Fig. 2

Expression of candidate sox9 down-stream targets analyzed by in situ hybridization analysis in 2 dpf embryos, the same stage as used for microarray analysis. Anterior to the left. Lateral views of crx (A–C), rs1 (D–F) and vsx1 (G–I) show that they are strongly expressed in developing retina; crx and rs1 but not vsx1 were expressed in the outer nuclear layer. Expression was severely reduced in sox9b mutants and double mutants; only a small ventral-nasal patch of retinal expression (black arrowhead) was detected for crx (B, C) and rs1 (E, F), and ventro-nasal expression in the retina (white arrowhead) was observed for vsx1 (H, I). Ventral view of neurod (J–L) and sox4a (M–O) showing neurod expression in the outer nuclear layer and inner nuclear layer of the retina and telencephalon (J) and sox4a expression in inner part of retina, telencephalon and diencephalon (M). Retinal expression of neurod was reduced in sox9b and double mutant embryos, but the expression in the telencephalon was not affected in these mutants, demonstrating specificity (K, L). Expression of sox4a in the retina was lost in sox9b mutants and double mutants, whereas expression in telencephalon and diencephalon was not affected in the mutants (N, O). At 2 dpf, sox9b (S, T), but not sox9a (P, Q), was expressed in retina, consistent with the reduced gene expression pattern shown above. Lateral view (P, S) and ventral view (Q, T). At 68 hpf, sox9a is expressed in the inner nuclear layer (R), and sox9b is expressed in ciliary marginal zone (U). These results confirmed the microarray result that crx, rs1, vsx1, neurod and sox4a are down-regulated in sox9b mutant embryos, and revealed that sox9b is required for expression of these candidate genes in retina at 2 dpf. cmz, ciliary marginal zone; d, diencephalon; onl, outer nuclear layer; pa, pharyngeal arches; r, retina; t, telencephalon; tc, tectum.