Ccm3, a gene associated with cerebral cavernous ......Ccm3, a gene associated with cerebral cavernous malformations, is required for neuronal migration Angeliki Louvi1,*, Sayoko Nishimura1

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© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1404-1415 doi:10.1242/dev.093526

ABSTRACTLoss of function of cerebral cavernous malformation 3 (CCM3) resultsin an autosomal dominant cerebrovascular disorder. Here, weuncover a developmental role for CCM3 in regulating neuronalmigration in the neocortex. Using cell type-specific gene inactivationin mice, we show that CCM3 has both cell autonomous and cell non-autonomous functions in neural progenitors and is specificallyrequired in radial glia and newly born pyramidal neurons migratingthrough the subventricular zone, but not in those migrating throughthe cortical plate. Loss of CCM3 function leads to RhoA activation,alterations in the actin and microtubule cytoskeleton affectingneuronal morphology, and abnormalities in laminar positioning ofprimarily late-born neurons, indicating CCM3 involvement in radialglia-dependent locomotion and possible interaction with theCdk5/RhoA pathway. Thus, we identify a novel cytoplasmic regulatorof neuronal migration and demonstrate that its inactivation in radialglia progenitors and nascent neurons produces severe malformationsof cortical development.

KEY WORDS: Radial glia, Neocortex, Nascent neurons, Cellautonomous function, Mouse, CCM3 (PDCD10)

INTRODUCTIONNeuronal migration is one of the fundamental processes governingcentral nervous system development. In the mammalian cerebralneocortex, projection neurons are generated by progenitor cellsresiding in the ventricular and subventricular zones (VZ and SVZ,respectively) of the dorsal telencephalon and segregate by radialmigration, eventually settling into distinct cell layers according totheir birthdates (Angevine and Sidman, 1961). To migrate, early-born neurons send their leading processes directly to the marginalzone (MZ) whereas late-born neurons depend on radial glia, atransient cell type whose cell soma is in the VZ and whose longbasal process spans the cerebral wall. Radial glia cells serve as bothneural progenitors and a dynamic scaffold for migrating neurons(Rakic, 1972; Miyata et al., 2001; Malatesta et al., 2003; Noctor etal., 2004; Yokota et al., 2010).

Impaired neuronal migration invariably leads to corticallamination defects and has been associated with mutations in severalgenes (e.g. Deuel et al., 2006; Koizumi et al., 2006; Bilgüvar et al.,2010), many of which encode components of two well-investigatedpathways. The first consists of reelin, its receptors Vldlr and Lrp8(also known as ApoER2), and its effector Dab1 (Rice and Curran,2001); the second comprises Cdk5 and its neural-specific activatorsp35 (Cdk5r1 – Mouse Genome Informatics) and p39 (Cdk5r2 –

RESEARCH ARTICLE

1Departments of Neurosurgery and Neurobiology, Yale Program onNeurogenetics, Yale School of Medicine, New Haven, CT 06520, USA.2Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.

*Author for correspondence ([email protected])

Received 20 December 2012; Accepted 22 January 2014

Mouse Genome Informatics) (Dhavan and Tsai, 2001; Su and Tsai,2011) and appear to regulate, respectively, radial glia-independentsomal translocation and radial glia-guided locomotion (Gupta et al.,2003; Ohshima et al., 2007; Franco et al., 2011).

Cerebral cavernous malformation 3 [CCM3; also known asprogrammed cell death 10 (PDCD10)], is one of three genes [krev1interaction trapped gene 1 (KRIT1) (also known as CCM1), CCM2and PDCD10] (Riant et al., 2010) that are mutated in familial CCM,a common neurovascular disorder characterized by the presence ofcerebrovascular lesions (Russell and Rubinstein, 1989; Ozturk et al.,2011). CCM3 is a cytoplasmic protein that can be found in acomplex with CCM1 and CCM2 (Hilder et al., 2007; Voss et al.,2007), and is thought to promote assembly of the Golgi apparatus innon-neuronal cell lines (Fidalgo et al., 2010; Kean et al., 2011).CCM1, CCM2 and CCM3 are expressed in endothelial cells and areessential for vascular development (Whitehead et al., 2004; Bouldayet al., 2009; Kleaveland et al., 2009; Whitehead et al., 2009; He etal., 2010; Zheng et al., 2010; Chan et al., 2011; Yoruk et al., 2012).They are also expressed in pyramidal neurons and astrocytes(Guzeloglu-Kayisli et al., 2004; Seker et al., 2006; Tanriover et al.,2008); in cultured neurons, CCM3 is found throughout the cell bodyand processes, but is not enriched in the Golgi (Lin et al., 2010).Loss of CCM3 in neural progenitors has cell-autonomous (astrocyteactivation) and non-autonomous effects (diffusely dilatedcerebrovasculature and lesion formation) in postnatal brain (Louviet al., 2011). CCM3 downregulation in cell lines and primaryastrocytes enhances proliferation and cell survival (Chen et al.,2009; Louvi et al., 2011). For an overview of current models ofCCM protein function, see Draheim et al. (Draheim et al., 2014).

We examine the role of CCM3 in cortical development byanalyzing conditional mutants that either lack CCM3 in neuralprogenitors and their progeny (hGfap-Cre;Ccm3lox/lox and Emx1-Cre;Ccm3lox/lox) or retain CCM3 in radial glia and SVZ progenitorsbut not in postmitotic neurons in the IZ and cortical plate (NEX-Cre;Ccm3lox/lox). We show that CCM3 is specifically required inneural progenitors and nascent pyramidal neurons that migratethrough the SVZ and uncover cell-autonomous and non-autonomousfunctions in neuronal migration. Cortical lamination defects areunderlain by radial glia abnormalities and resemble those of Cdk5and p35 mutants, suggesting a possible involvement of CCM3 inradial glia-dependent locomotion. We further demonstrate thatCCM3 is necessary for cytoskeletal remodeling of postmitoticneurons, and that loss of CCM3 in neural progenitors results inRhoA activation in the brain.

RESULTSCCM3 is necessary for cortical neuronal migrationIn embryonic forebrain, Ccm3 mRNA is expressed in the VZ andSVZ, which contain progenitor cells and newly postmitotic neuronsmigrating through the SVZ towards the cortical plate (Fig. 1A,B;supplementary material Fig. S1A-E,K,M,N). We crossed Ccm3lox/lox

Ccm3, a gene associated with cerebral cavernous malformations,is required for neuronal migrationAngeliki Louvi1,*, Sayoko Nishimura1 and Murat Günel1,2

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RESEARCH ARTICLE Development (2014) doi:10.1242/dev.093526

animals (He et al., 2010; Louvi et al., 2011) with Emx1-Cre (Gorskiet al., 2002) and hGfap-Cre (Zhuo et al., 2001; Malatesta et al.,2003) mice to target neural progenitors and their progeny,respectively, in dorsal telencephalon at E9.5, or broadly in the CNSstarting at E13.5. Loss of CCM3 expression in the conditionalmutant animals was confirmed by in situ hybridization,immunostaining and western blotting (supplementary material Fig.S1E-G and data not shown) (Louvi et al., 2011).

