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Fig. S1. Phenotypic severity increases with decreasing gene dose. (A-K) Cresyl Violet-stained sagittal sections through the cerebellar vermis of P20 mice. Black arrowheads and arrows indicate the primary and preculminate fissures, respectively. Zic1+/- (B) and Zic4+/- (C) and Zic4−/− (E) cerebella had normal foliation. Eighty-five percent (n=28) of Zic1+/-;Zic4+/- cerbella (D) also had normal foliation, while the remaining 15% (n=5) were severely affected (H) with hypoplasia the loss of one folium anterior to the preculminate fissure. Similarly, in Zic1+/-;Zic4−/− mice, 60% (n=10) had normal foliation (F), while the remaining 40% (n=6) had extremely hypoplastic cerebella that were missing an anterior folium (G). Zic1−/− (I) Zic1−/−;Zic4+/- (J) and Zic1−/−;Zic4−/− (K) cerebellar vermis sections were indistinguishable and resembled the severe phenotypes characterized above (G,H) with hypoplasia and anterior foliation defects. Scale bar: 500 µm.
Fig. S2. E17.5 patterning abnormalities correlate with abnormal foliation and are Shh independent. (A-H,M-P) Anterior views of in situ hybridization for Wnt7b and Epha4 showed that stripes were normal in wild type (A,E) and Zic4−/− (C,G) cerebella, but disrupted in Zic1−/− (B,F) and Zic1−/−;Zic4−/− (D,H) cerebella. d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral. (I-L) Diagrams depict normal (I,K) and disrupted (J,L) expression patterns. Notably, stripe patterns were also normal in Zic1+/-;Zic4+/-;Shh+/- (M,O) and Gli2-cko (N,P) cerebella. Regions of stripe differences are indicated by asterisks and brackets in A-H and M-P. Scale bars: 500 µm A-H and M-P.
Fig. S3. Epha4 expression is reduced in Zic mutant cerebella. Relative expression of Epha4 was significantly reduced in Zic1−/− and Zic1−/−;Zic4−/− cerebella at E17.5 (*P<0.05), though not at P1.
Fig. S4. Zic mutant cerebella are mispatterned at P3. (A-D) Anterior views of whole-mount antibody staining with Plcβ4 on P3 cerebella. d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral. (A′-D′) Magnified vermis views from A-D. Four major parasagittal stripes of expression (brackets; M, medial; L, lateral) that ran perpendicular to folia (roman numerals) were visible in wild-type (A′) and Zic4−/− (B′) cerebella. Fissures (primary and preculminate indicated by dotted lines A′-D′) excluded antibody penetration and produced areas with no visible staining between lobes creating a checkerboard appearance. Zic1−/− (C′) and Zic1−/−;Zic4−/− (D′) cerebella had abnormally narrow lateral stripes in lobe II/III and badly disrupted stripes across lobe IV/V. Scale bars: 500 µm in A-D; 250 µm in J,K.