Studying the role of N-Myc in cochlear development using a gain-of-function approach

Abstract

The mammalian cochlea is a complex organ for sound detection. The sensory component, the Organ of Corti, consists of hair cells and supporting cells which originate from a common pool of progenitors. The cell cycle exit and differentiation of these progenitor cells progress along the cochlear duct in opposite directions. The temporal regulation of the cell cycle exit and differentiation processes is not fully understood. Sonic hedgehog (Shh) signaling has been shown to regulate the progression of hair cell differentiation. Suppressor of fused (Sufu) is an important mediator of Shh signaling. The progression of hair cell differentiation was delayed in Sufu-deficient cochlear epithelium. However, it is not clear which factors regulate the maintenance of cochlear sensory progenitors downstream of Sufu. Previous studies showed that loss of N-Myc in the inner ear resulted in reduced proliferation of the prosensory cells and delayed differentiation. The expression of N-Myc was increased in Sufu-deficient cochlea. The aim of this project is to study the effects of N-Myc overexpression in the formation of cochlear sensory hair cells. Specifically, the roles of N-Myc on prosensory specification, proliferation, cell cycle exit and differentiation have been investigated. The doxycycline-inducible system was used to overexpress N-Myc in the murine inner ear at various stages. The expression pattern of the transgene was evaluated by staining of β-gal reporter activity. Following doxycycline treatment, the N-Myc transgene was expressed broadly in the cochlear epithelium within 24 hours. To understand whether N-Myc regulates the formation of prosensory domain, doxycycline was administered at E10.5, expression of the prosensory markers Sox2 and Jag1 was examined by immunostaining. Both Sox2 and Jag1 were expressed in the ventral cochlear epithelium, but the Sox2-positive domain was expanded at E13.5 in the N-Myc gain-of-function cochlea. BrdU labeling showed that there were more BrdU-positive progenitors in the E13.5 mutant, when compared to the control. Thus, N-Myc promotes proliferation of cochlear prosensory cells, which might explain the enlarged population of Sox2-positive sensory progenitors. To study whether prosensory cell cycle exit was affected in N-Myc gain-of-function mutant, p27Kip1 staining was performed. In the medial and apical part of the mutant at E13.5, p27Kip1 was not expressed. However, by E14.5, it was upregulated at the basal and medial turn but not in the apical region. The results suggested that the cell cycle withdrawal was delayed and occurred in an altered direction. Wholemount staining of hair cell marker indicated that the differentiation of sensory progenitors was also delayed after N-Myc upregulation. To investigate whether N-Myc regulates hair cell differentiation directly, N-Myc overexpression was induced at E14.5, the stage when prosensory cell cycle exit was largely completed. Myo7a staining showed that the timing of hair cell differentiation of this gain-offunction mutant was largely normal at E16.5. In conclusion, N-Myc contributes to the spatio-temporal control of the proliferation and cell cycle exit of cochlear sensory progenitors, which is crucial to the correct timing of hair cell differentiation at later stage.