Genetic dissection of plexin signaling in vivo · Genetic dissection of plexin signaling in vivo Thomas Worzfelda,b,1, Jakub M. Swiercza, Aycan Sentürkc,d, Berit Genze, Alexander

Genetic dissection of plexin signaling in vivoThomas Worzfelda,b,1, Jakub M. Swiercza, Aycan Sentürkc,d, Berit Genze, Alexander Korostylevf,2, Suhua Dengf,3,Jingjing Xiaa, Mikio Hoshinog, Jonathan A. Epsteinh, Andrew M. Chani,4, Brigitte Vollmare, Amparo Acker-Palmerc,d,Rohini Kunerf, and Stefan Offermannsa,b

aDepartment of Pharmacology, Max-Planck-Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany; bMedical Faculty, University of Frankfurt,60590 Frankfurt am Main, Germany; cInstitute of Cell Biology and Neuroscience and Buchmann Institute for Molecular Life Sciences, Goethe UniversityFrankfurt, 60438 Frankfurt am Main, Germany; dFocus Program Translational Neurosciences, University of Mainz, 55131 Mainz, Germany; eInstitute forExperimental Surgery, University of Rostock, 18057 Rostock, Germany; fInstitute of Pharmacology, University of Heidelberg, 69120 Heidelberg, Germany;gDepartment of Biochemistry and Cellular Biology, National Institute of Neuroscience, Kodaira, Tokyo 187-8502, Japan; hDepartment of Cell andDevelopmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and iIcahn Medical Institute, Mount Sinai School ofMedicine, New York, NY 10029

Edited by David E. Clapham, Howard Hughes Medical Institute, Boston Childrens Hospital, Boston, MA, and approved January 2, 2014 (received for reviewMay 16, 2013)

Mammalian plexins constitute a family of transmembrane recep-tors for semaphorins and represent critical regulators of variousprocesses during development of the nervous, cardiovascular, skel-etal, and renal system. In vitro studies have shown that plexinsexert their effects via an intracellular R-Ras/M-Ras GTPase-activatingprotein (GAP) domain or by activation of RhoA through interactionwith Rho guanine nucleotide exchange factor proteins. However,which of these signaling pathways are relevant for plexin func-tions in vivo is largely unknown. Using an allelic series of trans-genic mice, we show that the GAP domain of plexins constitutestheir key signaling module during development. Mice in whichendogenous Plexin-B2 or Plexin-D1 is replaced by transgenic versionsharboring mutations in the GAP domain recapitulate the phenotypesof the respective null mutants in the developing nervous, vascular,and skeletal system. We further provide genetic evidence that, un-expectedly, the GAP domain-mediated developmental functions ofplexins are not brought about via R-Ras and M-Ras inactivation. Incontrast to the GAP domain mutants, Plexin-B2 transgenic micedefective in Rho guanine nucleotide exchange factor binding areviable and fertile but exhibit abnormal development of the livervasculature. Our genetic analyses uncover the in vivo context-dependence and functional specificity of individual plexin-mediatedsignaling pathways during development.

neural tube | cerebellum | outflow tract

Plexins constitute a family of transmembrane proteins thatserve as receptors for semaphorins (1). They function as key

regulators of a multitude of developmental processes, includingaxon guidance, pattern and synapse formation in the nervoussystem (2), vasculogenesis and angiogenesis (3, 4), and mor-phogenesis of the heart, kidney, and skeletal system (5). In theadult organism, plexins play crucial roles in the physiology andpathophysiology of the immune and cardiovascular system, as wellas in bone homeostasis and in cancer (6–9). Nine plexins have beenidentified in the mammalian system, which are grouped into foursubfamilies, A–D, according to sequence homologies.The activation of plexins by their semaphorin ligands triggers

