Multiple developmental programs are altered by loss of Zic1 and Zic4 to cause Dandy-Walker malformation cerebellar pathogenesis

Abstract

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. Here, we show that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, we show that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1(+/-);Zic4(+/-) background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1(-/-);Zic4(-/-) mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlated with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype was Shh independent. In Zic1(+/-);Zic4(+/-);Shh(+/-), we observed normal cerebellar anlage patterning and foliation. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, our data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM.

Fig. 1.

Zic1 and Zic4 mutants have cerebellar size and foliation defects. (A-D) Dorsal views of whole P20 cerebella show that Zic4^−/− (B) cerebella were slightly smaller than wild type (A). Zic1^−/− (C) and Zic1^−/−;Zic4^−/− (D) cerebella were much smaller than wild type along the anterior-posterior (AP) axis and Zic1^−/−;Zic4^−/− cerebella were also smaller along the medial-lateral (ML) axis. (E-H) Cresyl Violet-stained sagittal sections through the cerebellar vermis. Black arrowheads and arrows indicate the primary and preculminate fissures, respectively, and folia are labeled with roman numerals (II-X). Wild-type (E) and Zic4^−/− (F) cerebella had three folia anterior to the primary fissure, whereas Zic1^−/− (G) and Zic1^−/−;Zic4^−/− (H) cerebella were dramatically hypoplastic, were missing one anterior folium and lacked a fissure between folia VIa and VIb. Scale bars: 500 μm in E-H.

Fig. 2.

Zic1 and Zic4 have dynamic expression patterns throughout cerebellar development. (A,B) P1 cerebellar sagittal sections: Zic1 and Zic4 were expressed in the cerebellar external granule layer (egl, arrow) and white matter (wm), and Zic4 was also expressed in the internal granule cell layer (igl, arrowhead). (C,D) Anterior view of whole cerebellum at E17.5; Zic1 and Zic4 were broadly expressed in the embryonic cerebellum. d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral. (E-H) Whole-mount lateral (E,G) and dorsal (F,H) views of the E12.5 cerebellar region (cb, brackets) with dotted lines (F,H) indicating the level of E12.5 sagittal sections in I,J. (I,J) Zic1 and Zic4 were both expressed in the cerebellar rhombic lip (rl) and mesenchyme (mes), with Zic1 also expressed in the ventricular zone (vz). Scale bars: 500 μm in A-H; 100 μm in I,J.

Fig. 3.

Zic1 and Zic4 cerebellar size and foliation defects arise postnatally. (A-D) Cresyl Violet stained sections showed no gross cerebellar morphological defects at E17.5 in single or double homozygous mutants (B-D) compared with wild type (A). (E-L) At P0, each genotype had the correct number of cardinal fissures, but while the Zic4^−/− mutant (F) resembled wild type (E), in Zic1^−/− (F) and Zic1^−/−;Zic4^−/− (G) mutants the preculminate fissure was shifted anteriorly and the anterobasal lobe (asterisk) was smaller than wild type. By P5, both the wild-type (I) and Zic4^−/− mutant (J) cerebella had grown considerably, whereas Zic1^−/− (F) and Zic1^−/−;Zic4^−/− (G) cerebella remained small. Black arrowheads and arrows indicate the primary and preculminate fissures, respectively. Scale bars: 500 μm.

Fig. 4.

Reduced cerebellar size and granule cell proliferation correlate with expression of Shh target genes. (A) Cross-sectional area of cerebellar vermis was similar for all genotypes at P0. By P1, Zic1^−/− and Zic1^−/−;Zic4^−/− cerebellar vermis areas were significantly smaller than those of wild-type or Zic4^−/− animals (**P<0.01). (B) Mitotic index was calculated by total number of BrdU-positive (GCP)/total GCP. By P1, Zic1^−/− and Zic1^−/−;Zic4^−/− had significantly reduced EGL proliferation compared with wild-type or Zic4^−/− animals (*P<0.05). (C) Expression of Shh target genes Gli1, Ptch1 and Mycn was significantly reduced in Zic1^−/− and Zic1^−/−;Zic4^−/− whole cerebella at E17.5, whereas Shh, Calb1 and Rora expression levels remained normal. Expression of Gli1 and Ptch1 was significantly reduced for all mutants at P1 (*P<0.05, **P<0.01). Data are mean±s.e.m.

