The inner ear disorders of hearing and balance that affect over 30 million Americans can be caused by a variety of factors including damage to mechanosensory hair cells, developmental abnormalities, and genetic mutations. Proposed research aims to gain an integrated view of mechanisms that underlie inner ear development by means of anatomical, physiological, and genetic approaches. The Xenopus acoustico-vestibular system will be used to investigate sensory endorgan formation with the long term goal of understanding how hair cells of the inner ear differentiate and acquire their unique structural and functional phenotypes during development. The experimental approach will leverage the augmented potential of Xenopus laevis and Xenopus (Silurana) tropicalis for developmental and genetic studies due to national resource sharing initiatives and genome sequencing efforts. A key objective of this research is to determine how developmental regulation of ion channel gene expression confers the distinctive electrical phenotypes that typify hair cells of auditory and vestibular endorgans.
The specific aims are: (1) to compare the structural and genetic development of auditory and vestibular endorgans, (2) to determine genetic regulation of ion channel expression during inner ear development, (3) to characterize the development of the electrical phenotype of mechanosensory hair cells, and (4) to investigate hair cell differentiation and regeneration in vitro using an immature (larval) inner ear culture preparation. Anatomical and molecular data gathered as part of this research will be prepared for dissemination in the public domain via online databases. The experimental plan will incorporate diverse methods including immunohistochemistry, multiphoton and confocal microscopy, electrophysiology (patch clamp), RT-PCR, molecular cloning, yeast two hybrid analysis, transient transfection, and cell culture. As part of this effort, putative promoter sequences for ion channels will be cloned and used to produce transgenic Xenopus lines. Experimental results are intended to advance fundamental understanding of mechanosensory hair cell differentiation during organogenesis, and of the genetic control of acoustico-vestibular development. Results of these investigations also may be valuable in design of treatments for hereditary and environmentally-induced sensory disorders of hearing and balance.

National Institute of Health (NIH)
National Institute on Deafness and Other Communication Disorders (NIDCD)
Research Project (R01)
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Special Emphasis Panel (ZRG1-SMI (08))
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Freeman, Nancy
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New Mexico State University Las Cruces
Schools of Arts and Sciences
Las Cruces
United States
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Trujillo-Provencio, Casilda; Powers, TuShun R; Sultemeier, David R et al. (2016) RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays. Methods Mol Biol 1427:73-92
Powers, TuShun R; Virk, Selene M; Trujillo-Provencio, Casilda et al. (2012) Probing the Xenopus laevis inner ear transcriptome for biological function. BMC Genomics 13:225
Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V Bleu et al. (2011) Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes. In Vitro Cell Dev Biol Anim 47:640-52
Powers, Tushun R; Virk, Selene M; Serrano, Elba E (2010) Strategies for enhanced annotation of a microarray probe set. Int J Bioinform Res Appl 6:163-78
Trujillo-Provencio, Casilda; Powers, TuShun R; Sultemeier, David R et al. (2009) RNA isolation from Xenopus inner ear sensory endorgans for transcriptional profiling and molecular cloning. Methods Mol Biol 493:3-20
Quick, Quincy A; Serrano, Elba E (2007) Cell proliferation during the early compartmentalization of the Xenopus laevis inner ear. Int J Dev Biol 51:201-9