Genomic organization of zebrafish cone-rod homeobox gene and exclusion as a candidate gene for retinal degeneration in niezerka and mikre oko

Purpose: To determine the genomic organization of the zebrafish crx gene and to evaluate if mutations in crx are responsible for the retinal degeneration phenotype in the zebrafish (Danio rerio) mutants niezerka (nie^m743) and mikre oko (mok^m632). Methods: Overlapping fragments were PCR amplified from genomic DNA isolated from homozygous mutant embryos and wild-type siblings (sibs). Amplicons were sequenced and sequence data assembled into contigs. Genomic organization was determined by alignment of contigs with published cDNA sequences and zebrafish genomic sequence from Sanger and Ensembl databases. Linkage analysis used DNA from mapping panels of single homozygous mutant animals with mixed genetic backgrounds. Results: The analysis indicated that the zebrafish crx gene consisted of three exons and 2 introns, and spans 3.8 kb of genomic DNA. The splice junctions were all located within the coding region. Highly repetitive sequences present in non-coding regions of crx and extended tetra-nucleotide repeats in intronic regions were associated with sequence variation between different strains. Homozygous mok^m632 or nie^m743 mutants and their respective wild-type sibs, showed identical patterns of heterozygosity and sequence variations within each line. No mutation in crx were identified in homozygous mok^m632 or nie^m743. Consistent with the absence of identified mutations, linkage analysis excluded linkage of the mutant phenotypes to crx. Conclusions: Despite the presence of sequence variations in their respective genetic backgrounds, within each line the sequence of crx was identical. Consistent with the absence of mutations, further analysis excluded linkage of the mutant phenotypes to crx. Analysis is in progress to map these loci and identify the genes responsible for the retinal degeneration phenotype in these mutant lines.