Fig. 4.

Hes/Hey binding sites are required to rerepress Atoh1 in supporting cells after Notch signaling is first inhibited, allowing Atoh1 levels to rise, and then restored. (A) At P1, the expression of both wild-type and mutant promoters is limited to hair cells, indicating that low levels of ATOH1 (activator insufficiency) are sufficient to maintain silencing from the mutant transgene. Scale bar: 20 µm. (B) Schematic shows experimental time course: P1 cochlear cultures from the double-transgenic mouse line were treated with DAPT for 18 h, after which DAPT was washed out from half of the explants, and the explants were then collected after an additional 6 h in culture. Expression analysis of endogenous Atoh1, GFP and tdTomato by qPCR shows that endogenous Atoh1 and the wild-type (WT) transgene are actively repressed by returning Notch signaling, but the mutant transgene fails to be rerepressed in the same time period (6 h). Values are mean±s.e.m.; n=3. *P<0.05.