibronectin. Phalloidin staining of wild-type (A-E) and p190 RhoGAP mutant

(F-J) MEFs at 10 minutes (A,F), 30 minutes (B,G), 60 minutes (C,H), 90 minutes (D,I) and 180 minutes (E,J) after plating. MEFs lackingfunctional p190 RhoGAP are not impaired in attachment to fibronectin. (K) Number of fibroblasts attached to fibronectin (10 g/ml) at timepoints between 10 and 60 minutes after plating. (L) Number of fibroblasts attached to fibronectin, at concentrations ranging from 0.1 to 10g/ml, at 60 minutes after plating. As a control for the detection of differences in cell adhesion, fibroblasts that do (Rat-2+GAP-N) or do not(Rat-2) express the amino-terminal region of RasGAP were compared. GAP-N overexpression has been shown to dramatically reduce adhesionof cells to fibronectin (McGlade et al., 1993). In addition, p190 RhoGAP wild-type MEFs (2044-4, 2025-8) andp190 RhoGAP mutant MEFs(2044-3, 2025-5) were assayed.


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structure is readily seen (35 examined) (Fig. 6A,B). Callosalaxons in mutant mice extend normally to the midline but failto cross. Instead, they remain ipsilateral and form neuromas,called Probst bundles (Probst, 1901). In addition to agenesis ofthe corpus callosum, formation of other forebrain midlinestructures is disrupted inp190 RhoGAP mutants. The anteriorand hippocampal commissures are absent at the midline or

minimally formed in all mutants (Fig. 6B and data not shown).Furthermore, an abnormal morphology of the third ventricle isseen in all of the mutants (Fig. 6C,D).

Agenesis of the corpus callosum can be caused by defectsin axon guidance and/or midline fusion of the cerebralhemispheres. To distinguish between thesepossibilities, we compared brain sections fromwild-type and p190 RhoGAP/ embryos atseveral developmental stages. We found thatoutgrowth of callosal axons towards themidline is normal in mutant mice (not shown).However, fusion of the cerebral hemispheres,which is completed at E17 in wild-typeembryos, is obviously delayed in the mutant

mice, as indicated by the persistence of thecerebral longitudinal fissure (Fig. 6E,F).Furthermore, at E16.5, when hemisphere fusionis underway, p190 RhoGAP protein isspecifically enriched in cells along the midlineof the forebrain, together with F-actin, althoughno obvious differences were seen in theappearance of actin in the corresponding regionof mutant embryos (data not shown).

In addition to the midline fusion defect in theforebrain of p190 RhoGAP mutant animals,these mice display a markedly disorganizedlayering of the cerebral cortex. Whereas inwild-type embryos, neurons in the cortical plateare densely packed (Fig. 6G,I), the neurons inthe same region in the mutant animals are muchmore diffusely organized (Fig. 6H,J),suggesting a defect in neuronal migration.Birth-dating of cortical neurons by BrDuincorporation during embryonic developmentdid not reveal any major defect in migrationassociated with particular layers of thedeveloping cortex (data not shown). In addition,the density of radial glial fibers, which are usedto guide the migration of cortical neurons fromthe ventricular zone, appears to be essentiallynormal in the mutants (Fig. 6K,L). Other areasof the brain, including the brain stem andcerebellum, do not display any obviousmalformations inp190 RhoGAP/animals.

Neural tube closure defects in p190RhoGAPmutant mice

Although most p190 RhoGAP/ newbornanimals appear grossly normal externally,approximately 30% of the mutant animalsexhibit exencephaly (Fig. 7A and 7B).Exencephaly typically reflects a defect inclosure of the anterior neural tube, whichnormally occurs between E8.5 and E9.5 (Copp

et al., 1990). Therefore, E10.5 embryos were examined byscanning electron microscopy for neural tube defects.Approximately 30% of p190 RhoGAP/ embryos (but nowild-type or p190 RhoGAP+/ embryos) display a severecranial neural tube closure defect. These embryos have an openneural tube extending from the developing forebrain to thepresumptive hindbrain (Fig. 7C-F). Complete closure of the

caudal neural tube was observed in all p190 RhoGAP mutantanimals, although the morphology of the roof plate wasabnormal, suggesting a mild closure defect in the spinal cordas well (data not shown).

