Teleost fish have a remarkable capacity to regenerate their central nervous system (CNS) following damage. Although successful regeneration has been observed for decades, little is known of the molecular events that govern it. Alpha1-tubulin (alpha1T) is a neuron-specific microtubule protein whose expression is induced in the developing and regenerating CNS. By investigating how expression of an early marker of regeneration such as alpha1T is regulated, our lab hoped to identify genes involved in regeneration. Transgenic zebrafish harboring an alpha1T promoter fragment driving GFP reporter expression were created to study alpha1T gene regulation during successful CNS regeneration. This promoter mediates gene induction in retinal ganglion cells during optic nerve regeneration and in a subset of Muller glia that proliferate following retinal injury.;To further characterize transgene expressing Muller glia, we generated transgenic fish harboring an alpha1T promoter fragment that is specifically induced in these cells following retinal damage. Transgene expression, BrdU-labeling and stem cell marker expression suggested that alpha1T-expressing Muller glia dedifferentiate and become multipotent progenitors in response to retinal injury. In addition, GFP and BrdU-mediated lineage tracing combined with retinal gene expression analysis indicated that alpha1T-expressing Muller glia are capable of generating retinal neurons and glia. These data strongly suggest alpha1T-expressing Muller glia dedifferentiate and mediate regeneration of the injured zebrafish retina.;Because alpha1T expression is induced in proliferating Muller glia following retinal injury, we searched for DNA elements that mediate alpha1T transgene expression in these cells. We report the identification of an E-box in the alpha1T promoter that is necessary for its activity in proliferating Muller glia. In a search for E-box binding proteins that may mediate alpha1T induction during retina regeneration, we found that the basic helix-loop-helix transcription factor ascl1a is induced in Muller glia within four hours following injury and regulates alpha1T promoter activity via the E-box in vitro. Knockdown of ascl1a expression in the regenerating retina confirmed that ascl1a regulates alpha1T transgene expression and is necessary for the generation of retinal progenitors and their differentiating progeny in vivo. These data suggest ascl1a is a key regulator of retina regeneration in zebrafish.
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