Abstract
Purpose: :
To test whether Müller glia in the adult zebrafish function as injury induced stem cells and are able to produce new neurons in response to retinal injury.
Methods: :
Retinas were injured by inserting a 30 gauge needle through the sclera of the zebrafish eye to a depth of ∼5mm. Two methods were used to lineage–trace injury–responsive Müller glia: 1) Transgenic zebrafish harboring a 1.0 kb {alpha}1–tubulin promoter fragment that drives GFP expression ({alpha}1T–GFP) exclusively in injury–responsive Müller glia; and 2) BrdU–labeling to identify Müller glia undergoing karyokinesis. Low–density cell culture of dissociated retinas was used to evaluate if injury–responsive Müller glia undergo karyokinesis without cytokinesis. Immunohistochemistry and in situ hybridization was used to assay the fate of injury–responsive Müller glia at various times post injury.
Results: :
In response to injury, {alpha}1T–GFP expression is induced exclusively in a subpopulation of Müller glia that re–enter the cell cycle. Consistent with previous studies, Müller glia represent the majority of dividing cells during the first week post injury, and our data indicate that almost all dividing cells are also GFP+. Groups of proliferating cells known as ‘neurogenic clusters’ express retinal stem cell markers following injury and are known to be a source of new neurons. Following retinal injury, GFP+ Müller cells also induce expression of retinal stem cell markers such as pax6 and vsx–1 and our data demonstrates that clusters of pax6+ cells correspond to GFP+ Müller glia, suggesting that Müller glia are the source of neurogenic clusters. Clusters of proliferating nuclei appear to be within a single Müller glia, and multiple nuclei can be identified in GFP+ Müller glia in low–density culture, suggesting that these cells undergo karyokinesis without cytokinesis. Cells labeled with BrdU at 4dpi become rod and cone photoreceptors as well as amacrine, bipolar, ganglion, horizontal, and Müller cells, indicating that Müller glia are a source of retina regeneration.
Conclusions: :
Our data suggests that Müller glia are injury induced retinal stem cells that give rise to new neurons following retinal injury.
Keywords: regeneration • retina • Muller cells