Delta-like 1 and lateral inhibition during hair cell formation in the chicken inner ear: evidence against cis-inhibition.

The formation of the salt-and-pepper mosaic of hair cells and supporting cells in the sensory epithelia of the inner ear is regulated by Notch signalling and lateral inhibition, but the dynamics of this process and precise mode of action of delta-like 1 (Dll1) in this context are unclear. Here, we transfected the chicken inner ear with a fluorescent reporter that includes elements of the mammalian Hes5 promoter to monitor Notch activity in the developing sensory patches. The Hes5 reporter was active in proliferating cells and supporting cells, and Dll1 expression was highest in prospective hair cells with low levels of Notch activity, which occasionally contacted more differentiated hair cells. To investigate Dll1 functions we used constructs in which Dll1 expression was either constitutive, regulated by the Hes5 promoter, or induced by doxycycline. In support of the standard lateral inhibition model, both continuous and Hes5-regulated expression of Dll1 promoted hair cell differentiation cell-autonomously (in cis) and inhibited hair cell formation in trans. However, some hair cells formed despite contacting Dll1-overexpressing cells, suggesting that some progenitor cells are insensitive to lateral inhibition. This is not due to the cis-inhibition of Notch activity by Dll1 itself, as induction of Dll1 did not cell-autonomously reduce the activity of the Hes5 reporter in progenitor and supporting cells. Altogether, our results show that Dll1 functions primarily in trans to regulate hair cell production but also that additional mechanisms operate downstream of lateral inhibition to eliminate patterning errors in the sensory epithelia of the inner ear.