Characterization of Light-Induced Zebrafish Retinal Regeneration and the Role of Stat3, CNTF, and Olig2 During Light-Induced Zebrafish Retinal Regeneration

The zebrafish retina is an excellent model to study the molecular basis of neural cell regeneration. Dark-treated albino zebrafish exposed to intense light undergo rod and cone photoreceptor apoptosis, which triggers MÌÄå_ller glia proliferation and the generation of neuronal progenitor cells. Next, multiple neuronal progenitor cells will either migrate to the outer nuclear layer and regenerate lost rods and cones or will remain in the inner nuclear layer to repopulate lost Müller glia. To identify candidate genes encode critical proteins required during photoreceptor regeneration, I performed a time course microarray of six time points representing different cellular events during the regeneration response. qRT-PCR verified the expression profiles for selected genes in the microarray data set. To examine genes involved in Müller glial cell proliferation and progenitor cell differentiation, I performed temporal and functional gene cluster analysis. I selected three genes for further analysis: i) Stat3, which functions through the Jak/Stat pathway, ii) CNTF, a growth factor that is known to activate the Jak/Stat pathway, and iii) Olig2, a transcription factor that regulates neuron and glial cell fates. Immunoblots verified increased total and activated Stat3 protein expression during the time course. Immunolocalization demonstrated increased Stat3 expression in Müller glial cells by 31 hours, and some Stat3-positive Müller glial cells expressed PCNA. To determine protein function during regeneration, I electroporated morpholinos into the adult retina to knockdown expression of target proteins. Stat3 knockdown significantly reduced the number of PCNA-labeled Müller glial cells during the light treatment, which demonstrated a role for Stat3 during light-induced retinal regeneration. Intravitreal CNTF injections suppressed light-induced photoreceptor apoptosis. Injection of CNTF into the undamaged zebrafish eye induced Stat3 expression in Müller cells, and Müller glia proliferation. However, knockdown of Stat3 in the CNTF-injected undamaged eye still induced Müller glia proliferation, which demonstrates CNTF's mode of action is independent of Stat3. These data suggest Stat3 and CNTF possess independent roles in the damaged and undamaged zebrafish retina. Olig2 was expressed in dedifferentiated Müller glia and INL neuronal progenitor cells between 51 and 96 hrs of light treatment. Electroporation of anti-olig2 morpholinos knocked down Olig2 during zebrafish retinal regeneration. However, knockdown of Olig2 did not effect either cell proliferation, migration, or photoreceptor cell differentiation.