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Krista Marie Angileri, Jeffrey Gross; Dnmt1 is required for stem cell maintenance in the Ciliary Marginal Zone of the zebrafish retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1472.
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© ARVO (1962-2015); The Authors (2016-present)
Our lab previously reported that mutation in zebrafish DNA methyltransferase 1 (dnmt1), results in a failure to maintain progenitor cells of the lens epithelium (Tittle et al. 2011). dnmt1 copies DNA methylation from parent to daughter strand during DNA replication. dnmt1-/- retinae develop normally through 3 days post fertilization (dpf), but by 4dpf, the dnmt1-/- retinal stem cell (RSC) population, located in the ciliary marginal zone (CMZ), appears to be reduced. This study determines if dnmt1 activity is required to maintain RSCs in the CMZ.
Proliferation was quantified at 3, 4, and 5dpf via Bromodeoxyuridine (BrdU) incorporation assays and phosphohistone-3 Ser10 (pH3) immunohistochemistry. Apoptosis was quantified via activated caspase3 immunohistochemistry. Cell counts were normalized to total retinal and/or CMZ cell number for statistical analyses. Gene expression domains within the CMZ were quantified at 3 and 4dpf using col15a1b, cyclinD1, atoh7, and cdkn1c as markers for unique CMZ domains.
Compared to sibling controls, the dnmt1-/- CMZ at 4dpf is reduced in size and cell number by 32%, and it is absent by 5dpf (p<1e-8). This result is unique to the CMZ as the overall proportions of retinal cell types are unaffected. The dnmt1-/- CMZ displays an 85% reduction in the proportion of BrdU+ cells at 5dpf (p<0.05), however the percent of pH3+ cells is not significantly changed from controls (p>0.1). Active caspase3 expression suggests that there is no change in apoptotic cell numbers within the dnmt1-/- CMZ between 3dpf and 5dpf. Preliminary analysis of gene expression domains within the CMZ at 4dpf show a reduction in total area of gene expression (col15a1b, cyclinD1, atoh7, and cdkn1c; p<0.0001) within the dnmt1-/- CMZ, and domains are shifted toward the distal retina.
These results indicate that dnmt1 function is required to maintain the global DNA methylation pattern within RSCs of the CMZ and is essential for proper gene regulation. Ongoing experiments will identify gene regulatory networks required for RSC maintenance by comparing DNA methylation with gene expression in defined populations RSCs in the CMZ of wild-type and dnmt1-/- retinae.
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