several intracellular signaling cascades, most of which modulatethe activity of small GTPases (10). The intracellular domain of allplexins shares homology with GTPase-activating proteins (GAPs)and confers the deactivation of R-Ras, M-Ras, and Rap1 (11–17).The GAP activity toward R-Ras and M-Ras, but not toward Rap1,requires binding of Rnd GTPases to the plexin receptor (11, 12,15, 16). Plexins of the B-subfamily differ from all other plexins inthat they carry a C-terminal PDZ domain interaction motif thatmediates a stable interaction with the Rho guanine nucleotideexchange factor (RhoGEF) proteins PDZ-RhoGEF and LARG(18–20). Activation of B-plexins by semaphorin ligands results inactivation of the RhoGEF proteins and subsequent activation ofRhoA and RhoC (18–21). This process and the GAP function ofB-plexins are independent of each other, because mutations in the

GAP domain do not interfere with the activation of RhoA, anddeletion of the PDZ domain interaction motif does not influenceGAP activity (11).The plexin-mediated signal transduction pathways have been

identified and characterized mainly in in vitro systems usingrecombinant proteins, purified membranes, and cell culture models.These in vitro approaches have often yielded conflicting results withrespect to the functional relevance of individual plexin-mediatedsignaling pathways. For example, the axonal growth cone collapseof primary neurons has been suggested to be caused by plexin-mediated deactivation of R-Ras (11, 12), activation of RhoA (20,22), and deactivation of Rap1 (15). Which plexin signaling path-ways are functionally relevant in vivo is largely unknown.Here we report on the generation of an allelic series of BAC

transgenic mice carrying subtle mutations in the Plexin-B2 andPlexin-D1 gene, which specifically affect particular downstreamsignaling functions of these plexins. Our genetic analyses reveal akey function for the GAP domain of plexins during mouse de-velopment, which is independent of R-Ras and M-Ras inactivation.

Significance

Plexins, a family of transmembrane receptors for semaphorins,control diverse biological processes during mouse devel-opment. However, it is largely unknown through which sig-naling pathways they exert their functions in vivo. Using anallelic series of transgenic mice, we show that the GTPase ac-tivating protein domain of plexins constitutes their key sig-naling module during development, which is required forproper formation of the nervous, cardiovascular, and skeletalsystem. In contrast, development of the liver vasculature spe-cifically depends on the activation of the small GTPase RhoAby the plexin family member Plexin-B2. This study uncoversthe in vivo context-dependence and functional specificityof individual plexin-mediated signaling pathways duringmouse development.

Author contributions: T.W., R.K., and S.O. designed research; T.W., J.M.S., A.S., B.G., A.K.,S.D., and J.X. performed research; M.H., J.A.E., and A.M.C. contributed new reagents/analytic tools; T.W., A.S., B.G., B.V., A.A.-P., R.K., and S.O. analyzed data; and T.W. andS.O. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: [email protected].

2Present address: Institute of Stem Cell Research, German Research Center for Environ-mental Health, Helmholtz Zentrum München, 85764 Neuherberg, Germany.

3Present address: Department of Pathology, Stanford University Medical School, Stanford,CA 94305.

4Present address: School of Biomedical Sciences, Faculty of Medicine, The Chinese Univer-sity of Hong Kong, Hong Kong.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1308418111/-/DCSupplemental.

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Furthermore, we identify a requirement of Plexin-B2–mediatedRhoA activation for the development of the liver vasculature.

