The roles of Irx3 and Irx5 in mouse cochlear epithelial cell patterning and differentiation

Abstract

The mammalian cochlea develops from an otocyst into a complex structure consisting of both sensory and nonsensory epithelium for its function of sound detection. Although the sensory portion is inherently important for sound detection, the nonsensory portion constitutes a majority of the cochlear duct and maintains a correct homeostatic environment also necessary for hearing. Irx3 and Irx5 are important genes in cochlear morphogenesis and sensory epithelial specification, leading to an enlarged sensory portion and altered cochlear morphogenesis in knockout mice. How Irx3 and Irx5 are able to expand the Sox2 sensory expression remains unknown. Additionally, the roles of the two genes in the development of nonsensory structures need to be explored. The aim of this study is to determine the roles of Irx3 and Irx5 in the cochlear cell patterning and differentiation, specifically studying the Sox2 positive prosensory epithelium expansion and nonsensory structures until postnatal stages. To better understand the characteristics of the expanded Sox2 prosensory region upon the lack of both Irx3 and Irx5, Sox2 expression was analyzed in different Cre recombinase mutants. Temporal knockouts and lineage tracing of Sox2 prosensory cells were conducted by tamoxifen induced recombination. A series of timed deletion of Irx3 in an Irx5 null background was aimed to pinpoint a certain time requirement of the two genes but exemplified the gene dosage requirement of Irx3 and Irx5 in generating the expansion of the prosensory epithelium. Sensory cell specification is dependent on both Irx3 and Irx5 before cell cycle exit and the start of differentiation at E14.5. Moreover, Sox2 lineage tracing directly depicts that Irx3 and Irx5 are necessary for normal sensory cell fates by controlling the downregulation of Sox2. To explore how Irx3 and Irx5 can affect the patterning of cochlear nonsensory cells, expression patterns of various gene markers were surveyed. Stria markers of Nr3b2, Kcnq1, Cxn26, and Cd44 were examined. Otx2, Aqp5, and Fgf10 were used to mark the Reissner’s membrane, outer sulcus, and the greater epithelial ridge, respectively. Cell patterning has been altered due to the great morphological changes of the cochlear duct in the double mutant although cell fates remain committed. Further examination of Irx3 and Irx5’s roles in the postnatal stages to observe important postnatal development of nonsensory cells were achieved through generation of Emx2Cre conditional mutants. In neonatal samples, Irx3 and Irx5 are significantly expressed in the outer sulcus which bridges the endolymph to the perilymph for ion recirculation. Upon the removal of Irx3 and Irx5, the development of root cells within the outer sulcus is significantly arrested, potentially affecting the cochlear homeostatic environment. The gene expression analysis suggests that Irx3 and Irx5 are necessary for cell patterning and are continually required postnatally for root cell development. Irx3 and Irx5 are crucial genes for determining the appropriate sensory cell fate through suppression of Sox2 and have additional roles in the nonsensory cell patterning and differentiation.