Injury-Dependent Müller Glia and Ganglion Cell Reprogramming during Tissue Regeneration Requires Apobec2a and Apobec2b

Abstract

Unlike mammals, adult zebrafish are able to regenerate multiple tissues including those of the CNS. In the zebrafish retina, injury stimulates Müller glia dedifferentiation into a multipotent retinal progenitor that is capable of regenerating all lost cell types. This dedifferentiation is driven by the reactivation of gene expression programs that share many characteristics with those that operate during early development. Although the mechanisms underlying the reactivation of these programs remain unknown, it is likely that changes in DNA methylation play a significant role. To begin investigating whether DNA demethylation may contribute to retina regeneration, we characterized the expression of genes associated with DNA demethylation in the uninjured and injured retina. We found that two cytidine deaminases (apobec2a and apobec2b) were expressed basally in the uninjured retina and that they were induced in proliferating, dedifferentiated Müller glia. The maximal induction of apobec2b required Ascl1a, but was independent of Lin28, and therefore defines an independent signaling pathway stemming from Ascl1a. Strikingly, when Apobec2a or Apobec2b was knocked down by antisense morpholino oligonucleotides, the proliferative response of Müller glia following injury was significantly reduced and injury-dependent induction of ascl1a and its target genes were inhibited, suggesting the presence of a regulatory feedback loop between Apobec proteins and ascl1a. Finally, Ascl1a, Apobec2a and Apobec2b were found to be essential for optic nerve regeneration. These data identify an essential role for Apobec proteins during retina and optic nerve regeneration and suggest DNA demethylation may underlie the reprogramming of cells to mount a regenerative response.

Analysis of DNA methylation reveals a partial reprogramming of the Müller glia genome during retina regeneration

“…MGPC formation is accompanied by the increased expression of pluripotency factors (14) and other key signaling molecules (15)(16)(17)(18)(19)(20)(21). The induction of pluripotency genes, along with the finding that the putative cytidine deaminases Apobec2a and Apobec2b (Apobec2a,2b) are necessary for MGPC formation (22), is consistent with the idea that active modification of the DNA methylation landscape may underlie the reprogramming of MG to MGPCs.…”

“…The predominance of DNA demethylation as MG transition to an MGPC is intriguing, and we suspected that Apobec2 proteins were involved in this process based on previous studies showing that their knockdown inhibits retina regeneration (22) and that they contribute to DNA demethylation in zebrafish embryos and adult mouse brains (28,32). Surprisingly, our studies suggest that they do not have a significant impact on site-specific active DNA demethylation during MG reprogramming.…”

“…This transition is accompanied by the increased expression of pluripotency and other regeneration-associated genes (14)(15)(16)(17)(19)(20)(21)(22). Previously studied somatic cell reprogramming events found strong correlations between the increased expression of pluripotency factors and decreases in their promoter methylation levels (7,27,28).…”

“…Interestingly, certain aspects of this basal methylation program are shared with mammals, suggesting they too may acquire progenitor properties under appropriate circumstances. the injured retina (22). To assess whether these genes were specifically induced in MGPCs, we used FACS to isolate MG from uninjured gfap:gfp transgenic fish retinas and MGPCs from injured 1016 tuba1a:gfp transgenic fish retinas at 4 dpi ( Fig.…”

“…Apobec2-Independent DNA Demethylation Predominates as MG Transition to MGPCs. We previously showed that Apobec2a,2b are required for MGPC formation and hypothesized that they participate in active DNA demethylation of the MG genome following injury (22). We sought to use RRBS to directly test whether they regulate DNA demethylation.…”