We previously reported large brains or neocortices in hGfap-Cre;Ccm3lox/lox and Emx1-Cre;Ccm3lox/lox postnatal and adult mice(henceforth hGfap/Ccm3 cKO and Emx1/Ccm3 cKO) (Louvi et al.,

2011). Brain weights of cKO animals were indistinguishable fromcontrols in the first postnatal week [weight average±s.d.: control,0.168±0.01789 g versus hGfap/Ccm3 cKO, 0.155±0.00707 g(P>0.05, P0)]; control, 0.284±0.01819 g versus hGfap/Ccm3 cKO,0.29±0.01732 g (P>0.05, P6)]. No differences in cortical thicknessbetween cKO and control animals were noticed at birth (P>0.05),which significantly increased from P7 onwards (Emx1/Ccm3 cKO,P7; P=0.01034, n=3; hGfap/Ccm3 cKO; P=0.01332, n=3). In Nissl-stained preparations, total neuron number in matched areas ofsomatosensory cortex (SSC) was similar in control and Emx1/Ccm3cKO (P7 and P15, n=2 per age, P>0.05). To estimate neuronal cell

Fig. 1. Cortical laminationabnormalities in Emx1/Ccm3 cKOneocortex. (A) Ccm3 mRNA isexpressed in VZ/SVZ progenitorsand newly postmitotic neuronsmigrating through the VZ/SVZ, butnot in those migrating through thecortical plate at E13.5. (B) mRNAexpression of Pax6 (radial glia), Tbr2(SVZ progenitors) and Tbr1(newborn neurons) in serial sectionsfrom the same embryo as in A. Insitu hybridization as indicated. (C-L) In situ hybridization of brainsections of control (C,E,G,I,K) andEmx1/Ccm3 cKO (D,F,H,J,L)littermates. Emx1/Ccm3 cKOmutants lack clearly defined corticallayers: subplate neurons (Ctfg; C,D)are in the middle of the neocortex;L6 neurons (Tle4; E,F) aresuperficial to the subplate in a broaddiffuse area; L5 neurons (Er81; G,H)are dispersed in upper cortex; L4neurons (Rorb; I,J) are distributed inthe upper half of the neocortical walland L2-4 neurons (Cux2; K,L) in itsentire depth. Control brains areCcm3lox/lox (C and inset) orEmx1/Ccm3lox/+ (E,G,I,K). (C,D) P7;insets show high magnification ofsubplate neurons (P15); (E-L) P21.(M) The radial distribution of neuronsexpressing each marker is shown tothe right of the respective in situhybridization panels. Bars withbroken lines indicate that all neuronsin the bin are labeled and could notbe counted. In Emx1/Ccm3 cKOneocortex, early-born neurons areshifted towards more superficialpositions (bins 1-5), whereas late-born neurons are distributed acrossthe neocortical wall (bins 1-10);many remain in deep positions (bins6-10). Scale bars: 0.5 mm.

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size, we measured cell density in a defined area of SSC andcalculated the mean cell area (field area per number of neurons); inEmx1/Ccm3 cKO brains, the number of neurons wasindistinguishable from controls (P7 and P15; n=2 per age) (e.g.mean neuronal cell area±s.d.: P7 control, 361.28291±53.37218 μm2

versus cKO, 346.97748±42.51494 μm2; P=0.51575).Cortical organization was visualized using Nissl staining

(supplementary material Fig. S1O-R) and in situ hybridization withlayer-specific markers [Ctgf (subplate); Tle4 (subplate and L6); Er81(Etv1 – Mouse Genome Informatics) (L5); Rorb (L4) and Cux2 (L2-4)] indicated neuronal migration defects. Cortical lamination wasseverely disrupted in Emx1/Ccm3 cKO mutants: subplate neuronsoccupied a diffuse band near the middle of the neocortex(Fig. 1C,D,M); L5 and L6 neurons were displaced to the upper half(Fig. 1E-H, Fig. 2B,E), and those of L2-4 were dispersed eitheracross almost its entire depth (L4) (Fig. 1I,J) or in the bottom two-thirds (L2/3) (Fig. 1K,L, Fig. 2H,K). These abnormalities wereexacerbated in the significantly thickened cingulate cortex,persisting in mature animals (supplementary material Fig. S2A-J),suggesting that they were not due to developmental delay. Notably,the positions of deep layer neurons relative to each other were as inwild type, with L5 neurons located superficially to those of L6(Fig. 1E-H); however, L2-4 neurons failed to migrate past them.Although layer order was maintained in hGfap/Ccm3 cKO mutants,where recombination is initiated after formation of the cortical plateand generation of early-born neurons from Ccm3-expressingprogenitors, many late-born L2-3 neurons were broadly distributedand, consequently, layer borders were blurred (Fig. 2A,D,G,J;supplementary material Fig. S3A-J).

To examine the molecular properties of the Emx1/Ccm3 cKOneocortex, we analyzed whether Satb2 and Ctip2 (Bcl11b – MouseGenome Informatics), which mark non-overlapping populations ofupper layer and L5 neurons (Arlotta et al., 2005; Alcamo et al.,2008; Britanova et al., 2008), became abnormally co-expressed. Thenumber of cells positive for either marker was comparable in controland cKO at P0, P3 and P8 (n=2; P>0.05 for each age); however,their laminar distribution varied significantly [e.g. the upper one-third of the mutant neocortex at P3 contained fewer Satb2+ cells(P=0.02242) but more Ctip2+ cells (P=0.00801) than the control].Nevertheless, in mutants, as in controls, only a small fraction of cells

co-expressed both [respectively, 3.68% versus 5.02% (P3); 0.7%versus 0.33% (P8)], indicating that, despite ectopic positioning,mutant neurons were not molecularly respecified. Reelin-expressingCajal-Retzius cells that could potentially contribute to corticallamination abnormalities were normal (not shown).

We also generated hGfap/Ccm3Delta/lox and Emx1/Ccm3Delta/lox

animals, carrying one null (Ccm3Delta) and one conditional allele, bycrossing Ccm3Delta/lox (obtained by using ACTB-Cre) with hGfap- orEmx1-Cre;Ccm3lox/+ mice. Cre is expected to target more efficientlythe single floxed allele, decreasing mosaicism. Lamination defectswere more pronounced in hGfap/Ccm3Delta/lox and Emx1/Ccm3Delta/lox, respectively, compared with hGfap/Ccm3 cKO andEmx1/Ccm3 cKO mutants (supplementary material Fig. S2K-T, Fig.S3K-T), representing an allelic series of different strengths(hGfap/Ccm3 cKO <hGfap/Ccm3Delta/lox <Emx1/Ccm3 cKO <Exm1-Ccm3Delta/lox). These observations suggest that CCM3 is necessaryfor neuronal migration and that the severity of cortical laminationdefects depends on developmental timing and extent of Ccm3inactivation in neural progenitors.

