Ascl1a/Dkk/β-catenin signaling pathway is necessary and glycogen synthase kinase-3β inhibition is sufficient for zebrafish retina regeneration

Abstract

Key to successful retina regeneration in zebrafish are Müller glia (MG) that respond to retinal injury by dedifferentiating into a cycling population of retinal progenitors. Although recent studies have identified several genes involved in retina regeneration, the signaling mechanisms underlying injury-dependent MG proliferation have remained elusive. Here we report that canonical Wnt signaling controls the proliferation of MG-derived retinal progenitors. We found that injury-dependent induction of Ascl1a suppressed expression of the Wnt signaling inhibitor, Dkk, and induced expression of the Wnt ligand, Wnt4a. Genetic and pharmacological inhibition of Wnt signaling suppressed injury-dependent proliferation of MG-derived progenitors. Remarkably, in the uninjured retina, glycogen synthase kinase-3β (GSK-3β) inhibition was sufficient to stimulate MG dedifferentiation and the formation of multipotent retinal progenitors that were capable of differentiating into all major retinal cell types. Importantly, Ascl1a expression was found to contribute to the multipotential character of these progenitors. Our data suggest that Wnt signaling and GSK-3β inhibition, in particular, are crucial for successful retina regeneration.pyrvinium | XAV939 | transgenic zebrafish | heat shock | frizzled

Analysis of DNA methylation reveals a partial reprogramming of the Müller glia genome during retina regeneration

“…S1A). MGPC formation is accompanied by the increased expression of pluripotency factors (14) and other key signaling molecules (15)(16)(17)(18)(19)(20)(21). The induction of pluripotency genes, along with the finding that the putative cytidine deaminases Apobec2a and Apobec2b (Apobec2a,2b) are necessary for MGPC formation (22), is consistent with the idea that active modification of the DNA methylation landscape may underlie the reprogramming of MG to MGPCs.…”

“…S1 B and C) (16). We have shown previously that mechanical lesion with a 30-gauge needle results in the activation of MG only at the site of injury and that 1016 tuba1a:gfp transgenic fish specifically label MGPCs at 4 dpi (16). Quantitative real-time PCR showed increased expression of genes associated with DNA demethylation, such as gadd45β, gadd45βl, gadd45γ, gadd45γl, apobec2a, apobec2b, tdg, and tet3 in MGPCs (Fig.…”

“…To assess whether these genes were specifically induced in MGPCs, we used FACS to isolate MG from uninjured gfap:gfp transgenic fish retinas and MGPCs from injured 1016 tuba1a:gfp transgenic fish retinas at 4 dpi ( Fig. S1 B and C) (16). We have shown previously that mechanical lesion with a 30-gauge needle results in the activation of MG only at the site of injury and that 1016 tuba1a:gfp transgenic fish specifically label MGPCs at 4 dpi (16).…”

The zebrafish eye—a paradigm for investigating human ocular genetics

“…Blocking β-catenin accumulation by injection of XAV939 or pyrvinium at the time of retinal injury resulted in reduced proliferation of Müller glial-derived progenitors indicating that β-catenin is required for retinal progenitor cell proliferation. 100 Importantly, β-catenin signalling can be enhanced by injection of the GSK-3β-inhibitor, lithium chloride in injured and uninjured zebrafish retinas, stimulating Müller glial dedifferentiation and proliferation into multiple retinal neuronal subtypes. 100 Reprogramming of endogenous Müller glial cells to a stem cell function, or transplantation of stem cells or progenitor cells into diseased retinas may provide therapeutic potential for degenerative retinal disorders.…”

“…100 Importantly, β-catenin signalling can be enhanced by injection of the GSK-3β-inhibitor, lithium chloride in injured and uninjured zebrafish retinas, stimulating Müller glial dedifferentiation and proliferation into multiple retinal neuronal subtypes. 100 Reprogramming of endogenous Müller glial cells to a stem cell function, or transplantation of stem cells or progenitor cells into diseased retinas may provide therapeutic potential for degenerative retinal disorders. Inspiration for Müller-cell therapies comes from findings that suggest Müller glia function as multipotent retinal stem cells, which can generate retinal neurons in response to loss of photoreceptors in the differentiated zebrafish retina.…”