Analysis of Human Samples Reveals Impaired SHH-Dependent Cerebellar Development in Joubert Syndrome/Meckel Syndrome

Analysis of human samples reveals impaired SHH-dependent cerebellar development in Joubert syndrome/Meckel syndrome

Andrea 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 Paris Cedex 5, France; cCentre National de la Recherche Scientiﬁque, Unité Mixte de Recherche 8197, F-75005 Paris, France; dDépartement de Génétique, Hôpital Necker-Enfants Malades, 75015 Paris, France; and eInstitut National de la Santé et de la Recherche Médicale U781, Hôpital Necker-Enfants Malades,Université de 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 pleiotropic expansion might explain the cerebellar abnormalities observed in JS ciliopathies characterized by severe defects of the cerebellar vermis, (7–9). In contrast, Shh-dependent GCP proliferation and cerebellar ranging from hypoplasia to aplasia. Interestingly, ciliary conditional structure were only mildly affected in Ahi1 or Cep290 KO mice, in mutant mice have a hypoplastic cerebellum in which the prolifera- which cilia formation is not modified (10). Thus, the analysis of tion of cerebellar granule cell progenitors (GCPs) in response to Sonic ciliary mutants and JS/MKS mice models yields antagonistic hy- hedgehog (SHH) is severely reduced. This suggests that Shh potheses on the involvement of Shh-driven GCP proliferation in the signaling defects could contribute to the vermis hypoplasia ob- etiology of the human forms of the syndromes. served in the human syndromes. As existing JS/MKS mutant mouse To investigate the relation between human ciliopathies and Shh- dependent GCP proliferation, we first analyzed Cep290-associated models suggest apparently contradictory hypotheses on JS/MKS – etiology, we investigated Shh signaling directly on human fetal JS in eight human fetal samples and Cep290-RNAi induced down- regulation mouse cell culture models, and demonstrate the locali- samples. First, in an examination of human cerebellar development, zation of the CEP290 protein at the base of cilia and its requirement we linked the rates of GCP proliferation to the different levels and for cilium formation. We then analyzed GCP proliferation and Shh localizations of active Shh signaling and showed that the GCP signaling in these fetal human JS/MKS samples and age-matched possessed a primary cilium with CEP290 at its base. Second, we controls, and show that the high GCP proliferation rate is correlated found that the proliferation of GCPs and their response to SHH were to detectable levels of Shh mRNA expression by Purkinje cells severely impaired in the cerebellum of subjects with JS/MKS and (PCs), and is strongly reduced in Cep290-associated JS/MKS sam- Jeune syndrome. Finally, we showed that the defect in GCP pro- ples. Surprisingly, however, in JS/MKS, the reduction in the rate of liferation was similar in the cerebellar vermis and hemispheres in all proliferation of GCP is similar in the vermis and the cerebellar patients with ciliopathy analyzed, suggesting that the specificcause hemispheres, suggesting that it may be responsible for a global BIOLOGY of vermal hypo-/aplasia precedes this defect. Our results, obtained cerebellar phenotype in the disease. The vermal hypoplasia/aplasia DEVELOPMENTAL from the analysis of human samples, show that the hemispheres and would most probably result from a pathological event occurring the vermis are affected in JS/MKS and provide evidence of a defec- earlier in development. Remarkably, similar GCP proliferation tive cellular mechanism in these pathologic processes. defects and impaired Shh signaling were also found in three other JS/MKS cases (TMEM67/MKS3, CC2D2A,unidentified mutation), cilia | granular neuron | hindbrain | developmental pathology as well as in a Jeune asphyxiating thoracic dystrophy (JATD) case. These findings show that impaired Shh signaling and defective GCP oubert syndrome (JS; Online Mendelian Inheritance in Man no. proliferation underlie JS/MKS syndromes and suggest that these J213300) and Meckel syndrome (MKS; Online Mendelian In- defects might