CCM3 is required in radial glia cells and nascent neurons forproper migrationOur findings do not address whether CCM3 is independentlyrequired in radial glia and/or postmitotic neurons because,differences in onset of recombination notwithstanding, hGfap-Creand Emx1-Cre lead to gene inactivation in both. We used NEX-Cre(Goebbels et al., 2006) to target postmitotic neurons in dorsaltelencephalon. NEX-Cre;Ccm3lox/lox (NEX/Ccm3 cKO) embryosmaintained Ccm3 expression in VZ/SVZ progenitors(supplementary material Fig. S1H-N); the NEX-Cre and Ccm3expression domains overlap in the outer SVZ at E13.5 suggestingthat in early phases of NEX-Cre action, at least some Cre-positive,presumably nascent, neurons retain residual Ccm3 expression(supplementary material Fig. S1H,K,L). From E14.5 onwards, Ccm3mRNA levels were clearly reduced away from the VZ(supplementary material Fig. S1M,N). NEX/Ccm3 cKO andNEX/Ccm3Delta/lox mice were born at expected Mendelian ratios;brain size and cortical thickness (NEX/Ccm3 cKO, P14, n=3) wereindistinguishable from controls (P>0.05) and lamination was normal(Fig. 2C,F,I,L; supplementary material Fig. S4A-H), suggesting that

Fig. 2. Migration defects of upper and deeplayer cortical neurons in Ccm3 cKOmutants. (A-L) In situ hybridization of brainsections of control (A-C,G-I) and Ccm3 cKOmutant littermates [(D,J) hGfap/Ccm3 cKO;(E,K) Emx1/Ccm3 cKO; (F,L) NEX/Ccm3 cKO]with markers of deep [Er81 (A-F)] and upper[Cux2 (G-L)] layers at ages indicated. (A-F) L5neurons are displaced superficially in Emx1/Ccm3 cKO (E), but at deep positions inhGfap/Ccm3 cKO (D) and NEX/Ccm3 cKO (F)mutants, comparable with controls (A-C). (G-L) Subsets of L2-4 neurons reside inectopic deep positions in hGfap/Ccm3 cKO (J)but in upper cortex in NEX/Ccm3 cKO (L)mutants, comparable with controls (G-I). InEmx1/Ccm3 cKO, the majority of L2-4neurons are in deep positions, indicatingseverely disrupted migration (K). Scale bar:0.5 mm.

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CCM3 is required in radial glia progenitors but is dispensable inpostmitotic neurons in the IZ and cortical plate for migration andpositioning (Fig. 2). To further test this, we inactivated Ccm3 inmigrating neurons by in utero electroporation (IUE) using NeuroD-CreERT2 to drive expression selectively in neurons (supplementarymaterial Fig. S5A,B). IUE of the neocortical wall of floxed Ccm3(Ccm3lox/lox) embryos at E13.5 with NeuroD-CreERT2 and a Cre-responsive GFP-expressing construct (Stop-GFP) (Shim et al., 2012)indicated that co-electroporated neurons migrated normally, similarto those co-electroporated with pCAG-Cre (Matsuda and Cepko,2007), driving expression in all cells, and with Stop-GFP, whichserved as controls (supplementary material Fig. S5C,D), consistentwith observations in NEX/Ccm3 cKO animals. Therefore, CCM3deficiency in radial glia proper and likely nascent neurons in theVZ/SVZ is largely responsible for the migrational defects in hGfap/and Emx1/Ccm3 cKO mutants.

Cell-autonomous function of CCM3 in maintaining radial gliamorphologyIn Emx1/Ccm3 cKO mutants positioning of late-born neurons, whichdepend on radial glia for migration, was disrupted, prompting us toexamine the radial glia scaffold. Wild-type radial glia fibers labeledwith rat-401 (nestin) spanned the cortical wall and were alignedperpendicular to the surface where end-feet anchored, but wereirregular, wavy, less dense (often crisscrossing each other) and hadfewer surface contacts in cKO mutants (Fig. 3A-D). DiI applicationonto the surface to label radial glia processes and their somata in theVZ (Malatesta et al., 2000) showed that fibers spanned the corticalwall at E13.5 in hGfap/Ccm3 cKO mutants (onset ofrecombination), but were disorganized at E15.5; hardly any cellbodies were labeled, suggesting that not many processes contactedthe MZ (Fig. 3E-G); in the VZ, radial glia appeared normal (Fig.3J,K). Staining of filamentous actin (F-actin) with falloidin revealedthe characteristic honeycomb pattern at the ventricular surface inhGfap/Ccm3 cKO and Emx1/Ccm3 cKO. Electron microscopyshowed that the basal lamina was intact in Emx1/Ccm3 cKO(supplementary material Fig. S6A,B); however, radial glia fibersoriented towards the surface were difficult to identify in uppercortical plate (Fig. 3H,I; supplementary material Fig. S6G,H), whereneuronal cell bodies were irregularly arranged (supplementarymaterial Fig. S6C,D,I,J). Mitotic figures in the VZ/SVZ andadherens junctions between VZ progenitors were normal(supplementary material Fig. S6E,F and Fig. S7A-C). Theseobservations suggest that CCM3 regulates the morphology of radialglia basal processes.

To further test that CCM3 acts cell-autonomously in neuralprogenitors, we performed IUE of Ccm3lox/lox mice at E14.5 withpCAG-Cre and pCAG-GFP. In electroporated, GFP-expressing,cells and their descendants, Ccm3 is inactivated as a consequence ofCre expression; non-electroporated cells retain Ccm3 expression.Analyses of mice at E16.5 (2 days post-IUE) revealed many GFP+

cells still in the VZ, suggesting that CCM3 is required for propermigration of newborn neurons; in embryos electroporated withpCAG-GFP, the majority of GFP+ cells migrated away from the VZ(Fig. 4A-C). Furthermore, IUE with pCAG-Cre and Stop-GFP atE13.5 showed that fewer neurons migrated into the cortical plate 3days post-IUE compared with those electroporated with pCAG-GFP(Fig. 4D-F), likely due to morphological defects in surrounding Cre-expressing radial glia. The majority of cells electroporated only withpCAG-GFP developed leading processes directed towards thesurface, whereas Ccm3 mutant cells had short irregular processes(Fig. 4G-J). These observations suggest that CCM3 is cell-

autonomously required for development of radial glia processes, andthat migration of neurons associated with defective radial glia is cellnon-autonomously affected.