Unlike in some mouse mutants displaying defects in neural


Fig. 5. Abnormal eye development inp190 RhoGAP mutant mice. (A-D) Whole-

mount eyes from E18.5 embryos at low (A,B) and high magnification (C,D). In thep190 RhoGAP mutants (B,D), hyperplasia of the RPE and a persistent optic fissure(arrowhead in D) can be seen, both of which are never observed in eyes dissectedfrom wild-type animals (A,C). (E-J) Phalloidin-stained transverse retinal sectionsfrom E11.5 (E,H), E12.5 (F,I) and E14.5 (G,J) wild-type (E-G) andp190 RhoGAP/

(H-J) embryos, revealing the invagination of retinal tissue associated with theabnormally fused neuroepithelial lips (arrowheads in H, I, J) inp190 RhoGAPmutants. (K-O) Retinal sections of E12.5 (K,L) and E14.5 (M-O) wild-type (K,M)and mutant (L-O) embryos stained with an anti-cortactin antibody revealing noabnormalities in cellular polarity in retinal cells in p190 RhoGAP mutant mice.Arrowheads indicate the presence of the optic fissure at E12.5 and the persistence ofthe fissure at E14.5 in mutant tissue. N and O are adjacent regions of the same mutantretina, demonstrating apparently normal cellular polarity outside the optic fissure.RPE, retinal pigmented epithelium.


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tube closure, no enhanced apoptosis was observed in theneuroepithelium of p190 RhoGAP mutants at E9.5 (data notshown). However, phalloidin staining of E10.5 wild-typeand mutant spinal cord sections revealed a substantiallyincreased accumulation of F-actin along the basal edge ofneuroepithelial cells at the presumptive floor plate in themutants (Fig. 7G,H). In some mutant animals, a reduced

apical constriction of neuroepithelial cells in this region isalso seen (Fig. 7H). Thus, the neural tube defect in p190RhoGAP/ mice appears to be associated with abnormalcell shape changes and actin assembly within theneuroepithelium. At E8.25, p190 RhoGAP mRNA isexpressed specifically in the neural folds (Fig. 7I), consistentwith a role for the protein in cells that participate directly inthis midline fusion event. At slightly later stages, expressionis more widespread, with a higher level of mRNA present inthe dorsal neural crest at E8.5 (Fig. 7K) and in the developingeye at E9.0 (Fig. 7J).

p190 RhoGAP localization is regulated by PKC

The midline fusion defects seen in p190 RhoGAP/

animals are strikingly similar to those previously reportedin mice lacking the major PKC substrate, MARCKS,including agenesis of the corpus callosum, a defect infusion of the cerebral forebrain, an open anterior neuraltube, eye defects, and occasional omphalocele (Stumpoet al., 1995). Since p190 RhoGAP is prominentlyphosphorylated on serine residues (Ellis et al., 1990), weinvestigated whether p190 RhoGAP, like MARCKs, isregulated by PKC-mediated phosphorylation. First, wedetermined that purified baculovirus-produced p190RhoGAP is a direct substrate of PKC in vitro (Fig. 8A). Inaddition, we found that p190 RhoGAP undergoes a

significant increase in its overall phosphorylation statefollowing exposure of cells to the PKC activator, 12-O-tetradeconoyl-phorbol-13-acetate (TPA) (Fig. 8B). To assessthe physiological relevance of this phosphorylation in vivo, weexamined the distribution of p190 RhoGAP in culturedfibroblasts before and after stimulation with TPA. Interestingly,we observed that p190 RhoGAP is rapidly relocalized from the

cytoplasm to regions of plasma membrane ruffling followingexposure of cells to TPA (Fig. 8C,E). Moreover, p190 RhoGAPis colocalized with polymerized actin in the TPA-inducedruffles (Fig. 8F). Therefore, it appears that PKC canphosphorylate p190 RhoGAP in vivo, and that thisphosphorylation, like the phosphorylation of MARCKS byPKC (Thelen et al., 1991), regulates the subcellularlocalization of p190 RhoGAP.

