Molecular Regulators of Innervation and Patterning in the Developing Chicken Inner Ear

Mary Katherine Scott, Purdue University

Abstract

Normal hearing and balance relies on the detection of sound, orientation and acceleration by sensory hair cells (HCs) located in the inner ear. Once sound is detected, that information must be transmitted to the brain by sensory neurons. Damage to the HCs and/or neurons in the auditory or vestibular organs of the inner ear can result in hearing loss or balance disorders. In mammals, these disorders can be permanent, as HCs do not regenerate after damage. While hearing aids and cochlear implants can restore some ability to hear, there are currently no molecular therapies for hearing loss. By examining genes involved in HC development and innervation, basic science can identify candidate genes for potential molecular therapies. This dissertation focuses on molecular regulators involved in establishing and/or maintaining innervation in the chicken inner ear during embryonic development. The basilar papilla (BP) is the auditory sensory organ in the chicken and is homologous to the mammalian organ of Corti (oC). The BP houses two types of sensory HCs – tall HCs and short HCs. On the neural side of the BP, tall HC receive primarily afferent innervation (neural-side identity). On the abneural side, short HC receive primarily efferent innervation (abneural-side identity). The patterning of these two identities along the radial axis is dependent upon the precise spatiotemporal expression of certain genes during embryonic development. One such gene is Wingless/integrated (Wnt)9a. Previous work has shown that Wnt9a is expressed on the neural edge of the BP and is likely secreted in a gradient across the prosensory domain during crucial time points when proliferation, differentiation, and innervation are occurring. When Wnt9a was overexpressed, we observed an increase in the width of the BP as well as an expansion of the neural-side identity, likely at the expense of the abneural-side identity. RNA sequencing of Wnt9a-overexpressing and control BPs identified genes involved in the Wnt signaling pathway, cytoskeletal remodeling, and axon guidance signaling that were differentially expressed. This dissertation focuses on axon guidance genes, specifically those involved in Slit/Robo (Roundabout), Contactin (Cntn), and Semaphorin (Sema) signaling, that were differentially expressed in this RNA sequencing data set. Slits typically act as repulsive cues for neurites expressing Robo receptors. RNA sequencing data indicates that Slit2 transcripts increased by 1.2 fold when Wnt9a was overexpressed. When examining Slit2 spatial expression pattern in Wnt9a-overexpressing BPs, we did not observe an upregulation of Slit2 but rather an expansion of the Slit2-expression domain that is likely due to increased proliferation in response to Wnt9a. To better understand the role of Slit/Robo signaling in the developing BP, we examined the radial expression patterns of Slit2, Robo1, and Robo2. Slit2 is expressed on the anterior and posterior walls of the cochlear duct (CD). Robo1 and Robo2 had graded expression in the prosensory domain of the BP, highest on the abneural side. Robo1 is also present in the auditory ganglion. While only a small population of cochleovestibular ganglion neurites have been previously shown to respond to Slits, Slit-Robo has also been shown to activate TCF transcription factor by non-canonically activating β-catenin through Abl kinase. We examined Abl kinase-activated -catenin in Slit2- and Wnt9a-overexpressing BPs but did not observe a change in phosphorylated -catenin. We also overexpressed a dominant-negative Robo1.

Degree

Ph.D.

Advisors

Fekete, Purdue University.

Subject Area

Audiology|Biochemistry|Bioinformatics|Cellular biology|Genetics|Surgery

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