CCM3 does not affect neural progenitor proliferationWe examined whether Ccm3 deletion affected division of radial gliaby labeling dividing progenitors in late G2/M or S phase forphosphorylated histone H3 (PH3) or BrdU incorporation,respectively, at E12.5 to E15.5. At all stages, PH3+ cells were liningthe ventricle, and, from E13.5 onwards, were also in the neocorticalwall. No statistically significant differences in number of progenitorsdividing in the VZ or at basal positions were found between controland Emx1/Ccm3 cKO or hGfap/Ccm3 cKO brains (n=3 per genotypeand stage) (supplementary material Fig. S7A-C). The total number

Fig. 3. Radial glia defects in Ccm3 cKO mutants.(A-D) Immunofluorescent staining of radial glia (nestin) reveals disorganizedprocesses in Ccm3 cKO mutants (B,D) compared with controls (A,C). Radialglia processes contact the pial surface (A,C), but are irregular in hGfap/Ccm3cKO (B, E16.5) and Emx1/Ccm3 cKO mutants (D, E14.5). Arrows in Dindicate abnormally fasciculated axons. (E-G) DiI labeling of cortical radialglia. At E15.5 (E,F) DiI-labeled cell bodies can be visualized at the VZ incontrol (E, arrow) but not in hGfap/Ccm3 cKO (F) embryos (asterisk indicatesthe ventricular surface). At E13.5 (approximate onset of hGfap-Cre action)labeled radial glia fibers span the cortical wall (G). (H,I) Electron micrographsshow radial glia fibers in upper cortical plate in Ccm3lox/+ (H, arrow), but not inEmx1/Ccm3 cKO littermates at E14.5 (I). (J,K) Immunofluorescent staining ofradial glia (nestin) reveals normal arrangement at the ventricular surface ofE14.5 Emx1/Ccm3 cKO (K) and control littermates (J). Scale bars: 20 μm inA-D; 50 μm in E-G; 10 μm in H,I; 20 μm in J,K.

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of BrdU+ cells (1-hour pulse) was similar in control and Emx1/Ccm3cKO embryos at E13.5 (n=5 per genotype) and E15.5 (n=3)(Student’s t-test, P>0.05), and in control and hGfap/Ccm3 cKOembryos at E13.5 (1 or 3 hours, n=3), and their radial distributionwas similar across all littermates analyzed (P>0.05, Mann-WhitneyU test) (supplementary material Fig. S7D,E,H,I,L). Neuronalbirthdating [hGfap/Ccm3 cKO: E14.5 to P10 (n=2), E14.5 to 7months (n=1), E14.5 to P7 (n=2), E16.5 to P2 (n=2); Emx1/Ccm3cKO: E13.5 to P7 (n=2), E14.5 to P0 (n=2), E17.5 to P7 (n=4),E18.5 to P7 (n=2)] revealed comparable numbers of BrdU+ cells incontrol and cKO brains (P>0.05 for all comparisons betweengroups), but significantly altered laminar distribution, in agreementwith in situ hybridization analysis (supplementary material Fig.S7F,G,J-L). Staining with Satb2 or Ctip2 and BrdU demonstratedthat the percentage of BrdU+ cells co-expressing either marker didnot differ significantly between brains from control and cKOlittermates, suggesting that production of different neuronal types isnot altered in the Emx1/cKO mutants [E13.5 to P0 (n=4); E15.5 toP4 (n=5); P>0.05 for all comparisons] (supplementary material Fig.S7M). Staining with β-tubulin type III indicated no delays inneurogenesis (not shown). In cKO neocortex, therefore, progenitorproliferation and neurogenesis are not disrupted, neurons with the

same birthdate occupy different relative positions compared withnormal, and the cytoarchitectonic defects are independent of thetime of neuron origin. Our observations, which are indeed consistentwith classic studies of the reeler mouse, the archetype of corticallaminar defects (Caviness and Sidman, 1973), further suggest thatincreased neocortical (Emx1/Ccm3 cKO) or overall brain(hGfap/Ccm3 cKO) size – and the absence of such effects inNEX/Ccm3 cKO mutants – is likely to be due to overproliferation ofastrocytes postnatally, a notion supported by significant andprogressive increase of Gfap mRNA and protein levels (Louvi et al.,2011).

CCM3 affects neuronal morphology in vivo and in vitroDefects in radial glia morphology, adhesion or polarity may or maynot be associated with changes in neuronal and glial differentiation(Rašin et al., 2007; Cappello et al., 2012). We used IUE to introducepCAG-GFP in the neocortical wall of littermates from crossesbetween Ccm3Delta/lox and Emx1/Ccm3lox/+ animals at E13.5 andanalyzed neuronal morphology 3 days post-IUE by fluorescencemicroscopy. GFP+ cells were localized in the IZ and cortical platein control embryos; however, in cKO mice, most arrested in theSVZ/IZ and only a few reached the lower cortical plate with leading

Fig. 4. Cell-autonomous function of CCM3 in neural progenitors. (A-C) In utero electroporation (IUE) in the neocortical wall of Ccm3lox/lox embryos (E14.5)with CAG-GFP (A) or CAG-Cre and CAG-GFP (B) plasmids, analyzed at E16.5. In the presence of Cre (B), migration of electroporated cells is delayed.(C) Quantification of radial distributions of GFP-expressing cells. In the presence of Cre, the distribution of labeled cells is shifted toward the VZ (bin 1 is at theventricular surface and bin 10 at the MZ). Error bars represent s.e.m. Student’s t-test, *P<0.05. (D-J) IUE in the neocortical wall of Ccm3lox/lox embryos (E13.5)with CAG-GFP (D,G,H) or CAG-Cre and Cre-responsive GFP (Stop-GFP) (E,I,J) plasmids, analyzed at E16.5. Following recombination, only a few GFP-expressing cells (thus, Cre-expressing) are in the cortical plate (cp), indicating delayed migration (E,I), compared with cells expressing GFP only (D,G).Quantification of the radial distribution of GFP-expressing cells from the outer IZ (bin 1) to the MZ (bin 5) (F). Significantly more Cre-expressing cells remaindeep; GFP-only-expressing cells are in superficial positions. Error bars represent s.e.m. Student’s t-test, *P<0.05. High magnification of the leading processesof cells that have migrated into the cortical plate; compared with GFP-only-expressing cells, a large proportion of which extend long processes toward thecortical surface (89.43±6.44% in upper cortical plate), the leading processes of Cre-expressing cells are shorter and irregular, with a significant fraction(36.33±8.7%) displaying very short processes (H,J). Insets in G,I are low magnification views of G,H and I,J. Scale bars: 0.05 mm in A,B,D,E; 20 μm in G-J;0.2 mm in insets (G,I).

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processes extending toward the pia (Fig. 5A-D). Control neuronswere multipolar at the pre-migratory zone, and bipolar at the upperIZ and cortical plate (Tabata and Nakajima, 2003; Noctor et al.,2004). A large proportion of Ccm3 cKO neurons (56.36±8.83%,n=3) in the migratory zone were abnormal, many failing to transitioninto bipolar morphology, displaying multiple very thin processes(Fig. 5E,F). GFP-expressing axons extended tangentially within theIZ in controls, but were stunted in cKO lacking defined trajectories(Fig. 5G,H).

We established primary cortical neuronal cultures fromhGfap/Ccm3 cKO and Emx1/Ccm3 cKO at E13.5 to E15.5 andexamined at 3, 5 and 7 days in vitro (DIV) (Fig. 5I-P; supplementarymaterial Fig. S8). cKO neurons had multiple short neurites thatresembled filopodia, revealed by Tuj1 or falloidin staining; manyexpressed MAP2, identifying them as dendrites, and weresignificantly shorter in the cKO neurons (Fig. 5I-N; supplementary

material Fig. S8A-G); doublecortin (DCX) staining indicated defectsin the microtubule cytoskeleton (Fig. 5O,P). Golgi morphology anddeployment, in part regulated by the CCM3 interactor STK25(Matsuki et al., 2010; Ceccarelli et al., 2011; Xu et al., 2012), werenormal (not shown).

