EDNRB2 is a novel marker for hair cell precursors in chick auditory epithelia.

Although mammalian cochleae have no capacity for hair cell (HC) regeneration, the avian auditory epithelium, namely basilar papilla (BP), can regenerate HCs through direct conversion of supporting cells (SCs) or mitotic proliferation of SCs. To explore molecular mechanisms for HC regeneration in chick BP, we previously established an explant culture model of chick BP, in which HC regeneration occurs predominantly via direct conversion of SCs (Matsunaga et al., 2020) and performed single-cell RNA sequencing (Matsunaga et al., 2023) by using our model. A pseudotime trajectory analysis for the process of SC-to-HC conversion revealed that SCs were once reprogrammed to the precursor state, followed by differentiation into HCs (Matsunaga et al., 2023). In addition, we observed temporal upregulation of EDNRB2 encoded en-dothelin receptor b2 in reprogrammed SCs as differentially expressed genes during SC-to-HC conversion. In the current study, we examined the expression patterns of EDNRB2 in the developing chick BP to determine which stage progenitor or precursor populations express EDNRB2. The expression of EDNRB2 in BP was found only in the time points when HC differentiation was initiated, not in common progenitors for HCs and SCs or immature HCs and SCs after fate determination. The result supports our hypothesis that EDNRB2 is a marker for HC precursors and indicates that SCs are re-programmed to the precursor state just before fate determination, not to the common progenitor state. We also assessed the roles of EDNRB signaling during SC-to-HC conversion in regenerating chick BP explants using a selective inhibitor for EDNRB signaling. The pharmacological inhibition of EDNRB signaling significantly reduced the number of regenerated HCs, not reduced the number of SCs, which indicates that EDNRB signaling may play a role in the differentiation of precursors into HCs. Further, RNA sequencing is underway to identify the critical molecules downstream of EDNRB signaling. In conclusion, EDNRB2 is a novel marker for HC precursors in chick auditory epithelia and may be involved in HC differentiation. [ABSTRACT FROM AUTHOR]