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Travis J. Bailey, Catherine M. Groden, Kristin M. Ackerman, David R. Hyde; Retinal Developmental Defects In The good effortnt2 Mutant Zebrafish Are Correlated With Elevated Cell Death. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3958.
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© ARVO (1962-2015); The Authors (2016-present)
In a screen to identify mutations resulting in eye defects, we found the good effortnt2 (gef) mutant. The retina of gef embryos is characterized by the successful initiation of the optic primordium and normal retinal development over the first two days post fertilization. The mutant retina, however, fails to continue to grow. This study further characterized the gef mutation to gain a molecular understanding of cause of the gef phenotype.
Meiotic mapping was used to localize the mutation interval. Embryos from gef heterozygous incrosses were analyzed for cell death by acridine orange and by TUNEL labeling at 2 days post fertilization. Genes found in the linkage interval were analyzed by quantitative real-time PCR and in situ hybridization. One-cell embryos were injected with morpholino oligonucleotides against the candidate gene with or without in vitro transcribed mRNA of the human orthologous gene to either mimic or rescue the gef mutant phenotype, respectively.
The number of dying cells in gef mutant embryos at 2 days post fertilization that labeled with TUNEL and acridine orange was significantly greater than wild type embryos. This time was earlier than any observable gross morphological differences, which suggested that this cell death led to the gross morphological defects. Meiotic linkage mapping found a one-megabase interval on chromosome 9 that contained eleven potential genes. Of these eleven genes, only itgbl1 transcripts were found at reduced levels in acridine orange labeled embryos at 2 days post fertilization. The expression of itgbl1 was detected in the brain and retina with weak or no expression in the lens. High itgbl1 expression was also found in the developing meninges and vasculature cells. The gef mutants also exhibited reduced circulation relative to wild-type siblings. Morpholino inhibition of Itgbl1 protein expression in wild-type embryos phenocopied the gef mutant and this phenotype was partially rescued by injection of in vitro transcribed human ITGBL1.
The failure of the gef mutant retina to increase in size was due to elevated retinal cell death, which resulted from decreased levels of itgbl1 and abnormal vasculature. This suggests that the requirement for Itgbl1 activity in proper zebrafish retina development might be due to effects on patterning of the periocular and brain vasculature. This will be further examined using a variety of zebrafish mutants and transgenic lines.
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