We analyzed the role of Stat3, Ascl1a, and Lin28a in Müller glia reentry into the cell cycle following damage to the zebrafish retina. Immunohistochemical analysis was employed to determine the temporal and spatial expression of Stat3 and Ascl1a proteins following rod and cone photoreceptor cell apoptosis. Stat3 expression was observed in all Müller glia, whereas Ascl1a expression was restricted to only the mitotic Müller glia. Knockdown of Stat3 protein expression did not affect photoreceptor apoptosis, but significantly reduced, without abolishing, the number of proliferating Ascl1a-positive Müller glia. Knockdown of Ascl1a protein also did not change the extent of photoreceptor apoptosis, but did yield significantly fewer Müller glia that reentered the cell cycle relative to the stat3 morphant and significantly decreased the number and intensity of Stat3-expressing Müller glia. Finally, introduction of lin28a morpholinos resulted in decreased Müller glia expression of Stat3 and Ascl1a, significantly reducing the number of proliferating Müller glia. Thus, there are three populations of Müller glia in the light-damaged zebrafish retina: 1) Stat3-expressing Ascl1a-nonexpressing nonproliferating (quiescent) Müller glia; 2) Stat3-dependent Ascl1a-dependent proliferating Müller glia; and 3) Stat3-independent Ascl1a-dependent proliferating Müller glia. Whereas Ascl1a and Lin28a are required for Müller glia proliferation, Stat3 is necessary for the maximal number of Müller glia to proliferate during regeneration of the damaged zebrafish retina.
This work describes three different populations of Müller glia in the light-damaged retina. One population is Stat3-positive and Ascl1a-negative and does not proliferate. A second population (primary proliferating Müller glia [PPMg]) is stimulated by dying neurons to increase Lin28, which in turn activates Ascl1a, allowing these cells to proliferate. Ascl1a also activates Stat3 expression in the PPMg, which stimulates the same signaling pathway in nearby secondary proliferating Müller glia (the third population of Müller glia) and induces their proliferation.