ResultsGeneration of Allelic Series of Plexin-B2 and Plexin-D1 BAC TransgenicMice. Plxnb2 knockout mice (plxnb2−/−) display neural tube clo-sure defects, which result in exencephaly and perinatal lethality(23, 24). A small proportion of mice (∼5%) bypasses this neuraltube closure phenotype and demonstrates abnormalities in cer-ebellar granule cell migration and defects in corticogenesis andmigration of neuroblasts in the subventricular zone (23–26). Inour effort to understand which signaling functions of Plexin-B2are relevant in vivo, we first generated BAC transgenic mice thatexpress triple-myc-tagged wild-type Plexin-B2 (BAC B2WT).This mouse line was crossed twice with mice heterozygous for theplxnb2 knockout allele (plxnb2+/−) to obtain animals in which thetransgenic BAC-encoded plxnb2 allele was the only functionalcopy of the plxnb2 gene (BAC B2WT; plxnb2−/−). These micewere fully rescued from phenotypes observed in plxnb2−/− mice,were viable and fertile, and produced offspring with the expectedMendelian frequencies (Fig. 1A). On the basis of the BACencoding wild-type Plexin-B2, we generated an allelic series ofmice expressing Plexin-B2 mutants (Fig. 1B). These mutantsincluded Plexin-B2 versions that carry mutations in all threearginines critical for GAP activity (BAC R1395A/R1396G/R1691A) (11), in the two N-terminal arginines (BAC R1395A/R1396G), or in the C-terminal arginine of the GAP domain(BAC R1691A), and a version that lacks the PDZ domain-binding motif (the four most C-terminal amino acids, VTDL)critical for interaction with RhoGEF proteins (20) (SI Appendix,Figs. S1 and S2A). In contrast to the Plexin-B2 versions carryingmutations of all three arginines or of the two N-terminal argi-nines of the GAP domain, the Plexin-B2 version with a mutationof the C-terminal arginine retained the ability to bind R-Ras (SIAppendix, Fig. S2 B–D). We extended our analysis of plexin-dependent signaling to the prototypic member of another plexinsubfamily, Plexin-D1, which is mainly expressed in endothelial

cells and neurons during embryonic development (27). Plexin-D1–deficient mice exhibit defects in vascular patterning, an-giogenesis and outflow tract septation of the heart, skeletalmalformations, errors in axonal projections and synapse for-mation, and defects in thymocyte migration (28–36). Micelacking Plexin-D1 have only one great vessel arising from theheart (persistent truncus arteriosus) instead of two (aorta andpulmonary artery) and therefore die shortly after birth. Inanalogy to the approach with Plexin-B2, we first generateda BAC transgenic mouse line expressing triple-myc-taggedwild-type Plexin-D1 (BAC D1WT), which fully rescued thedevelopmental defects observed in plxnd1−/− mice: the inter-somitic vessels, the outflow tract of the heart, and the skeletonappeared indistinguishable from wild-type mice (Fig. 1C), andthe mice were viable and fertile and produced offspring withthe expected Mendelian frequencies. On the basis of the BACencoding wild-type Plexin-D1, we established mouse lines har-boring mutations of the three arginines required for the GAPfunction of Plexin-D1 (BAC R1484A/R1485A/R1770A, BACR1484A/R1485A, BAC R1770A) (Fig. 1D).The expression levels and the expression pattern of all BAC-

encoded plexin proteins were comparable to the endogenous wild-type proteins (SI Appendix, Supporting Information and Figs. S3–S7).

The GAP Domain of Plexin-B2 Is Required for Neural Tube Closure andCerebellar Granule Cell Migration.To assess the biological relevanceof individual Plexin-B2–dependent signaling pathways during de-velopment, we analyzed embryos of the Plexin-B2 mutant mouselines at embryonic day (E) 9.5 and 15.5. The neural tube closuredefect observed in plxnb2 knockout mice was fully rescued byexpression of a mutant Plexin-B2 version lacking the PDZ do-main-binding motif required for interaction with RhoGEFproteins (Fig. 2A). In contrast, mice in which the endogenousPlexin-B2 was replaced by a Plexin-B2 version with mutationsin the three arginines required for GAP activity phenocopiedthe plxnb2 knockout and displayed an open neural tube at E9.5and exencephaly at E15.5 (Fig. 2A). Similarly, expression of

