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B.A. Link, G.B. Willer, J.M. Fadool, J.E. Dowling, R.G. Gregg; Positional Cloning of the Zebrafish Young Mutation Identifies an Essential Role for the Brahma Chromatin Remodeling Complex in Coordinating Retinal Differentiation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2041.
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Purpose: To study genes and molecular pathways that coordinate retinal differentiation we sought to identify the gene responsible for the young phenotype. Zebrafish with the young mutation show normal cell cycle withdrawal and cell-type specification within the retina, but retinal cells do not undergo differentiation and lack morphogenesis. To further our analysis, we have investigated candidate signaling pathways in wild type and mutant retinas. Methods: Positional cloning coupled with BAC rescue techniques were used to identify the gene altered in young mutants. Morpholino antisense knock-down and cDNA transgene rescue was then used to confirm that the altered gene caused the young phenoptype. To test whether known signaling pathways function during retinogenesis in young eyes, in situ hybridization, immunohistochemistry, and small molecule inhibitors were employed. Results: We have identified a single base substitution that introduces a stop codon early within the coding region of the zebrafish homologue for brahma related gene 1 (brg1). This locus normally encodes a transcriptional regulator that can mediate chromatin remodeling and activation of specific genes. During retinogenesis brg1 is first expressed throughout the optic cup and subsequently becomes restricted to the inner retina and marginal zones. Candidate differentiation pathways including components of the hedgehog pathway and ath5 are expressed normally in young retinas; however, the wave of MAP kinase activity normally observed prior to retinal differentiation does not manifest in the mutant retinas. To test the significance of MAP kinase activation, we injected inhibitors of this pathway into wild type eyes at the time when MAP kinase is normally activated. Such manipulations blocked retinal differentiation. Conclusions: Our analyses show that the young mutation disrupts the zebrafish homologue of brg1. Expression of this gene is essential to initiate a wave of MAP kinase activity across the retina, and this wave is required for normal retinal differentiation.
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