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Developmental Patterning of the Inner Ear

Thiede, Benjamin
Thesis/Dissertation; Online
Thiede, Benjamin
Corwin, Jeffrey
Verbal communication depends on the discrimination of sounds across a wide range of frequencies. The cochlea provides that capacity through mechanical tuning due to the basilar membrane’s resonance properties and through responses of specialized sensory receptor cells called hair cells (HCs). Gradients of HC phenotypes parallel the longitudinal, or tonotopic, axis. Projecting from the apical surfaces of HCs are F-actin rich structures, called stereocilia, which are arranged in close-packed rows of increasing length. In the chicken cochlea, proximal-end HCs contain 250 or more stereocilia that reach a maximum length of 1.5 μm, while distal-end HCs contain 50 or fewer stereocilia that reach a maximum length of 5.5 μm. At locations between those ends, the number and length of stereocilia grade from HC to HC. As a result of the graded HC phenotypes and the mechanical properties of the basilar membrane that differ along the longitudinal axis, the cochlea’s proximal (or basal) end is tuned for high frequencies of sound and its distal (or apical) end is tuned for low frequencies. The hundreds of individual cellular phenotypes that are characterized by each HC’s integer number of stereocilia provide a uniquely quantifiable and accessible readout of the topographic cellular patterning in the nervous system. A similar spatial gradient occurs in mammalian cochleae. The structural phenotypic differences between HCs also have functional importance in their frequency tuning – shorter bundles respond optimally to high frequency sounds and longer bundles respond better to low frequency sounds. The graded differences in the number and maximum length of stereocilia produced along the longitudinal axis of the cochlea led me to hypothesize that diffusible, extracellular signaling molecules form morphogenetic gradients along this axis that pattern the HCs during development. In the first series of experiments I found that early in cochlea morphogenesis there are opposing gradients of expression for enzymes that synthesize and degrade retinoic acid (RA), a signaling molecule, across the tonotopic axis of the chicken cochlea. The expression gradients of these enzymes reverse as cochlear development proceeds and HCs begin to differentiate and acquire location-specific phenotypes. This presumptively leads to the formation of a morphogenetic gradient of RA across the distal-to-proximal axis. Through a series of in vitro experiments, I found that RA is necessary and sufficient to induce a distal-like HC phenotype throughout the chicken BP. In other experiments, I found that promoting RA signaling enhanced expression of Espin and Fscn2, actin crosslinking genes involved in stereocilia elongation. In another series of experiments I found that fluorophore-conjugated phalloidin permeates living HCs and that this permeation requires metabotropic P2Y receptor signaling. These findings provide a novel method for vitally labeling sensory cells in developmental studies, which overcomes some of the limitations of visualizing live HCs with fluorescent styryl dyes, which have broad, fixed absorption spectra.
University of Virginia, Department of Neuroscience, PHD, 2014
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