Fig. 2

Type IV collagen gene col4a6 is required for axogenesis of the GCs in caudolateral lobes.

(A-D) Staining with an anti-Vglut1 antibody, which marks the presynaptic termini of GC axons, revealed that the GCs in the caudolateral lobes of 5-dpf homozygous shiomaneki (sio^rk18/rk18) mutants had abnormal axons, which formed abnormally branched bundles (C, D). Dorsal projection views of wild-type control (A, B) and sio mutant (C, D) cerebella. (E, F) Labeling of the caudolateral GCs in the GC-specific Gal4 line hspGFFDMC90A also showed aberrant axogenesis (marked by arrowheads in F). Dorsal views of the control (E) and sio mutant (F) cerebella. (G) Schematic representation of sio phenotypes. Dorsal views. (H) The sio locus was mapped to chromosome 7, between 41.69 and 43.12 Mb from the telomere. The type IV collagen genes col4a6 and col4a5 are located in this region. The numbers of recombinations in the mutant genomes are indicated. (I) The mutant contained a T-to-C point mutation in a splicing donor site of the col4a6 gene. (J) Detection of mutant transcripts by RT-PCR with primers (P1, P2) shown in K. (K) Structures of the wild-type and mutant transcripts. (L) Structures of the wild-type and mutant proteins. ‘Collagenous’ and ‘NC1’ represent the collagenous domain and non-collagenous domain 1, respectively. The mutant transcripts and proteins in K and L correspond to the PCR bands indicated by the same number in J. (Ma-Pb) Expression of col4a6 (Ma, Na) and col4a5 (Oa, Pa) in wild-type (Ma, Mb, Oa, Ob) and sio (Na, Nb, Pa, Pb) larvae at 3 dpf. Lateral views (Ma-Pa) and cross sections of the dorsal hindbrain region (Mb-Pb). (Q-W) The abnormal axogenesis of the caudolateral GCs in the sio mutants was suppressed by expressing wild-type col4a6. col4a6 RNA (50 pg) was injected into one-cell-stage embryos from a cross between sio heterozygotes (T-W). The resultant 5-dpf larvae were stained with an anti-Vglut1 antibody. Uninjected controls for wild-type (Q) and sio^rk18/rk18 (R, S) larvae are also shown. Some of the injected sio larvae showed normal GC axons (U), and others showed a relatively weak abnormality in the GC axons (V). The number of abnormal GC axon branches in a larva is indicated (W). The abnormal branch points were counted in the uninjected wild-type (4 larvae) and sio mutants (3 larvae) and in the col4a6 RNA-injected wild-type (3 larvae) and sio mutants (6 larvae), and the means were calculated. The injection of col4a6 RNA significantly reduced the number of abnormal branch points (*, P<0.05). Scale bars: 50 μm in A, C; 20 μm in B, D; 500 μm in Pa (applied to Ma, Na, Oa); 25 μm in Pb (applied to Mb, Nb, Ob); 50 μm in V (applied to Q, R, S, T, U).