The impact of timing and injury mode on induced neurogenesis in the adult mammalian retina

Regeneration of neurons has enormous implications for human health and is a topic of interest both from the biological and translational perspective. The retina poses an excellent system to study the potential of replacing neurons following injury and the impact different lesions can have on this process. We have established mouse models where proneural transcription factors are expressed in Müller glia to stimulate neurogenesis, which is analogous to how zebrafish regenerate the retina after injury. Our previous studies have shown that Müller glia overexpressing either Ascl1 or Ascl1-Atoh1 will respond to inner retinal damage, caused by a neurotoxic dose of NMDA, by acquiring a progenitor-like state and giving rise to bipolar cells or retinal ganglion-like cells, respectively. It was not known however whether neurogenesis would still occur if the retina suffered outer retinal injury and if the timing of injury relative to the expression of proneural factors was critical for the process. To address these questions, we explored whether timing or mode of injury affect the induced neurogenesis from MG. We show that reprogramming MG with Ascl1 is not impacted by the mode or timing of injury, since all paradigms resulted in similar ratios of new bipolar neurons. By contrast, MG that express Ascl1-Atoh1 respond to outer retinal damage by producing a new type of retinal ganglion-like cell that is not generated after NMDA injury. Therefore, although the fate of reprogrammed neurons is mostly dictated by the proneural transcription factors, there is a context-dependent effect of the injured retinal microenvironment. Overall design: The aim of this work was to study the impact of timing and injury mode on induced neurogenesis from Müller glia in the adult mammalian retina. We used transgenic mice in which the reporter GFP and transcription factor Ascl1 or combination of factors Ascl1-Atoh1 are expressed in Müller glia in a Cre-inducible manner. Following different injury modes (light-damage or NMDA injection in the vitreal cavity of the eye) GFP+ cells were harvested from retinas and processed for scRNA-seq using the Chromium Next GEM Single Cell 3' v3.1 (dual index) protocol.