Molecular Interactions of Basic Helix Loop Helix (bHLH) Genes in Inner Ear Development
The inner ear originates from an ectodermal thickening, the otic placode, which invaginates and forms the otic vesicle. The otic placode gives rise to the hearing (cochlea) and balance (vestibular) organs, comprising the sensory hair cells and sensory neurons of the cochlear (spiral) and vestibular ganglia. In mammals, inner ear sensory cells undergo terminal mitosis during embryogenesis and do not regenerate. Therefore, during postnatal life, sensory cell loss is a progressively deteriorating phenomenon leading to permanent hearing impairment. This work focuses on the molecular mechanisms regulating the cascade of events that lead to sensory cell formation during inner ear embryogenesis. Major players in these events are basic helix-loop-helix (bHLH) genes: Neurogl, Atohl and Neurodl. We investigated whether co-expression of Neurogl and Atohl in common neurosensory precursors could explain the loss of hair cells in Neurogl null mice. Analysis of terminal mitosis, using BrdU, supports previous findings regarding timing of exit from cell cycle. Specifically, we show that cell cycle exit occurs in spiral sensory neurons in a base to apex progression followed by cell cycle exit of hair cells in the organ of Corti in an apex to base progression, with some overlap of cell cycle exit in the apex for both hair cells and spiral sensory neurons. Hair cells in Neurogl null mice show cell cycle exit in an apex to base progression about 1-2 days earlier. Atohl is expressed in an apex to base progression rather then a base to apex progression as in wildtype littermates. We tested the possible expression of Atohl in neurosensory precursors using two Atohl-Cre lines. We show Atohl-Cre mediated ß-galactosidase expression in delaminating sensory neuron precursors as well as undifferentiated epithelial cells at El 1. PCR analysis shows expression of Atohl in the otocyst as early as E10.5, prior to any histology based detection techniques. Combined, these data suggest that low levels of Atohl exist much earlier in precursors of hair cells and sensory neurons, possibly including neurosensory precursors. Analysis of Atohl-Cre expression in El 8.5 embryos and P31 mice reveal ß- galactosidase stain in all hair cells but also in vestibular and cochlear sensory neurons and some supporting cells. A similar expression of Atohl -LacZ exists in postnatal and adult vestibular and cochlear sensory neurons, and Atohl expression in vestibular sensory neurons is confirmed with RT-PCR. We propose that absence of NEUROG1 protein leads to loss of sensory neuron formation through a phenotypic switch of cycling neurosensory precursors from sensory neuron to hair cell fate. Neurogl null mice show a truncation of clonal expansion of hair cell precursors through temporally altered terminal mitosis, thereby resulting in smaller sensory epithelia. This in vivo analysis of the molecular biology of neurosensory epithelia formation provides valuable information that might help to design strategies to induce therapeutically hair cell regeneration in mammals.
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