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

<p>(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 <i>shiomaneki</i> (<i>sio</i>^{<i>rk18/rk18</i>}) mutants had abnormal axons, which formed abnormally branched bundles (C, D). Dorsal projection views of wild-type control (A, B) and <i>sio</i> 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 <i>sio</i> mutant (F) cerebella. (G) Schematic representation of <i>sio</i> phenotypes. Dorsal views. (H) The <i>sio</i> locus was mapped to chromosome 7, between 41.69 and 43.12 Mb from the telomere. The type IV collagen genes <i>col4a6</i> and <i>col4a5</i> 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 <i>col4a6</i> 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 <i>col4a6</i> (Ma, Na) and <i>col4a5</i> (Oa, Pa) in wild-type (Ma, Mb, Oa, Ob) and <i>sio</i> (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 <i>sio</i> mutants was suppressed by expressing wild-type <i>col4a6</i>. <i>col4a6</i> RNA (50 pg) was injected into one-cell-stage embryos from a cross between <i>sio</i> heterozygotes (T-W). The resultant 5-dpf larvae were stained with an anti-Vglut1 antibody. Uninjected controls for wild-type (Q) and <i>sio</i>^{<i>rk18/rk18</i>} (R, S) larvae are also shown. Some of the injected <i>sio</i> 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 <i>sio</i> mutants (3 larvae) and in the <i>col4a6</i> RNA-injected wild-type (3 larvae) and <i>sio</i> mutants (6 larvae), and the means were calculated. The injection of <i>col4a6</i> 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).