Project summary Our proposed study aims to address the function of LIN28B and follistatin in supporting cell reprogramming and hair cell regeneration in the murine cochlea. Loss of auditory hair cells (HCs) due to disease or trauma is permanent and is a leading cause for hearing impairments and deafness in humans. Immature auditory supporting cells (SCs) do regenerate HCs in response to damage but their ability to regenerate lost HCs rapidly declines as SCs undergo maturation and little to no HC regeneration is observed in adult animals. We recently uncovered that the RNA binding protein LIN28B and its paralog LIN28A control the regenerative capacity of cochlear SCs in neonatal cochlear organoids and explants. Whether LIN28A/B has a similar role in cochlear HC regeneration in vivo has yet to be tested. Furthermore, our recent in vitro studies suggest that LIN28B promotes HC regeneration through reprogramming SCs into progenitor-like cells and that such state transition can be further enhanced by the co-activation of the Activin antagonist follistatin. However, whether SCs truly activate a transitional progenitor-like state during HC regeneration and if so, how LIN28B and FST may influence such state transition are still unresolved. In our proposed study we will use mouse genetic tools to address whether LIN28A/B regulates spontaneous cochlear HC regeneration in the immature cochlea in vivo (aim1). Furthermore, we will use single cell RNA sequencing and single molecule FISH to determine whether LIN28B reprograms cochlear SCs into progenitor-like cells during HC regeneration (aim2). Moreover, to establish how LIN28B and FST enhance SC reprogramming and subsequent HC regeneration we will manipulate the function of potential LIN28B and FST effector genes using lentiviral overexpression and CRISPR-Cas9-mediated knockout strategies in cochlear organoids (aim3). Finally, we will determine whether co-expression of LIN28B and FST with Atoh1 enables SCs to regenerate cochlear HCs in the mature cochlea in vivo (aim4).