Analysis of human samples reveals impairedSHH-dependent cerebellar development inJoubert syndrome/Meckel syndromeAndrea Aguilara,b,c,1,2, Alice Meuniera,b,c, Laetitia Strehla,b,c,3, Jelena Martinovicd, Maryse Bonniered,Tania Attie-Bitachd,e, Féréchté Encha-Razavid,e, and Nathalie Spasskya,b,c,1

aInstitut de Biologie de l’Ecole Normale Supérieure, F-75005 Paris, France; bInstitut National de la Santé et de la Recherche Médicale U1024, F-75230 ParisCedex 5, France; cCentre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, F-75005 Paris, France; dDépartement de Génétique, HôpitalNecker-Enfants Malades, 75015 Paris, France; and eInstitut National de la Santé et de la Recherche Médicale U781, Hôpital Necker-Enfants Malades,Universitéde Paris René Descartes, 75006 Paris, France

Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY 10065, and approved September 6, 2012 (received for review January 25, 2012)

Joubert syndrome (JS) and Meckel syndrome (MKS) are pleiotropicciliopathies characterized by severe defects of the cerebellar vermis,ranging from hypoplasia to aplasia. Interestingly, ciliary conditionalmutant mice have a hypoplastic cerebellum in which the prolifera-tionof cerebellar granule cell progenitors (GCPs) in response to Sonichedgehog (SHH) is severely reduced. This suggests that Shhsignaling defects could contribute to the vermis hypoplasia ob-served in the human syndromes. As existing JS/MKS mutant mousemodels suggest apparently contradictory hypotheses on JS/MKSetiology, we investigated Shh signaling directly on human fetalsamples. First, in an examination of human cerebellar development,we linked the rates of GCP proliferation to the different levels andlocalizations of active Shh signaling and showed that the GCPpossessed a primary cilium with CEP290 at its base. Second, wefound that the proliferation of GCPs and their response to SHHwereseverely impaired in the cerebellum of subjects with JS/MKS andJeune syndrome. Finally, we showed that the defect in GCP pro-liferation was similar in the cerebellar vermis and hemispheres in allpatients with ciliopathy analyzed, suggesting that the specific causeof vermal hypo-/aplasia precedes this defect. Our results, obtainedfromthe analysis of human samples, showthat the hemispheres andthe vermis are affected in JS/MKS and provide evidence of a defec-tive cellular mechanism in these pathologic processes.

cilia | granular neuron | hindbrain | developmental pathology

Joubert syndrome (JS; OnlineMendelian Inheritance inManno.213300) and Meckel syndrome (MKS; Online Mendelian In-

heritance in Man no. 249000) are believed to represent the twoextremes of the samemultisystemic disorder. JS is characterized bya complex malformation of the cerebellum/brainstem, the moststriking feature of which is hypoplasia or complete aplasia of thecerebellar vermis (1, 2). InMKS,which is lethal in utero, this defectis associated with occipital encephalocele or anencephaly, severelycystic kidneys, and abnormal liver and skeleton. To date, 16 genesresponsible for JS (NPHP1,AHI1,ARL13B, INPP5E,KIF7,OFD1,TCTN1, and CEP41), MKS (MKS1, B9D1, and B9D2), or both(RPGRIP1L,CEP290,CC2D2A, TMEM216, TMEM67/MKS3, andTCTN2) have been identified (3–6). Although all JS/MKS genesencode a ciliary/basal body (BB) protein, which is compellingevidence that the primary cilium is involved in these pathologicconditions, the cellular mechanisms by which cilium dysfunctionleads to severe brain malformations remain a mystery.Because the use of human subjects to study human pathologies is

problematic, insight into diseases usually comes from the analysis ofmurine models. However, none of the currently available murinemodels of JS/MKS faithfully recapitulate the cerebellar phenotypeseen in JS/MKS. Interestingly, in ciliary conditional mutant mice,the cerebellum is hypoplastic and Sonic hedgehog (Shh)-dependentproliferation of granule cell progenitors (GCPs) is severely reduced,as in mice in which the JS-associated gene RPGRIP1L has beenabrogated, suggesting that cilia-related defects in Shh-inducedGCP

expansionmight explain the cerebellar abnormalities observed in JS(7–9). In contrast, Shh-dependentGCP proliferation and cerebellarstructure were only mildly affected in Ahi1 or Cep290 KO mice, inwhich cilia formation is not modified (10). Thus, the analysis ofciliary mutants and JS/MKS mice models yields antagonistic hy-potheses on the involvement of Shh-drivenGCP proliferation in theetiology of the human forms of the syndromes.To investigate the relation between human ciliopathies and Shh-

dependent GCP proliferation, we first analyzed Cep290-associatedJS in eight human fetal samples and Cep290-RNAi–induced down-regulation mouse cell culture models, and demonstrate the locali-zation of the CEP290 protein at the base of cilia and its requirementfor cilium formation.We then analyzed GCP proliferation and Shhsignaling in these fetal human JS/MKS samples and age-matchedcontrols, and show that the highGCPproliferation rate is correlatedto detectable levels of Shh mRNA expression by Purkinje cells(PCs), and is strongly reduced in Cep290-associated JS/MKS sam-ples. Surprisingly, however, in JS/MKS, the reduction in the rate ofproliferation of GCP is similar in the vermis and the cerebellarhemispheres, suggesting that it may be responsible for a globalcerebellar phenotype in the disease. The vermal hypoplasia/aplasiawould most probably result from a pathological event occurringearlier in development. Remarkably, similar GCP proliferationdefects and impaired Shh signaling were also found in three otherJS/MKS cases (TMEM67/MKS3, CC2D2A, unidentified mutation),as well as in a Jeune asphyxiating thoracic dystrophy (JATD) case.These findings show that impaired Shh signaling and defectiveGCPproliferation underlie JS/MKS syndromes and suggest that thesedefects might be common to a number of ciliopathies.