Fig. 1. Generation of BAC transgenic mice ex-pressing triple-myc-tagged versions of Plexin-B2 andPlexin-D1. (A) (Upper) Pictures of E15.5 embryos;arrow points to the open cephalic neural tube(exencephaly). (Lower) Nissl-stained adult cerebella;boxed areas are magnified below, arrows point toclusters of ectopic granule cells at the cerebellarsurface. (Scale bars, 3 mm in Upper, 300 μm inLower.) (B) Schematic illustration of the allelic seriesof plxnb2 BAC transgenes. Purple, triple-myc-tag;blue, Sema domain; yellow, PSI domains; red, IPT/TIG domains; dark blue, split GAP domain; orange,Rnd/RhoD/Rac1 binding site; green, PDZ domaininteraction motif. (C) (Top) WhoIe-mount immuno-histochemistry on E11.5 embryos using an anti-CD31antibody. Arrowheads point to intersomitic vessels,and arrow points to disorganized intersomitic ves-sels. (Middle) Pictures of the cardiac outflow tractof E18.5 embryos. Ao, Aorta; PA, pulmonary artery;TA, truncus arteriosus. (Bottom) Alcian blue/Alizarinred staining of P0 skeletons. Arrow points to fusionof vertebral bodies. (Scale bars, 300 μm in Top andMiddle, 1 mm in Bottom.) (D) Schematic illustrationof the allelic series of plxnd1 BAC transgenes. Pur-ple, triple-myc-tag; blue, Sema domain; yellow, PSIdomains; red, IPT/TIG domains; dark blue, split GAPdomain; orange, Rnd/RhoD/Rac1 binding site.

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Plexin-B2 versions with mutations in either the N-terminal orthe C-terminal arginines of the split GAP domain of Plexin-B2also failed to rescue the neural tube closure defects (Fig. 2A). Totest the functional significance of individual Plexin-B2–mediatedsignaling pathways in the developing cerebellum, we crossedmice carrying floxed alleles of Plexin-B2 (plxnb2flox/flox) with miceexpressing Cre specifically in cerebellar granule cell precursorcells under the control of the Atoh1 promoter (Atoh1-Cre;plxnb2flox/flox) (23, 37). These mice phenocopied the cerebellarabnormalities observed in surviving plxnb2 global knockout mice(Fig. 2B). Whereas expression of transgenic wild-type Plexin-B2fully rescued the cerebellar defects of Atoh1-Cre;plxnb2flox/floxmice, expression of mutant Plexin-B2 versions defective in GAPactivity failed to do so (Fig. 2B). Mice expressing Plexin-B2 de-fective in RhoGEF binding showed a normal development of thecerebellum (SI Appendix, Fig. S8). These data indicate thatneural tube closure and migration of cerebellar granule cellprecursors require a functional GAP domain of Plexin-B2 andare independent of its ability to interact with RhoGEF proteins.

Outflow Tract Septation and Skeletal Morphogenesis Require the GAPDomain of Plexin-D1. To evaluate the specific contribution ofPlexin-D1–dependent signaling events during development, weexamined mouse lines at E18.5 with respect to the heart and theskeletal system. Plexin-D1 mutant mouse lines, in which theendogenous Plexin-D1 was replaced by mutant Plexin-D1 pro-teins defective in GAP activity, displayed defects in outflow tract

septation, with a persistent truncus arteriosus as observed inPlexin-D1–deficient animals (Fig. 3A). These animals also phe-nocopied the axial skeletal morphogenesis defects observed inPlexin-D1 knockout mice (Fig. 3B) and became cyanotic anddied shortly after birth. These results indicate that outflow tractseptation of the heart and axial skeletal patterning depend onthe GAP domain of Plexin-D1.

