Investigating Early Regulators of Injury and Regeneration in the Zebrafish Retina

Zebrafish possess the ability to regenerate all retinal cell types after damage. Following injury, Müller glia dedifferentiate and divide asymmetrically, producing a Müller glia and a neuronal progenitor cell (NPC). These NPCs then continue to proliferate, and eventually migrate towards the site of damage, at which point they differentiate into the lost cell types, allowing for full recovery of retinal function.

Numerous pathways regulating zebrafish retinal regeneration have been identified, however despite promising studies within the mammalian retina, none have induced a response equal to that of the zebrafish. Therefore, I analyzed the zebrafish retina during regeneration at the single cell level. This led to identification of multiple novel transcription factors which regulate retinal regeneration, providing further targets for study within mammalian systems. Furthermore, I identified several targets for study that may regulate very early Müller glia responses to damage.

Multiple iron homeostasis genes are differentially regulated within Müller glia prior to retinal cell death suggesting that iron might be a key regulator of retinal cell death and the Müller glia response. I show that ferrous iron can induce significant retinal damage, that induces a particularly strong regenerative response. Additionally, I demonstrate that iron contributes to retinal cell death, with chelation resulting in significantly less photoreceptor death during light treatment, and decreased Müller glia proliferation. Despite this contribution of iron to retinal cell death, I demonstrate the ferroptosis is not a major contributor to retinal cell death in the light-treated retina. I also show that a key regulator of iron transport, Tfr1b, and a regulator of ferroptosis, Gpx4b, are required for a full proliferative response from Müller glia.

The alternative Ciliary neurotrophic factor receptor (Cntfr) ligands Cardiotrophin-like cytokine factor 1 (Clcf1) and Cytokine-like factor receptor 1a (Crlf1a) are significantly upregulated in Müller glia prior to cell death. I show that these ligands are neuroprotective, decreasing photoreceptor cell death during light treatment. I also show their requirement for Müller glia proliferation following injury, as knockdown of the receptor, or associated ligands results in significantly fewer proliferating Müller glia. Additionally, I show that CLCF1/CRLF1 can induce rod progenitor proliferation in an Igf-1r -dependent manner.