rapidly decreased by 10 days. Application of recombinant purpurin protein promoted neurite outgrowth from adult goldfish retinal explant culture (Matsukawa et al. 2004). During zebrafish retinal development, purpurin mRNA appeared in the ventral part of the retina at 40 hours post fertilization (hpf). The expression was located in the photoreceptor cells at 3–5 days after fertilization (dpf) and decreased by 10 dpf (Tanaka et al. 2007). Purpurin protein was secreted into all retinal layers. In the present study, we aimed to investigate the structure of purpurin genomic DNA and its function during the early stage of zebrafish retinal development. At first, we cloned 3.7-kbp genomic DNA fragment which included 1.4-kbp 5 -flanking region and 2.3-kbp of full-length of coding region of purpurin from zebrafish genomic DNA library. Purpurin gene had 6 exons and 5 introns. Secondly, we characterized 1.4-kbp 5 -flanking region. Transgenic zebrafish embryos injected with a constructed DNA of 1.4-kbp promoter region and GFP reporter gene showed photoreceptor specific expression of GFP. This region contained some cone-rod homeobox (crx) protein binding motifs, which found in promoter region of several photoreceptor-specific genes. Finally, we made purpurin and crx knock down zebrafish embryos by injection of morpholino antisense oligonucleotides. Phenotypes of these morphant (purpurin and crx) were very similar, small size of eyeball and lacking of differentiated retinal lamination. The role of purpurin during the early development of zebrafish retina and its transcriptional regulation by crx were thus discussed.

59.2 Materials and Methods

59.2.1 Experimental Animals

Zebrafish (Danio rerio) were reared in a water tanks at 28˚C with a 12:12 h lightdark cycle. Fertilized embryos were collected after natural spawning, and were kept in embryo medium containing 0.003% phenylthiourea.

59.2.2 Screening of Genomic DNA for Zebrafish Purpurin

An amplified zebrafish brain genomic DNA library in the Lambda FixII/XhoI partial Fill-in vector (Stratagene Inc.) was screened with radiolabeled cDNA derived from zebrafish purpurin cDNA. Escherichia coli cells (XL-1 Blue MRN) were infected with recombinant phages, and 1.0 × 106 phage plaques were screened. After hybridization with 32P-labeled cDNA probes, duplicate filters were washed four times and exposed to X-ray films. Positive plaques were then selected three times. The nucleotide sequences of the obtained genomic DNA clones on both strands were determined using a dye terminator kit and Ampli TagDNA polymerase (Applied Biosystems) on a DNA sequencer (ABIPRISM 310 Genetic Analyzer; Applied Biosystems).

59.2.3 Construction of the pur-GFP Reporter Vector

The purpurin promoter-GFP (pur-GFP) vector was composed of 1.4-kbp 5 -flanking region of zebrafish purpurin genomic DNA and pAcGFP 1-1 vector (Clontech). A 1428-bp 5 -flanking region of zebrafish purpurin genomic DNA was generated by PCR (forward primer: 5 -TGG CAA TAA AGC TCG ACG TA-3 ; reverse primer: 5 -GTG GTT CTC CAC AAG GCT GT-3 ). The 1428-bp fragment was cloned into pGEM-T Easy vector (Promega), and subsequently excised by ApaI/SacI digestion, and ligated into the corresponding sites of the pAcGFP 1-1 vector. The linearized pur-GFP vector was injected into one-cell stage of zebrafish embryos.

59.2.4 Morpholino and Microinjections

Morpholinos were obtained from Gene Tools. Purpurin morpholino was composed of MO1: 5 -TCA CAA AGC ATA CAA CAT ACC CTC T-3 . Crx gene morpholino was composed of 5 -ATG TAG GAC ATC ATT CTT GGG ACG G-3 (Shen and Raymond 2004). A standard morpholino (5 -CCT CTT ACC TCA GTT ACA ATT TAT A-3 ) was used as control. The morpholinos were diluted in distilled water at 0.5 mM in 0.1% phenol red, and were microinjected into 1–2 cell stage of embryos.

59.2.5 In Situ Hybridization

Whole mount in situ hybridization was performed as previously described (Sugitani et al. 2006) with a slight modification. In brief, embryos fixed and stored in 100% methanol at –20 ˚C were rehydrated, acetylated, and permeabilized with 10 μg/ml proteinase K at room temperature for 20–30 min. After refixation in 4% paraformaldehyde solution, embryos were prehybridized in hybridization buffer for 30 min. Hybridization was performed with 100 ng of probe in 200 μl hybridization solution overnight at 70˚C. On the next day, the embryos were washed and treated with 20 mg/ml RNase at 37˚C for 30 min. To detect the signals, the samples were incubated with alkaline phosphatase-conjugated anti-DIG antibody (Roche) overnight at 4˚C. Visualization of the signals was done using tetrazolium-bromo-4 chloro-3-indolyl-phosphate (Roche).

59.2.6 RNA Isolation, RT-PCR and mRNA Synthesis

Total RNA was isolated from 30 embryos at 3 dpf using Sepasol-RNA I super (Nacalai tesque). RT-PCR was performed using Reverse Transcriptase XL (AMV) (TaKaRa) and TaKaRa LA Taq (TaKaRa). Primer sequences (exon 1 forward primer: 5 -CAT TCA TTC AGG ACA TCA TC-3 ; exon 5 reverse primer: