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Rebecca Chowdhury, Jillian Goetz, Mary Horton, Johanna Kowalko, Lauren Laboissonniere, Jeffrey Trimarchi; Using zebrafish to examine vertebrate retinal development. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1469.
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© ARVO (1962-2015); The Authors (2016-present)
During retinal development, multipotent progenitor cells produce a variety of cell types. Our goal is to understand the intrinsic gene expression patterns that control retinal cell fate decisions and neuronal maturation processes. Ath5 is a highly conserved transcription factor that is essential for retinal ganglion cell (RGC) development in the developing mouse and zebrafish retina. Using single cell transcriptome profiling, we previously characterized individual mouse Math5+ cells and found several genes with similar expression patterns as Math5. We hypothesize that these candidate genes (Plk3, Trim9, Ebf3 and Rassf4) will play important roles in retinal cell fate determination and the maturation of early retinal neurons. We are studying the roles of these genes using genome editing techniques.
To generate mutations in our candidate genes, CRISPRs (Clustered regularly interspaced short palindromic repeats) and TALENS (Transcription activator-like effector nucleases) were designed specific to these genes, injected into single-cell zebrafish embryos and genotyped to identify the induced mutations. Immunohistochemistry and in situ hybridizations (ISH) were used to detect any changes to different populations of retinal neurons that result from the loss of these genes in the zebrafish retina.
From our Math5+ single cell transcriptome data, we selected Trim9, Rassf4, Ebf3 and Plk3 as candidate genes involved in retinal development. We observed the expression of these genes in single Ath5:GFP zebrafish cells at various stages of development, indicating that they may play a regulatory role in retinal neuronal development. This finding was supported by the expression of these genes in the zebrafish retina by section and whole-mount ISH. We successfully mutated these genes in zebrafish using either the TALEN or CRISPR-Cas9 system and are now examining the phenotypic changes associated with these mutations. To characterize these phenotypic changes, we are currently using probes and antibodies that stain the early generated cell types of the retina such as RGCs and amacrine cells.
We have identified a cohort of genes with a similar expression pattern as Math5 during mouse retinal development. We further characterized the expression of these in zebrafish development and engineered loss of function mutations. Using these mutants we are studying the specific role(s) of these genes in retinal cell fate acquisition.
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