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

ABSTRACT: 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.

SUBMITTER: Otteson DC

PROVIDER: S-EPMC2879142 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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

Otteson Deborah C DC Tsujikawa Motokazu M Gunatilaka Tushara T Malicki Jarema J Zack Donald J DJ

Molecular vision 20051117

<h4>Purpose</h4>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)).<h4>Methods</h4>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 determ ...[more]

PMID: 16319818

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Project description:Purpose. XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model to investigate the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild type and XOPS-mCFP retinas in order to identify genes that may contribute to the regeneration of the rods. Methods. Wild type and XOPS-mCFP retinal mRNA was subjected to microarray analysis using the Agilent platform. The Ingenuity Pathway Analysis program was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization, immunohistochemistry, and using a transgenic fluorescent reporter line. Results. Over 600 genes displayed significant expression changes in XOPS-mCFP retinas compared to wild type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, cell development and cell death. RT-PCR analysis of a subset of these genes confirmed the microarray results. Three transcription factors (sox11b, insm1a, and c-myb) displaying increased expression in XOPS-mCFP retinas were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone. Conclusions. This study identified numerous gene expression changes in response to rod degeneration in zebrafish, and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration. Two-condition experiment: wild-type vs. XOPS-mCFP retinas. Four biological replicates for each condition. RNA was prepared from one retina for each sample. Each hybridization was accompanied by a dye-swap control, for a total of eight array hybridizations.

Dataset Information

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

Publications

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

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