Reprogramming Muller glia to regenerate ganglion-like cells in adult mouse retina with developmental transcription factors.

Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in non-mammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. In this report, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in MG, including Ascl1, Pou4f2, Islet1 and Atoh1. Using IHC, scRNA-seq, scATAC-seq, and electrophysiology we find retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental RGC-transcription factors. Overall design: Included in this submission are scRNA-seq and scATAC-seq data from overexpression experiments of Islet1/Pou4f2/Ascl1 (IPA) or Ascl1-only in murine Muller glia, both in vivo and in vitro. For in vivo datasets, overexpression of transgenes alongside GFP in Glast-Cre mice was combined with intravitreal injections of NMDA and TSA. After >3 weeks, retinas from 4-8 mice were pooled and dissociated and the GFP+ cells were FACS purified and then processed with the 10x genomics platform for scRNA-seq or scATAC-seq. Additionally, retinas dissociated from E14 mice were processed for scATAC-seq. For the in vitro experiments, transgenes were under control of rtTA. Muller glia cultured from 3-5 mice and treated with doxycycline to induce transgenes for 5 days, alongside untreated control glia, were processed with the 10x platform for scRNA-seq.