be common to a number of ciliopathies. heritance in Man no. 249000) are believed to represent the two Results extremes of the same multisystemic disorder. JS is characterized by a complex malformation of the cerebellum/brainstem, the most Human GCP Possess Primary Cilia. In mice, GCPs, located in the striking feature of which is hypoplasia or complete aplasia of the external granular layer (EGL), actively proliferate in response to cerebellar vermis (1, 2). In MKS, which is lethal in utero, this defect Shh, leading to massive cerebellar growth and generation of the largest neuronal population in the brain, the granule cells (11– is associated with occipital encephalocele or anencephaly, severely 15). We and others have previously shown that murine GCP have cystic kidneys, and abnormal liver and skeleton. To date, 16 genes NPHP1 AHI1 ARL13B INPP5E KIF7 OFD1 a primary cilium, which is required for their proliferation in re- responsible for JS ( , , , , , , sponse to Shh (8, 9). In JS/MKS, ciliary genes mutations lead to TCTN1,andCEP41), MKS (MKS1, B9D1,andB9D2), or both (RPGRIP1L, CEP290, CC2D2A, TMEM216, TMEM67/MKS3,and TCTN2) have been identified (3–6). Although all JS/MKS genes Author contributions: A.A. and N.S. designed research; A.A., A.M., L.S., and N.S. per- encode a ciliary/basal body (BB) protein, which is compelling formed research; J.M., M.B., T.A.-B., and F.E.-R. contributed new reagents/analytic tools; evidence that the primary cilium is involved in these pathologic A.A., A.M., and N.S. analyzed data; and A.A. and N.S. wrote the paper. conditions, the cellular mechanisms by which cilium dysfunction The authors declare no conflict of interest. leads to severe brain malformations remain a mystery. This article is a PNAS Direct Submission. Because the use of human subjects to study human pathologies is 1 problematic, insight into diseases usually comes from the analysis of To whom correspondence may be addressed. E-mail: [email protected] or nathalie. [email protected]. murine models. However, none of the currently available murine 2Present address: Department of Molecular and Cellular Physiology, Stanford University models of JS/MKS faithfully recapitulate the cerebellar phenotype School of Medicine, Stanford, CA 94305. seen in JS/MKS. Interestingly, in ciliary conditional mutant mice, 3Present address: Université Pierre et Marie Curie Paris 6, Institut National de la Santé et the cerebellum is hypoplastic and Sonic hedgehog (Shh)-dependent de la Recherche Médicale, Unité Mixte de Recherche S975, Brain and Spinal Cord In- proliferation of granule cell progenitors (GCPs) is severely reduced, stitute, Hôpital de la Salpêtrière, 75013 Paris, France. RPGRIP1L as in mice in which the JS-associated gene has been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. abrogated, suggesting that cilia-related defects in Shh-induced GCP 1073/pnas.1201408109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1201408109 PNAS | October 16, 2012 | vol. 109 | no. 42 | 16951–16956 Downloaded by guest on September 30, 2021 a drastic hypoplasia of the cerebellar vermis that could be caused around BB rootlets in mouse and human GCPs (Fig. 1B), as by defects in cilia function in the GCP. previously described in hTERT-RPE cells (5, 19). CEP290 was To determine whether control human GCP possess a primary also found at the ciliary transition zone of cultured primary mouse cilium, we immunostained paraffin or fresh frozen cerebellar sec- neural progenitors, as reported in hTERT-RPE1 cells and IMCD3 tions from human fetuses aged 12 to 36 gestational weeks (gw) with cells (3, 5), as well as at the centrosome, as in IMCD3 cells (Fig. cilia-specific antibodies (Arl13B or Adenylate cyclase 3). Because 1C and Fig. S1) (3, 20, 21). no specific staining was observed in cilia, probably as a result of CEP290 is involved in the assembly of primary cilium in sev- antigen destruction by late postmortem fixation of the tissue and eral established cell lines (3, 19, 22–24), but no data are available paraffin embedding, we examined a human gw 21 cerebellar regarding the brain. Therefore, we