Since PKC, like p190 RhoGAP, has been implicated inintegrin-mediated signaling to the actin cytoskeleton (Defilippiet al., 1997), we next examined the distribution of p190 RhoGAPin cells during spreading on fibronectin. As was seen in TPA-

Fig. 6. Abnormal forebrain development inp190 RhoGAPmutant mice. (A-D) Haematoxylin and Eosin stained coronalforebrain sections from a wild-type (A,C) and ap190

RhoGAP/(B,D) newborn mouse. (A) A well-developed corpuscallosum (arrow) and anterior commissure (arrowhead) can beseen in the wild-type brain. (B) In thep190 RhoGAP mutant,instead of traversing the midline, callosal axons remainipsilateral and form Probst bundles (arrow). In addition, theanterior commissure is not formed in the mutant forebrain(arrowhead at the region corresponding to arrow in A).(C,D) The third ventricle, which has a characteristic morphologyin wild-type newborn mice (C), exhibits an abnormalmorphology inp190 RhoGAP mutant animals (D), and isfrequently aberrantly folded (arrow in D). (E,F) Toluidine Blue-stained coronal plastic sections of E17.5 wild-type andp190

RhoGAP/

brains highlighting the midline region where thecerebral hemispheres normally fuse. By E17.5, the cerebralhemispheres have fused in the wild-type brain (E), whereas inthep190 RhoGAP/brain (F), the cerebral longitudinal fissureis still present, as indicated by the arrowheads. (G-J) Nissl-stained coronal forebrain sections from E18.5 embryos at low(G,H) and high magnification (I,J) showing the compactappearance of the cortical plate of wild-type mice(G,I; horizontal bars demarcate the cortical plate (CP)) asopposed to the diffuse organization of the cortical plate in themutant mice (H,J). (K,L) Rat-401 staining of E16.5 wild-type(K) and mutant (L) coronal forebrain sections, demonstrating thenormal appearance of radial glial fibers (indicated by arrows) inthe developing cortex of p190 RhoGAP mutant embryos.


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treated cells, in fibroblasts plated on fibronectin, p190 RhoGAPrapidly re-localizes to membrane ruffles, where it co-localizeswith polymerized actin (Fig. 8I,J). By contrast, cells spreadingon poly-L-lysine exhibit no redistribution of p190 RhoGAPprotein (Fig. 8G,H). Thus, integrin-mediated cell spreadinginduces the same redistribution of p190 RhoGAP as does PKCactivation. The translocation of p190 RhoGAP observed in both

cases can be completely blocked by pretreatment of cells withPKC inhibitors (data not shown). The observed redistributionof p190 RhoGAP, as seen by immunostaining with a p190RhoGAP antibody, was confirmed in TPA-treated cellsfollowing transfection with a plasmid that expresses an epitope-tagged p190 RhoGAP (Fig. 8K,L). As described earlier,fibroblasts derived from p190 RhoGAP mutant mice appear tospread normally on fibronectin, potentially reflectingredundancy provided by p190-B, which is also a substrate forPKC and relocalizes to membrane ruffles upon PKC activation(data not shown). We also found that in Cos cellsexpressing a recombinant form of p190RhoGAP that mimics the truncation producedin the mutant mice, TPA can induce its

redistribution to the plasma membrane,suggesting that the response to PKC ismaintained in the altered protein (Fig. 8M,N).However, there is a difference in the appearanceof immunostained retinal sections from wild-type and mutant p190 RhoGAP mice, suggestingthat subcellular distribution may be regulated byadditional factors (Fig. 8O,P).

DISCUSSION

Using a gene targeting strategy, we haveinvestigated the biological function of p190RhoGAP in mammalian development. Previousstudies in cultured cells have revealed that theRho GTPase plays an important role inregulating cell shape and that p190 RhoGAP, apotent Rho inhibitor, mediates a signal to theactin cytoskeleton in response to integrinactivation. Our in vivo studies demonstrate anessential role for p190 RhoGAP in regulating avariety of morphogenetic events in neuraldevelopment that are believed to requireadhesion molecule-dependent modulation ofthe actin cytoskeleton. While some recentstudies have implicated Rho GTPase signalingin tissue morphogenesis in invertebrateorganisms, including Drosophila and C.elegans (Settleman, 1999), these are the firstgenetic studies that establish a critical role forRho GTPase regulation in mammalianembryonic morphogenesis.

Although we cannot conclude that thetargeted p190 RhoGAP allele that was used inthese studies is a null allele, the fact that it isdefective for all of the measurable in vivofunctions of the p190 RhoGAP protein suggeststhat it is, minimally, a severely defective loss-of-function allele. However, it remains formally

possible that a complete deletion of the gene might result inadditional or more severe phenotypes. As is the case wheninterpreting the results of many gene targeting experiments inmice, the analysis of the p190 RhoGAP mutant phenotype issomewhat complicated by the potential redundancy provided bythe closely related p190-B molecule, which is also expressedthroughout the nervous system (Burbelo et al., 1998; M. B., S.

