Defective Wnt-dependent cerebellar midline fusion in a mouse model of Joubert syndrome

Abstract

The ciliopathy Joubert syndrome is marked by cerebellar vermis hypoplasia, a phenotype for which the pathogenic mechanism is unclear. To investigate Joubert syndrome pathogenesis, we have examined mice with mutated Ahi1, the first identified Joubert syndrome-associated gene. These mice show cerebellar hypoplasia with a vermis-midline fusion defect early in development. This defect is concomitant with expansion of the roof plate and is also evident in a mouse mutant for another Joubert syndrome-associated gene, Cep290. Furthermore, fetal magnetic resonance imaging (MRI) of human subjects with Joubert syndrome reveals a similar midline cleft, suggesting parallel pathogenic mechanisms. Previous evidence has suggested a role for Jouberin (Jbn), the protein encoded by Ahi1, in canonical Wnt signaling. Consistent with this, we found decreased Wnt reporter activity at the site of hemisphere fusion in the developing cerebellum of Ahi1-mutant mice. This decrease was accompanied by reduced proliferation at the site of fusion. Finally, treatment with lithium, a Wnt pathway agonist, partially rescued this phenotype. Our findings implicate a defect in Wnt signaling in the cerebellar midline phenotype seen in Joubert syndrome that can be overcome with Wnt stimulation.

Figure 1. Reduced cerebellum size and foliation defects in Ahi1^−/− mice

(a) Whole mount images of representative mutant and littermate control brains. The vermal folia are outlined with dashed lines and the visible folia V-IX are labeled. Scale bar, 1 mm. (b) Midline sagittal cresyl-violet (C-V) stained sections from representative littermates. Folia are labeled and arrows indicate foliation defects: decreased size of V and fusion of VI and VII. Scale bar, 1 mm. Quantification of vermis area at the right showing average area measurements of midline sections. *P<0.05, Student’s t-test, n = 3 for each genotype. (c) Quantification of foliation defects in Ahi1 mutant mice. Lobules were identified and counted from six mice for each geneotype, Ahi1^−/− and control, and the average is shown below. *P<0.05, Student’s t-test. Scale bar, 1 mm. (d) Transverse C-V stained sections from P5 littermates revealing specific vermis folia size defect particularly in folia V (arrows). Scale bar, 1 mm. (e) Sagittal midline C-V stained sections from representative littermates aged P4 and E18.5 revealing the presence of a mild size defect (bars) and foliation defects (arrows). Scale bar, 1 mm (upper panels), 200 μm (lower panels). (f) Average vermis area at E18.5 and P4 in Ahi1^−/− and control littermates. *P<0.05, Student’s t-test, n = 3 for each genotype. Error bars are S.E.M.

Figure 2. Early proliferation defect and the absence of a postnatal Shh defect

(a) BrdU stained (green) sections from representative littermates aged E16.5 and E18.5. Dashed lines demarcate external granule layer and Hoechst labels nuclei. Scale bar, 50 μm. (b) Quantification of BrdU labeled CGNs indicated as average number of BrdU positive cells as a ratio of total CGNs. E16.5, and to a lesser extent P5, reveal a significant decrease in relative BrdU stained cells. *P<0.05, **P<0.005 Student’s t-test, n = 3 for each genotype. (c) N-myc staining (green) in midline sagittal sections from P5 littermates. Hoechst labels nuclei. Scale bar, 20 μm (d) qRT-PCR analysis of Shh target genes, N-Myc, Gli1, and Ptc1. Values are average expression levels relative to actin. NS=not significant, Student’s t-test, n = 3 for each genotype and gene tested. Error bars are S.E.M.

Figure 3. Midline fusion defect in mice and humans with Joubert syndrome

(a) Transverse C-V stained sections from littermates at E16.5 and E14.5 revealing midline fusion defect (arrows). Scale bar, 0.5 mm. (b) Whole mount images of littermate embryos at E12.5 imaged laterally (top) and dorsally (bottom). Arrows point to the expanded roof plate and fourth ventricle. Dashed lines demarcate roof plate while asterisks point to cerebellar vermis anlage. Anterior (A) and posterior (P) directionality is depicted by a double arrow. Scale bar, 2 mm (upper panels), 1 mm (lower panels). (c) Midline and medial sagittal C-V stained sections from E12.5 littermates revealing elongated roof plate (bars). Scale bar, 0.5 mm. (d) Transverse cresyl-violet stained sections of representative Cep290 mutant and littermate control embryos at E16.5. Arrows point to mild midline fusion defect, which is more pronounced on posterior sections. Scale bar, 0.5 mm. (e) Fetal MRIs from a representative subject with Joubert syndrome and a control fetus both 25 weeks gestational age (GA). Sagittal image reveals superiorly tilted fourth ventricle roof (white arrowhead), while axial series reveals a gap between cerebellar hemispheres (arrows) that is more severe on inferior sections. The MTI is also already visible in the fetus with Joubert syndrome (black arrowhead). Scale bar, 1 cm.

Figure 4. Defective Wnt signaling and lithium rescue in Ahi1 mutant cerebella

(a) Top, X-gal stained whole mount dorsal view of E13.5 BATgal+ littermates revealing Wnt activity (arrowheads) at the site of hemisphere fusion (asterisk). Bottom, X-gal stained transverse sections of superior cerebellar anlage from BATgal+ littermates. White asterisks depict roof plate directly abutting the site of midline fusion. Arrows point to cerebellar hemisphere cells surrounding the midline with decreased Wnt activity and defective fusion in Ahi1 null. Scale bar, 100 μm. (b) β-galactosidase (green) and BrdU (red) antibody staining of transverse superior cerebellar sections of Ahi1 null and littermate control BATgal+ embryos at E13.5. Dashed line indicates the five-cell layer boundary used for BrdU counting in c. Hoechst (blue) labels nuclei. Scale bar, 20 μm. (c) Quantification of BrdU positive cells adjacent to ventricle relative to total cell number as determined by Hoechst. *P<0.05, Student’s t-test, n = 3 for each genotype. Error bars are S.E.M. (d) C-V stained transverse sections of Ahi1 mutant and littermate control embryos. First row: E14.5 control NaCl treated littermates. Dashed lines represent measurement area for panel e. Second row: E14.5 LiCl treated mutant and control littermates. Arrows point to expansion of cerebellar hemispheres. Third row: E16.5 LiCl treated mutant and control littermates with complete midline fusion (arrows). Scale bar, 0.5 mm. (e) Degree of separation of cerebellar hemispheres as measured by average inter-hemispheric space. *P<0.05, Student’s t-test, n = 3 for each genotype (littermates) and condition. Error bars are S.E.M. NS=not significant. (f) Average percentage of Ki67⁺ cells relative to total cell number (Hoechst). *P<0.05, Student’s t-test, n = 3 for each genotype (littermates) and condition. Error bars are S.E.M. NS=not significant. (g) Sagittal C-V stained sections from P4 Ahi1 mutants and littermate controls from a dam treated with LiCl or untreated. Note the similarity between Ahi1^−/− treated with LiCl (second panel) to untreated Ahi1^+/+ (third panel). Scale bar, 1 mm.