The GAP Domain-Dependent Developmental Functions of Plexin-B2and Plexin-D1 Are Independent of R-Ras and M-Ras Inactivation. Inin vitro assays, the GAP domain of plexins has been shown toexert its effects on cellular functions through inactivation ofR-Ras (11, 12, 38). If the GAP domain-dependent develop-mental functions of plexins in vivo also rely on their ability toinactivate R-Ras, the developmental defects observed in plexinknockout mice should result from the failure to inactivate R-Ras.We reasoned that in plexin knockout mice this failure to in-activate R-Ras could be compensated for by genetic inactivationof R-Ras, which should at least partially rescue the develop-mental defects. To test this hypothesis, we crossed Plexin-B2-and Plexin-D1–deficient mice with R-Ras–deficient mice, whichare viable and fertile and devoid of morphological abnormalities(39) (Fig. 4). Surprisingly, genetic inactivation of R-Ras amelio-rated neither neural tube closure failure (Fig. 4A) nor cerebellargranule cell migration defects observed in Plexin-B2 knockoutmice (Fig. 4B); furthermore, it failed to rescue the outflowtract septation defects in Plexin-D1 knockout mice (Fig. 4C).

Fig. 2. Neural tube closure and cerebellar granulecell migration depend on the GAP domain of Plexin-B2. (A) (Upper) Scanning electron microscopy pic-tures of E9.5 embryos. Arrow points to nonfusedneural head folds. (Lower) Pictures of E15.5 em-bryos. Arrow points to the open cephalic neuraltube (exencephaly). (Scale bars, 200 μm in Upper, 3mm in Lower.) (B) (Upper) Nissl staining of adultcerebella. (Lower) Magnification of the boxed areasin Upper. Arrows point to clusters of ectopic gran-ule cells. (Scale bar, 300 μm.)

Fig. 3. Outflow tract septation and skeletal de-velopment depend on the GAP domain of Plexin-D1. (A) Pictures of the cardiac outflow tract of E18.5embryos. Ao, Aorta; PA, pulmonary artery; TA, trun-cus arteriosus. (Scale bar, 300 μm.) (B) Alcian blue/Alizarin red staining of E18.5 skeletons. Arrow pointsto splitting of vertebral bodies. (Scale bar, 1 mm.)

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Given that plexins have been reported to influence cellular be-havior also by inactivation of M-Ras (14), we also crossed Plexin-B2- and Plexin-D1–deficient mice with mice carrying a geneticinactivation of M-Ras, which are viable and develop normally(40). However, neither M-Ras deficiency nor R-Ras-/M-Rasdouble deficiency protected from defects in neural tube closure(Fig. 4A) or cerebellar (Fig. 4B) or heart development (Fig. 4C)in Plexin-B2 or Plexin-D1 knockout mice, respectively. A bio-chemical analysis revealed that R-Ras-GTP levels are unchangedin Plexin-B2 transgenic mice harboring mutations in the GAPdomain (SI Appendix, Fig. S9). These results strongly suggest thatthe GAP domain-dependent developmental functions of Plexin-B2 and Plexin-D1 are independent of their ability to inactivateR-Ras and M-Ras.

Plexin-B2–Mediated RhoA Activation Is Dispensable for NeocorticalDevelopment. During development of the nervous system, Plexin-B2 is strongly expressed in the telencephalic ventricular and sub-ventricular zone, as well as in the neuroepithelium of the medialand lateral ganglionic eminences, and mice lacking Plexin-B2 thatbypass the neural tube closure defect display severe abnormalitiesin corticogenesis, including abnormal cortical layering and de-fective migration and differentiation of several subtypes of corticalneurons (25, 41). The small GTPase RhoA plays a key role in thedevelopment of the nervous system by regulating a multitude ofcellular functions, including cell migration, polarity, and survival(42, 43), and regulation of RhoA activity is required for cortico-genesis (44, 45). To test whether Plexin-B2 exerts its functions incorticogenesis via activation of RhoA, we examined mice in whichthe endogenous Plexin-B2 was replaced by a Plexin-B2 versiondefective in RhoGEF binding. A detailed analysis of neocorticaldevelopment over critical embryonic stages using typical markerproteins for cortical layering, neuronal processes, and glia cellsdid not reveal abnormalities in the morphology, number, andpositioning of neurons. Tbr1-positive early-born neurons, Brn1-positive later-born neurons, CSPG-positive preplate neurons,MAP2-positive neuronal processes, Cajal-Retzius cells (Reelin-positive), and glia (GFAP-positive astrocytes and RC2-positiveradial glia) at embryonic stages E15.5 (SI Appendix, Fig. S10A)and E17.5 (SI Appendix, Fig. S10B) appeared indistinguishablebetween mutants and controls, indicating that Plexin-B2–mediatedRhoA activation is not essential for corticogenesis.