ResultsHuman GCP Possess Primary Cilia. In mice, GCPs, located in theexternal granular layer (EGL), actively proliferate in response toShh, leading to massive cerebellar growth and generation of thelargest neuronal population in the brain, the granule cells (11–15). We and others have previously shown that murine GCP havea primary cilium, which is required for their proliferation in re-sponse to Shh (8, 9). In JS/MKS, ciliary genes mutations lead to

Author contributions: A.A. and N.S. designed research; A.A., A.M., L.S., and N.S. per-formed research; J.M., M.B., T.A.-B., and F.E.-R. contributed new reagents/analytic tools;A.A., A.M., and N.S. analyzed data; and A.A. and N.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: aguilara@stanford.edu or nathalie.spassky@ens.fr.

2Present address: Department of Molecular and Cellular Physiology, Stanford UniversitySchool of Medicine, Stanford, CA 94305.

3Present address: Université Pierre et Marie Curie Paris 6, Institut National de la Santé etde la Recherche Médicale, Unité Mixte de Recherche S975, Brain and Spinal Cord In-stitute, Hôpital de la Salpêtrière, 75013 Paris, France.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1201408109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1201408109 PNAS | October 16, 2012 | vol. 109 | no. 42 | 16951–16956

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

020

a drastic hypoplasia of the cerebellar vermis that could be causedby defects in cilia function in the GCP.To determine whether control human GCP possess a primary

cilium, we immunostained paraffin or fresh frozen cerebellar sec-tions fromhuman fetuses aged 12 to 36 gestational weeks (gw) withcilia-specific antibodies (Arl13B or Adenylate cyclase 3). Becauseno specific staining was observed in cilia, probably as a result ofantigen destruction by late postmortem fixation of the tissue andparaffin embedding, we examined a human gw 21 cerebellarsample, post mortem, by transmission EM. At this developmentalstage, human GCP had primary cilia with the usual features: alarsheets, a basal foot, and a rootlet (Fig. 1A, arrows). Interestingly,every cilium analyzed (n = 18) grew from a ciliary pocket (16) andits base was coated with electron-dense material (Fig. 1A, arrow-head). These results thus show that control human GCPs possessa primary cilium. Unfortunately, we could not determine whetherhumanGCPs possess a primary cilium in JS/MKS cases because allJS/MKS samples used in this study (as described later) are paraffin-embedded for clinical diagnosis and therefore not suitable fortransmission EM analysis.

CEP290 Is Localized at Centrosome of Human GCP and Is Involved inMurine Neural Cilium Assembly. So far, the largest number of cil-iopathies is caused by mutations in CEP290/NPHP6 (17), andgenetic abrogation of Cep290 in mice leads to mild cerebellarhypoplasia (10). The Cep290 transcript is expressed in GCPs andtheir progeny (18), but the subcellular location of the protein inmouse and human cerebellum is still unknown. By immunohisto-chemistry, we detected CEP290 in granular structures scattered

around BB rootlets in mouse and human GCPs (Fig. 1B), aspreviously described in hTERT-RPE cells (5, 19). CEP290 wasalso found at the ciliary transition zone of cultured primary mouseneural progenitors, as reported in hTERT-RPE1 cells and IMCD3cells (3, 5), as well as at the centrosome, as in IMCD3 cells (Fig.1C and Fig. S1) (3, 20, 21).CEP290 is involved in the assembly of primary cilium in sev-

eral established cell lines (3, 19, 22–24), but no data are availableregarding the brain. Therefore, we down-regulated Cep290 byRNAi in cultured primary mouse neural progenitors (the shRNAswere previously tested for efficiency in HEK cells; Fig. S1). Cellswere transfected with shRNA and plated at high density to rap-idly reach confluence. Three days later, almost 70% of the controlcells, but only 20% of the CEP290-depleted cells, had primarycilia (Fig. 1 C and D). Thus, our results show that CEP290 isinvolved in the assembly of primary cilia in neural cells. Inpatients with CEP290 mutations, JS might therefore result fromciliary defects.