down-regulated Cep290 by sample, post mortem, by transmission EM. At this developmental RNAi in cultured primary mouse neural progenitors (the shRNAs stage, human GCP had primary cilia with the usual features: alar were previously tested for efficiency in HEK cells; Fig. S1). Cells sheets, a basal foot, and a rootlet (Fig. 1A, arrows). Interestingly, were transfected with shRNA and plated at high density to rap- every cilium analyzed (n = 18) grew from a ciliary pocket (16) and idly reach confluence. Three days later, almost 70% of the control its base was coated with electron-dense material (Fig. 1A, arrow- cells, but only 20% of the CEP290-depleted cells, had primary head). These results thus show that control human GCPs possess cilia (Fig. 1 C and D). Thus, our results show that CEP290 is a primary cilium. Unfortunately, we could not determine whether involved in the assembly of primary cilia in neural cells. In human GCPs possess a primary cilium in JS/MKS cases because all patients with CEP290 mutations, JS might therefore result from JS/MKS samples used in this study (as described later) are paraffin- ciliary defects. embedded for clinical diagnosis and therefore not suitable for transmission EM analysis. GCP Proliferation Is Impaired in Cerebellar Vermis and Hemispheres in JS/MKS. We and others have previously shown that, in mice, se- CEP290 Is Localized at Centrosome of Human GCP and Is Involved in lective genetic ablation of genes required for cilia formation (Kif3A, Murine Neural Cilium Assembly. So far, the largest number of cil- Ift88,orFtm) in GCP leads to ataxia and cerebellar hypoplasia iopathies is caused by mutations in CEP290/NPHP6 (17), and caused by impaired Shh-dependent GCP proliferation (8, 9, 25). genetic abrogation of Cep290 in mice leads to mild cerebellar Cep290 KO mice, however, have only a mild cerebellar phenotype hypoplasia (10). The Cep290 transcript is expressed in GCPs and that mainly results from Shh-independent mechanisms (10). their progeny (18), but the subcellular location of the protein in Given the prominent role of cilia in Shh signaling in most mouse and human cerebellum is still unknown. By immunohisto- organs analyzed so far (26, 27), we quantified GCP proliferation chemistry, we detected CEP290 in granular structures scattered in the cerebellum of 12 cases of JS/MKS caused by mutations in

Fig. 1. CEP290 is enriched at the base of murine and human GCPs; shRNA-mediated down-regulation of Cep290 reduces the number of ciliated cells. (A) Transmission EM images of a gw 21 sample of human EGL. Left: Three GCPs have a primary cilium (yellow arrow) that extends from the BB (black asterisk) near the centriole (blue asterisk). The primary cilium has a basal foot (black arrow) and a ciliary pocket, the base of which is coated by electron-dense material (yellow arrowhead). (B) Immunohistofluorescence imaging of CEP290 (green) and a BB rootlet component rootletin (red) on cryosections of mouse (Left) and human cerebellum (Right). In murine EGL, dots of CEP290 surround the rootlet; this pattern is observed from embryonic day 16 to postnatal day 14. In human EGL at gw 21, CEP290 is also localized in dots surrounding the BB rootlet. (C)Immunostainingof CEP290 (white), GFP (shRNA transfected cells, green), and the ciliary and BB marker polyglutamylated tubulin (GT335, red) in adherent astroglial stem cells, transfected with GFP-expressing plasmids driving the expression of scramble or CEP290 shRNA (green). Most control transfected cells express CEP290 and have a primary cilium. When CEP290 is no longer detectable in CEP290 shRNA-expressing cells, they do not possess a primary cilium. (D)Quantification of primary cilia in scramble or CEP290 shRNA-transfected cells. For CEP290 shRNA-transfected cells, only those in which CEP290 was no longer immunode- tectable were quantified. CEP290 down-regulation leads to a 69% reduction in cells presenting a primary cilium. The result shown is the mean of three replicates. Error bars indicate SD. (Scale bars: A,left to right, top to bottom, 10 μm, 2.5 μm, and 0.5 μm; B, 5 μm; C,lefttoright,2μmand1μm.)

