Auditory Hair Cell Regeneration and Gene Expression in Noise-Exposed Zebrafish (Danio rerio)

Fishes are capable of regenerating sensory hair cells in the inner ear after exposure to excessive noise. However, a time course of auditory hair cell regeneration has not been characterized for zebrafish, nor has gene expression following noise exposure. To establish a time course of hair cell regeneration, adult zebrafish were exposed to a 100 Hz pure tone at 179 dB re 1 |u.Pa RMS for 36 hours, and then allowed to recover for 0 to 14 days before morphological analysis. Hair cell loss and recovery were determined using phalloidin and DAPI labeling to visualize hair cell bundles and nuclei, respectively. Cell proliferation was quantified through BrdU labeling. Immediately following noise exposure, zebrafish saccules exhibited significant hair cell bundle loss and reduced DAPI staining in the caudal region. Hair cell bundle counts increased over the course of the experiment, reaching pre-treatment levels at 14 days post-noise exposure. Cell proliferation peaked two days post-noise exposure in the caudal region, and to a lesser extent in the rostral region. Low levels of proliferation were also observed in untreated controls, indicating that cells of the zebrafish saccule are mitotically active in the absence of a damaging event. To characterize gene expression in the zebrafish inner ear following noise exposure, fish were noise-exposed as above, and then allowed ta recover for 2 or 4 days. The inner ears were then removed, and their RNA extracted and subjected to microarray analysis. Genes putatively involved in cell proliferation, wound healing, and apoptosis were identified, as were genes previously noted as highly expressed in hair cells. Understanding the pathways in which these genes participate during the process of hair cell regeneration may provide direction in the development of treatments for deafness in the future

The Effects of Growth Hormone in the Inner Ear of Zebrafish (\u3ci\u3eDanio rerio\u3c/i\u3e) during Hair Cell Regeneration

Although deafness is a universal problem, effective treatments have remained elusive. In order to develop potential treatments, an overall understanding of the cellular process of auditory hair cell regeneration, which occurs in fish but not mammals, must be established. A previous microarray analysis and qRT-PCR validation of noise-exposed zebrafish showed that growth hormone (GH) was significantly upregulated during the process of auditory hair cell regeneration. Thus, GH may play an important role during hair cell regeneration. However, cellular effects of exogenous GH in the zebrafish auditory hair cell regeneration have not been examined after noise exposure. To understand the effect of GH in hair cell regeneration, adult zebrafish were exposed to a 150 Hz pure tone at a source level of 179 dB re 1 μPa RMS for 36 hours. Afterward the fish were immediately injected intraperitoneally with carp recombinant GH (20 μg/gram of body mass) or buffer (0.1 M, pH 7.4 phosphate buffer) and then placed in a recovery tank. The effect of GH on apoptosis in fish inner ear end organs were examined using TUNEL-labeling. Cell proliferation was measured by BrdU incorporation assay. Hair cell regeneration was determined by phalloidin-labeling to allow visualization of hair cell stereociliary bundles. After GH injection, the numbers of TUNEL-labeled cells showed a significant decrease in all three inner ear end organs (saccule, lagena, utricle), suggesting GH may suppress hair cell death induced by acoustic trauma. Higher levels of cell proliferation were also observed in the ears of GH-injected fish, indicating that GH is capable of activating cell mitosis in the zebrafish auditory system. Following sound exposure, the GH-injected group exhibited greater numbers of saccular hair cell bundles compared to the buffer-injected group. These results indicate that GH promotes hair cell regeneration following acoustic damage. Future studies are needed to examine the potential therapeutic benefits of GH in the mammalian ear

Growth Hormone Promotes Hair Cell Regeneration in the Zebrafish (Danio rerio) Inner Ear following Acoustic Trauma