GCP Proliferation Is Impaired in Cerebellar Vermis and Hemispheres inJS/MKS. We and others have previously shown that, in mice, se-lective genetic ablation of genes required for cilia formation (Kif3A,Ift88, or Ftm) in GCP leads to ataxia and cerebellar hypoplasiacaused by impaired Shh-dependent GCP proliferation (8, 9, 25).Cep290 KOmice, however, have only a mild cerebellar phenotypethat mainly results from Shh-independent mechanisms (10).Given the prominent role of cilia in Shh signaling in most

organs analyzed so far (26, 27), we quantified GCP proliferationin the cerebellum of 12 cases of JS/MKS caused by mutations in

Fig. 1. CEP290 is enriched at the base of murine andhuman GCPs; shRNA-mediated down-regulation ofCep290 reduces the number of ciliated cells. (A)Transmission EM images of a gw 21 sample of humanEGL. Left: Three GCPs have a primary cilium (yellowarrow) that extends from the BB (black asterisk) nearthe centriole (blue asterisk). The primary cilium hasa basal foot (black arrow) and a ciliary pocket, thebase of which is coated by electron-dense material(yellow arrowhead). (B) Immunohistofluorescenceimaging of CEP290 (green) and a BB rootlet compo-nent rootletin (red) on cryosections of mouse (Left)and human cerebellum (Right). In murine EGL, dotsof CEP290 surround the rootlet; this pattern is ob-served from embryonic day 16 to postnatal day 14. Inhuman EGL at gw 21, CEP290 is also localized in dotssurrounding the BB rootlet. (C) Immunostaining ofCEP290 (white), GFP (shRNA transfected cells, green),and the ciliary and BB marker polyglutamylated tu-bulin (GT335, red) in adherent astroglial stem cells,transfected with GFP-expressing plasmids driving theexpression of scramble or CEP290 shRNA (green).Most control transfected cells express CEP290 andhave a primary cilium. When CEP290 is no longerdetectable in CEP290 shRNA-expressing cells, they donot possess a primary cilium. (D) Quantification ofprimary cilia in scramble or CEP290 shRNA-trans-fected cells. For CEP290 shRNA-transfected cells, onlythose in which CEP290 was no longer immunode-tectable were quantified. CEP290 down-regulationleads to a 69% reduction in cells presenting a pri-mary cilium. The result shown is the mean of threereplicates. Error bars indicate SD. (Scale bars: A, leftto right, top to bottom, 10 μm, 2.5 μm, and 0.5 μm; B,5 μm; C, left to right, 2 μm and 1 μm.)

16952 | www.pnas.org/cgi/doi/10.1073/pnas.1201408109 Aguilar et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

020

the ciliary genes CEP290, CC2D2A, or TMEM67/MKS3 or byunidentified mutations (Table S1) and 11 age-matched controlsselected for their lack of cerebral involvement. Fetal cerebellarsections were stained with anti-Ki67 to label proliferating GCPs,which were quantified in the EGL of the vermis and the cerebellarhemispheres, and normalized to the EGL surface (Fig. 2 A and B).In control cases, the rate of GCP proliferation decreased greatlyfrom age 12 gw to age 14 to 15 gw (P < 0.05). It then increasedgreatly from age 16 gw to age 21 gw (P < 0.005), after which itstabilized (Fig. 2B). GCP proliferation in the pathological casesampled before gw 16 was similar to GCP proliferation in controls.In contrast, in 10 of 11 cases sampled after gw 16, the rate of GCPproliferation was significantly reduced compared with the controlcases (Fig. 2B). These results show that defective GCP pro-liferation after gw 16 is common to almost all cases of JS/MKS.

GCP Proliferation Is Also Disrupted in JATD.Ataxia, which frequentlyresults from cerebellar dysfunction, and cerebellar abnormalitieshave been reported in several ciliopathies [Bardet-Biedl syn-drome (BBS), orofaciodigital syndrome (OFD), nephronophtisis(NPHP), JATD] (28–31). We have quantified GCP expansion ina gw 17 JATD sample, in which postaxial polydactyly, short lowerlimbs, a pear-shaped thorax with short ribs, and retrognathismwere observed, but development of the cerebellar hemispheresand vermis appeared normal on neuropathological examination.GCP proliferation was severely impaired in this subject (Fig. 2B),supporting the hypothesis that defective GCP expansion might becommon to a number of ciliopathies.

GCP Proliferation Is Similar in Cerebellar Vermis and Hemispheres inJS/MKS and Controls. To test whether the vermis was more affectedthan the hemispheres in JS/MKS, given the particularly severehypo-/aplasia in this territory in the disease, proliferation rateswereevaluated separately in each structure and compared with controls.Although the vermis can be completely lost in JS/MKS, the struc-ture was present in all 12 of our JS/MKS cases. Nevertheless, onlyseven of the 12 cases could be analyzed quantitatively because thestructure was damaged or lost as a result of sectioning in the sagittalplane (Fig. S2). Surprisingly, in five of seven JS cases, proliferationrates in the vermis and in the hemispheres were similar, as seen inall the control cases analyzed (Fig. 2C). As the cerebellar hemi-spheres are affected to the same extent as the vermis in JS/MKS,vermal hypo-/aplasiamost probably results from apathogenic eventoccurring earlier in cerebellar development, not from impairedGCP proliferation.