Selectively labeled (Golgi-Cox impregnation) adult controlneurons were polarized, with a long, thick apical dendrite extendingtoward the pia and multiple smaller dendrites arising from the soma,whereas neuronal arrangement lacked a recognizable pattern anddendritic development was abnormal in Emx1/Ccm3 cKO and to alesser degree in hGfap/Ccm3 cKO mutants (Fig. 5Q-V and notshown). Finally, SMI32 and MAP2 staining revealed abnormaldendrites in NEX/Ccm3 cKO neocortex (not shown).

These observations indicate a defect of multipolar to bipolartransition in mutant neurons in vivo, consistent with morphologicalabnormalities in primary cultures. They also suggest that CCM3

Fig. 5. Abnormal cortical neuronal morphology in Ccm3 cKO mutants. (A-H) In utero electroporation (IUE) in the neocortical wall of Emx1/Ccm3 cKO andcontrol littermates (E13.5) with the CAG-GFP plasmid, analyzed at E16.5 by fluorescence microscopy. (A,B) In control embryos (A), postmitotic neuronsmigrate into the cortical plate (cp) 3 days post-IUE; by contrast, many Emx1/Ccm3 cKO neurons remain in IZ (B). (C,D) In upper cortical plate, Emx1/Ccm3cKO neurons are irregular and their leading processes do not reach the pial surface (D); control neurons are regularly arranged (C). (E,F) Migratory postmitoticneurons in upper IZ have transitioned into bipolar morphology (E; arrow indicates a leading process oriented towards the neocortical surface); however,Emx1/Ccm3 cKO neurons either remain multipolar or retain multiple short processes that are not oriented towards the pial surface [F; each of the four panelsrepresents a different mutant neuron with distinct morphology; note multiple short processes (arrowheads)]. Neocortical surface is towards the top. (G,H) Low-magnification views of lateral neocortex showing axonal projections of electroporated neurons in control (G) and Emx1/Ccm3 cKO (H) littermates. Midline istowards the left. Axons extend towards the midline (G), but have irregular trajectories in the mutants (H). Insets are longer exposure views of axonal projectionssuperimposed onto short exposure images of the electroporated hemisphere. (I-P) Morphology of primary cortical neurons. Immunofluorescent staining ofneurons from Emx1/Ccm3 cKO (J,N) and hGfap/Ccm3 cKO (L,P) embryos, and control (I,K,M,O) littermates cultured for 3-5 days in vitro (DIV). (I,J) TUJ1 (I,control; J, Emx1/Ccm3Delta/lox); neurons isolated from E14.5 embryos at 5 DIV. (K,L) Falloidin staining of F-actin (K, control; L, hGfap/Ccm3 cKO); neuronsisolated from E13.5 embryos at 5 DIV. (M,N) MAP2 (M: control; N: Emx1/Ccm3 cKO); neurons isolated from E14.5 embryos at 3 DIV. cKO neurons (N) developmultiple shorter dendrites when compared with controls (M). See also supplementary material Fig. S8G. (O,P) DCX (O, control; P, hGfap/Ccm3 cKO); neuronsisolated from E15.5 embryos at 5 DIV. (Q-V) Golgi-Cox staining of adult Emx1/Ccm3lox/+ control (Q,S,T) and Emx1/Ccm3 cKO (R,U,V) brains. Dendritic treesare evident throughout the control neocortex, especially in upper layers (Q,S,T), but are underdeveloped in cKO (R,U,V). (Q,R) Symmetrical views ofdorsomedial neocortex oriented towards the midline; (S,U) medial parietal cortex; (T,V) SSC. In all panels, the cortical surface is towards the top. Scale bars:0.05 mm in A,B; 20 μm in C,D; 10 μm in E,F; 0.05 mm in G,H; 0.02 mm in I-P; 0.2 mm in Q,R; 0.05 mm in S-V.

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function is required for dendrite formation and that its loss disrupts the actin and microtubule cytoskeletons. This furtherindicates that, in addition to being essential in radial glia forneuronal migration, CCM3 is required cell-autonomously forneurite outgrowth.

Abnormal axonal trajectories in Ccm3 cKO mutantsStaining of axonal projections with 2H3 (Fig. 6A-H) revealed thin,ectopic and abnormally bifurcated bundles in Emx1/Ccm3 cKO (Fig.6D,G,H) compared with controls (Fig. 6B,E,F). Cytochrome oxidasehistochemistry (Fig. 6I-K) and Nissl staining (Fig. 6L-N) showed adisorganized (hGfap/Ccm3 cKO) or nearly absent (Emx1/Ccm3cKO) barrel field (Fig. 6J,K,M,N), suggesting that post-synaptic L4neurons failed to segregate. Thalamocortical axons immunostainedwith vGlut2 formed barrel-shaped bundles in control, were irregularin hGfap/Ccm3 cKO and failed to cluster in Emx1/Ccm3 cKO (Fig.6O-Q). These observations suggest abnormal axonal projections.

RhoA activation in Ccm3 cKO mutant neocortexCCM3 loss results in defects in neuronal migration and in actin andmicrotubule cytoskeletal remodeling, processes partly regulated byRho GTPases (Heng et al., 2010; Govek et al., 2011; Kawauchi,2011). Several observations suggest a possible interaction betweenRhoA and CCM (Crose et al., 2009; Whitehead et al., 2009;

Borikova et al., 2010; Stockton et al., 2010; Zheng et al., 2010;Louvi et al., 2011; McDonald et al., 2011; Cappello et al., 2012;McDonald et al., 2012). RhoA and Ccm3 have similar expression inneocortex and are required in radial glia for neuronal migration (thisstudy) (Cappello et al., 2012). Active, GTP-bound Rho was elevated2.7-fold in hGfap/Ccm3 cKO mutants (E15.5, n=3) and 1.4-fold inhGfap/Ccm3lox/+ heterozygotes compared with controls (n=2) (Fig.7A,B); whole-genome microarray analysis indicated a 1.43553-fold(P=0.00103957) RhoA upregulation in hGfap/Ccm3 cKO overcontrol neocortex (n=3; P2), suggesting that RhoA activation mayunderlie the migrational and cytoskeletal defects in cKO neocortex.