Development of Liver Vasculature Requires Plexin-B2–Mediated RhoAActivation. Plexin-B2 is strongly expressed in the endothelial cellsof the hepatic sinusoids and portal veins (Fig. 5A) (46). Weobserved that mutant mice defective in RhoGEF bindingexhibited malformations in the liver vasculature. These abnor-malities included venous ectasias and misorganization of thevascular architecture (Fig. 5 B and C). Albeit not fully penetrant,this phenotype was found both under physiological conditions aswell as in regenerating liver tissue after partial hepatectomy (Fig.5 B and C). The vascular malformations were predominantly butnot exclusively located at or in the vicinity of portal veins (Fig. 5B and C). Consistent with this, intravital fluorescence microscopyrevealed that sinusoidal blood flow and perfusion was largelyunaffected (SI Appendix, Fig. S11 A–C), and signs of hepatocytedamage were not detectable (SI Appendix, Fig. S11 D and E). In

Fig. 4. R-Ras and M-Ras deficiency do not rescuePlexin-B2 and Plexin-D1 knockout phenotypes. (A)(Upper) Scanning electron microscopy pictures ofE9.5 embryos. Arrow points to nonfused neuralhead folds. (Lower) Pictures of E15.5 embryos. Ar-row points to the open cephalic neural tube(exencephaly). (Scale bars, 200 μm in Upper, 3 mm inLower.) (B) (Upper) Nissl staining of adult cerebella.(Lower) Magnification of Insets in Upper. Arrowspoint to disorganized cerebellar folia. (Scale bars,300 μm.) (C) Pictures of the cardiac outflow tract ofE18.5 embryos. Ao, Aorta; PA, pulmonary artery;TA, truncus arteriosus. (Scale bar, 300 μm.)

Fig. 5. Plexin-B2–mediated RhoA signaling is required for development ofthe liver vasculature. (A) Immunofluorescence staining of adult liver usinganti-Plexin-B2 and anti-CD31 antibodies. (Scale bar, 50 μm.) (B) H&E-stainedsections derived from untreated liver (Upper) or regenerating liver 7 d after2/3 partial hepatectomy (Lower). Arrows point to bile ducts. (Scale bars, 100μm.) (C) 3D reconstructions of Z stacks of CD31-stained sections derived fromuntreated liver (Upper) or regenerating liver 7 d after 2/3 partial hepatec-tomy (Lower). (Scale bar, 50 μm.) (D) Intravital fluorescence microscopy pic-tures of adult livers. (Scale bar, 200 μm.)

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addition to the venous ectasias, the livers of some mutant miceexhibited blood vessels with abnormally wide diameter, whichwere already visible macroscopically (Fig. 5D). These resultsshow that Plexin-B2–mediated activation of RhoA is involved inthe formation of the liver vasculature.

