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Pawat Seritrakul, Jeffrey M Gross; Tet-mediated DNA hydroxymethylation is required for retinal neurogenesis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5567.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal neurons are derived from a common pool of progenitor cells (RPCs), serving as an ideal structure in which to study the epigenetic regulation of cellular differentiation. Tet enzymes convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) epigenetic marks that regulate transcription. How 5mC and 5hmC facilitate gene expression that underlies retinal cell differentiation has not been resolved. This study tested the hypothesis that Tet activity is required for neurogenesis during retinal development.
We generated zebrafish mutants for tet2 and tet3 and inducible transgenic tet overexpression lines, and analyzed retinal development using immunohistological and gene expression assays. To determine whether tet2 and tet3 activity was required cell autonomously or non-autonomously, we generated chimeric embryos whose retinae were composed of clones of wild-type and tet2;tet3 mutant cells.
Homozygous tet2 or tet3 mutant embryos develop normally. However, double mutant (tet2;tet3) embryos show ocular defects; they are microphthalmic, and possess severely reduced numbers of differentiated retinal cells such as amacrine, photoreceptor and Muller glial cells, and while possessing differentiated RGCs, they nonetheless lack an optic nerve. Gene expression analyses reveal that while the normal cascade of proliferation is slightly prolonged, retinal cells are properly specified and exit the cell cycle but fail to terminally differentiate. RNA-seq analyses of dissected eye tissues identified reduced expression of markers of terminally differentiated retinal cell types and upregulation of non-ocular genes, suggesting that subsets of RPCs may be mis-specified when tet protein function is disrupted. Similarly, transgenic embryos with elevated Tet2 activity showed defects in photoreceptor terminal differentiation. Mosaic analyses demonstrate that tet activity regulates cell non-autonomous pathways required for normal retinal neuron differentiation.
This work is the first to determine the role of tet enzymes during retinal development and shows that tet-mediated 5hmC conversion is required for retinal neurogenesis. Ongoing work is aimed at deciphering the mechanistic details underlying the neuronal differentiation defects observed in tet2;tet3 mutants and determining how tet activity regulates extrinsic pathways required for normal retinal development.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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