PROJECT SUMMARY Cochlear Hair Cell (HC) loss is a leading cause of noise-induced and age-related hearing loss worldwide. Regeneration of HCs in response to damage has been observed in nonmammalian species, such as birds and fish, but is not observed in the mature mammalian cochlea. Although Supporting Cells (SCs), a diverse population of cells offering metabolic and structural support to hair cells, show the capacity to produce hair cells in neonatal mice, this plasticity is lost by postnatal day 5. Previous work from our lab has identified the de-differentiation of SCs into a more progenitor-like state as a key component of the HC regenerative process, as hair cells and supporting cells arise from a common progenitor pool during cochlear development. Supporting cell de-differentiation, the downregulation in expression of supporting cell-specific genes and upregulation in expression of, allows the progenitor-like cells to respond to HC fate-inducing cues. Preliminary studies have identified members of the NFI and ZBTB families of transcription factors as potential regulators of SC identity, maintaining SCs in a terminally differentiated state and preventing endogenous reprogramming. The NFI factors have previously been studies in the context of retinal regeneration, where they have been shown to regulate retinal Müller glial cell differentiation. Additionally, disruption of NFI factor function has been shown to promote conversion of Müller glia into retinal neurons. Zbtb20 has been implicated in astrocytogenesis, and has been linked to Primrose syndrome, a rare developmental disorder which is known to cause hearing loss. The overarching hypothesis of this grant proposal is that Nfia/b/c/x and Zbtb20 are crucial in maintaining SC identity, and that loss of one or more of these factors will enable SC reprogramming and HC regeneration in stage P5 mouse tissue. This hypothesis will be tested using in vitro cochlear organoid culture derived from stage P2 and P5 murine cochlear sensory epithelia. Additionally, the function of Zbtb20 will be studied in vivo and in a HC damage model. I will use RT-qPCR, fluorescence imaging of a HC reporter line, immunolabeling, EdU pulse experiments, and scRNA-sequencing in Aim 1 to determine if gain of function of NFIA/B/C/X or ZBTB20 inhibits cochlear SC reprogramming and SC-based HC formation at stage P2 in vitro and in Aim 2 to determine if loss of function of NFIA/B/C/X or ZBTB20 enhances cochlear SC plasticity and SC-based HC formation at stage P5 in vitro. In Aim 3, I will use immunolabeling, RT-qPCR, EdU pulse experiments, and a HC damage model to define the role of Zbtb20 in maintaining SCs in a terminally differentiated state. These studies will expand our knowledge of SC identity maintenance and the HC regenerative process and will aid future translational research using regenerative therapies to treat hearing loss in humans.