Shh Pathway Is Active in Early Fetal Human Cerebellum. In themousecerebellum, massive proliferation of GCPs and consequent cere-bellar growth aredrivenby apotentmitogen, SHH,which is secretedby PCs starting at embryonic day 17.5 and inducesGli1 expression inthe GCPs (11–15). Until very recently, the precise timing of Shhpathwayactivation and its functional relevance toGCPproliferationand cerebellar growth had not been described in humans.To establish the cellular and temporal patterns of SHH ex-

pression in the human cerebellum, we assessed SHH mRNA ex-pression by in situ hybridization from 12 to 34 gw. Before gw 17,SHH mRNA levels were undetectable in CaBP+ cells and in theEGL (Fig. 3A). Unlike mouse PCs that start expressing SHHwhenthey integrate the PC cell layer (14, 15), at gw 17, staining wasdetected in a subset of PCs that were still migrating (30) (Fig. 3A).As PCs aligned into a multicellular layer, SHH expression in-tensified and was observed in all PCs at 33 gw (Fig. 3A).To gain insight into the timing of the GCP response to SHH sig-

naling, we performed in situ hybridization to evaluateGLI1mRNAexpression in human GCPs on the same samples previously assessedfor SHHmRNAexpression.Althoughwewere not able to detect theexpression of SHH in PCs before 17 gw, we observed that GCPsexpressed GLI1 at all stages analyzed (Fig. 3B), suggesting that anadditional source of SHH might exist before 17 gw (32, 33). How-ever, SHH mRNA expression detected in the PCs from 17 gwcoincides with a peak in GCP proliferation (Figs. 2B and 3); thus,these results strongly suggest that massive GCP proliferation corre-lates with active Shh signaling in the developing human cerebellum.

SHH Expression Is Correlated with Cerebellar Growth. In mice, it hasbeen proposed that the potent mitogenic effect of SHH is not re-quired for the basal proliferation of GCPs but rather for themassive postnatal proliferation of these cells that is necessary forlobe growth (14). To investigate whether SHH expression is alsocorrelated with the surface growth necessary for lobulation in thehuman cerebellum, we measured the surface area of the EGLthroughout normal fetal development on histologically stainedhorizontal sections. The surface of theEGL increased exponentiallywith time. The curve diverged from the linear at approximately gw16, indicating an important increase in the rate of expansion of theEGL surface at this point in development (Fig. 3B). These results

Fig. 2. GCP proliferation is severely impaired in the cerebellar vermis andhemispheres of most JS/MKS cases. (A) Immunolabeling of cycling cellsexpressing the marker Ki67 (red) in the EGL in the gw 23 JS/MKS case 070151and a gw 21 control. In all panels, a dashed line outlines the EGL. Note thatthere are fewer labeled cells in the EGL of the JS/MKS case (Right) comparedwith control (Left), although thickness of the EGL is comparable in bothcases. (B) Quantification of Ki67-positive cells per 100 μm2 in the EGL of 12cases of JS/MKS (CEP290, MKS3, CC2D2A, and mutations still under in-vestigation), one case of JATD, one case of HLS, and 11 age-matched con-trols. In the controls, GCP proliferation is high at 12 gw, greatly decreases atapproximately 14 to 15 gw (P < 0.05), increases again from gw 15 to gw 21(P < 0.005), and then stabilizes at a significantly higher plateau. The numberof Ki67-positive cells in the cerebellum (vermis and hemispheres) is dramati-cally decreased in the majority of JS/MKS cases (10 of 12) and in the JATD case,compared with controls. (C) Ki67-positive cells per 100 μm2 in the cerebellarvermis and hemispheres in the control and JS cases in B. As in all control cases(green), in five of seven of the JS/MKS cases (blue) studied, the proliferationdefect was similar in the vermis (light green/blue) and the hemispheres (darkgreen/blue). (Scale bar: 10 μm.) ***P < 0.001, **P < 0.01, and *P < 0.05.

Aguilar et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 16953

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

020

suggest that strong levels of PC-derived SHH drive the massiveGCP proliferation necessary for lobe growth.To assess the contribution of the early activation of Shh in the

EGL (Fig. 3A), we evaluated GCP proliferation at 15 gw ina case of JS/hydrolethalus syndrome (HLS) caused by an inac-tivating mutation in KIF7, the human orthologue of DrosophilaCostal2, a key component of the Hedgehog signaling pathway.Although Shh signaling was compromised (Fam 1, Fetus 3) (34),GCP proliferation was not impaired at gw 15 in this case (Fig.2B). This supports the possibility that SHH pathway activationbefore 16 gw only mildly contributes to the massive GCP pro-liferation driving lobe growth.

Response of GCP to Shh Signaling Is Impaired in JS/MKS. Shh path-way activation can be impaired as a result of defects in SHHexpression or in the GCP response to this signal. Faulty migrationof PCs might contribute to a deficit in Shh signaling, as suggestedby the heterotopic PC and local interruptions in the PC layerobserved in all our JS/MKS cases (Fig. S2). However, combinedin situ hybridization of SHH and calbindin labeling of PCs onsections from eight JS/MKS and four control cases showed that,when PCs reached their destination, they expressed normal levelsof SHH (Fig. 4 A and B). On the contrary, the levels of GLI1mRNA and PTC in the GCP were reduced in most analyzed JS/MKS cases in which GCP expansion was defective, indicating animpaired response of GCPs to SHH (Fig. 4).