16952 | www.pnas.org/cgi/doi/10.1073/pnas.1201408109 Aguilar et al. Downloaded by guest on September 30, 2021 the ciliary genes CEP290, CC2D2A,orTMEM67/MKS3 or by GCP Proliferation Is Also Disrupted in JATD. Ataxia, which frequently unidentified mutations (Table S1) and 11 age-matched controls results from cerebellar dysfunction, and cerebellar abnormalities selected for their lack of cerebral involvement. Fetal cerebellar have been reported in several ciliopathies [Bardet-Biedl syn- sections were stained with anti-Ki67 to label proliferating GCPs, drome (BBS), orofaciodigital syndrome (OFD), nephronophtisis – fi which were quantified in the EGL of the vermis and the cerebellar (NPHP), JATD] (28 31). We have quanti ed GCP expansion in hemispheres, and normalized to the EGL surface (Fig. 2 A and B). a gw 17 JATD sample, in which postaxial polydactyly, short lower limbs, a pear-shaped thorax with short ribs, and retrognathism In control cases, the rate of GCP proliferation decreased greatly P < were observed, but development of the cerebellar hemispheres from age 12 gw to age 14 to 15 gw ( 0.05). It then increased and vermis appeared normal on neuropathological examination. P < greatly from age 16 gw to age 21 gw ( 0.005), after which it GCP proliferation was severely impaired in this subject (Fig. 2B), stabilized (Fig. 2B). GCP proliferation in the pathological case supporting the hypothesis that defective GCP expansion might be sampled before gw 16 was similar to GCP proliferation in controls. common to a number of ciliopathies. In contrast, in 10 of 11 cases sampled after gw 16, the rate of GCP proliferation was significantly reduced compared with the control GCP Proliferation Is Similar in Cerebellar Vermis and Hemispheres in cases (Fig. 2B). These results show that defective GCP pro- JS/MKS and Controls. To test whether the vermis was more affected liferation after gw 16 is common to almost all cases of JS/MKS. than the hemispheres in JS/MKS, given the particularly severe hypo-/aplasia in this territory in the disease, proliferation rates were evaluated separately in each structure and compared with controls. Although the vermis can be completely lost in JS/MKS, the structure was present in all 12 of our JS/MKS cases. Nevertheless, only seven of the 12 cases could be analyzed quantitatively because the structure was damaged or lost as a result of sectioning in the sagittal plane (Fig. S2). Surprisingly, in five of seven JS cases, proliferation rates in the vermis and in the hemispheres were similar, as seen in all the control cases analyzed (Fig. 2C). As the cerebellar hemispheres are affected to the same extent as the vermis in JS/MKS, vermal hypo-/aplasia most probably results from a pathogenic event occurring earlier in cerebellar development, not from impaired GCP proliferation.

Shh Pathway Is Active in Early Fetal Human Cerebellum. In the mouse cerebellum, massive proliferation of GCPs and consequent cerebellar growth are driven by a potent mitogen, SHH, which is secreted by PCs starting at embryonic day 17.5 and induces Gli1 expression in the GCPs (11–15). Until very recently, the precise timing of Shh

pathway activation and its functional relevance to GCP proliferation BIOLOGY

and cerebellar growth had not been described in humans. DEVELOPMENTAL To establish the cellular and temporal patterns of SHH expression in the human cerebellum, we assessed SHH mRNA expression by in situ hybridization from 12 to 34 gw. Before gw 17, SHH mRNA levels were undetectable in CaBP+ cells and in the EGL (Fig. 3A). Unlike mouse PCs that start expressing SHH when they integrate the PC cell layer (14, 15), at gw 17, staining was detected 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 signaling, we performed in situ hybridization to evaluate GLI1 mRNA expression in human GCPs on the same samples previously assessed for SHH mRNA expression. Although we were not able to detect the expression of SHH in PCs before 17 gw, we observed that GCPs expressed GLI1 at all stages analyzed (Fig. 3B), suggesting that an Fig. 2. GCP proliferation is severely impaired in the cerebellar vermis and additional source of SHH might exist before 17 gw (32, 33). How- hemispheres of most JS/MKS cases. (A) Immunolabeling of cycling cells ever, SHH mRNA expression detected in the PCs from 17 gw expressing the marker Ki67 (red) in the EGL in the gw 23 JS/MKS case 070151 coincides with a peak in GCP proliferation (Figs. 2B and 3); thus, and a gw 21 control. In all panels, a dashed line outlines