Patterning of the Cochlear Apex-to-Base Axis - ABSTRACT Morphogens such as sonic hedgehog and retinoic acid form gradients that pattern the apex-to-base axis of the mammalian and avian embryonic cochlea, thereby resulting in differential hair cell size and stereociliary organization. Although much has been learned about how these individual gradients are established, how they are integrated and translated into region-specific characteristics at the cellular level remains unclear. Therefore, the objective of this proposal is to define the molecular mechanisms that encode tonotopic identity at the transcriptional and epigenetic levels. The central hypothesis of this proposal is that hedgehog signaling acts upstream of a cascade involving retinoic acid, miRNAs, and the chromatin modifier HMGA2, thus imprinting positional information in the cochlea. To test this hypothesis, three Specific Aims will be pursued. First, the hypothesis that hedgehog signaling controls retinoic acid signaling activity will be tested by mapping the retinoic acid signaling activity in hedgehog gain or loss of function experiments. Furthermore, hedgehog effectors (GLI1- 3) will be classified into transcriptional activators and repressors, and GLI target genes will be identified through a gene regulatory network analysis. Also, genes modulating retinoic acid activity among the hedgehog targets, such as Cyp26b1, will be identified using a hedgehog loss of function paradigm. Second, the hypothesis that retinoic acid induces expression of miRNAs, thus shaping opposing mRNA gradients will be tested. Initial experiments will determine whether let7 is a critical factor in shaping the cochlear Hmga2 gradient by blocking of specific let7 family members. Next, miRNAs and mRNAs among the retinoic acid regulated genes will be identified by using a retinoic acid loss of function allele. Third, the hypothesis that HMGA2 controls tonotopic maturation of postnatal HCs will be tested. Initially, the temporal requirement for Hmga2 in hearing acquisition will be determined by deletion of Hmga2 before and after the onset of hearing. Cell type specific functions for Hmga2 will be elucidated by characterizing hearing and cell type specific markers upon conditional loss of Hmga2 in prosensory progenitors and hair cells. Finally, the impact of HMGA2 on the chromatin landscape will be determined upon conditional deletion of Hmga2. Our expected outcomes after successful completion of the proposed project include comprehensive characterization of the gene regulatory network mediating apex-to-base patterning in the cochlea. The results should explain the transcriptional and epigenetic mechanisms guiding tonotopic development in the auditory periphery. Learning how the auditory sensor is constructed will be critical to enable future studies, including those aimed at regenerating lost hearing.
