A hypoplastic retinal lamination in the purpurin knock down embryo in zebrafish

Recently, we cloned a photoreceptor-specific purpurin cDNA from axotomized goldfish retina. In the present study, we investigate the structure of zebrafish purpurin genomic DNA and its function during retinal development. First, we cloned a 3.7-kbp genomic DNA fragment including 1.4-kbp 5'-flanking region and 2.3-kbp full-length coding region. In the 1.4-kbp 5'-upstream region, there were some cone-rod homeobox (crx) protein binding motifs. The vector of the 1.4-kbp 5'-flanking region combined with the reporter GFP gene showed specific expression of this gene only in the photoreceptors. Although the first appearance time of purpurin mRNA expression was a little bit later (40 hpf) than that of crx (17-24 hpf), the appearance site was identical to the ventral part of the retina. Next, we made purpurin or crx knock down embryos with morpholino antisense oligonucleotides. The both morphants (purpurin and crx) showed similar abnormal phenotypes in the eye development; small size of eyeball and lacking of retinal lamination. Furthermore, co-injection of crx morpholino and purpurin mRNA significantly rescued these abnormalities. These data strongly indicate that purpurin is a key molecule for the cell differentiation during early retinal development in zebrafish under transcriptional crx regulation.