Differential regulation of col11a2 expression by sox9a and sox9b. (A–D) Lateral view and (E–H) ventral view of 2 dpf embryos. Expression of col11a2 appeared in neurocranium (ethmoid plate, arrowhead), pharyngeal arches (mandibular and ceratohyal, arrows), otic vesicle and in the pectoral fin bud (A, E). Expression in neurocranium and pharyngeal arches was lost in the sox9a mutant (B, F). In the sox9b mutant, expression disappeared from the pharyngeal cartilages, but expression in the neurocranium (arrowhead) remained (C, G). Expression almost disappeared in sox9a;sox9b double mutant embryos (D, H). At 2dpf, sox9a was expressed in the neurocranium (arrowhead) and pharyngeal arches (arrows) in locations corresponding to col11a2 expression (I), but sox9b was only expressed in mandibular and ceratohyal (arrows), not in the ethmoid plate and trabeculae (J). These results confirmed microarray results showing that col11a2 is down-regulated in sox9 mutant embryos, and revealed that sox9a and sox9b regulate col11a2 expression in a tissue specific manner according to sox9 expression patterns. ch, ceratohyal; ep, ethmoid plate; ma, mandibular; ov, otic vesicle; op, opercle; pf, pectoral fin bud.

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.

Candidate target genes calb2a and calb2b are co-orthologs of tetrapod Calb2 and their expression was lost in the sox9b mutant retina. (A) Phylogenetic analysis of Calbindin family proteins using the neighbor-joining method. Numbers on the branches are bootstrap values for the group in a thousand runs. Zebrafish Calb2a and Calb2b branched together as co-orthologs of tetrapod Calb2 with very high bootstrap support. Ascidian Calbindin-related protein was used as an outgroup. Cin: ascidian, Ciona intestinalis; Dre: zebrafish, Danio rerio; Hsa: human, Homo sapiens; Mmu: mouse, Mus musculus. NCBI accession numbers, Hsa_CALB1; NP_004920, Mmu_Calb1; NP_033918, Dre_Calb1; XP_00134160, Hsa_CALB2; NP_001731, Mmu_Calb2; NP_031612, Dre_Calb2a; NP_957005, Dre_Calb2b; NP_957012, and Ensembl peptide ID, Cin_Calb-r; ENSCINP00000011776. (B) A conserved synteny around Human CALB2 on Homo sapiens chromosome 16 (Hsa16) with zebrafish calb2a on Danio rerio chromosome 7 (Dre7) and calb2b on Dre18. Red dots represent zebrafish genes orthologous to human genes in the position 60–80 Mb of Hsa16. Dorsal view of calb2a (C–E) and calb2b (F–H) expression. Expression of calb2a appeared in the retina, cranial ganglia and hindbrain neurons (C). Expression in the retina was not observed in sox9b mutants (D) and double mutants (E), but other expression domains were not affected, demonstrating specificity (D, E). The expression of calb2b was observed in the olfactory placode, tegmentum and ganglion cell layer (F). Expression in the retina was not detected in sox9b mutants (G) and double mutants (H), but expression in the other tissues was not affected (G, H). These results showed that zebrafish calb2a and calb2b are co-orthologs of the vertebrate Calb2 gene, that they are differently expressed in retina, and that both genes depend on sox9b function. cg, cranial ganglia; gcl, ganglion cell layer; op, olfactory placode; r, retina; tg, tegmentum.

Candidate genes were expressed in different layers in the developing retina. (A–C), vsx1; (D–F), rs1; (H–I), calb2a; (J–L), calb2b. Expression of vsx1 was observed in the outer part of the inner nuclear layer (A). Expression was reduced especially in the dorsal part in sox9b mutants (B) and double mutants (C). In wild-type embryos, rs1 was expressed in the outer part of the inner nuclear layer and in the outer nuclear layer (D). In sox9b mutants and sox9a;sox9b double mutant embryos, expression was severely reduced or nearly gone except for the ventral ciliary marginal zone (E, F). Expression of calb2a was observed in the inner nuclear layer (G) and calb2b was expressed in the ganglion cell layer (J). In each case, expression was severely reduced in sox9b mutants (H, K) and sox9a;sox9b double mutant embryos (I, L). White bar in (A, D), inlo, outer part of inner nuclear layer; yellow bar in (D), onl, outer nuclear layer; green bar in (G), inl, inner nuclear layer; orange bar in (J), gcl, ganglion cell layer.

