Background Hair cells (HCs) are the sensory receptors in both the auditory and the vestibular organs of the inner ear. Supporting cells (SCs) are non-sensory cells embracing the HCs. Injuries of the HCs by aging, acoustic trauma or ototoxic drugs (mainly aminoglycosides, e.g. gentamicin) and cisplatin, often cause permanent impairment of hearing and balance. Birds and amphibians can regenerate their auditory and vestibular HCs after injury through proliferation of SCs or direct transdifferentiation of a SC into a HC. For mammals this ability is limited and spontaneous HC regeneration occurs only in the vestibular sensory epithelia. The utricle is one of the five vestibular organs and contributes to our balance by registering linear acceleration and head tilts. The aim of this PhD thesis was to investigate morphological and morphometric events during spontaneous HC regeneration following gentamicin exposure in neonatal rat utricular explants. Methods Long-term organ culture of macula utriculi, which is stable and reproducible for up to 28 days in vitro (DIV), was used in all papers in the thesis. HC damage was induced by gentamicin. On 2 DIV the explanted utricular maculae were divided into two groups, a control group and a gentamicin-exposed group. In the latter group macular explants were exposed to gentamicin for 48 hours during 2-3 DIV and then allowed to recover. Morphologic and morphometric evaluations were done from utricles harvested at various time points during 28 DIV. Imaging techniques used were light microscopy, including immunohistochemistry, and transmission electron microscopy. Results In the control group the epithelia were well preserved with a slight decline in HC density after 14 DIV. In the gentamicin-exposed group there was an initial substantial decline in HC density and thereafter the proportion of HCs in relation to SCs increased significantly. Using BrdU as a proliferation marker and myosin 7a as a HC marker, we found no cells that were double marked. At the ultrastructural level, the apical occlusion of the explanted epithelia was intact in both the control and the gentamicin exposed group during the entire in vitro period. Cells that seemed to be in a transitional state, transforming from SCs into HCs were observed in the gentamicin-exposed group. These cells had cytoplasmic extensions basally i.e. foot processes, an assembly of mitochondria basally in the cell or in these foot processes, and often apical SC extensions covering the HC. HCs classified as transitional cells had an increased number of SC connections to their basal parts compared to mature HCs. Conclusions  In these neonatal rat utricular explants: - The morphological structure of the sensory epithelia was well preserved during long-term culture. - The renewal of hair cells after gentamicin exposure occurred through direct transdifferentiation of supporting cells into hair cells. - There was also a proliferative response by the supporting cells, but this supporting cell proliferation did not contribute to the generation of new hair cells. - Cells in a transitional state, showing a characteristic morphology, were observed during the process of transdifferentiation from supporting cells into hair cells. - The tight junctional seal of the epithelia stayed morphologically intact also after gentamicin exposure. - Gap junctions were observed in between supporting cells but not found in between hair cells and supporting cells or between transitional cells and supporting cells.

Prenatal alcohol exposure is known to have many profound detrimental effects on human fetal development (fetal alcohol spectrum disorders), which may manifest into lifelong disabilities. Although hearing deficiency is a recognized effect, how alcohol affects the auditory/vestibular systems has not been well studied. This is the first study that used the zebrafish, Danio rerio, as a model organism to investigate morphological effects of alcohol on the developing octavolateral system (auditory, vestibular and lateral line). Zebrafish embryos of two hours post fertilization (hpf) were treated in 2% alcohol for 48 hours and screened at 72 hpf for morphological defects of the inner ear and lateral line neuromasts. Inner ear size and otoliths of zebrafish from both alcohol-treated and control groups were examined using light microscopy. Zebrafish were stained with fluorescent vital dyes to visualize lateral line hair cells using confocal microscopy. The size of neuromasts and length of kinocilia were measured using scanning electron microscopy. Results reveal that alcohol treatment during the early development impairs the formation of otoliths, neuromasts and their hair cells, as well as ear size and kinocilium length. We observed several otolith phenotypes including zero, one, two abnormal, two normal and multiple otoliths for alcohol-treated zebrafish. Fetal alcohol exposure appears to severely impact the size of both anterior and posterior lateral line neuromasts. Our results indicate that early fetal alcohol exposure most likely results in functional defects of the octavolateral system due to inner ear and lateral line dysmorphology.

The inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.

The vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helixloop- helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a. The developing sensory epithelia express several factors that regulate differentiation and maintenance of hair cells. We show that pax5 is differentially expressed in the anterior utricular macula (sensory epithelium). Knockdown of pax5 function results in utricular hair cell death and subsequent loss of vestibular (balance) but not auditory (hearing) defects. SAG neurons are formed normally in these embryos but show disorganized dendrites in the utricle following loss of hair cells. Lastly, we examine the development of SAG. SAG precursors (neuroblasts) are formed in the floor of the ear by another basic helix-loophelix transcription factor neurogenin1 (neurog1). We show that Fgf emanating from the utricular macula specifies neuroblasts, that later delaminate from the otic floor and undergo a phase of proliferation. Neuroblasts then differentiate into bipolar neurons that extend processes to hair cells and targets in the hindbrain. We show evidence that differentiating neurons express fgf5 and regulate further development of the SAG. As more differentiated neurons accumulate, increasing level of Fgf terminates the phase of neuroblast specification. Later on, elevated Fgf stabilizes the transit-amplifying phase and inhibits terminal differentiation. Thus, Fgf signaling regulates SAG development at various stages to ensure that proper number of neurons is generated.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Age-related hearing loss is an acute health problem affecting people worldwide, often arising due to defects in the proper functioning of sensory hair cells in the inner ear. The apical surface of sensory hair cells contains actin-based protrusions known as stereocilia, which detect sound and head movements. Since hair cells are not regenerated in mammals, it is important to maintain the functioning of stereocilia for the life of an organism to maintain hearing ability. The actin filaments within a stereocilium are extensively crosslinked by various actin crosslinking proteins, which are important for stereocilia development and maintenance. Multiple studies have shown that the stereocilia actin core is exceptionally stable whereas actin is dynamic only at the tips of stereocilia. However, whether the actin crosslinking proteins, which are nearly as abundant as actin itself, are similarly stable or can freely move in and out of the core remains unknown. Loss or mutation of crosslinkers like plastin-1, fascin-2, and XIRP2 causes progressive hearing loss along with stereocilia degeneration while loss of espin prevents stereocilia from even developing properly. Do these phenotypes stem from an unstable stereocilia core? Does crosslinking confer stability to the core? To address these questions, we generated novel transgenic reporter lines to monitor the dynamics of actin in mice carrying fascin-2R109H mutation and espin null mice and also to study the dynamics of actin crosslinkers, in vivo and ex-vivo. We established that actin crosslinkers readily exchange within the highly stable F-actin structure of the stereocilia core. In addition, we determined that stereocilia degeneration in mice carrying fascin-2R109H mutation and espin null mice could possibly occur due to a less stable actin core. These studies suggest that dynamic crosslinks stabilize the core to maintain proper stereocilia functioning. Future work warrants understanding the reason behind the importance of dynamic crosslinks within a stable stereocilia core. Actin stability not only depends on actin crosslinkers, but also on actin filament composition as evident from distinct stereocilia degeneration and progressive hearing loss patterns in hair-cell specific knockout of actin isoforms. Although beta- and gamma- actin polypeptide sequences differ by only 14 four amino acids, whether the latter determine the unique function of each cytoplasmic actin isoform was previously unknown. Here we determined that these four critical amino acids determine the unique functional importance of beta-actin isoform in sensory hair cells. Taken together, our study demonstrates that actin cytoskeletal proteins are important for the morphogenesis and maintenance of stereocilia.

No description available.

No description available.

No description available.