Distinct cerebellar and inner-ear phenotypes in Atoh1 promoter-proximal deletion mice.

Abstract

OBJECTIVE: Atoh1 is essential for the development of the cerebellum and inner ear, but the in vivo role of its promoter-proximal region remains incompletely understood. We generated deletion lines targeting the Atoh1 promoter-proximal region containing C sites and examined their phenotypes in the cerebellum and inner ear. METHODS: Using the CRISPR-Cas9 genome editing method, mice with deletions in the Atoh1 promoter-proximal region containing C sites were generated, and four independent deletion lines were established. Gross morphology and histology of the cerebellum and inner ear were examined in 2-month-old mice. RESULTS: Four independent deletion alleles were obtained and designated line A to line D in ascending order of deletion size. In the cerebellum, gross examination and sagittal sections showed progressively more severe hypoplasia from line A to line D, accompanied by reduced foliation and disorganization of cortical architecture. The granule cell layer was progressively reduced, whereas Purkinje cells were relatively preserved. Behavioral abnormalities were detected only in the most severely affected line. In the inner ear, the maculae of the otolith organs were relatively preserved across all lines. The ampullary cristae were relatively preserved in lines A-C but showed hair-cell loss in line D. Cochlear phenotypes were more severe: line A retained relatively preserved inner hair cells with reduced outer hair cells, whereas lines B-D showed near-complete loss of hair cells and marked disruption of the organ of Corti in the analyzed regions. Descriptive quantitative analyses of available specimens supported progressive cerebellar hypoplasia, relative macular preservation, line D crista involvement, and severe cochlear hair-cell loss in lines B-D. CONCLUSION: Deletion of the Atoh1 promoter-proximal region produced distinct tissue-specific phenotypes in vivo. These findings suggest that the Atoh1 promoter-proximal region is differentially required in the cerebellum and inner ear and that its contribution varies among vestibular and cochlear sensory organs.