the EGL. Note that these results strongly suggest that massive GCP proliferation corre- there are fewer labeled cells in the EGL of the JS/MKS case (Right) compared lates with active Shh signaling in the developing human cerebellum. with control (Left), although thickness of the EGL is comparable in both cases. (B) Quantification of Ki67-positive cells per 100 μm2 in the EGL of 12 SHH Expression Is Correlated with Cerebellar Growth. In mice, it has cases of JS/MKS (CEP290, MKS3, CC2D2A, and mutations still under in- been proposed that the potent mitogenic effect of SHH is not re- vestigation), one case of JATD, one case of HLS, and 11 age-matched con- quired for the basal proliferation of GCPs but rather for the trols. In the controls, GCP proliferation is high at 12 gw, greatly decreases at massive postnatal proliferation of these cells that is necessary for approximately 14 to 15 gw (P < 0.05), increases again from gw 15 to gw 21 SHH < fi lobe growth (14). To investigate whether expression is also (P 0.005), and then stabilizes at a signi cantly higher plateau. The number correlated with the surface growth necessary for lobulation in the of 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, human cerebellum, we measured the surface area of the EGL compared with controls. (C) Ki67-positive cells per 100 μm2 in the cerebellar throughout normal fetal development on histologically stained vermis and hemispheres in the control and JS cases in B. As in all control cases horizontal sections. The surface of the EGL increased exponentially (green), in five of seven of the JS/MKS cases (blue) studied, the proliferation with time. The curve diverged from the linear at approximately gw defect was similar in the vermis (light green/blue) and the hemispheres (dark 16, indicating an important increase in the rate of expansion of the green/blue). (Scale bar: 10 μm.) ***P < 0.001, **P < 0.01, and *P < 0.05. EGL surface at this point in development (Fig. 3B). These results

Aguilar et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 16953 Downloaded by guest on September 30, 2021 Fig. 3. SHH signaling is active in control cases in which the exponential increase in the EGL surface begins. (A) Combined in situ hybridization of SHH or GLI1 mRNA and calbindin immunostaining at gw 12, 17, and 33. At gw 12, neither the PCs (arrows) nor the EGL express SHH, whereas the EGL expresses GLI1. At 17 gw, around the observed peak of GCP proliferation, some calbindin-positive PCs still migrating toward the pial surface express detectable levels of SHH mRNA (arrows) whereas others do not (arrowheads). At the same time, GLI1 mRNA is expressed in the EGL. At gw 33, PCs, aligned in a single-cell row beneath the EGL, all express high levels of SHH mRNA (arrows). (B) Quantiﬁcation of the EGL surface on horizontal H&E-stained sections (green dots). From the onset of detectable SHH expression (i.e., gw 17, blue arrow), the exponential curve ﬁtting all the data points starts to diverge from the linear regression calculated from the data points at gw 10 to gw 15. The divergence corre- sponds with the timing of the GCP proliferation peak (Fig. 2, purple arrows), suggesting that SHH- dependent increase in the rate of GCP proliferation is responsible for the exponential increase in the surface of the EGL. (Scale bars: 20 μm.)

suggest that strong levels of PC-derived SHH drive the massive Discussion GCP proliferation necessary for lobe growth. Our study shows that SHH-dependent GCP proliferation is im- To assess the contribution of the early activation of Shh in the paired in fetuses with JS/MKS in whom ciliary genes are mu- EGL (Fig. 3A), we evaluated GCP proliferation at 15 gw in tated. This cellular mechanism, which we demonstrate here in a case of JS/hydrolethalus syndrome (HLS) caused by an inac- humans, is found in patients with both JS and MKS, as well as in tivating mutation in KIF7, the human orthologue of Drosophila cilia or RPGRIP1L mutant mice (8, 9), and is thus conserved Costal2, a key component of the Hedgehog signaling pathway. through evolution. GCP expansion and the response to SHH Although Shh signaling was compromised (Fam 1, Fetus 3) (34), were also defective in a case of JATD, raising the possibility that GCP proliferation was not impaired at gw 15 in this case (Fig. impaired Shh-dependent GCP proliferation might be common to 2B). This supports the possibility that SHH pathway activation a number of ciliopathies. before 16 gw only mildly contributes to the massive GCP pro- Previous