Reprogramming adult murine Müller glia via L-Myc expression

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PROJECT SUMMARY In response to damage from injury or disease, lost neurons in the adult mammalian retina are irreplaceable which can lead to visual impairment including blindness. Some non-mammalian vertebrates, such as teleost fish, can regenerate the retina in response to damage via the Müller glia. In teleost fish, Müller glia will respond to signals from damaged neurons by asymmetrically dividing to produce retinal progenitor cells. These progenitor cells then differentiate into neuronal cell types and functionally restore tissue. Mammalian Müller glia are quiescent and do not display regenerative capacity in response to damage. However, mammalian Müller glia demonstrate some cell state plasticity and can be reprogrammed into induced pluripotent stem cells (iPSC) in vitro. Mammalian Müller glia likely have the capacity for reprogramming but lack the cell intrinsic- or extrinsic-signaling necessary to do so. I have found that overexpression of L-Myc, a robust reprogramming factor used to reprogram many somatic cell types to iPSCs, leads to cell cycle re-entry in adult mammalian Müller glia in ex vivo retinal tissue. Using murine retinal explant tissue as an injury model, I will characterize the effects of L-Myc overexpression on proliferation and dedifferentiation in mammalian Müller glia. I hypothesize that L-Myc overexpression in Müller glia leads to direct and indirect changes in gene expression and chromatin accessibility that reprogram Müller glia toward a progenitor-like state. To test this hypothesis, I will first characterize the effects of L-Myc expression on proliferation. I will do so by defining the interval of proliferation as it relates to L-Myc expression and determining if Müller glia or their descendants are able to divide repeatedly following L-Myc overexpression. Next, I will characterize the effect of L-Myc overexpression in Müller glia on cell identity and gene regulation. Using data integration of RNA-sequencing, ATAC-sequencing, and CUT&Tag of FACS sorted L-Myc overexpressing Müller glia, I will identify gene regulatory networks affected directly or indirectly by L-Myc overexpression and identify changes in cell state. Combining Müller glia, a highly promising population within the mammalian retina for reprogramming, and L-Myc, a potent reprogramming factor to promote proliferation and dedifferentiation, creates an ideal scenario to reveal targets to promote regeneration in the mammalian retina through the analysis of gene regulatory networks activated or inhibited by L-Myc overexpression. Regardless of the cell state changes that occur in response to L-Myc overexpression in Müller glia, this project will reveal what signaling barriers exist that prevent tissue regeneration via Müller glia in the mammalian retina. This study is designed to identify future targets for genetic therapies to promote restoration of tissue and visual function after injury in the mammalian retina, which one day could lead to treatments for currently untreatable visual diseases.