M. and J. S., unpublished observation). This may account for thefact that the p190 RhoGAP mutant phenotype is restricted to asubset of neural tissues, despite widespread expression of theprotein throughout the nervous system. In fact, the regionalrestriction of phenotypes in p190 RhoGAP mutant mice hasmade it difficult to isolate and compare cell populations fromwild-type and mutant animals in order to further explore thecellular nature of the observed tissue defects. Thus far, we havenot been able to identify cellular defects in such brain-derivedcultures (M. B., S. M., and J. S., unpublished observations).


Fig. 7. Defective closure of the anterior neural tube inp190 RhoGAP mutant embryos.(A,B) Heads from E15.5 wild-type (A) and mutant (B) embryos. Arrow in B indicatesbrain tissue outside of the calvarium in this exencephalic embryo. (C-F) Scanningelectron micrographs of E10.5 wild-type (C) andp190 RhoGAP mutant (D-F) embryos,demonstrating the open anterior neural tube inp190 RhoGAP mutant embryos.(C,D) Frontal/dorsal view of the head region, (E) lateral view of ap190 RhoGAPmutant embryo, (F) dorsal view of the head region of ap190 RhoGAP mutant embryo.(G,H) Transverse sections of E10.5 wild-type (G) and mutant (H) embryos, stainedwith phalloidin to label F-actin in the developing neural tube. Large arrowheadsindicate the basal edge of neuroepithelial cells at the floor plate. (I-L) Whole-mount insitu hybridizations of wild-type embryos with antisense (I-K) or sense (L) p190RhoGAP-specific probes (I: E8.25, J: E9.0, K,L: E8.5). Arrow in I indicates theanterior neural folds; arrowhead in J indicates the developing eye; arrow in K indicatesthe dorsal neural crest. f, forebrain; h, hindbrain; m, midbrain.


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Significantly, p190-B accounts for the majority of total RhoGAPactivity in fibroblasts (Vincent and Settleman, 1999), indicatingthat in some cell types, p190-B may be the major regulator ofRho GTPase activity. Notably, we did not find evidence ofcompensatory up-regulation of p190-B expression in cells ortissues derived from thep190 RhoGAP mutant mice (M. B. andJ. S., unpublished observation).

Consistent with the predominantly nervous system-restrictedexpression profile of the p190 RhoGAP gene duringembryogenesis, we found that mice lacking a functional p190RhoGAP protein exhibit defects that appear to be largelyrestricted to the developing nervous system. A detailedhistological analysis of the mutant mice revealed that threetemporally and spatially distinct midline fusion events inneural development require a functional p190 RhoGAPprotein: namely, closure of the cranial neural tube, fusion ofthe hemispheres at the midline of theforebrain, and closure of the optic fissure.It is possible that there is also a requirementfor p190 RhoGAP in adult tissues, where itis widely expressed (Settleman et al.,

1992a), but the perinatal death of themutant mice precludes such an analysis.Presently, the specific cause of death in thep190 RhoGAP mutant animals is unknown.

The presence of multiple midline fusiondefects inp190 RhoGAP/animals suggeststhat a similar cellular mechanism, whichrequires p190 RhoGAP function, underliesthe morphogenetic process that normallyleads to these distinct fusion events. Indeed,previous observations support a mechanisticrelationship among the various midlinefusion events associated with normalmammalian neural development. Forexample, mice lacking the transcriptionfactor Pax2 exhibit a neural tube defect anda persistent optic fissure (Torres et al., 1996),and mice lacking the PKC substrate,MARCKS, as mentioned earlier, exhibitdefects in neural tube closure, fusion of theforebrain hemispheres, and formation of thecorpus callosum (Stumpo et al., 1995). Inaddition, many human patients that exhibitcoloboma of the eye also have agenesis ofthe corpus callosum (Denslow and Robb,1979), and approximately 5% of patientsidentified with a midline fusion defect alsoexhibit at least one additional midline fusiondefect (Khoury et al., 1989).

