59.3 Results

Fig. 59.1 The structure of purpurin gene and photoreceptor-specific expression of reporter gene. (a) Structures of 1.4-kbp 5 -flanking region and 2.3-kbp transcriptional region of the purpurin gene are shown in minimized length. The transcription start site is positioned at +1. The thin vertical bars in the 5 -promoter region show crx recognition motifs (three OTX and four OTX-like) and the TATA box. The black squares in the transcriptional region show the exons. The purpurin gene has 6 exons and 5 intorns. The translation start codon is within exon 2 and the stop codon is within exon 6. A polyadenylation signal AATAAA is positioned at +2314 of exon 6. (b) Construction of the pur-GFP reporter vector. (c) GFP expression in pur-GFP transgenic zebrafish at 5 dpf. (d) In situ hybridization study of purpurin mRNA at 5 dpf. Scale = 50 μm. Le: lens

recognition and binding motif in zebrafish and mammals (Kawamura et al. 2005). Crx is a photoreceptor-specific transcriptional factor, and the expression and the function of crx in the zebrafish retina are well studied by Raymond group (Liu et al. 2001; Shen and Raymond 2004). During zebrafish retinal development, crx mRNA first appears in ventral part of the retina at 17–24 hpf. At 52 hpf, the expression is expanded to the photoreceptor layer and the outermost inner nuclear layer. Knock down of crx gene using morpholino resulted in small size of eyeball and lacking of retinal lamination (Fig. 59.2b) compared to the control retina (Fig. 59.2a). During zebrafish retinal development, purpurin mRNA first appears in ventral part of the retina at 40 hpf and the expression is spread to the photoreceptor layer at 48–72 hpf (Tanaka et al. 2007). Knock down of purpurin gene using morpholino also resulted in small size of eyeball and lacking of retinal lamination (Fig. 59.2c). Similar phenotypes of purpurin and crx morphants are shown in Table 59.1.

Fig. 59.2 Toluidine blue staining of control (a), crx morphant (b), purpurin morphant (c) and crx morphant co-injected with purpurin mRNA (d) retina at 3 dpf. (a) Retinal lamination is completely formed in the control retina at 3 dpf. (b, c) Crx or purpurin morphants do not show any retinal lamination at 3 dpf. (d) Injection of purpurin mRNA into the crx morphant rescues the small size of eyeball and lacking of retinal lamination. An arrow indicates the inner plexiform layer in (d). Scale bar = 50 μm

Table 59.1 Similar phenotypes of purpurin and crx morphant at 3 dpf

aRefer to Shen and Raymond (2004); N/A: not analyzed.

59.4 Rescuing Effect of Purpurin mRNA to the Crx Morphant

The most notable effect of purpurin or crx knock down is small eye and lacking of retinal lamination. Therefore, we tested effect of co-injection of purpruin mRNA and crx morpholino. In the control eye at 3 dpf, the size of eyeball was more than 230 μm and retinal lamination was complete (Table 59.1, Fig. 59.2a). In the crx morphant, about 80% of embryos showed small eyeball (<200 μm, Table 59.1) and lacking of retinal lamination (Fig. 59.2b). Co-injection of 0.5 mM crx morpholino and 44 ng/μl purpurin mRNA notably rescued these abnormal phenotypes (Fig. 59.2d).

59.5 Discussion

In this study, we demonstrated the possibility that crx is a transcriptional regulator for purpurin gene during early development of zebrafish retina; (1) Purpurin promoter region contained crx binding motifs. (2) The appearance time of crx mRNA was a little bit earlier than that of purpurin, and initial appearance site of both mRNAs was just identical. (3) Crx knock down embryos generated the small size of eyeball and lacking of retinal lamination as seen as in the purpurin morphant.

(4) Injection of purpurin mRNA into crx morphant rescued these abnormalities. And our preliminary study showed that knock down of crx markedly reduced purpurin expression at 3 dpf, but not vise versa. Although it is still unclear that crx directly binds to the purpurin promoter region, crx might be a transcriptional factor for purpurin gene.

Crx is a member of otx/otd gene family of paired-like homeobox gene (Royet and Finkelstein 1995; Bally-cuif and Boncinelli 1997). In developing mouse retina, crx expression begins by day E 12.5 and peaks at day P 3 (Chen et al. 1997; Furukawa et al. 1997). The day E12.5 is approximately the time of cone cell genesis and the day P3 is near the time of maximal genesis of rod cells (Carter and LaVail 1979). In mice homozygous for targeted null mutation of crx, photoreceptors fail to form outer segment and eventually become degenerative (Furukawa et al. 1999). Mutations in human crx gene have been identified in three kinds of photoreceptor degeneration diseases (cone-rod dystrophy, retinitis pigmentosa and Leber congenital amaurosis) (Freund et al. 1997; Sohocki et al. 1998; Silva et al. 2000). Therefore, crx is essential for differentiation and maintenance of photoreceptors in mammals. In contrast, zebrafish crx regulates retinal neurogenesis, not only photoreceptor cells but also inner retinal cells. There is many photoreceptor specific genes regulated by crx, such as rhodopsin, arrestin, phosdusin and cGMP phosphodiesterase (Livesey et al. 2000; Zhu and Craft 2000; Pittler et al. 2004). However they are all involved in photo-signal transduction. The secreted purpurin protein might act as a retinol binding protein or an adhesive molecule for cell differentiation. Although mammalian purpurin is not yet identified, we propose that fish purpurin offers a solving cue for photoreceptor degeneration and the determination of cell fate in the retinal development.

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