In response to light-induced photoreceptor apoptosis, the zebrafish retina undergoes a robust regenerative response that results in recovery of lost or damaged rod and cone photoreceptors. This response initiates with a cell reprogramming event, in which of a subset of Müller glia divide asymmetrically to produce a transiently amplifying neuronal progenitor cell (NPC) population. NPCs continue to proliferate and migrate from the inner nuclear layer to the outer nuclear layer, where they differentiate into new rod and cone photoreceptors. Müller glia reprogramming is an essential, rate limiting step in the regeneration program, but the molecular mechanisms that control this process are just starting to be elucidated. Induction of several pluripotency factors necessary for reprogramming of iPS cells, including lin28, sox2, klf4, myc, oct4 and nanog (Ramachandran et al., 2010), has been observed during the early stages of retina regeneration, but of these, only Lin28 function has been studied. Sox2 is a well established neuronal stem cell-associated transcription factor that regulates neural development and adult neurogenesis in vertebrates. We found that Sox2 expression is maintained in the Müller glia and a subset of amacrine cells in the undamaged adult zebrafish retina, recapitulating that of other well-studied vertebrates. Expression of sox2 transcript increased significantly in retinas following 31 hours of light treatment, when Müller glia begin proliferating. This correlated with increased Sox2 protein expression in proliferating Müller glia. Knockdown of Sox2 expression decreased the number of proliferating Müller glia, demonstrating that Sox2 is required for Müller glia reprogramming and proliferation. In contrast, induced overexpression of Sox2 stimulated proliferation of Müller glia in the absence of retinal damage. We investigated the role of Wnt/β-catenin signaling, which is required for Müller glia reprogramming and is a known regulator of sox2 expression during vertebrate retinal development. Contrary to previous reports, we observed differential expression and non-redundant roles of the two zebrafish β-catenin paralogs. While β-catenin 2, but not β-catenin 1, was required for Müller glia proliferation, neither β-catenin paralog was required for sox2 expression following retinal damage. We also determined that Sox2 regulates expression of ascl1a and lin28a, but not stat3 expression following retinal damage. Additionally, sox2 expression was not dependent upon Ascl1a, suggesting Sox2 lies exclusively upstream of ascl1a. Finally, we observed differential expression of Sox2 during later stages of regeneration. Specifically, all early NPCs express Sox2 at 51 hours, but later stage progenitors express Sox2 heterogenously. This study is the first to identify a functional role for Sox2 during Müller glial-based regeneration of the vertebrate retina.
|Author||Ryne A Gorsuch|
|Advisor||David R. Hyde|
|Contributor||David R. Hyde, Committee Chair|
|Degree Level||Doctoral Dissertation|
|Degree Discipline||Biological Sciences|
|Departments and Units|