BACKGROUND: Previous microarray analysis showed that growth hormone (GH) was significantly upregulated following acoustic trauma in the zebrafish (Danio rerio) ear suggesting that GH may play an important role in the process of auditory hair cell regeneration. Our objective was to examine the effects of exogenous and endogenous GH on zebrafish inner ear epithelia following acoustic trauma. METHODOLOGY/PRINCIPAL FINDINGS: We induced auditory hair cell damage by exposing zebrafish to acoustic overstimulation. Fish were then injected intraperitoneally with either carp GH or buffer, and placed in a recovery tank for either one or two days. Phalloidin-, bromodeoxyuridine (BrdU)-, and TUNEL-labeling were used to examine hair cell densities, cell proliferation, and apoptosis, respectively. Two days post-trauma, saccular hair cell densities in GH-treated fish were similar to that of baseline controls, whereas buffer-injected fish showed significantly reduced densities of hair cell bundles. Cell proliferation was greater and apoptosis reduced in the saccules, lagenae, and utricles of GH-treated fish one day following trauma compared to controls. Fluorescent in situ hybridization (FISH) was used to examine the localization of GH mRNA in the zebrafish ear. At one day post-trauma, GH mRNA expression appeared to be localized perinuclearly around erythrocytes in the blood vessels of the inner ear epithelia. In order to examine the effects of endogenous GH on the process of cell proliferation in the ear, a GH antagonist was injected into zebrafish immediately following acoustic trauma, resulting in significantly decreased cell proliferation one day post-trauma in all three zebrafish inner ear end organs. CONCLUSIONS/SIGNIFICANCE: Our results show that exogenous GH promotes post-trauma auditory hair cell regeneration in the zebrafish ear through stimulating proliferation and suppressing apoptosis, and that endogenous GH signals are present in the zebrafish ear during the process of auditory hair cell regeneration

Transcriptomic analysis of the zebrafish inner ear points to growth hormone mediated regeneration following acoustic trauma

Background: Unlike mammals, teleost fishes are capable of regenerating sensory inner ear hair cells that have been lost following acoustic or ototoxic trauma. Previous work indicated that immediately following sound exposure, zebrafish saccules exhibit significant hair cell loss that recovers to pre-treatment levels within 14 days. Following acoustic trauma in the zebrafish inner ear, we used microarray analysis to identify genes involved in inner ear repair following acoustic exposure. Additionally, we investigated the effect of growth hormone (GH) on cell proliferation in control zebrafish utricles and saccules, since GH was significantly up-regulated following acoustic trauma. Results: Microarray analysis, validated with the aid of quantitative real-time PCR, revealed several genes that were highly regulated during the process of regeneration in the zebrafish inner ear. Genes that had fold changes of \u3e = 1.4 and P values \u3c = 0.05 were considered significantly regulated and were used for subsequent analysis. Categories of biological function that were significantly regulated included cancer, cellular growth and proliferation, and inflammation. Of particular significance, a greater than 64-fold increase in growth hormone (gh1) transcripts occurred, peaking at 2 days post-sound exposure (dpse) and decreasing to approximately 5.5-fold by 4 dpse. Pathway Analysis software was used to reveal networks of regulated genes and showed how GH affected these networks. Subsequent experiments showed that intraperitoneal injection of salmon growth hormone significantly increased cell proliferation in the zebrafish inner ear. Many other gene transcripts were also differentially regulated, including heavy and light chain myosin transcripts, both of which were down-regulated following sound exposure, and major histocompatability class I and II genes, several of which were significantly regulated on 2 dpse. Conclusions: Transcripts for GH, MHC Class I and II genes, and heavy-and light-chain myosins, as well as many others genes, were differentially regulated in the zebrafish inner ear following overexposure to sound. GH injection increased cell proliferation in the inner ear of non-sound-exposed zebrafish, suggesting that GH could play an important role in sensory hair cell regeneration in the teleost ear

IDENTIFICATION OF THE MOLECULAR MECHANISMS OF ZEBRAFISH INNER EAR HAIR CELL REGENERATION USING HIGHTHROUGHPUT GENE EXPRESSION PROFILING