studies in which GCP cilia were ablated in mouse fi liferation driving lobe growth. models provided the rst evidence that defects in Shh-dependent GCP proliferation may contribute to the cerebellar phenotype Response of GCP to Shh Signaling Is Impaired in JS/MKS. Shh path- observed in JS and MKS (23, 24). It was unclear, however, way activation can be impaired as a result of defects in SHH whether these defects were responsible for hypo-/aplasia of the expression or in the GCP response to this signal. Faulty migration vermis or affected cerebellar development globally. In the present study, we evaluated GCP proliferation at different developmental of PCs might contribute to a deficit in Shh signaling, as suggested stages in 12 cases of JS/MKS, one case of JS/HLS, and one case of by the heterotopic PC and local interruptions in the PC layer JATD. Interestingly, although defective GCP proliferation and observed in all our JS/MKS cases (Fig. S2). However, combined SHH vermis hypoplasia were observed in most of our JS/MKS cases (11 in situ hybridization of and calbindin labeling of PCs on of 12), vermis hypoplasia was also found to be present in case sections from eight JS/MKS and four control cases showed that, 18485, which does not present a GCP proliferation defect. Fur- when PCs reached their destination, they expressed normal levels thermore, in five of seven analyzed fetuses, defective GCP pro- of SHH (Fig. 4 A and B). On the contrary, the levels of GLI1 liferation affected the vermis and the cerebellar hemispheres to mRNA and PTC in the GCP were reduced in most analyzed JS/ the same extent (Fig. 2). These results suggest that defective GCP MKS cases in which GCP expansion was defective, indicating an expansion is not responsible for vermal hypo-/aplasia in JS/MKS, impaired response of GCPs to SHH (Fig. 4). but, most probably, for the global cerebellar phenotype. In a recent

16954 | www.pnas.org/cgi/doi/10.1073/pnas.1201408109 Aguilar et al. Downloaded by guest on September 30, 2021 correlation between the mutated gene and the proliferation defect. Additional cases of JS/MKS with TMEM67/MKS3 mutations need to be analyzed to confirm this observation. In mice, such a genotype–phenotype correlation is not observed. Rather, a correlation might exist between ciliary function and GCP proliferation: mouse models in which GCP cilia were abrogated (Kif3a, Ift88, or RPGRIP1L mutants) showed very severe defects in GCP proliferation (23, 24). In contrast, Cep290 and Ahi1 KO mice and mice with spontaneous deletion of the mouse orthologue Tmem67/Mks3, had no defects in either cilia formation or Shh- dependent GCP proliferation (10, 38). Such strong differences in phenotype between humans and mice further outline the necessity of studies addressing the cellular and molecular mechanisms of human pathologic conditions in actual human tissue samples. Materials and Methods Tissue Samples. The human fetal material was obtained from the tissue col- lections of the Histology, Embryology and Cytogenetics Department of the Necker Children’s Hospital, Paris, France. The tissue was obtained from spontaneous or medically induced abortions, with appropriately informed, written maternal consent for brain autopsy, the use of the brain tissue, and access to medical records for research purposes. The study was approved by the Ethics Boards of Hôpital Necker-Enfants Malades. Unstained paraffin- embedded JS and control tissues, as well as H&E-stained sections, came from the tissue archives. We also obtained unfixed control cerebellar samples. The mice were C57Bl6/J or mixed backgrounds purchased from Janvier.

Fluorescence Microscopy and in Situ Hybridization. Unfixed human and mouse tissue was flash-frozen, embedded in Tissue-Tek optimal cutting temperature compound (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 °C methanol. For the other antigens or in situ hybridization, tissue sections were dewaxed and the antigen retrieved by incubation for 30 min at 96 °C in preheated retrieval solution (S2367; Dako), pH 9, and then allowed to cool at room temperature for another 30 min in the same solution. The sections were

