Identification and Characterization of miRNA Expression During Zebrafish Retinal Regeneration

Unlike most other vertebrates, zebrafish are able to regenerate many tissues including retina. Light-induced photoreceptor cell death causes inner nuclear layer Müller glia to dedifferentiate, reenter the cell cycle and produce proliferating progenitor cells. These progenitor cells continue to proliferate and migrate to the appropriate retinal layer and differentiate into functional photoreceptors. While a number of genes essential to retinal regeneration have been identified, the mechanisms regulating these genes have been little studied.

Many post-transcriptional regulatory mechanisms exist in the cell including miRNAs, short RNA sequences encoded in the genome. To determine if miRNAs play a role in retinal regeneration, I reduced global miRNA expression by Dicer knockdown, an enzyme essential to miRNA maturation. Dicer knockdown prior to light damage lead to decreased proliferation in regenerating retinas without affecting the number of Müller glia during retinal regeneration or cell death. These studies demonstrate an essential role for global miRNA expression during retinal regeneration. To identify which of the 346 miRNAs known in zebrafish were likely to play an important role in retinal regeneration, both a microarray and RNA deep sequencing experiment were conducted. These analyses identified 63 and 104 miRNAs respectively, that had a greater than 2-fold change in expression during retinal regeneration. Nine candidate miRNAs were knocked down prior to light damage, resulting in three distinct phenotypes, reducing proliferation 1) during progenitor production and proliferation 2) only progenitor production or 3) only during progenitor proliferation. The different effects on proliferation caused by these individual miRNA knockdowns suggest that individual miRNAs have distinct roles in retinal regeneration.

One miRNA, miR-7a, was further characterized through knockdown prior to light damage and analyzed for effects on proliferation. This knockdown reduced proliferation during progenitor production, but did not affect the number of proliferating progenitors at later time points. Nor did miR-7 knockdown alter the number of Müller glia or affect cell death. These data suggest that miR-7 plays a specific temporal role in the production of progenitor cells. This study demonstrates that global and individual miRNA expression play specific and essential roles in gene regulation during retinal regeneration.