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Ralph Nelson, Sophia Rizvi, Paul Cohen, Victoria Connaughton, Sachihiro Suzuki, Hiba Codore, Sadie Bergeron, Rachel Wong, Harold Burgess; Transgene Insertion near Musashi1 Disrupts Zebrafish Short-Wavelength Cone Development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2494.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the retinal function of Musashi1 (Msi1), an RNA binding protein involved in neural development.
We screened a library of transgenic zebrafish Gal4 enhancer trap lines for expression of a UAS:Kaede reporter gene in adult retina. Examination of retinal slices indicated that line Et(SCP1:Gal4ff)y245 (y245) selectively marked photoreceptors. The transgene insertion site was mapped with linkage-mediated PCR. To determine the cone types labeled, y245 was crossed with transgenic lines marking red cones (trβ2:td-Tomato), blue cones (SWS2:GFP, Takechi et al, 2008) or UV cones (SWS1:GFP, Takechi et al, 2003). Antibodies against GFP or DSred confirmed the selected cone marking.
The transgene insertion mapped to chromosome 8, in the promoter region of msi1. Rod and double-cone containing patches were seen in heterozygous adult y245 retinas. In 5 dpf heterozygous and homozygous larvae, diffuse label of the entire photoreceptor layer was punctuated by ‘doughnut-like’ putative nuclear regions within elongate cone-like morphologies. Analysis of y245 ; UAS:Kaede ; SWS1:GFP. triple transgenics revealed normal UV-cone density in y245 larvae, but the UV cone layer was irregular, and often without clearly defined pedicle and nuclear stratification. GFP and Kaede were co-expressed in only 5% (4 of 79) of UV cones. In y245 ; UAS:Kaede ; SWS2:GFP triple transgenics, blue cone density was low with only 10 (N=10) blue cones per section compared to 56 (N=1) in SWS2:GFP controls. Cone positioning was spotty and irregular. Kaede was not localized in blue cones (3%, 3 of 104). The y245 ; UAS:Kaede ; trβ2:td-Tomato triple transgenic revealed a regular, dense mosaic of red cones. 39% (120 of 309) of kaede cells co-localized with these red cones. Other kaede cells were located within the dark spaces of the red cone mosaic.
Blue cone density is greatly reduced, and UV-cone layering is disrupted in y245 transgenic larvae, indicating that msi1 is critical to normal development of SWS1/2 cones.
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