Fig. 3

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.