Conditional Deletion of Atoh1 Reveals Distinct Critical Periods for Survival and Function of Hair Cells in the Organ of Corti

Abstract

Atonal homolog1 (Atoh1) encodes a basic helix-loop-helix protein that is the first transcription factor to be expressed in differentiating hair cells. Previous work suggests that expression of Atoh1 in prosensory precursors is necessary for the differentiation and survival of hair cells, but it is not clear whether Atoh1 is required exclusively for these processes, or whether it regulates other functions later during hair cell maturation. We used EGFP-tagged Atoh1 knock-in mice to demonstrate for the first time that Atoh1 protein is expressed in hair cell precursors several days before the appearance of differentiated markers, but not in the broad pattern expected of a proneural gene. We conditionally deleted Atoh1 at different points in hair cell development and observe a rapid onset of hair cell defects, suggesting that the Atoh1 protein is unstable in differentiating hair cells and is necessary through an extended phase of their differentiation. Conditional deletion of Atoh1 reveals multiple functions in hair cell survival, maturation of stereociliary bundles, and auditory function. We show the presence of distinct critical periods for Atoh1 in each of these functions, suggesting that Atoh1 may be directly regulating many aspects of hair cell function. Finally, we show that the supporting cell death that accompanies loss of Atoh1 in hair cells is likely caused by the abortive trans-differentiation of supporting cells into hair cells. Together our data suggest that Atoh1 regulates multiple aspects of hair cell development and function.

β-Catenin Is Required for Hair-Cell Differentiation in the Cochlea

“…Furthermore, Lgr5 acts as a receptor for R-spondins. At postnatal stages, addition of Rspo1 to dissociated Lgr5-expressing cochlear cells induces expression of hair cell markers (Chai et al, 2012;Shi et al, 2012), and we show that addition of Rspo1 to embryonic cochleae leads to an increase in hair cells. This implies that the Lgr5-positive cells respond to stimulation with the Wnt potentiator, Rspo1, during development.…”

“…Using similar reporters based on multimerized TCF sites upstream of other promoters, TOP-gal and TCF/LEF:H2B-GFP, we and others have found that Wnt activity parallels the course of hair-cell development (DasGupta and Fuchs, 1999;Jacques et al, 2012;Shi et al, 2013). Lgr5-GFP, a downstream Wnt target gene which successfully identified Wnt-responsive stem cells in intestine (Barker et al, 2007), reveals a similar pattern of Wnt activity in the developing cochlea (Chai et al, 2012;Shi et al, 2012).…”

“…␤-Catenin is required for hair-cell specification but not for maintaining hair-cell fate Atoh1 is a key transcription factor in hair cell development, with roles in the initial differentiation as well as the maturation and stability of hair cells (Bermingham et al, 1999;Cai et al, 2013;Chonko et al, 2013). Overexpressing Atoh1 in developing and young organ of Corti induces generation of hair cells (Zheng and Gao, 2000;Gubbels et al, 2008).…”

“…The initial signal that specifies hair cells from these common progenitors is unknown; however, Wnt signaling was suggested to play a role, because Atoh1 is a direct target of the Wnt pathway . Lgr5, a member of the Wnt-potentiating positive-feedback regulatory loop, was found to mark inner ear stem cells, and Wnt signals had dual roles in the control of their proliferation and differentiation (Chai et al, 2012;Shi et al, 2012). In cochlear explants treated with Wnt inhibitors and activators (Jacques et al, 2012), Wnt was shown to play dual roles in both proliferation and hair-cell differentiation in the developing organ of Corti.…”

Organ of Corti and Stria Vascularis: Is there an Interdependence for Survival?

“…Atoh1, a transcription factor belonging to the family of basic helix-loop-helix (bHLH)-containing proteins, plays a critical role for hair cell differentiation and survival during development and adulthood [15,17,18]. The Atoh1 -CKO mouse self-terminates expression of Atoh1 in hair cells around birth using an Atoh1 enhancer to drive cre to conditionally delete the floxed Atoh1 .…”