The observation that a small percentageof the p190 RhoGAP/ animals (like theMARCKS mutants) exhibit omphalocele, amidline fusion defect of the abdominal wall,also supports the conclusion that the distinctphenotypes observed in these animals are allrelated to a similar problem with tissuefusion. Clinical studies have revealed anassociation between the presence ofomphalocele and other midline defects,including an open neural tube, coloboma of

the eye, and agenesis of the corpus callosum (Calzolari et al.,1997; Martinez-Frias 1995). Thus, it seems likely that many, ifnot all, midline tissue fusion events in mammalian developmentare mediated by a common morphogenetic mechanism, and thatp190 RhoGAP is an important regulator of such fusion events.

A shared feature of these various morphogenetic processesof neural development is the regulated alteration of cell

morphology that is associated with dynamic rearrangements ofthe cytoskeleton. For example, a role for actin reorganization inneural tube closure is well documented (Schoenwolf and Smith,1990), and several mouse mutants lacking actin regulatoryproteins, such as Mena, Abl, Vinculin and MARCKS, exhibitanterior neural tube closure defects (Koleske et al., 1998; Lanieret al., 1999; Stumpo et al., 1995; Xu et al., 1998). TheMARCKS protein, which like p190 RhoGAP is regulated byPKC, associates directly with the actin cytoskeleton, and can

Fig. 8. Regulation of the subcellular localization of p190 RhoGAP by PKC. (A) Lipid-dependent in vitro phosphorylation of purified p190 RhoGAP by PKC. (B) In vivo

phosphorylation of p190 RhoGAP by PKC, as examined by immunoprecipitation of p190RhoGAP protein from phospholabeled MEFs, treated with the PKC activating phorbol esterTPA for 30 minutes. (C-F) Translocation of p190 RhoGAP from the cytoplasm in untreatedNIH 3T3 fibroblasts (C) to regions of membrane ruffling in cells exposed to TPA for 30minutes (E) as detected by p190 RhoGAP immunostaining. Co-localization with actin wasrevealed by phalloidin staining (D,F). A similar translocation of p190 RhoGAP protein isdetected in cells during spreading on fibronectin (I,J), in contrast with cells adhering topoly-L-lysine for 30 minutes (G, H), as detected by p190 RhoGAP immunostaining (G,I)and actin staining by phalloidin (H,L). (K-N) Immunofluorescence of Cos-7 cells,transiently expressing HA-tagged p190 RhoGAP (K,L), using an anti-HA-tag antibody, orthe R6 amino-terminal truncation mutant (M,N), using a p190 RhoGAP antibodyindicates a similar TPA-induced (L,N) translocation of p190 RhoGAP from cytoplasm tomembrane ruffles as seen with endogenous protein. Arrows indicate areas of membraneruffling. Magnifications: (C-J) 125, (K,L) 250.


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regulate membrane ruffling and cell spreading (Myat et al.,1997), suggesting that its role in neural tube closure may alsoinvolve the regulation of actin organization. Thus, it is likelythat Rho GTPase regulation of the actin cytoskeleton plays asignificant role in neural tube closure.

In comparisons of the neural tubes of wild-type and p190RhoGAP/embryos, we observed a substantial increase in F-

actin along the basal edge of neuroepithelial cells in mutantembryos, and defects in apical constrictions in these cells,suggesting abnormal regulation of actin polymerization at themedian hinge point. Significantly, the neural tube defect inp190RhoGAP mutants is largely restricted to the anterior neural tube,and it was recently reported that actin microfilamentreorganization in the apical region of neuroepithelial cells isrequired for closure of the cranial neural tube but not the spinalneural tube (Ybot-Gonzalez and Copp, 1999). Possibly, p190RhoGAP regulates the distribution of Rho-mediated actinpolymerization, thereby affecting neuroepithelial cell shapechanges. In light of the observation that PKC activation leads toa redistribution of p190 RhoGAP within cells, it is conceivablethat signals from activated adhesion molecules, via PKC, direct

p190 RhoGAP to subcellular sites of actin reorganization.Indeed, it has been observed that p190 RhoGAP rapidlyaccumulates at sites of antibody-mediated cross-linking ofintegrins in cultured cells (Burbelo et al., 1995). Loss of p190RhoGAP activity is likely to result in Rho activation, and it hasbeen observed in Drosophila that either a decrease or increasein Rho GTPase signaling is detrimental to several morphogeneticprocesses in embryonic development (Settleman, 1999),suggesting that disruption of Rho GTPase pathways inmammalian embryos may have similar consequences.

