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Travis Bailey, David Hyde; Retinal degenerative defects in the good effort mutant zebrafish are due to aberrant splicing of the chaf1b RNA. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5149.
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© ARVO (1962-2015); The Authors (2016-present)
We discovered the good effortnt2 (gef) mutant in a genetic screen to identify mutations resulting in eye defects. The gef embryo successfully develops an optic cup and retinoblast layer, as well as early differentiating retinal ganglion cells. However, the retinoblast layer quickly degenerates. This study characterized the gef mutation to gain a molecular understanding of cause of the gef mutant phenotype.
Meiotic mapping was used to localize the mutation interval. Expression of candidate proteins were knocked down by morpholinos and assayed for cell death by acridine orange and TUNEL labeling at 2 days post fertilization (dpf) and immunostained for the expression of retinal neuron-specific proteins. One-cell embryos were injected with either morpholinos against candidate genes to phenocopy the gef mutant phenotype or coinjected with in vitro transcribed mRNA of the orthologous human gene to rescue the gef mutant phenotype. The chaf1b cDNA and genomic DNA were cloned from gef mutants, sequenced, and compared to wild-type sequences. Candidate genes downstream of Chaf1b activity were analyzed by qRT-PCR.
The chaf1b mRNA transcripts lacked an early exon. Genomic DNA sequencing revealed that the gef mutant had a 3 bp deletion that disrupted a splice donor site and resulted in a premature stop codon in the altered transcript. The cell death in gef mutant embryos at 2 dpf was phenocopied by morpholinos against the chaf1b transcript. Transcripts of tp53 and its target gene bax were upregulated in gef mutant embryos, but the tp53 target gene p21(waf1) was downregulated. At 3 dpf, gef mutant embryos injected with tp53 morpholino displayed reduced cell death compared with gef mutant embryos injected with control morpholinos.
The failure of the gef mutant retina to increase in size was due to elevated retinal cell death, which resulted from decreased levels of Chaf1b and disruption of the cell cycle that increased Tp53 and Bax activity. The good effort mutant likely represents a null allele of the chaf1b gene.
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