Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti

Abstract

Atonal homolog1 (Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell deaths. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti (Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), most of the developing organ of Corti is lost through embryonic cell deaths, with the remaining cells transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre)::Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, leaving only few basal and a more prominent apical innervation. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.

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

“…Similarly, three rows of OHCs and one row of IHCs together with supporting cells were also observed in the cochlear section preparation (Fig 2B). In Atoh1 -CKO mice, no Myo7a-positive cells were observed in the entire length of the cochlea and the organ of Corti was replaced by a flat epithelium consisting of BMP4 positive Claudius cells [15,35]. An example of the lack of Myo7a-positive cells from the apical turn is shown in Fig 2C.…”

“…This K + recycling loop is assumed to be important for maintaining the EP and homeostasis of the ion composition in the endolymph [4]. In the Atoh1 -CKO mice, this pathway is no longer present since the entire organ of Corti degenerates and the basilar membrane only contains one to two layers of epithelial cells [35]. One would expect that the EP magnitude would drop if this recycling loop is no longer present.…”

Atoh1, an Essential Transcription Factor in Neurogenesis and Intestinal and Inner Ear Development: Function, Regulation, and Context Dependency

“…It has been observed, however, that cells that look histologically similar to SCs are present at P0 (Bermingham et al 1999), and the structure of the tissue is consistent with wild-type patterning at E16.5, E18.5, and P1 Dabdoub et al 2008;Pan et al 2010). It has been demonstrated that specification of the sensory epithelium is independent of Atoh1 (Chen et al 2002;Dabdoub et al 2008); this would mean that, during early development of the OC, SC markers such as Sox2, Prox1, and Jagged1 would be detected in Atoh1-null animals, as these factors are required for early patterning.…”

“…The cochlea of a homozygous Atoh1 mutant mouse lacks differentiated HCs. Histological analysis and electron micrographs of cochlear tissue throughout development and at P0 show that, within the zone usually assigned to the OC, HCs are absent (Bermingham et al 1999;Pan et al 2010). Further analysis using immunohistochemistry and ISH to identify individual cell types at P0 showed that not only are there no cells expressing HC markers such as myosin 6 and calretinin, but that there are also no cells expressing markers of the various organ of Corti supporting cells (SCs), including inner pillar cells (probed for Jagged1, Fgfr3, TC2, and p75 ntr ), Deiters' cells (probed for Jagged1, glial fibrillary acidic protein, TC2, and Fgfr3) and phalangeal cells (probed for Jagged1 and glial fibrillary acidic protein) (Woods et al 2004).…”

Neurog1 can partially replace Atoh1 to differentiate and maintain hair cells in a disorganized organ of Corti

“…Other loss-of-function studies showed that Atoh1 drives HC differentiation (Bermingham et al, 1999;Fritzsch et al, 2005;Pan et al, 2011), and overexpression of Atoh1 transformed non-sensory cells into HCs (Kelly et al, 2012;Zheng and Gao, 2000). Atoh1 differentiates HCs in the ear, and the level and duration of Atoh1 expression regulate different types of HCs and their viability Sheykholeslami et al, 2013).…”