Placing CCM3 in context: a connection with the Cdk5/p35pathway?Emx1/Ccm3 cKO, Cdk5 and p35 mutants (this study) (Ohshima etal., 1996; Chae et al., 1997; Gilmore et al., 1998; Kwon and Tsai,1998; Ohshima et al., 2007; Hoerder-Suabedissen et al., 2009) havenearly identical cortical lamination defects and strikingly similarCtgf, Er81 and Cux2 profiles (Fig. 1D,H,L) (Ohshima et al., 2007;Hoerder-Suabedissen et al., 2009) raising the possibility of CCM3interaction with the Cdk5/p35 pathway, which regulates actin andmicrotubule dynamics during neuronal migration, as well asdendritic development of pyramidal neurons (Xie et al., 2003;Kawauchi et al., 2006; Ohshima et al., 2007), processes also

Fig. 6. Abnormal axonal trajectories inCcm3 cKO mutants. (A-D) Immunofluorescentstaining of axons (2H3) in control (A,B) andEmx1/Ccm3 cKO (C,D) or Emx1/Ccm3Delta/lox

littermates (A-D: E14.5; E-H: E16.5). Indorsomedial mutant cortex, axonal bundlesappear thinner (C) and aberrant axonal tractsare ectopically located (arrow in D) near the piaand abnormally fasciculated (D) compared withcontrols (B). Abnormalities in fasciculation andbifurcation of afferent and efferent axons (E-H).F,H are high-magnification views of E,G. (I-Q) The barrel field (I,L,O) is poorly formed inhGfap/Ccm3 cKO (J,M,P) and absent fromEmx1/Ccm3 cKO (K,N,Q) mutants. Tangentialsections through L4 of flattened P15 (I-K), P22(L-N) or P8 (O-Q) cortices processed forcytochrome oxidase (CO) histochemistry (I-K),Nissl staining of postsynaptic L4 neurons (L-N)and immunofluorescent staining (vGlut2) ofthalamocortical afferent terminals (O-Q). Scalebars: in A, 500 μm for A,C; in B, 20 μm for B,D;in E,F, 50 μm for E-H; in I, 200 μm for I-N; in O,200 μm for O-Q.

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disrupted in the Ccm3 cKO mutants. Cdk5 modulates actinreorganization via RhoA suppression and regulation of cofilinactivity (Kawauchi et al., 2006), and controls microtubuleorganization via FAK phosphorylation (Xie et al., 2003). A principaltarget of FAK is paxillin, to which CCM3 has been shown to bind(Li et al., 2010), and we previously detected highly abundant cofilinin lesions by RNA-Seq (FPMK average±s.d.: 101.6±7.3) (Louvi etal., 2011). Cofilin activity is regulated through phosphorylation (Ser3); we observed an increase in inactive, phosphorylated cofilin inneocortical lysates from Emx1/Ccm3 cKO (1.2-fold at E13.5, n=3;1.6-fold at P3, n=5) compared with controls, consistent with RhoAactivation (Fig. 7C,D). Immunostaining showed increased p-cofilinimmunoreactivity in the Emx1/Ccm3 cKO neocortex compared withcontrol (Fig. 7E,F). By contrast, paxillin levels or phosphorylationwere unaffected (not shown). These findings suggest that theCdk5/RhoA/cofilin pathway is disturbed in the Ccm3 cKO mutants.

DISCUSSIONHere, we demonstrate that CCM3 is a crucial regulator of neuronalmigration and that its inactivation in radial glia and nascent neuronsproduces severe neocortical malformations in a developmental time-dependent manner. CCM3 impacts on the actin and microtubulecytoskeletons, and is required for neurite outgrowth in vivo and invitro. These functions appear to be mediated, at least partly, byRhoA activation.

The allelic series of Ccm3 cKO mutants display a spectrum ofneuronal migration and positioning abnormalities (Fig. 8A-F). Thenormal cortical lamination of NEX/Ccm3 cKO and NEX/Ccm3Delta/lox

mutants implies that CCM3 is not required in late-migrating neuronsin the IZ and cortical plate for radial glia-independent migration, orassociation with and detachment from CCM3+ radial glia. Inagreement with genetic findings, NeuroD-Cre-mediated deletion ofCcm3 in postmitotic neurons has no obvious consequences on

migration. Because at early stages of NEX-Cre action, Cre+

(presumably newborn) neurons retain some CCM3 due to the delayin the effects of recombination on protein levels, whether CCM3action is required in early-migrating neurons in the SVZ remainsunknown. However, our finding that neuronal morphology is cell-autonomously affected suggests a role for CCM3 in neuriteoutgrowth, a process indeed initiated in early-migrating pyramidalneurons in the SVZ (Shoukimas and Hinds, 1978; Polleux andSnider, 2010), lending support to the notion that they require CCM3.

Despite broad defects in Emx1/Ccm3 cKO and Emx1/Ccm3Delta/lox

mutants, L5 neurons still migrate past their predecessors, implyingthat migration of early-born neurons is not disturbed, even thoughboth radial glia and neurons lack CCM3. By contrast, most L2-4neurons, which largely employ glia-guided locomotion, fail tomigrate past earlier-born neurons, pointing to CCM3-deficient radialglia as the underlying cause of migration defects. Considering thatCCM3 is not required in late-migrating neurons, our findingssuggest that CCM3 also has a cell non-autonomous function inradial glia, affecting interactions with postmitotic neurons duringradial glia-guided migration, which are reminiscent of its cell non-autonomous functions in the neurovascular unit (Louvi et al., 2011).However, CCM3 also acts cell-autonomously, regulating themorphology of embryonic radial glia (this study) and of radial glia-derived postnatal astrocytes (Louvi et al., 2011). These cell-autonomous functions warrant further investigation, taking intoconsideration that CCM3 overexpression in vitro and in vivo causescell death (Chen et al., 2009; Lin et al., 2010). Genetic rescueexperiments that control for cell-type specific conditional re-expression of CCM3 at levels comparable to endogenous levels inmutant neocortex would need to be contrasted to the effects of focaland controlled overexpression of floxed CCM3 constructsintroduced into the Ccm3 cKO neocortex by IUE. The latterapproach will also allow us to observe the behavior of sparse

Fig. 7. RhoA activation in Ccm3 cKO mutant neocortex. (A) Western blot of neocortical protein extracts following a RhoA pull-down assay showing thatactivated Rho is elevated in hGfap/Ccm3 cKO neocortex at E15.5, compared with control samples. Total RhoA was used as loading control. Quantitativeanalysis indicates that activated RhoA is elevated 1.4-fold in hGfap/Ccm3lox/+ heterozygotes and 2.7-fold in hGfap/Ccm3 cKO mutants when compared withcontrols. Bars correspond to 25 kDa. (B) Histogram showing the quantification of the normalized signals obtained from A. Error bars represent s.e.m.(C) Western blot of neocortical protein extracts showing elevated ratio of inactive (Ser3 phosphorylated) cofilin to total cofilin in Emx1/Ccm3 cKO mutants.Quantitative analysis indicates that phospho-cofilin is increased 1.2-fold (E13.5; n=3) and 1.6-fold (P3; n=5) in Emx1/Ccm3 cKO mutants when compared withcontrols. Bars correspond to 25 kDa. (D) Histogram showing the quantification of the normalized signals obtained from (C). Error bars represent s.e.m.(E,F) Immunofluorescent staining of cortical neurons with phosphorylated cofilin shows more intense immunoreactivity in the neocortex of Emx1/Ccm3 cKO (D)when compared with control (C) (P3). Nuclei are counterstained with propidium iodide. Scale bar: 10 μm.

