The zebrafish is an excellent model organism to use in the study of molecular pathways important in both retinal development and neuronal regeneration. The zebrafish shares similarities in eye development and morphology with all vertebrate eyes. However, unlike the mammalian retina, the zebrafish completely regenerates retinal neurons following injury . The source of regeneration are M?ller glial cells that dedifferentiate into a more primordial stem cell-like state, divide, and yield transiently amplifying progenitor cells that migrate to the site of damage and replenish the lost neuronal cell types. Dedifferentiating M?ller glia in the adult zebrafish retina experience reprogramming in DNA methylation and expression of transcription factors that are involved during normal retinal development (Pollak et al., 2013; Powell et al., 2013). Therefore, studying the cellular and molecular pathways important during retinal development may provide insight into the roles these genes play during regeneration in the adult retina. In an effort to identify pathways involved in early retinal development in zebrafish, a gene microarray analysis was performed, which demonstrated a significant up regulation in components of the JAK/Stat3 pathway, including Socs3a and Socs1. Immunoblots, immunohistochemistry, and in situ hybridization data confirmed that Stat3, Socs3a, and Socs1 were up regulated in the zebrafish eye between 3-5 days post-fertilization, time points associated with significant maturation of vision. Stat3, Socs3a, and Socs1 are initially expressed ubiquitously throughout the developing retina before showing a distinct localization within M?ller glial cells. Morpholino-mediated knockdown of Stat3, Socs3a, or Socs1 demonstrated that a loss of JAK/Stat3 function caused abnormal retinal cell death during development. A loss of either Socs1 or Socs3a resulted in decreased rhodopsin expression near the retinal margins of the developing zebrafish, while and a loss of Stat3 affected cone photoreceptor cell and M?ller glial cell maintenance. These results indicated that JAK/Stat3 signaling is important to the development of photoreceptor and M?ller glial cells as well as the maintenance of a healthy retina.
The adult zebrafish retina regenerates following light-induced photoreceptor cell death. M?ller glial cells in light-damaged retinas divide yielding undifferentiated progenitors that migrate and differentiate to replace the lost rod and cone photoreceptors. We demonstrated previously that, following light-induced photoreceptor cell death, a loss of either TNFα or Stat3 reduced M?ller glial cell proliferation significantly, suggesting that the number of M?ller glia that reenter the cell cycle following retinal damage was dependent upon coordinated expression of TNFα and Stat3 (Nelson et al., 2013). Although it seems likely that TNFα initiates Stat3 signaling through a cytokine-receptor mediated pathway, a receptor linking the two proteins together has yet to be identified. Also, previous studies demonstrated that tyrosine phosphorylation of Stat3 and downstream activation of Stat3-activated genes, including the two zebrafish paralogues of Socs3 (socs3a and socs3b), was associated with neuronal regeneration (Elsaeidi et al., 2014; Liang et al., 2012). However, the kinase responsible for tyrosine phosphorylation of Stat3 and the role of Socs3a and Socs3b in regulating M?ller glial proliferation remains elusive. This report demonstrates that TNFα, Stat3, and Socs3a associate with TNFRSFa (tumor necrosis factor receptor superfamily member a) in the regenerating retina and that a loss of TNFRSFa decreases Stat3-mediated expression of Socs3a and TNFα, which causes a significant reduction in M?ller glia proliferation similar to what is observed when decreasing either TNFα or Stat3 expression alone. This work also demonstrates that JAK1 mediates M?ller glial proliferation through Y708 phosphorylation of Stat3 and that socs3a and socs3b are expressed in M?ller glia in the adult light-damaged retina. A knockdown of either socs3 gene alone has no effect on Stat3 expression or M?ller glial proliferation. However, a knockdown of socs3a and socs3b combined results in a significant increase in Stat3 expression as well as M?ller glial proliferation in response to light damage. Taken together, these studies demonstrate that TNFα likely induces activation of Stat3 through a TNFRSFa / JAK1 signaling cascade and that Socs3a and Socs3b function to dampen the proliferative response of M?ller glia caused by Stat3.