Abnormalities in actin-dependent cell shape changes mayalso account for the observed defects in ventriclemorphogenesis, forebrain fusion, and optic fissure closure inthe mutant mice. Indeed, we find that p190 RhoGAP proteinis enriched in cells along the forebrain longitudinal fissure,where it co-localizes with F-actin. However, mechanisms lessdirectly associated with cell shape change cannot be ruled out.For example, the optic fissure closure defect in Pax2 mutantmice is associated with a failure to degrade extracellularlaminin at the fissure (Torres et al., 1996). Interestingly, p190RhoGAP is required for local degradation of extracellularmatrix in melanoma cells exposed to laminin (Nakahara et al.,1998), suggesting that p190 RhoGAP may affect integrin-mediated tissue morphogenesis by regulating the formation ofcellular processes which secrete metalloproteases that remodelthe extracellular matrix.

Several cell culture studies point to a role for p190 RhoGAPin integrin-mediated signaling from the extracellular matrix(Burbelo et al., 1995; McGlade et al., 1993; Nakahara et al.,1998; Sharma, 1998). In fact, the defects observed in p190RhoGAP mutant mice are consistent with a role for p190RhoGAP in adhesion signaling not only via cell-matrixinteractions, but also through cell-cell adhesion. The cell-celladhesion molecules, N-cadherin and N-CAM (Bronner-Fraseret al., 1992), at least one integrin subunit (Kil et al., 1996), andthe laminin 5 chain (Miner et al., 1998) are required forproper neural tube closure. In addition, like p190 RhoGAP,integrin subunits 3 and v and neural adhesion molecules, L1and N-CAM, have each been implicated in neuronal migrationin the developing mouse brain (Anton et al., 1999; Asou et al.,

1992; Tomasiewicz et al., 1993). It is also worth noting thatseveral of the neural adhesion molecules have been directlyimplicated in the guidance and fasciculation of axons (Walshand Doherty, 1997), and we have begun to identify similardefects in a subset of axon tracts within the nervous system of p190 RhoGAP mutant mice (to be reported elsewhere).Activation of each of these various types of adhesion molecules

is known to affect actin re-organization (Burden-Gulley andLemmon, 1996), and it would therefore not be surprising tofind that their effects on the cytoskeleton are mediated by theRho GTPase and its regulators. Indeed, integrin-induced cellshape changes in fibroblasts require Rho GTPases (Clark et al.,1998; Hotchin and Hall, 1995).

Significantly, PKC has been implicated in the transduction ofsignals that affect cell shape both by integrins (Katz and Yamada,1997) and neural adhesion molecules, including L1, N-CAM andN-cadherin (Bixby and Jhabvala, 1990; Kolkova et al., 2000). Inaddition, the MARCKS-related protein, MacMARCKS, which isa PKC substrate, is also required for neural tube closure (Chenet al., 1996), and has been found to be required for integrin-dependent spreading of macrophages (Li et al., 1996). In light of

our observation that PKC as well as integrin activation induces asimilar re-localization of p190 RhoGAP, we propose that inresponse to engagement of diverse neural adhesion molecules,p190 RhoGAP functions to transduce signals via PKC activationthat direct the morphogenesis of neural tissues. Finally, it is worthnoting that all of the developmental defects observed in thep190RhoGAP mutant mice reflect defects that appear to beindependent of cell fate determination or cell proliferation. Thus,it appears that the role of the Rho GTPase in mammaliandevelopment may be restricted to the regulation of changes in cellshape and cell movements that determine the morphogenesis ofembryonic tissues.

We are grateful to Amin Fazeli, Piotr Sicinski, and Lena Du for

help in producing knockout cells and mice, and to Andrea McClatcheyfor critical reading of the manuscript. We also thank LawrenceCherkas for assistance with photography, Ed Seling for assistancewith electron microscopy, and Pradeep Bhide, Philippe Soriano, MarcTessier-Lavigne, and Ken Kosik for helpful discussions. We thankSheila Thomas for providing the cortactin antibody and Tony Pawsonfor providing Rat-2 GAP-N fibroblasts. This work is supported byNIH and ACS awards to J. S.

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Roles of p190 RhoGAP in mouse development. Development, 2000.

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