DiscussionOur study shows that SHH-dependent GCP proliferation is im-paired in fetuses with JS/MKS in whom ciliary genes are mu-tated. This cellular mechanism, which we demonstrate here inhumans, is found in patients with both JS and MKS, as well as incilia or RPGRIP1L mutant mice (8, 9), and is thus conservedthrough evolution. GCP expansion and the response to SHHwere also defective in a case of JATD, raising the possibility thatimpaired Shh-dependent GCP proliferation might be common toa number of ciliopathies.Previous studies in which GCP cilia were ablated in mouse

models provided the first evidence that defects in Shh-dependentGCP proliferation may contribute to the cerebellar phenotypeobserved in JS and MKS (23, 24). It was unclear, however,whether these defects were responsible for hypo-/aplasia of thevermis or affected cerebellar development globally. In the presentstudy, we evaluated GCP proliferation at different developmentalstages in 12 cases of JS/MKS, one case of JS/HLS, and one case ofJATD. Interestingly, although defective GCP proliferation andvermis hypoplasia were observed in most of our JS/MKS cases (11of 12), vermis hypoplasia was also found to be present in case18485, which does not present a GCP proliferation defect. Fur-thermore, in five of seven analyzed fetuses, defective GCP pro-liferation affected the vermis and the cerebellar hemispheres tothe same extent (Fig. 2). These results suggest that defective GCPexpansion is not responsible for vermal hypo-/aplasia in JS/MKS,but, most probably, for the global cerebellar phenotype. In a recent

Fig. 3. SHH signaling is active in control cases inwhich the exponential increase in the EGL surfacebegins. (A) Combined in situ hybridization of SHH orGLI1mRNA and calbindin immunostaining at gw 12,17, and 33. At gw 12, neither the PCs (arrows) northe EGL express SHH, whereas the EGL expressesGLI1. At 17 gw, around the observed peak of GCPproliferation, some calbindin-positive PCs still mi-grating toward the pial surface express detectablelevels of SHH mRNA (arrows) whereas others donot (arrowheads). At the same time, GLI1 mRNA isexpressed in the EGL. At gw 33, PCs, aligned ina single-cell row beneath the EGL, all express highlevels of SHH mRNA (arrows). (B) Quantification ofthe EGL surface on horizontal H&E-stained sections(green dots). From the onset of detectable SHHexpression (i.e., gw 17, blue arrow), the exponentialcurve fitting all the data points starts to divergefrom the linear regression calculated from the datapoints at gw 10 to gw 15. The divergence corre-sponds with the timing of the GCP proliferationpeak (Fig. 2, purple arrows), suggesting that SHH-dependent increase in the rate of GCP proliferationis responsible for the exponential increase in thesurface of the EGL. (Scale bars: 20 μm.)

16954 | www.pnas.org/cgi/doi/10.1073/pnas.1201408109 Aguilar et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

020

study in Cep290 and Ahi1 KO mice, as well as in three subjectswith JS, cerebellar midline fusion was incomplete (10). Accordingto this study, this cerebellar defect could explain vermian hypo-plasia and would originate from defective Wnt signaling (10).Therefore, it would be interesting, in a future work, to assess Wntpathway activation in JS/MKS fetal tissue samples by performingin situ hybridization of two direct Wnt targets, AXIN2 (35) andLEF1 (36, 37).In humans, genotype–phenotype correlations have previously

been established for some JS genes or pathological CEP290 alleles(35, 36). In our study, there were no obvious correlations betweenthe different CEP290 alleles responsible for JS and the degree towhich GCP proliferation was impaired: the two pairs of siblingsanalyzed (cases 16676 and 16290 and cases 18485 and 050158; Fig.2B and Table S1) exhibit radically different degrees of GCP pro-liferation compared with their age-matched controls, even thoughthey have the sameCEP290 alleles.We did observe, however, that,when TMEM67/MKS3 was mutated (case 060247), proliferationwas more severely impaired than in JS/MKS caused by other mu-tated genes (Fig. 2B), suggesting a possible phenotype–genotype

correlation between themutated gene and the proliferation defect.Additional cases of JS/MKSwith TMEM67/MKS3mutations needto be analyzed to confirm this observation. In mice, such a geno-type–phenotype correlation is not observed. Rather, a correlationmight exist between ciliary function andGCP proliferation: mousemodels in which GCP cilia were abrogated (Kif3a, Ift88, orRPGRIP1L mutants) showed very severe defects in GCP pro-liferation (23, 24). In contrast, Cep290 and Ahi1 KO mice andmice with spontaneous deletion of the mouse orthologueTmem67/Mks3, had no defects in either cilia formation or Shh-dependent GCP proliferation (10, 38). Such strong differences inphenotype between humans andmice further outline the necessityof studies addressing the cellular and molecular mechanisms ofhuman pathologic conditions in actual human tissue samples.

Materials and MethodsTissue Samples. The human fetal material was obtained from the tissue col-lections of the Histology, Embryology and Cytogenetics Department of theNecker Children’s Hospital, Paris, France. The tissue was obtained fromspontaneous or medically induced abortions, with appropriately informed,written maternal consent for brain autopsy, the use of the brain tissue, andaccess to medical records for research purposes. The study was approved bythe Ethics Boards of Hôpital Necker-Enfants Malades. Unstained paraffin-embedded JS and control tissues, as well as H&E-stained sections, came fromthe tissue archives. We also obtained unfixed control cerebellar samples. Themice were C57Bl6/J or mixed backgrounds purchased from Janvier.

Fluorescence Microscopy and in Situ Hybridization. Unfixed human and mousetissue was flash-frozen, embedded in Tissue-Tek optimal cutting temperaturecompound (1S-LB-4583-EA; Sakura Finetek), cut into 10-μm-thick sections,mounted, and dried.