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‘rescued’ cells in an otherwise mutant environment. Conversely, byoverexpressing Cre recombinase in Ccm3lox/lox embryos, themorphology and behavior of sparse Ccm3-mutant cells can beanalyzed within the wild-type cortex, an extension of experimentsreported herein. Performing IUE at various embryonic stages andanalyzing the outcome at different times thereafter, will addresswhether the rescued neurons eventually settle at normal positions,thus allowing us to evaluate cell-autonomous versus cell non-autonomous functions of CCM3 in greater detail. Finally, it wouldalso be informative to examine using mouse chimeras the relativecontributions of CCM3-positive and -negative neurons in corticallayer development and positioning.

In hGfap/Ccm3 cKO and hGfap/Ccm3Delta/lox mutants, Ccm3 isinitially maintained in progenitors and early corticogenesis isnormal. At the onset of recombination, and presumably briefly,radial glia become transiently mosaic for Ccm3 expression; as theexact timing of recombination (and, therefore, Ccm3 inactivation)for any given cell is unknown, a neuron will stochastically attach toa radial glia fiber that might, or might not, still express CCM3, anddo this until Ccm3 has been inactivated in all progenitors. With thecaveat of CCM3 protein stability unknown, the hGfap/Ccm3 cKOanimals will be useful for addressing interactions between newbornneurons and mosaic radial glia. Finally, we conclude that CCM3-dependent migration is required for laminar positioning per se,because in hGfap/Ccm3Delta/lox mutants, even though the preplate isproperly split and early-born neurons migrate normally, late-bornneurons are mislocalized, displacing upwards those born earlier.

The dual cell-autonomous and cell non-autonomous function of acytoplasmic protein in radial glia is counter-intuitive. Intracellularmediators may translate extracellular cues to modifications of theactin and microtubule networks, as well as cell-cell adhesion, bothof which are modulated during neuronal migration (Marín et al.,2010). Therefore, CCM3 could control radial-glia-dependentneuronal migration non-autonomously by transducing extracellularsignals to the intracellular migration machinery. Notably, LIS1,NDEL1 and their signaling partner 14-3-3ε, cytoplasmic proteinsthat regulate positioning of late-born neurons, have significant cell

non-autonomous functions in neuronal migration revealed viamosaic analysis with double markers (Hippenmeyer et al., 2010).

Ccm3 is one of few genes known to be required exclusively inneural progenitors for migration, notwithstanding that only selectkey players have been tested independently in radial glia versuspostmitotic neurons. For example, Cdk5 is required cell-autonomously in both (Hirasawa et al., 2004; Ohshima et al., 2007),Dab1 is required only in migrating neurons (Franco et al., 2011),whereas β1 integrins (Belvindrah et al., 2007) and RhoA (Cappelloet al., 2012) are radial glia specific. Ccm3 (this study) (Louvi et al.,2011) and Itgb1 mutants display neuronal migration and corticallamination defects (Graus-Porta et al., 2001; Schmid et al., 2004;Huang et al., 2006; Marchetti et al., 2010), pial detachment ofendfeet (Graus-Porta et al., 2001; Kwon et al., 2011) and reactivegliosis (Robel et al., 2009), suggesting a – possibly indirect –interaction that warrants investigation, especially given theassociation of CCM1 with ICAP1α, which binds the β1 integrincytoplasmic tail (Zawistowski et al., 2002; Faurobert et al., 2013).On the other hand, RhoA is necessary in radial glia to stabilize theactin and microtubule cytoskeletons, and to maintain the radial gliascaffold (Cappello et al., 2012); thus, our findings that RhoA isactivated in Ccm3 conditional mutants could partially explain themigration defects.

In future studies it will be important to test the hypothesis thatCCM3 may generate signals in radial glia, which are thentransmitted to neurons to facilitate migration, thus accounting for thecell non-autonomous effects we uncovered, to determine whetherextracellular cues and signaling cascades regulate CCM3 activity tocontrol radial glia-dependent locomotion and to elucidatemechanistic relationships with the Cdk5/p35 pathway.

MATERIALS AND METHODSAnimalsMice were maintained in compliance with National Institutes of Healthguidelines and approval of the Yale University Institutional Animal Care andUse Committee. Ccm3lox mutants were reported previously (He et al., 2010;Louvi et al., 2011). hGfap-Cre (Zhuo et al., 2001; Malatesta et al., 2003),

Fig. 8. Summary of neuronal migration and positioning defects in Ccm3 cKO mutant neocortex. (A-F) Schematic drawings of the distribution of corticalpyramidal neurons in wild-type and Ccm3 cKO mice (P21). (A) Laminar positioning in wild-type mouse. (B) In hGfap/Ccm3 cKO mutants, cortical lamination isgrossly normal. A subset of L2/3 neurons fails to migrate to the upper cortex. (C) In hGfap/Ccm3Delta/lox mutants, in which one Ccm3 allele is deleted in all cellsand the other only in the radial glia lineage, most L5 neurons are displaced upwards near the pial surface; L4 neurons are distributed across the depth of theneocortex medially or displaced to the upper half laterally; upper layer neurons are dispersed across the neocortex. (D) In Emx1/Ccm3 cKO mutants, deeplayer neurons are displaced upwards, whereas those of upper layers reside either across almost the entire depth of the neocortex (L4), or at its bottom two-thirds (L2/3); subplate neurons are confined to a diffuse median band. Most L2-4 neurons accumulate underneath L5/6. The relative positions of deep layerneurons are as in wild type, despite their ectopic location in upper cortex. (E) The Exm1/Ccm3Delta/lox mutants have a slightly more severe phenotype than theEmx1/Ccm3 cKO, reflected in a broad neuronal dispersion across all cortical layers. (F) Normal laminar positioning in NEX/Ccm3 cKO (and NEX/Ccm3Delta/lox,not shown) mutants. MZ, marginal zone; L, layer; SP, subplate; WM, white matter.

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Emx1-IRES-Cre (Gorski et al., 2002) and ACTB-Cre (Lewandoski et al.,1997) mice were purchased from JAX. NEX-Cre (Goebbels et al., 2006;Belvindrah et al., 2007) mice were a gift from Drs Schwab and Nave (MaxPlanck Institute for Experimental Medicine, Goettingen, Germany).

In situ hybridizationEmbryonic and postnatal brains were fixed, respectively, by immersion inor intracardial perfusion with 4% paraformaldehyde (PFA), post-fixed in30% sucrose in 4% PFA and sectioned on a cryomicrotome (LeicaMicrosystems, Wetzlar, Germany). Sections were processed for in situhybridization as described previously (Tanriover et al., 2008). RNA probescomplementary to mouse Ccm3 (Louvi et al., 2011), Pax6 (Götz et al.,1998), Tbr1 and Tbr2 (Englund et al., 2005), Tle4 (Yao et al., 1998), Etv1(Weimann et al., 1999), Rorb (Nakagawa and O’Leary, 2003), Cux2 (Nietoet al., 2004; Zimmer et al., 2004) and Ctgf (Heuer et al., 2003) were labeledwith digoxigenin-11-UTP. Sections were analyzed using a Stemistereomicroscope or AxioImager (Zeiss, Oberkochen, Germany) fitted withan AxioCam MRc5 digital camera. Images were captured using AxioVisionsoftware (Zeiss) and assembled in Adobe Photoshop.