DiscussionPlexins have been shown to regulate a number of different sig-naling pathways in diverse types of cells in vitro (10). Despite awealth of literature on the important roles of plexins in develop-ment and disease, the signaling events downstream of plexinsmediating their effects in vivo have been largely unknown. Herewe directly addressed this fundamental question by generatingan allelic series of BAC transgenic mice carrying subtle mutationsin the Plexin-B2 and Plexin-D1 genes at sites that encode residuescritical for distinct signaling pathways. Given that mouse nullmutants of most genes are available, our BAC transgenic rescueapproach represents an efficient alternative to conventional geneticapproaches relying on the knockin of mutations by homologousrecombination in embryonic stem cells, and is widely applicableto the analysis of other signaling pathways in vivo.A common feature of all plexins is their intracellular GAP

domain, which catalyzes the inactivation of R-Ras, M-Ras, andRap1 upon binding of semaphorin ligands (11, 14, 15). Surpris-ingly, although plexins have been shown to regulate several in-dependent signaling pathways in vitro, mutations in the GAPdomain were sufficient to fully phenocopy the null mutants in vivowith respect to development of the nervous, the vascular, and theskeletal system. This crucial importance of the GAP domain ofplexins during mouse development is in line with findings inDrosophila and Caenorhabditis elegans, where the GAP domainsof PlexA and PLX-1 have been shown to be critical for de-velopment of the nervous system (47, 48). Owing to the perinatallethality of Plexin-B2 and Plexin-D1 GAP domain mutant mice,we could not systematically address the functional significanceof the GAP domain at postnatal stages of development or inphysiological and pathophysiological processes in the adult [e.g.,for Plexin-B2 in the postnatal migration and proliferation ofneuroblasts (26) and in wound healing (49), or for Plexin-D1in postnatal development of the nervous system (34, 35) and inangiogenesis (36, 50)]. Further studies using mice with floxedalleles crossed to the BAC transgenic and specific Cre mice willbe required to address these open questions.In addition to uncovering the crucial role of the plexin GAP

domain in vivo, this study also revealed considerable insights intothe mechanism of GAP domain function during development.The functional significance of the enzymatic activity of the plexinGAP domain has not been resolved hitherto in studies per-formed in vitro, owing to controversial observations: whereasseveral reports have shown that GAP domain-mediated in-activation of R-Ras and M-Ras regulates cellular behavior,including migration, axonal growth cone collapse, and dendritemorphology (11, 14, 38), others suggest that binding and se-questration of active R-Ras, rather than R-Ras inactivation, isrequired for the biological effects of plexins (15, 51). We foundthat mutations in all three critical arginines or the two N-terminalarginines of the Plexin-B2 GAP domain block both R-Ras GAPactivity as well as R-Ras binding, whereas mutation of the C-ter-minal arginine results in the loss of GAP activity toward R-Raswhile retaining the ability to bind R-Ras, thereby allowing a dif-ferentiation between these two scenarios. Our in vivo data on thephenotypes of transgenic mice expressing these different Plexin-B2 mutant versions favor a model whereby the enzymatic in-activation of small GTPases, rather than binding and seques-tration, is the mechanism by which the plexin GAP domain exertsits biological effects.To further test the functional relevance of plexin-mediated

R-Ras and M-Ras inactivation in vivo, we performed a rescue ex-periment by genetically inactivating R-Ras and M-Ras in Plexin-B2- and Plexin-D1–deficient mice. Surprisingly, even the combinedinactivation of R-Ras and M-Ras did not ameliorate the defects

observed in the Plexin-B2- and Plexin-D1 null mutants, stronglysuggesting that R-Ras and M-Ras are not the major effectors ofPlexin-B2 and Plexin-D1 during development. Of note, plexinsexert GAP activity toward R-Ras and M-Ras only when Rndproteins are bound to their intracellular moieties (11, 14, 16).Interestingly, in vitro data show that the GAP domain of plexinscan regulate cellular effects even in the absence of Rnd expression(30, 51). Recently, it has been shown that Rap1 proteins are al-ternative substrates of the plexin GAP domain and that GAPactivity toward Rap1 does not require Rnd binding to plexins(15). Rap1 proteins are well-established regulators of inside-outsignaling to integrins, cell adhesion, cell proliferation, and celljunction formation (52), and it is tempting to speculate that theycould represent the critical substrates of the plexin GAP domainduring development. Alternatively, the combinatorial inactivationof R-Ras, M-Ras, and Rap1 or the inactivation of an as yet un-identified GTPase could account for the developmental functionsof plexins. The GTPase activity of plexins toward R-Ras andM-Rasmight also be important at later developmental stages of neuronaldevelopment, including dendrite remodeling (14), or under path-ophysiological conditions such as tumor angiogenesis, in whichactive R-Ras has been shown to improve vessel integrity andblood vessel perfusion (39, 53).In addition to the GAP function common to all plexins, B-