For CEP290/rootletin immunostaining, sections were fixed for 10 min in−20 °Cmethanol. For the other antigens or in situ hybridization, tissue sectionswere dewaxed and the antigen retrieved by incubation for 30 min at 96 °C inpreheated retrieval solution (S2367; Dako), pH 9, and then allowed to cool atroom temperature for another 30 min in the same solution. The sections werethen incubated for 12 to 72 h at 4 °C with the following primary antibodies:affinity-purified rabbit anti-human and anti-mouse CEP290 (1:500) (gift fromS. Saunier, Hôpital Necker-Enfants Malades, Paris, France) (7), goat anti-root-letin (sc67-824, 1:100; Santa Cruz), mouse anti-detyrosinated α-tubulin (clone1D5, 302011, 1:1,000; Synaptic Systems), rabbit anti-GFP (A11122, 1:1,000;Molecular Probes), mouse anti-Ki67 (MIB1 clone, M7240, 1:50; DAKO), rabbitanti-Pax6 (AB5409, 1:1,000; Chemicon), and rabbit anti-calbindin (CB38a,1:20,000; Swant). Species-specific secondary antibodies from Sigma-Aldrichwere then applied for 1 h to the slides. Finally, the sections were counter-stained with DAPI (10 μg/mL; Sigma) and mounted in Fluoromount.

For in situ hybridization on human tissue, antisense riboprobes were la-beled as described previously (8) by in vitro transcription of cDNAs encodingmouse Shh (gift from A. McMahon) or human GLI1 (gift from A. Ruiz iAltaba). In situ hybridization was then performed as described previously (8).Standard H&E histological staining was performed on paraffin sections.

Stained cilia, rootlet, and Ki67 were imaged with a Zeiss Observer Z1 mi-croscope equipped with an Apotome device and a Hamamatsu ORCA R2C10600 camera. Combined in situ hybridization and immunostaining wereimaged with a Leica DM 5500Bmicroscope and a Hamamatsu ORCA ER C4742-80 camera.

Transmission EM. Postmortem fetal tissue was dissected in PBS solution into1 × 2-mm blocks, which were then fixed in 2% PFA/1% glutaraldehyde so-lution in 0.1 M phosphate buffer for 3 h. After rinsing in PBS solution, thetissue was postfixed in 1% osmium tetroxide for 30 min on ice, protectedfrom light, with shaking. The tissue blocks were then dehydrated in 50% and70% ethanol baths in water for 7 min each, and then stained in 1% uranylacetate in methanol. After final dehydration, samples were immersed for 40min in a graded series of ethanol/Epon (2/1, 1/1, 1/2 ratios), then in pureEpon. Samples were then mounted in Epon blocks for 48 h at 60 °C to ensurepolymerization. Ultrathin sections (70 nm) were cut sagittally on an ultra-microtome (Ultracut E; Reichert-Jung) and analyzed with a Jeol 10–11transmission electron microscope.

Quantification of GCP Proliferation Rate and Cerebellar Surface. KI67-positiveGCP quantification was accomplished on half horizontal sections of controland human JS fetal cerebella, comprising half the vermis and one hemisphere

Fig. 4. Conserved SHH expression and reduced GLI1mRNA and PTC levels inmost JS/MKS cases with defective GCP proliferation. (A) GLI1, PTC, and SHHexpression in JS samples compared with controls. SHH, GLI1, and PTC levels ofexpression were assessed by in situ hybridization (SHH, GLI1) or immunohis-tochemistry (PTC). SHH mRNA expression is conserved in the vast majority ofJS/MKS cases with defective GCP proliferation, but GLI1mRNA and PTC levelsare reduced or undetectable, indicating a defective response to SHH. (∼,comparable toWT; ND, not determined; NE, not expressed;−, diminished.) (B)Combined SHH in situ hybridization and calbindin immunolabeling of JS case080144 and an age-matched control. SHHmRNA levels in JS calbindin-positivePCs (green) are comparable to control levels. The EGL and PC layer (PCL) aredelimited by a dashed line. (C) GLI1 in situ hybridization or PTC immunohis-tochemistry and DAPI staining in two JS cases and age-matched controls.GLI1mRNA and PTC expression are reduced in the JS EGL. (Scale bar: 20 μm.)

Aguilar et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 16955

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

020

(routine sectioning for diagnosis done at the Histology, Embryology andCytogenetics Department of the Necker Children’s Hospital).

For small samples (to 18 gw), the entire EGL was photographed with a 20×objective on a Zeiss Observer Z1 microscope equipped with an Apotomedevice and a Hamamatsu ORCA R2 C10600 camera. The EGL was thenmanually reconstituted and, based on EGL length, divided into 16 parts. Thenumber of Ki67+ cells and the EGL surface were manually quantified foreach part by using ImageJ (National Institutes of Health; on average, 3500Ki67+/Pax6+ cells were counted per fetus). For bigger samples, this methodbecame technically very challenging. We therefore sampled the EGL bytaking 16 to 20 equidistant images at 20× throughout the EGL. In eachpicture, the number of Ki67+ cells and the EGL surface were manuallyquantified. In both cases, either when the full EGL was quantified or when itwas sampled, the quantified regions are spatially comparable. The 16 to 20measurements of the proliferation rate per sample were then averaged toobtain the final proliferation rate for each fetus.

