The roles of Irx3 and Irx5 genes in mammalian inner ear development

Abstract

Iroquois genes encode a family of highly conserved TALE homeodomain transcription factors that are involved in multiple developmental processes. Physiological tests indicated that Irx3 and Irx5 mutant mice displayed hearing impairment. However, the functions of these two genes during inner ear development are not known. The aim of this study is to characterize the roles of Irx3 and Irx5 during mammalian inner ear development using mouse models, in order to reveal the underlying mechanism for the hearing abnormality in the mutants. Two mouse mutants, Irx3tauLacZ and Irx3flox5EGFP with β-gal and EGFP reporters, were analyzed to examine the expression of these two genes in the otic vesicle and cochlear epithelium. In the otocyst, both Irx3 and Irx5 were expressed in the ventral-medial region. Irx5 expression was restricted to the non-sensory domain of the cochlear epithelia, while Irx3 was widely expressed, including the auditory sensory organ, the organ of Corti. The overlapping expression patterns of Irx3 and Irx5 suggest that they may share redundant functions. To investigate the roles of Irx3 and Irx5 during inner ear development, phenotypic analysis was performed on Irx3-/-, Irx5-/- and Irx3/5-/- mutant embryos. As shown by paint-filling analysis, Irx3/5-/- displayed shortened cochlear duct, enlarged cochlear lumen with fused sensory organ. Whole-mount phalloidin staining of hair cell bundles showed that Irx3-/- displayed occasional ectopic inner hair cells. Moreover, only supernumerary vestibular hair cell-like cells were developed in Irx3/5-/- mutant. These results suggest that Irx3 and Irx5 are required for inner ear morphogenesis and the formation of organ of Corti. To understand the effect of Irx3 and Irx5 in the cellular patterning of the cochlea, mutant cochleae were analyzed with markers for different regions of the cochlear epithelia. Altered expression domain of MyoVIIa, Sox2 and Gata2 in Irx3/5-/- cochlea revealed that the boundary between the Kolliker’s organ and the organ of Corti was lost and the location of sensory and non-sensory region was shifted. These results imply that Irx3 and Irx5 function in the establishment of the sensory/non-sensory boundary. It is known that p27kip1 regulates the wave of cell cycle exit in the developing organ of Corti and Sox2 takes part in prosensory specification. To explore the underlying reason for the patterning defects in Irx3/5-/- mutant, cochlear duct from prosensory stages were analyzed. Irx3/5-/- showed altered Sox2 and p27kip1 expression, with expanded prosensory domain and disrupted cell cycle exit. Ectopic prosensory proliferation was detected in the middle turn of the cochlear duct at E13.5 by BrdU incorporation assay. Therefore, Irx3 and Irx5 may participate in the subdivision of sensory territory in developing cochlea by controlling prosensory proliferation. In summary, this study demonstrates that Irx3 and Irx5 cooperate in multiple aspects of inner ear development: an early role to regulate prosensory proliferation and cell cycle exit; a second role to regulate cellular patterning of the cochlear duct by controlling the setting of sensory/non-sensory boundaries in the cochlea; a later role to regulate inner ear morphogenesis. This study supports the idea that Irx3 and Irx5 act as patterning genes during vertebrate evolution.