Fig. 5.

Zic gene interaction with the Shh pathway controls cerebellar size, but not foliation. (A-F) Cresyl Violet-stained sagittal sections through the cerebellar vermis of P20 mice. Black arrowheads and arrows (A-F) indicate the primary and preculminate fissures, respectively. Compared with wild type (A), Shh^+/− (B) and Zic1^+/−;Zic4^+/− (C) cerebellar vermis sections had normal foliation and size. Zic1^+/−;Zic4^+/−;Shh^+/− (D) cerebella also had the correct number of folia, but vermis area was reduced, similar to that of the Zic1^−/−;Zic4^−/− mutant (E). The Gli2-cko (F) had an extremely hypoplastic cerebellum, but retained three anterior folia. Scale bars: 500 μm. (G) Representative horizontal MR image with a heat map indicating regions of size difference that were significant at a false discovery rate of 0.1% in wild type and Zic1^+/−;Zic4^+/−;Shh^+/− brains. (H) Quantification of voxels from wild-type, Zic1^+/−;Zic4^+/−, Shh^+/− and Zic1^+/−;Zic4^+/−; Shh^+/− cerebella (n=10 per genotype) showed that cerebellar size was significantly reduced in Zic1^+/−;Zic4^+/−; Shh^+/− mice (whole dataset comparison f>17, P<0.0001; paired t-test comparison of Shh^+/− with Zic1^+/−;Zic4^+/−;Shh^+/− **P=0.0024). Frontal lobe size did not vary significantly across genotypes (f=1.53, P=0.22). (I) Relative expression of Gli1, Ptch1 and Mycn at E17.5 (*P<0.05). Data are mean±s.e.m.

Fig. 6.

Embryonic and neonatal mispatterning of Zic mutant cerebella correlates with adult foliation defects. (A-D) Anterior views of in situ hybridization for Wnt7b (A,B) and Epha4 (C,D) in whole E17.5 cerebella. Wild-type parasagittal stripe patterns (A,C). Anterior stripes were reduced in size or absent in Zic1^−/−;Zic4^−/− cerebella (B,D). d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral. (E-H) Diagrams depict normal (E,G) and disrupted expression patterns (F,H). Regions of stripe differences are indicated by asterisks and brackets in A-H. (I) The anterior surface of a P3 cerebellum with vermis highlighted (red dotted box). (J,K) Anterior vermis views of anti-Plcβ4 whole-mount antibody staining of P3 cerebella. Wild-type cerebellar vermis (J) with four major parasagittal stripes of expression (brackets; M, medial; L, lateral) perpendicular to folia (roman numerals). Fissures (dotted lines in J and K) excluded antibody penetration and produced areas with no visible staining between lobes. Zic1^−/−;Zic4^−/− (K) cerebella lacked one anterior lobule and remaining stripes were abnormally patterned. The medial stripes of lobe II/III were relatively unaffected, but the lateral stripes were much narrower compared with wild type. Lobe IV/V patterns were completely disrupted with irregularly shaped and bilaterally asymmetric regions of expression. Scale bars: 500 μm in A-D; 250 μm in J,K.

Fig. 7.

Pattering of the cerebellar anlage is normal at E12.5. (A) Diagram of a sagittal section through dorsal rhombomere 1 at E12.5 showing relative positions of cell populations c1-4. (B-G) Antibody staining of sagittal sections showed populations of Lhx1/5-positive c2/c4 cells (B,C), Lmx1a positive c3 cells (D,E) and Atoh1 positive c1 (rl=rhombic lip) cells (F,G) to be the same in wild-type and Zic1^−/−;Zic4^−/− E12.5 cerebella. (H-J) BrdU incorporation analysis showed that proliferation was not grossly changed in the ventricular zone of Zic1^−/−;Zic4^−/− cerebella at E12.5. Scale bars: 500 μm in B-G,I,J. Data are mean±s.e.m. Outlined areas in I, J indicate regions of cell counts.