Expression of candidate genes in retina at 3 dpf. In situ hybridization on histological sections with calb2a (Aa, wild-type; Ab, sox9a mutant; Ac, sox9b mutant; Ad, double mutant), calb2b (Ba–Bd), crx (Ca–Cd), neurod (Da–Dd), rs1 (Ea–Ed), sox4a (Fa–Fd), vsx1 (Ga–Gd) and col2a1a (Ha–Hd). Expression of calb2a appeared in the ganglion cell layer and inner nuclear layer (Aa); calb2b was expressed in the ganglion cell layer and a cell population in the inner part of the inner nuclear layer (Ba); crx was expressed in the outer part of the inner nuclear layer and outer nuclear layer (Ca); neurod was expressed in the outer nuclear layer and a cell population in the inner nuclear layer (Da); rs1 was expressed in the outer part of the inner nuclear layer and the outer nuclear layer (Ea); sox4a was expressed in a cell population in the inner nuclear layer (Fa); and vsx1 was expressed in the outer part of the inner nuclear layer (Ga). The ciliary marginal zone did not express calb2a, calb2b, neurod, rs1 or sox4a (asterisk). In sox9b and double mutants, expression in dorsal margin and ventral margin was reduced for all seven genes. The expression of sox4a in the inner nuclear layer was stronger in sox9b and sox9a;sox9b double mutants than in wild-type and sox9a mutants (Fa–Fd). Expression of col2a1a appeared in iris (Ha), and was not affected in sox9 mutant embryos (Hb–Hd). No obvious defect was observed in the sox9a mutant retina. cmz, ciliary marginal zone; gcl, ganglion cell layer; inl, inner nuclear layer; inli, inner part of inner nuclear layer; inlo, outer part of inner nuclear layer; ir, iris; onl, outer nuclear layer.

Fluorescent antibody staining of retinal cell types in wild-type and sox9 single and double mutant larvae. (A–P) 5 dpf and (Q–T) 4 dpf. Eye morphology in sox9a mutants was generally similar to that in wild-type siblings. In contrast, sox9b mutants and sox9a;sox9b double mutants had smaller eyes and eye morphology was less organized than in wild-type siblings and sox9a mutants. (A, E, I, M) Retinal Müller glial cells labeled by anti-carbonic anhydrase showed that sox9b mutants (I) and sox9a;sox9b double mutants (M) had fewer Müller glial cells compared to wild-types (A) and sox9a (E) siblings. (B, F, J, N) Photoreceptor cells labeled by zpr-3 showed that fewer and thinner photoreceptor cells were present in sox9b mutants (J) and sox9a;sox9b double mutants (N) compared to wild-type (B) and sox9a (F) siblings. (C, G, K, O) Nuclear layers of the retina stained by nuclear dye (TO-PRO-3 iodide) showed that all the nuclear layers were present in sox9 mutants. (D, H, L, P) green: anti-carbonic anhydrase, red: zpr-3, and blue: nuclear staining. (Q–T) Retinal amacrine cells labeled by anti-GABA: green, photoreceptor cell labeled by zpr-3: red, and nuclear layers of the retina staining by nuclear dye (TO-PRO-3 iodide): blue. The results showed that sox9a mutants (R), sox9b mutants (S) and sox9a;sox9b double mutants (T) have fewer organized amacrine cells and photoreceptor cells compared to wild-type siblings (Q). Scale bar: 100 μm.

Expression of candidate genes in the pectoral fin bud. (A, B) Lateral view and (C, D) dorsal view of klf2b expression. In wild-type embryos, klf2b was expressed in the mesenchyme of the pectoral fin bud and in the adjacent cleithrum (A, C). Expression in the pectoral fin bud was not detected in double mutants (B, D). Expression of klf2b in the cleithrum was not affected in the mutant. Expression of EST AI722369 was observed in the presumptive muscle in the pectoral fin bud (E). AI722369 expression did not appear in sox9a;sox9b double mutant embryos (F). Lateral view of sox9a (G) and sox9b (H) expression. At this stage, sox9a was expressed in an L-shape in the pectoral fin bud (G), and sox9b expression appeared more broadly in the pectoral fin bud (H). cl, cleithrum; co, scapulocoracoid; ed, endochondral disc; pf, pectoral fin bud.