The formation of inner ear sensory epithelia is believed to occur in two steps, specification of sensory competent (prosensory) regions followed by determination of specific cell-types, such as hair cells (HCs) and supporting cells. Initially, Sox2 is required to specify prosensory competence, but subsequent down-regulation of Sox2 must occur to allow Atoh1 expression, most likely through a direct interaction with the Atoh1 promoter. These results implicate Sox2-mediated changes in prosensory cells as an essential step in their ability to develop as HCs. mutants, demonstrating a key role for Sox2 in prosensory formation. In contrast with Sox2, Atoh1 expression does not begin until prosensory cells have become post-mitotic and is thought only to directly regulate the formation of cells as hair cells (Woods et al., 2004; Zheng and Gao, 2000). Forced expression of Atoh1 is sufficient to induce hair cell formation both within the prosensory domain name and in adjacent non-sensory cells (Kelly et al., 2012; Woods et al., 2004; Zheng and Gao, 2000) and although ectopic hair cells will induce neighboring cells to develop as supporting cells, Atoh1 expression is not directly required for supporting cell formation (Woods et al., 2004). A key component of the prosensory hypothesis is the idea that only specific regions of the otocyst, the prosensory regions, become competent to develop as hair cells and supporting cells. Therefore, within the context of this hypothesis, factors that specify the prosensory region should do so by fundamentally changing the developmental competence KIAA1235 of HKI-272 supplier cells within those regions. However, the observation that ectopic expression of Atoh1 can lead to hair cell formation in regions of the inner ear that do not normally contain hair cells and are therefore not thought to contain prosensory cells raises the possibility that uniquely competent prosensory regions do not exist. Rather, these results suggest that expression of Atoh1 may be sufficient to induce hair cell formation and that the role of Sox2 HKI-272 supplier may, therefore be limited to induction or enhancement of the expression of Atoh1 in specific regions of the HKI-272 supplier developing inner ear. Since, as mentioned, developing hair cells have the ability to recruit surrounding cells to develop as supporting cells, regulation of the spatial expression pattern of Atoh1 could be sufficient to regulate patterning of sensory epithelia within the inner ear. To examine these hypotheses, the experiments described here addressed the ability of Atoh1 to induce hair cell formation in the absence of Sox2 with the results providing additional insights into the role of Sox2 in formation of inner ear sensory epithelia. A better understanding of the role of Sox2 and the presence and nature of the prosensory domain name, should provide valuable insights regarding inner ear formation and possible regenerative strategies. METHODS Mice All animal procedures were performed according to the guidelines and regulations of the Institutional Animal Care and Use Committee at the National Institutes of Health and The Medical University of South Carolina (MUSC). The generation and genotyping of mutant mice was described previously (Kiernan et al., 2005). Briefly, heterozygotes. Cochleae from wild-type CD1 (Charles River; Harlan Laboratories) or mice were collected from timed-pregnant females at specific time points between E13 and P0. Generation of plasmid constructs For transient expression of Sox2, a fusion construct was generated by cloning Sox2 (open reading frame and to introduce a stop codon at the end of sequence. The resulting fusion was confirmed by sequencing and then inserted into the vector to generate overexpression, we used HKI-272 supplier and as described previously (Puligilla et al., 2010; Woods et al., 2004). The expression vector, was used for continuous expression of fusion protein in both cell lines and cochlear HKI-272 supplier explants. Immunostaining Cochlear explants were fixed in 4% paraformaldehyde for 10 min, washed in PBS, permeabilized with 0.5% Tween-20 and then blocked with 10% serum. Samples were then incubated overnight in primary antibodies at 4 C with rocking followed by extensive rinsing. Binding of primary antibodies was detected.