Early Functions of Tbx2 and Tbx3 in inner ear development

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Abstract: The mature inner ear is responsible for the vestibular and auditory senses in mammals. A simple otic vesicle (OV) gives rise to all mature inner ear structures. However, much about the genetic controls regulating cell fate decisions required for morphogenesis of the complex inner ear structures are unknown. There are three T-box transcription factor genes, Tbx1, Tbx2 and Tbx3 expressed in the OV. With the use of Pax2-Cre/+, I inactivated Tbx2 and/or Tbx3 floxed alleles and I uncovered cochlear and saccular defects in Tbx2 conditional knockout (Tbx2cKO) embryos, semicircular canal defects in Tbx3 conditional knockout (Tbx3cKO) embryos and complete failure of the OV to undergo morphogenesis in Tbx2/3cKO double mutant embryos. From these preliminary results, I suggest that Tbx2 and Tbx3 have unique and shared functions in inner ear development. I have generated preliminary RNAscope in situ hybridization data using NeuroD1 (Neurogenic Differentiation 1) that marks neuronal precursors, Bmp4 (Bone Morphogenetic Protein 4) that marks sensory precursor cells and Otx2 (Orthodenticle Homeobox 2) that marks non-sensory precursor cells. We identified an ectopic domain of NeuroD1 expression in the posterior OV, an expansion of the Bmp4 expression in the posterior domain and reduction of Otx2 expression in the ventral domain in Tbx2/3cKO embryos. Our lab previously found that inactivation of Tbx1 results in failed morphogenesis of the OV with expansion of the neurogenic domain to the posterior OV, as in Tbx2/3cKO embryos. Based upon these expression changes, we hypothesize that Tbx2 and Tbx3 have early shared functions in restricting sensory and neuronal cell fates and promoting non-sensory cell fates. In Aim 1, I will carefully determine the spatiotemporal expression patterns of Tbx2 and Tbx3 and will further assess phenotypes of individual and combined Tbx2 and Tbx3 mutant embryos to gain a better understanding of when and where inner ear defects arise with respect to expression of the two genes. In Aim 2, I will take a candidate gene approach to determine the relative position as to where Tbx1, Tbx2 and Tbx3 fit into the genetic hierarchy of cell fate decisions to form the neuronal, sensory or non-sensory fates. I will also distinguish between changes in cell fate and changes in cell populations. Taken together, this project will elucidate the early shared functions of Tbx2 and Tbx3, and compare that with established Tbx1, Eya1 (EYA Transcriptional Coactivator And Phosphatase 1), and Six1 (Sine Oculis Homeobox Homolog 1) pathways, all required for morphogenesis of the inner ear.