Characterization of Danio rerio retinal regeneration and the role of TNFalpha, Stat3, and Ascl1a during the initiation of Muller glia proliferatio

Zebrafish is an excellent model organism to study the molecular basis of retinal regeneration. Damage-induced apoptosis of retinal neurons in adult zebrafish stimulates Muller glia within the inner nuclear layer to dedifferentiate and divide giving rise to undifferentiated transiently amplifying multipotent neuronal progenitor daughter cells. These cells continue to proliferate and migrate to the damaged retinal layer where they differentiate into functional and integrated retinal neurons. The mechanisms in which Muller glia are induced to divide by the apoptotic neurons during the initiation of regeneration are unknown.;Following retinal damage, the expression of transcription factors Signal transducer and activator of transcription 3 (Stat3) and Achaete-scute complex-like 1(Ascl1a) are upregulated in Muller glia and are each required for maximal Muller glia proliferation following retinal damage. To examine the requirement of each of these factors for regeneration and their hierarchal relationship, morpholino-mediated knockdowns were performed. This analysis revealed a complex signaling cascade involving three separate populations of Muller glia that have differential requirements for the expression of Stat3, Ascl1a and proliferation (Nelson et al., in press).;To examine the mechanism in which Muller glia are signaled to divide, retinal lysates were collected at the peak of photoreceptor apoptosis, homogenized and injected into the intravitreal space of undamaged eyes. The light-damaged retinal homogenates stimulated Muller glia proliferation, which demonstrated that a Muller glia proliferation-stimulating factor was present during the peak of photoreceptor apoptosis.;To identify candidate protein signaling pathways that could be responsible for the initiation of Muller glia proliferation, I performed a MALDI-TOF mass spectrometry proteomic analysis comparing proteins of undamaged retinas to those isolated during the peak of light-induced photoreceptor apoptosis. Tumor necrosis factor alpha (TNFalpha) signaling proteins were increased in expression in the light-damaged retinas. TNFalpha expression was increased by apoptotic photoreceptors and later in Muller glia. Knockdown of TNFalpha significantly decreased the expression of Ascl1a and Stat3 and significantly reduced the number of Muller glia stimulated to reenter the cell cycle, but did not affect levels of photoreceptor death. This study identifies the TNFalpha signaling pathway as a novel participant that is induced by retinal damage which acts upstream of Ascl1a and Stat3 during Danio rerio photoreceptor regeneration.