All nonmammalian vertebrates studied can regenerate inner ear mechanosensory receptors, i.e. hair cells, but mammals only possess a very limited capacity for regeneration after birth. As a result, mammals suffer from permanent deficiencies in hearing and balance once their inner ear hair cells are lost. The mechanisms of hair cell regeneration are poorly understood. Because the inner ear sensory epithelium is highly conserved in all vertebrates, we chose to study the hair cell regeneration mechanism in adult zebrafish, hoping the results would be transferrable to inducing hair cell regeneration in mammals. We defined the comprehensive network of genes involved in hair cell regeneration in the inner ear of adult zebrafish with the powerful transcriptional profiling technique, Digital Gene Expression (DGE), which leverages the power of next-generation sequencing. We also identified a key pathway, stat3/socs3, and demonstrated its role in promoting hair cell regeneration through stem cell activation, cell division, and differentiation. In addition, transient pharmacological up-regulation of stat3 signaling accelerated hair cell regeneration without over-producing cells. Taking other published datasets into account, we propose that the stat3/socs3 pathway is a key response in all tissue regeneration and thus an important therapeutic target not only for hair cell regeneration, but also for a much broader application in tissue repair and injury healing. The dissertation contains four supplemental files. Supplemental file 1 contains raw data of five expression profiles generated by DGE. It is a tab-delimited text file with six columns. The first column contains the sequences of the tags and the second to sixth columns contain the count of the corresponding tags in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 2 contains UniGene clusters identified from unambiguously mapped tags. It is a tab-delimited text file with six columns. The first column contains the UniGene IDs. The second to sixth columns contain the count of the corresponding UniGene clusters in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 3 contains candidate genes identified by comparison of the expression profiles during regeneration to the control profiles. It is a tab-delimited text file with 19 columns. The contents in each column are specified in the header. Supplemental file 4 contains a list of the candidate genes known to be expressed in the inner ear and/or the lateral line system during development. It is a tab-delimited text file with four columns which contain UniGene IDs, ZFIN IDs, Entrez Gene IDs, and gene symbols respectively

The Time-Course of the Effects of Growth Hormone During Zebrafish (\u3ci\u3eDANIO RERIO\u3c/i\u3e) Auditory Hair Cell Regeneration

Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the effects of GH on cellular proliferation; TUNEL-labeling was used to assess the effects of GH on apoptosis in the zebrafish inner ear following acoustic trauma. The time-course of hair cell bundle density, cell proliferation, and apoptosis was established by combining data for baseline fishes and sound-exposed fishes at post-sound exposure day 1 (psed1), psed2, and psed3. GH-injected fish exhibited greater densities of hair cells than bufferinjected controls. In addition, GH-injected fish had higher levels of cell proliferation and lower levels of apoptosis than buffer-injected controls. This suggests that GH may play an important role in zebrafish inner ear hair cell regeneration by stimulating cellular proliferation and inhibiting cellular apoptosis

Prophylactic Effect if Growth Hormone on Zebrafish Auditory Hair Cell Damage

Growth hormone (GH) has been shown to play a role in and improve hair cell regeneration when injected intraperitoneally in zebrafish post-sound exposure. The purpose of this study was to examine whether exogenous GH has a prophylactic effect on auditory hair cell damage when injected prior to acoustic trauma. Groups of zebrafish were injected with either GH or buffer. Immediately following the injection, auditory hair cell damage was induced through exposure to acoustic overstimulation. Hearing tests were then performed on the fish by measuring auditory evoked potentials. Then, the fish ears were dissected either immediately post-trauma or at one, two, and three days following acoustic exposure. The dissected zebrafish saccules were stained with fluorescein-conjugated phalloidin and visualized under flourescence microscopy. Hearing loss and hair cell damage was reduced following trauma in GH-treated fish in comparison to buffer-treated fish. The results show that exogenous growth hormone has a prophylactic effect on acoustically-induced zebrafish auditory hair cell damage