Introduction: Hearing loss affects over 500 million people worldwide and results from irreversible damage to inner ear hair cells. The only available treatment is cochlear implants, which may be unable to provide sensory input if neuronal connections are lost, as they are in mouse models. Thus, regeneration of hair cells offers the only permanent cure; however, such therapeutic intervention requires a detailed molecular understanding inner ear development and hair cell maintenance. During mouse development, there is a balance between proliferation and differentiation that not only determines the size of the ear, but also is needed to form a functional sensory unit. The fulcrum to this balance is N-Myc, a key transcription factor that acts as a node incorporating many upstream growth signaling pathways and funnels them to directly alter the cell cycle and at the same time inhibits differentiation. The loss of N-Myc results in major morphogenetic abnormalities, including a progressive loss of cochlear, despite their initial formation. Interestingly, N-Myc is present in inner ear hair cells after birth, long after proliferation in the inner ear ceased. In addition to N-Myc, L-Myc is co-expressed throughout development in the inner ear. This data suggests that N-Myc and L-Myc may play partially redundant roles both early during development and later in hair cells. Elucidating the relative importance of the Mycs and their interdependent roles in maintaining the balance between proliferation and differentiation may shed light on future hair cell regeneration avenues.
Methods: We generated two Cre-LoxP lines, knocking out both N-Myc and L-Myc before (Pax2-Cre) and after (Atoh1-Cre) hair cell formation. We assessed the possibility of Myc redundancy through 3D reconstructions generated from confocal image stacks from E10.5-E18.5 and the effects of early Myc loss on the balance between proliferation and differentiation through a quantitative PCR study that assessed relative changes in gene expression, using the Pax2-Cre N-Myc f/f L-Myc f/f mice. We assessed organ of Corti development and functionality at P21 and four months of age in the Atoh1-Cre N-Myc f/f L-Myc f/f mice.
Results: The development of the Pax2-Cre N-Myc f/f L-Myc f/f mutant ear was more severely impacted than the Pax2-Cre N-Myc f/f alone, as shown by an additional 50% reduction in size. Genes important to cell cycle maintenance were downregulated whereas differentiation transcription factors were initially downregulated but subsequently later upregulated to normal levels. In Atoh1-Cre N-Myc f/f L-Myc f/f mice, there were no defects in hair cell development.
Discussion: There appears to be redundancy between N-Myc and L-Myc with N-Myc playing a more important role in inner ear formation. The late-onset defects seen in the Pax2-Cre N-Myc f/f mice appear to be a result of abnormal formation of hair cells due to the disruption in the balance between proliferation and differentiation much earlier on. This is the first time such a late-onset hair cell loss has been shown to be due to a defect sustained much earlier and is an important finding as the majority of people suffer from late-onset hearing loss. Additionally, these findings highlight the continued therapeutic importance in elucidating the molecular interactions controlling the delicate shift from a proliferating precursor to a differentiating cell.