Fig. 4. Conserved SHH expression and reduced GLI1 mRNA and PTC levels in then incubated for 12 to 72 h at 4 °C with the following primary antibodies: BIOLOGY affinity-purified rabbit anti-human and anti-mouse CEP290 (1:500) (gift from most JS/MKS cases with defective GCP proliferation. (A) GLI1, PTC,andSHH DEVELOPMENTAL expression in JS samples compared with controls. SHH, GLI1, and PTC levels of S. Saunier, Hôpital Necker-Enfants Malades, Paris, France) (7), goat anti-root- expression were assessed by in situ hybridization (SHH, GLI1) or immunohis- letin (sc67-824, 1:100; Santa Cruz), mouse anti-detyrosinated α-tubulin (clone tochemistry (PTC). SHH mRNA expression is conserved in the vast majority of 1D5, 302011, 1:1,000; Synaptic Systems), rabbit anti-GFP (A11122, 1:1,000; JS/MKS cases with defective GCP proliferation, but GLI1 mRNA and PTC levels Molecular Probes), mouse anti-Ki67 (MIB1 clone, M7240, 1:50; DAKO), rabbit are reduced or undetectable, indicating a defective response to SHH. (∼, anti-Pax6 (AB5409, 1:1,000; Chemicon), and rabbit anti-calbindin (CB38a, comparable to WT; ND, not determined; NE, not expressed; −, diminished.) (B) 1:20,000; Swant). Species-specific secondary antibodies from Sigma-Aldrich Combined SHH in situ hybridization and calbindin immunolabeling of JS case were then applied for 1 h to the slides. Finally, the sections were counter- 080144 and an age-matched control. SHH mRNA levels in JS calbindin-positive stained with DAPI (10 μg/mL; Sigma) and mounted in Fluoromount. PCs (green) are comparable to control levels. The EGL and PC layer (PCL) are For in situ hybridization on human tissue, antisense riboprobes were la- delimited by a dashed line. (C) GLI1 in situ hybridization or PTC immunohis- beled as described previously (8) by in vitro transcription of cDNAs encoding tochemistry and DAPI staining in two JS cases and age-matched controls. GLI1 mouse Shh (gift from A. McMahon) or human GLI1 (gift from A. Ruiz i mRNA and PTC expression are reduced in the JS EGL. (Scale bar: 20 μm.) Altaba). 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- study in Cep290 and Ahi1 KO mice, as well as in three subjects croscope equipped with an Apotome device and a Hamamatsu ORCA R2 with JS, cerebellar midline fusion was incomplete (10). According C10600 camera. Combined in situ hybridization and immunostaining were to this study, this cerebellar defect could explain vermian hypo- imaged with a Leica DM 5500B microscope and a Hamamatsu ORCA ER C4742- plasia and would originate from defective Wnt signaling (10). 80 camera. Therefore, it would be interesting, in a future work, to assess Wnt pathway activation in JS/MKS fetal tissue samples by performing Transmission EM. Postmortem fetal tissue was dissected in PBS solution into in situ hybridization of two direct Wnt targets, AXIN2 (35) and 1 × 2-mm blocks, which were then fixed in 2% PFA/1% glutaraldehyde so- LEF1 (36, 37). lution in 0.1 M phosphate buffer for 3 h. After rinsing in PBS solution, the In humans, genotype–phenotype correlations have previously tissue was postfixed in 1% osmium tetroxide for 30 min on ice, protected been established for some JS genes or pathological CEP290 alleles from light, with shaking. The tissue blocks were then dehydrated in 50% and (35, 36). In our study, there were no obvious correlations between 70% ethanol baths in water for 7 min each, and then stained in 1% uranyl acetate in methanol. After final dehydration, samples were immersed for 40 the different CEP290 alleles responsible for JS and the degree to min in a graded series of ethanol/Epon (2/1, 1/1, 1/2 ratios), then in pure which GCP proliferation was impaired: the two pairs of siblings Epon. Samples were then mounted in Epon blocks for 48 h at 60 °C to ensure analyzed (cases 16676 and 16290 and cases 18485 and 050158; Fig. B polymerization. Ultrathin sections (70 nm) were cut sagittally on an ultra- 2 and Table S1) exhibit radically different degrees of GCP pro- microtome (Ultracut E; Reichert-Jung) and analyzed with a Jeol 10–11 liferation compared with their age-matched controls, even though transmission electron microscope. they have the same CEP290 alleles. We did observe, however, that, when TMEM67/MKS3 was mutated (case 060247), proliferation Quantification of GCP Proliferation Rate and Cerebellar Surface. KI67-positive was more severely impaired than in JS/MKS caused by other mu- GCP quantification was accomplished on half horizontal sections of control tated genes (Fig. 2B), suggesting a possible phenotype–genotype and human JS fetal cerebella, comprising half the vermis and one hemisphere

