Efficient stimulation of retinal regeneration from Muller glia in adult mice using combinations of proneural bHLH transcription factors.

Regenerative neuroscience aims to stimulate endogenous repair mechanisms in the nervous system to replace neurons that have been lost from degenerative diseases or trauma. In the retina, many non-mammalian vertebrate species spontaneously regenerate retinal neurons from glial cells, through an injury-induced reprogramming of the cells to a retinal progenitor state. Recently, we have reported that engineering mouse Muller glia to express a retinal progenitor gene, the proneural transcription factor, Ascl1, is sufficient to stimulate neurogenesis in the glia to generate functional neurons. However, this process is inefficient and only a third of the Ascl1-expressing glia generate new neurons. Here we test whether proneural transcription factors of the Atoh1/7 class can further promote the neurogenic capacity of MG. We report that the combination of Ascl1:Atoh1 is remarkably efficient at stimulating neurogenesis (>80% expressing cells), even in the absence of retinal injury or TSA. We use electrophysiology and scRNA-seq to demonstrate that Ascl1:Atoh1 generates a diversity of retinal neuron types with the majority expressing characteristics of retinal ganglion cells. Overall, our results provide a proof-of-principle that transcription factors that are normally sequentially active in cell fate specification, can when combined, substantially improve glial reprogramming to neurons and expand the repertoire of cell fates that can be regenerated in the adult mouse retina. The ability to stimulate the neurogenic potential of glia in adult mice with high efficiency is a key step towards translation of this approach to repair the central nervous system. Overall design: This submission includes 4 scRNA-seq samples. The Control cultured glia sample contains cultured mouse glia treated infected with a lentivirus containing RFP. The Atoh7 5day in vitro sample contains cultured mouse glia infected with pLOC-hATOH7-IRES-copGFP virus. The Ascl1 5day in vitro sample contains cultured glia infected with pLOC-hAscl1-IRES-turboGFP virus. Lastly there is a sample of retinal cells from Glast-CreER:LNL-tTA:tetO-mAscl1-tetO-Atoh1-ires-GFP mice. All samples were FACS sorted to identify affected cells.