family plexins can activate the small GTPase RhoA, a centralregulator of cytoskeletal dynamics and cell migration that is re-quired for neocortical development (44, 45). Our data indicatethat Plexin-B2–mediated RhoA activation is dispensable fordevelopment of the neocortex. Of note, the semaphorin ligandfor Plexin-B2 in the embryonic cortex, Sema4D, also binds to theclose Plexin-B2 homolog, Plexin-B1, with high affinity (1, 25).Although Plexin-B1 is strongly expressed in the developingneocortex, the nervous system of Plexin-B1–deficient mice de-velops normally (23, 41). It is plausible that the loss of Plexin-B1-or Plexin-B2–mediated regulation of RhoA activity could mu-tually be compensated for by the presence of the respective otherfamily member and that the combined inhibition of this pathwaycould reveal its biological role in the developing neocortex.In contrast to its redundant role in corticogenesis, we identi-

fied an involvement of Plexin-B2–mediated RhoA activation inthe formation of the liver vasculature, indicating that the require-ment for RhoA activation varies depending upon the tissue. Theobserved abnormalities are in line with the expression pattern ofPlexin-B2 in the highly specialized discontinuous endothelium ofthe liver. During both development and liver regeneration inthe adult, the hepatic portal veins and sinusoids derive frompreexisting vessels through angiogenesis (54, 55). There is evidencethat RhoA activation downstream of Plexin-B1 stimulates cellularprocesses in endothelial cells that are crucial for angiogenesis,including chemotactic migration and tubulogenesis (56). Of note,in liver sinusoidal endothelial cells, a critical role for RhoA in theregulation of the actin cytoskeleton has been described (57).Apart from its role in the liver vasculature, Plexin-B2–mediated

activation of RhoA may also be important under certain patho-physiological conditions. Indeed, there is evidence that RhoAsignaling via the Plexin-B2 homolog, Plexin-B1, is crucially in-volved in the suppression of osteoblast differentiation and in themetastatic dissemination of ErbB-2–positive cancer cells (21, 58).In summary, we demonstrate that Plexin-B2 and Plexin-D1

exert their developmental functions through the GAP domainindependently of the inactivation of R-Ras and M-Ras. In ad-dition, we show that Plexin-B2–mediated RhoA activation isrequired for development of the liver vasculature. This studyclarifies signaling pathways that account for the diverse biologicalfunctions of plexins in mouse development in vivo and underscoresthe context- and tissue-dependence of the recruitment of specificpathways downstream of an activated plexin.

Materials and MethodsGeneration of BAC Transgenic Mice. The following BAC clones were ob-tained from the BACPAC Resource Center (Children’s Hospital Oakland

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Research Institute, Oakland, CA): RP24-245O13 carrying the murine plxnb2gene, and RP23-396M22 carrying the murine plxnd1 gene. All BAC modi-fications were performed using the Counter Selection BAC ModificationKit (Gene Bridges). Further details are provided in SI Appendix, SI Materialsand Methods.

RNA Extraction and RT-PCR. RNA extraction was performed using an RNeasyKit (Qiagen) according to the manufacturer’s instructions. RT-PCR was doneusing standard reagents and protocols (Fermentas).

ACKNOWLEDGMENTS. Rras−/− mice were kindly provided by Erkki Ruoslahti.

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