Aguilar et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 16955 Downloaded by guest on September 30, 2021 (routine sectioning for diagnosis done at the Histology, Embryology and Primary Cortical Neural Stem Cell Culture and shRNA Assay. The cortex of Cytogenetics Department of the Necker Children’s Hospital). postnatal day 0 to 2 mice was dissected (exclusion of the choroid plexus, hip- × For small samples (to 18 gw), the entire EGL was photographed with a 20 pocampus, corpus callosum, olfactory bulbs, and meninges) and mechanically objective on a Zeiss Observer Z1 microscope equipped with an Apotome dissociated, digested with papain. The cells were plated at high density in 10% device and a Hamamatsu ORCA R2 C10600 camera. The EGL was then FCS-containing medium, and allowed to grow to confluence. The primary manually reconstituted and, based on EGL length, divided into 16 parts. The + cortical astroglial cells were then detached and nucleofected in suspension with number of Ki67 cells and the EGL surface were manually quantified for each part by using ImageJ (National Institutes of Health; on average, 3500 the expression arrest pGIPZ CEP290shRNAmir-GFP plasmid or with a pGIPZ-GFP Ki67+/Pax6+ cells were counted per fetus). For bigger samples, this method control plasmid (Abgene) using a Nucleofector device (Lonza) and the Basic became technically very challenging. We therefore sampled the EGL by Neuron Small Cell Number Nucleofector kit (VSPI-1003; Lonza). The cells were taking 16 to 20 equidistant images at 20× throughout the EGL. In each then plated at high density to form pure confluent monolayers of monociliated + picture, the number of Ki67 cells and the EGL surface were manually astroglia that were fixed for 1.5 min in 4% paraformaldehyde/0.33 M sucrose at fi fi quanti ed. In both cases, either when the full EGL was quanti ed or when it 4 °C, then for 10 min in −20 °C MeOH (40). After triple staining for GFP, CEP290, fi was sampled, the quanti ed regions are spatially comparable. The 16 to 20 and polyglutamylated tubulin (GT335; gift from C. Janke, Institut Curie, Orsay, measurements of the proliferation rate per sample were then averaged to France), as described earlier, cilia presence was assessed. In cells in which obtain the final proliferation rate for each fetus. CEP290 was down-regulated, only GFP+/CEP290− cells were quantified. The EGL surface area was measured on representative individual sections from each stage (n = 1 or 2 per fetus and n = 1–3 fetuses per stage), which were selected for being situated at the same anteroposterior level: they all Statistical Analysis. The results (i.e., average of the 16 to 20 measures of the ± encompass the dentate nucleus. The H&E-stained sections were scanned to proliferation rate obtained for each fetus) are presented as mean SD. A obtain low-magnification and high-resolution images. On each image, the two-sided Wilcoxon test was used for all statistical analyses except for EGL surface was manually outlined and measured by using ImageJ. comparison of GCP proliferation in the cerebellar hemispheres vs. the vermis, for which a two-sided averages test was used. Validation of RNAi-Mediated Down-Regulation of CEP290. To test the efficiency of CEP290 down-regulation by the CEP290 shRNA, HEK 293T cells were ACKNOWLEDGMENTS. We thank the Cellular Imaging Center of the Pitié- cotransfected with a mCEP290-GFP plasmid (gift from J. Gleeson, University of Salpêtrière Hospital and Dr. D. Langui for assistance with confocal and trans- California, San Diego, CA) and a pGIPZ CEP290shRNAmir-GFP plasmid or a pGIPZ- mission EM; M.-P. Muriel for excellent technical assistance with EM; GFP control plasmid (Abgene). After 24 h, cells lysates were run on a NuPage 4% A. McMahon for mouse Shh probe; T. Caspari for anti-Arl13B antibody; C. Janke to 12% Bis-Tris gel. After transfer, the membrane was cut below the 110-kDa for GT335 antibody; J. Gleeson for mCEP290-GFP plasmid; A. Ruiz i Altaba for the GLI1 riboprobe plasmid; and Dr. M. V. Nachury for support. This study was marker, and the upper half was blotted for GFP (homemade polyclonal anti-GFP α supported by grants from Agence Nationale de la Recherche, International antibody) (39) with the lower half blotted for -tubulin (Hybridoma Bank clone Human Frontier Science Program Organization, Institut de France, Mairie de 12G10, 1/5,000), following standard procedures. Protein bands were visualized Paris, Fondation pour la Recherche Médicale (to N.S.), a doctoral fellowship for with HRP-labeled protein A or anti-mouse antibody, followed by detection with the French Ministry of Research and Association pour la Recherche sur le Can- chemiluminescence and quantification with ImageJ software. cer (to A.A.).

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