ImmunostainingSectionsFixed brains were cryoprotected in 30% sucrose in PBS, sectioned andprocessed free-floating. For 3,3′-diaminobenzidine (DAB) staining, sectionswere treated with 1% H2O2, washed in PBS and pre-incubated in blockingsolution containing 5% normal donkey serum (Jackson ImmunoResearchLaboratories, West Grove, PA), 1% bovine serum albumin, 0.1% glycine,0.1% L-lysine and 0.03% Triton X-100. Primary antibodies were added for48 hours at 4°C, and secondary antibodies [raised in donkey (JacksonImmunoResearch Laboratories; 1:250 dilution)] for 2 hours at roomtemperature; sections were processed with Vectastain ABC Elite (VectorLaboratories, Burlingame, CA) for 2 hours and developed using the DABsubstrate kit for Peroxidase (Vector). For immunofluorescence, the H2O2

step was omitted; secondary antibodies were Alexa-Fluor conjugated(Molecular Probes; 1:500 dilution).

CellsPrimary neurons were fixed with 4% PFA for 15 minutes, washed withPBS, blocked, incubated with primary antibodies for 1 hour at roomtemperature, washed and incubated with Alexa-Fluor-conjugatedsecondary antibodies.

AntibodiesRat-401 (1:100; developed by S. Hockfield, MIT, Cambridge, MA, USA)and 2H3 (1:300; developed by T. Jessell and J. Dodd, Columbia University,New York, USA) were both obtained from DSHB developed under theauspices of the NICHD and maintained by the University of Iowa,Department of Biological Sciences, Iowa City, IA. Other antibodies usedwere TUJ1 (1:500; Covance, Emeryville, CA; #MMS-435), MAP2 (1:1000;Sigma-Aldrich, St Louis, MO; #M4403), PDCD10/CCM3 (1:200; Sigma;#027095), DCX (C-18) (1:100; Santa Cruz Biotechnology, Santa Cruz, CA;#sc-8066), vGlut2 (1:300; Millipore, Billerica, MA; ab2251), Satb2 (1:300;Santa Cruz; #sc-81376), Bcl11b/Ctip2 (1:500; Abcam, Cambridge, MA;#ab18465), Cux1 (1:150; Santa Cruz; #sc-13024), phospho-cofilin (Ser-3)(1:1000; Cell Signaling Technology, Danvers, MA; #3311), phospho-Histone H3 (Ser-10) (1:2000; Cell Signaling) and BrdU (1:200; BecktonDickinson, San Jose, CA; #347580).

Microarray analysisNeocortices were dissected at P2 (n=3) and RNA was isolated using theRNeasy lipid tissue kit (Qiagen, Valencia, CA) and amplified usingTotalPrep RNA Amplification kit (Applied Biosystems). Biotin-labeledcRNA for hybridization onto Illumina arrays was used according to Illuminaprotocols; arrays were scanned on the Illumina Iscan.

Cytochrome oxidase histochemistryPFA-fixed neocortices were flattened and sectioned tangentially, andincubated with cytochrome C type III in the presence of DAB.

Golgi-Cox stainingGolgi-Cox staining was performed using the FD Rapid GolgiStain Kit (FDNeuro Technologies, Ellicott City, MD) according to the manufacturer’sinstructions.

Western blotWestern blot was performed by standard methods. Antibodies used werecofilin (1:1000; Cell Signaling; #3312), phospho-cofilin (Ser-3) (1:1000;Cell Signaling; #3311) and PDCD10/CCM3 (1:300; Proteintech, Chicago,IL; #10294). ImageJ was used for quantification of bands.

BrdU labelingPregnant females were injected intraperitoneally with a solution of 5-bromo-2′-deoxyuridine (BrdU; 15 mg/ml in saline) at 20 mg/g of body weight. Forembryonic stages (n=3 litters per genotype), litters were analyzed only ifthey contained a minimum of two control and two cKO littermates. At leastthree (and up to six) matched sections were analyzed per embryo.

Layer distribution analysisTo quantify the distribution of neurons, the postnatal neocortex was dividedradially into 10 equal-sized bins from the pia to the upper edge of the whitematter. For embryonic analyses, the pia to the VZ (E13.5 and E14.5) or theupper edge of the intermediate zone to the ventricular zone (E15.5 and older)was divided radially into ten bins. The cells in each bin were quantified andreported as the percentage of total cells counted.

Quantitative analysisData were analyzed by two-tailed Student’s t-tests with a significance levelof at least P<0.05 for all statistical comparisons. Numbers of replicates aregiven in the main text or figure legends. ImageJ/NeurphologyJ (Ho et al.,2011) was used for quantification of neuronal morphology.

DiI tracingTo label radial glia, crystals of the lipophilic dye DiI (Molecular Probes/Invitrogen, Carlsbad, CA) were applied on the pial surface of fixed brains.The brains were stored in 4% PFA in the dark for 2-4 weeks to allowdiffusion of the dye, then sectioned at a thickness of 100 μm using avibratome.

RhoA activation assayActive GTP-bound Rho was determined with a pull-down assay (Millipore,Temecula, CA; #17-294) according to the manufacturer’s instructions.

Primary cortical neuronal cell culturesPrimary cultures were established as described (Šestan et al., 1999) atplating density of 75×103/cm2 on laminin/poly-L-ornithine-coated glasscoverslips in 24-well plates.

In utero electroporationAll surgeries were performed using sterile conditions as describedpreviously (Saito and Nakatsuji, 2001) and in accordance with an IACUCapproved protocol. Plasmids used were NeuroD-CreERT2 (a gift from N.Šestan, Yale University, New Haven, CT, USA), an expression vector thatconsists of a fragment of the mouse NeuroD promoter driving Creexpression in postmitotic premigratory neurons; Stop-GFP, a Cre-responsiveGFP-expressing construct (Shim et al., 2012); and pCAG-Cre (Matsuda andCepko, 2007).

AcknowledgementsThis article is dedicated to Marion Wassef (Régionalisation Nerveuse, Institut deBiologie de l’École Normale Supérieure, Paris, France) on the occasion of herrecent retirement. We are grateful to N. Šestan for insightful discussions andcomments, a critical suggestion, and for sharing the template for Fig. 8. We thankK. Kwan for discussions and comments on the manuscript; W. Han, K. Yasuno andS. Assimacopoulos for discussions; anonymous reviewers for suggestions; M.Schwab and K.-A. Nave for the NEX-Cre mice; K. Ishigame for excellent technicalassistance; and M. Graham at the Center for Cell and Molecular Imaging (YaleSchool of Medicine) for help with electron microscopy. D

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Competing interestsThe authors declare no competing financial interests.

Author contributionsA.L. designed and performed research, analyzed data and wrote the paper; S.N.performed research and analyzed data; M.G. initiated the studies on CCM andprovided criticism.

FundingThis work was supported by a grant from the National Institutes of Health [R01-NS046521] and the Yale Program on Neurogenetics. Deposited in PMC forrelease after 12 months.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.093526/-/DC1

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Ccm3, a gene associated with cerebral cavernous ......Ccm3, a gene associated with cerebral cavernous malformations, is required for neuronal migration Angeliki Louvi1,*, Sayoko Nishimura1