The EGL surface area was measured on representative individual sectionsfrom each stage (n = 1 or 2 per fetus and n = 1–3 fetuses per stage), whichwere selected for being situated at the same anteroposterior level: they allencompass the dentate nucleus. The H&E-stained sections were scanned toobtain low-magnification and high-resolution images. On each image, theEGL surface was manually outlined and measured by using ImageJ.

Validation of RNAi-Mediated Down-Regulation of CEP290. To test the efficiencyof CEP290 down-regulation by the CEP290 shRNA, HEK 293T cells werecotransfected with a mCEP290-GFP plasmid (gift from J. Gleeson, University ofCalifornia, SanDiego,CA)andapGIPZCEP290shRNAmir-GFPplasmidorapGIPZ-GFP control plasmid (Abgene). After 24h, cells lysateswere run onaNuPage4%to 12% Bis-Tris gel. After transfer, the membrane was cut below the 110-kDamarker, and theupper halfwas blotted forGFP (homemadepolyclonal anti-GFPantibody) (39) with the lower half blotted for α-tubulin (Hybridoma Bank clone12G10, 1/5,000), following standard procedures. Protein bands were visualizedwithHRP-labeledproteinA or anti-mouse antibody, followedbydetectionwithchemiluminescence and quantification with ImageJ software.

Primary Cortical Neural Stem Cell Culture and shRNA Assay. The cortex ofpostnatal day 0 to 2 mice was dissected (exclusion of the choroid plexus, hip-pocampus, corpus callosum, olfactory bulbs, and meninges) and mechanicallydissociated, digested with papain. The cells were plated at high density in 10%FCS-containing medium, and allowed to grow to confluence. The primarycortical astroglial cellswere thendetachedandnucleofected in suspensionwiththe expression arrest pGIPZ CEP290shRNAmir-GFP plasmid orwith a pGIPZ-GFPcontrol plasmid (Abgene) using a Nucleofector device (Lonza) and the BasicNeuron Small Cell Number Nucleofector kit (VSPI-1003; Lonza). The cells werethenplatedathighdensity to formpure confluentmonolayers ofmonociliatedastroglia thatwerefixed for1.5min in4%paraformaldehyde/0.33Msucroseat4 °C, then for 10min in−20 °CMeOH (40). After triple staining for GFP, CEP290,and polyglutamylated tubulin (GT335; gift from C. Janke, Institut Curie, Orsay,France), as described earlier, cilia presence was assessed. In cells in whichCEP290 was down-regulated, only GFP+/CEP290− cells were quantified.

Statistical Analysis. The results (i.e., average of the 16 to 20 measures of theproliferation rate obtained for each fetus) are presented as mean ± SD. Atwo-sided Wilcoxon test was used for all statistical analyses except forcomparison of GCP proliferation in the cerebellar hemispheres vs. the ver-mis, for which a two-sided averages test was used.

ACKNOWLEDGMENTS. We thank the Cellular Imaging Center of the Pitié-Salpêtrière Hospital and Dr. D. Langui for assistancewith confocal and trans-mission EM; M.-P. Muriel for excellent technical assistance with EM;A.McMahon formouse Shh probe; T. Caspari for anti-Arl13B antibody; C. Jankefor GT335 antibody; J. Gleeson for mCEP290-GFP plasmid; A. Ruiz i Altaba forthe GLI1 riboprobe plasmid; and Dr. M. V. Nachury for support. This study wassupported by grants from Agence Nationale de la Recherche, InternationalHuman Frontier Science Program Organization, Institut de France, Mairie deParis, Fondation pour la RechercheMédicale (to N.S.), a doctoral fellowship forthe French Ministry of Research and Association pour la Recherche sur le Can-cer (to A.A.).

1. Brancati F, Dallapiccola B, Valente EM (2010) Joubert syndrome and related disorders.Orphanet J Rare Dis 5:20.

2. Louie CM, Gleeson JG (2005) Genetic basis of Joubert syndrome and related disordersof cerebellar development. Hum Mol Genet 14 spec no 2:R235–R242.

3. Sang L, et al. (2011) Mapping the NPHP-JBTS-MKS protein network reveals ciliopathydisease genes and pathways. Cell 145:513–528.

4. Novarino G, Akizu N, Gleeson JG (2011) Modeling human disease in humans: Theciliopathies. Cell 147:70–79.

5. Garcia-Gonzalo FR, et al. (2011) A transition zone complex regulates mammalianciliogenesis and ciliary membrane composition. Nat Genet 43:776–784.

6. Lee JE, et al. (2012) CEP41 is mutated in Joubert syndrome and is required for tubulinglutamylation at the cilium. Nat Genet 44:193–199.

7. Delous M, et al. (2007) The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renalsyndrome (Joubert syndrome type B) and Meckel syndrome. Nat Genet 39:875–881.

8. Spassky N, et al. (2008) Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool. Dev Biol 317:246–259.

9. Chizhikov VV, et al. (2007) Cilia proteins control cerebellar morphogenesis by pro-moting expansion of the granule progenitor pool. J Neurosci 27:9780–9789.

10. Lancaster MA, et al. (2011) Defective Wnt-dependent cerebellar midline fusion ina mouse model of Joubert syndrome. Nat Med 17:726–731.

11. Dahmane N, Ruiz i Altaba A (1999) Sonic hedgehog regulates the growth and pat-terning of the cerebellum. Development 126:3089–3100.

