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Jeffrey M Gross, Pawat Seritrakul; Tet2 and Tet3 methylcytosine dioxygenases are required for retinal neurogenesis during zebrafish eye development. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
DNA methylation is an epigenetic mechanism that chemically modifies cytosine bases into 5-methylcytosines (5mC), and is thought to be critical for the regulation of gene expression. Tet dioxygenases convert 5mC into 5-hydroxymethylcytosine (5hmC). Little is known about the role of Tet proteins or 5hmC during the development of complex organs like the eye. The zebrafish retina is composed of neuronal and glial cells derived from a common pool of retinal progenitor cells (RPCs). Given its unique architecture and development, the zebrafish retina serves as an ideal structure in which to study the epigenetic regulation of cellular differentiation. Here, we determine the requirements and functions of Tet proteins during zebrafish retinal development.
Gene expression analyses reveal that both tet2 and tet3 are expressed in the developing retina. To determine the functions of these enzymes, we generated loss-of-function mutations using Transcription Activator-Like Effector Nucleases (TALENs), which resulted in loss of function alleles of both genes.
Homozygous mutant embryos deficient in either tet2 or tet3 are viable, fertile, and showed no visible phenotype. However, double mutant (tet2-/-; tet3-/-) embryos developed ocular defects; they were microphthalmic and possessed reduced numbers of terminally differentiated retinal neurons. BrdU incorporation assays showed elevated number of proliferative RPCs, and in situ hybridization revealed an expanded RPC population. Key transcription factors involved in the specification of retinal neuron subtypes were expressed relatively normally. Transcriptome analyses revealed numerous markers of retinal neuron differentiation and ectopic expression of genes not normally involved in ocular development.
Our results indicate that retinal neurons in tet2-/-; tet3-/- mutants are specified but not terminally differentiated. This supports a model in which tet2 and tet3 function redundantly to facilitate retinal development likely by regulating the expression of genes required for terminal differentiation and maturation. Parallel studies are underway to elucidate the epigenetic requirements for retinal neurogenesis at the whole genome level and how enzymes like tet2 and tet3, as well as the DNA methylation machinery, facilitate this process.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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