PgmNr Z568: Tet-mediated DNA hydroxymethylation is required for retinal neurogenesis.

Authors:
Pawat Seritrakul 1,2 ; Jeffrey Gross 1


Institutes
1) University of Pittsburgh, PA; 2) University of Texas at Austin, TX.


Abstract:

Tet dioxygenases convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), epigenetic marks that are thought to regulate gene expression.  How 5mC and 5hmC facilitate gene expression that underlies cellular differentiation during vertebrate eye development has not been resolved. The zebrafish retina is composed of neuronal and glial cells derived from a common pool of seemingly indistinguishable retinal progenitor cells (RPCs), serving as an ideal structure in which to study the epigenetic regulation of cellular differentiation. Gene expression analyses reveal that tet2 and tet3 are expressed in the developing retina. To functionally test the roles of these enzymes, we generated loss-of-function mutations in these genes. Homozygous mutant embryos deficient in either tet2 or tet3 showed no visible phenotype. However, double mutant (tet2-/-; tet3-/-) embryos developed ocular defects; they are microphthalmic, lack an optic nerve, and possess reduced numbers of differentiated neurons. BrdU incorporation assays showed elevated numbers of proliferative RPCs, and in situ hybridization revealed expanded RPC marker gene expression. Immunohistological and gene expression analyses revealed that the neurogenesis phenotype is likely due to failure of retinal neurons to terminally differentiate. RNA-seq analyses of gene expression in dissected eye tissues revealed reduced expression of terminal differentiation markers and upregulation of genes not normally expressed in the eye, suggesting that RPCs may be mis-specified when tet protein function is disrupted.  To independently test this model, we generated inducible transgenic tet overexpression lines to artificially drive 5mC to 5hmC conversion. Embryos expressing the transgenes show increased global levels of 5hmC and defects in RPC differentiation. At the genome-wide level, virtually nothing is known about how the 5mC/5hmC landscape changes during the RPC to neuron transition. To profile these marks at single nucleotide resolution, we performed bisulfite sequencing, hydroxymethylation profiling, and RNA-seq using pure RPCs isolated from vsx2:GFP embryos, which express GFP in RPCs prior to differentiation. Initial analyses revealed reduced 5mC levels and enrichment of 5hmC at regulatory regions of known RPC genes, while genes expressed in other cell types maintain high 5mC levels. This correlates with RNAseq data, where RPC-specific genes are highly expressed. Ongoing work is aimed at profiling both 5hmC and 5mC at a genome-wide level during neurogenesis, utilizing a similar strategy to isolate pure populations of differentiated neuron types. Together, our work is the first evidence that tet proteins and 5hmC are required for normal retinal development.



ZFIN Genetics Index
1. tet2
2. tet3
3. vsx2