12. Wallace VA (1999) Purkinje-cell-derived Sonic hedgehog regulates granule neuronprecursor cell proliferation in the developing mouse cerebellum. Curr Biol 9:445–448.

13. Wechsler-Reya RJ, Scott MP (1999) Control of neuronal precursor proliferation in thecerebellum by Sonic Hedgehog. Neuron 22:103–114.

14. Corrales JD, Rocco GL, Blaess S, Guo Q, Joyner AL (2004) Spatial pattern of sonichedgehog signaling through Gli genes during cerebellum development. De-velopment 131:5581–5590.

15. Lewis PM, Gritli-Linde A, Smeyne R, Kottmann A, McMahon AP (2004) Sonic hedge-hog signaling is required for expansion of granule neuron precursors and patterningof the mouse cerebellum. Dev Biol 270:393–410.

16. Molla-Herman A, et al. (2010) The ciliary pocket: an endocytic membrane domain atthe base of primary and motile cilia. J Cell Sci 123:1785–1795.

17. Lee JE, Gleeson JG (2011) Cilia in the nervous system: Linking cilia function andneurodevelopmental disorders. Curr Opin Neurol 24:98–105.

18. Valente EM, et al.; International Joubert Syndrome Related Disorders Study Group(2006) Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropicforms of Joubert syndrome. Nat Genet 38:623–625.

19. Kim J, Krishnaswami SR, Gleeson JG (2008) CEP290 interacts with the centriolar sat-ellite component PCM-1 and is required for Rab8 localization to the primary cilium.Hum Mol Genet 17:3796–3805.

20. Sayer JA, et al. (2006) The centrosomal protein nephrocystin-6 is mutated in Joubertsyndrome and activates transcription factor ATF4. Nat Genet 38:674–681.

21. Chang B, et al. (2006) In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal de-generation in the rd16 mouse. Hum Mol Genet 15:1847–1857.

22. Graser S, et al. (2007) Cep164, a novel centriole appendage protein required forprimary cilium formation. J Cell Biol 179:321–330.

23. Tsang WY, et al. (2008) CP110 suppresses primary cilia formation through its in-teraction with CEP290, a protein deficient in human ciliary disease. Dev Cell 15:187–197.

24. Lopes CA, et al. (2011) Centriolar satellites are assembly points for proteins implicatedin human ciliopathies, including oral-facial-digital syndrome 1. J Cell Sci 124:600–612.

25. Han YG, et al. (2009) Dual and opposing roles of primary cilia in medulloblastomadevelopment. Nat Med 15:1062–1065.

26. Goetz SC, Ocbina PJ, Anderson KV (2009) The primary cilium as a Hedgehog signaltransduction machine. Methods Cell Biol 94:199–222.

27. Goetz SC, Anderson KV (2010) The primary cilium: A signalling centre during verte-brate development. Nat Rev Genet 11:331–344.

28. Waters AM, Beales PL (2011) Ciliopathies: An expanding disease spectrum. PediatrNephrol 26:1039–1056.

29. Baskin E, et al. (2002) Cerebellar vermis hypoplasia in a patient with Bardet-Biedlsyndrome. J Child Neurol 17:385–387.

30. Tobin JL, Beales PL (2009) The nonmotile ciliopathies. Genet Med 11:386–402.31. Lehman AM, et al. (2010) Co-occurrence of Joubert syndrome and Jeune asphyxiating

thoracic dystrophy. Am J Med Genet A 152A:1411–1419.32. Huang X, et al. (2010) Transventricular delivery of Sonic hedgehog is essential to

cerebellar ventricular zone development. Proc Natl Acad Sci USA 107:8422–8427.33. Haldipur P, et al. (2012) Expression of Sonic hedgehog during cell proliferation in the

human cerebellum. Stem Cells Dev 21:1059–1068.34. Putoux A, et al. (2011) KIF7 mutations cause fetal hydrolethalus and acrocallosal

syndromes. Nat Genet 43:601–606.35. Jho EH, et al. (2002) Wnt/beta-catenin/Tcf signaling induces the transcription of

Axin2, a negative regulator of the signaling pathway. Mol Cell Biol 22:1172–1183.36. Porfiri E, et al. (1997) Induction of a beta-catenin-LEF-1 complex by wnt-1 and

transforming mutants of beta-catenin. Oncogene 15:2833–2839.37. Hsu SC, Galceran J, Grosschedl R (1998) Modulation of transcriptional regulation by

LEF-1 in response to Wnt-1 signaling and association with beta-catenin. Mol Cell Biol18:4807–4818.

38. Cook SA, et al. (2009) A mouse model for Meckel syndrome type 3. J Am Soc Nephrol20:753–764.

39. Nachury MV, et al. (2007) A core complex of BBS proteins cooperates with the GTPaseRab8 to promote ciliary membrane biogenesis. Cell 129:1201–1213.

40. Bellion A, Baudoin JP, Alvarez C, Bornens M, Métin C (2005) Nucleokinesis in tan-gentially migrating neurons comprises two alternating phases: Forward migration ofthe Golgi/centrosome associated with centrosome splitting and myosin contraction atthe rear. J Neurosci 25:5691–5699.

16956 | www.pnas.org/cgi/doi/10.1073/pnas.1201408109 Aguilar et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

020