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

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

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

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

Next Generation Sequencing Reveals Gene Expression Patterns in the Zebrafish Inner Ear Following Growth Hormone Injection

Loss of hair cells due to acoustic trauma results in the loss of hearing. In humans, unlike other vertebrates, the mechanism of hair cell regeneration is not possible. The molecular mechanisms that underlie this regeneration in nonmammalian vertebrates remain elusive. To understand the gene regulation during hair cell regeneration, our previous microarray study on zebrafish inner ears found that growth hormone (GH) was significantly upregulated after noise exposure. In this current study, we utilized Next Generation Sequencing (NGS) to examine the genes and pathways that are significantly regulated in the zebrafish inner ear following sound exposure and GH injection. Four groups of 20 zebrafish each were exposed to a 150 Hz tone at 179 dB re 1μPa RMS for 40 h. Zebrafish were injected with either salmon GH, phosphate buffer or zebrafish GH antagonist following acoustic exposure, and one baseline group received no acoustic stimulus or injection. RNA was extracted from ear tissues at 1 and 2 days post-trauma, and cDNA was synthesized for NGS. The reads from Illumina Pipeline version SCS 2.8.0 were aligned using TopHat and annotated using Cufflinks. The statistically significant differentially expressed transcripts were identified using Cuffdiff for six different pairwise comparisons and were analyzed using Ingenuity Pathway Analysis. I found significant regulation of growth factors such as GH, prolactin and fibroblast growth factor receptor 2, different families of solute carrier molecules, cell adhesion molecules such as CDH17 and CDH23, and other transcription factors such as Fos, FosB, Jun that regulate apoptosis. Analysis of the cell proliferation network in the GH-injected condition compared to buffer-injected day 1 showed significant up-regulation of GH while downregulation of apoptotic transcription factors was found. In contrast, the antagonist-injected condition compared to the GH-injected condition showed an opposite pattern in which up-regulation of apoptotic transcription factors were found while GH was down-regulated. A number of other transcripts (e.g., POMC, SLC6A12, TMEM27, HNF4A, CDH17 and FGFR2) that showed up-regulation in GH-injected condition showed down-regulation in antagonist-injected condition. These results strongly suggest that injection of exogenous GH potentially has a protective role in the zebrafish inner ear following acoustic trauma

Growth Hormone and the Auditory Pathway: Neuromodulation and Neuroregeneration

Growth hormone (GH) plays an important role in auditory development during the embryonic stage. Exogenous agents such as sound, noise, drugs or trauma, can induce the release of this hormone to perform a protective function and stimulate other mediators that protect the auditory pathway. In addition, GH deficiency conditions hearing loss or central auditory processing disorders. There are promising animal studies that reflect a possible regenerative role when exogenous GH is used in hearing impairments, demonstrated in in vivo and in vitro studies, and also, even a few studies show beneficial effects in humans presented and substantiated in the main text, although they should not exaggerate the main conclusionsS

Transcriptomic analysis of the developing and adult mouse cochlear sensory epithelia

International audienceThe adult mammalian cochlea lacks regenerative ability and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Previous data show that early postnatal cochlea harbors stem/progenitor-like cells and shows a limited regenerative/repair capacity. These properties are progressively lost later during the postnatal development. Little is known about the genes and pathways that are potentially involved in this difference of the regenerative/repair potentialities between early postnatal and adult mammalian cochlear sensory epithelia (CSE). The goal of our study is to investigate the transcriptomic profiles of these two stages. We used Mouse Genome 430 2.0 microarray to perform an extensive analysis of the genes expressed in mouse postnatal day-3 (P3) and adult CSE. Statistical analysis of microarray data was performed using SAM (Significance Analysis of Microarrays) software. We identified 5644 statistically significant differentially expressed transcripts with a fold change (FC) >2 and a False Discovery Rate (FDR) ≤0.05. The P3 CSE signature included 3,102 transcripts, among which were known genes in the cochlea, but also new transcripts such as, Hmga2 (high mobility group AT-hook 2) and Nrarp (Notch-regulated ankyrin repeat protein). The adult CSE overexpressed 2,542 transcripts including new transcripts, such as Prl (Prolactin) and Ar (Androgen receptor), that previously were not known to be expressed in the adult cochlea. Our comparative study revealed important genes and pathways differentially expressed between the developing and adult CSE. The identification of new candidate genes would be useful as potential markers of the maintenance or the loss of stem cells and regenerative/repair ability during mammalian cochlear development

Plasticity within the Auditory Systems of Fishes

Fishes inhabit incredibly cacophonous environments and experience functional, morphological, and transcriptional auditory system plasticity in reproductive state-dependent and auditory experiential contexts. In contrast to the comprehensive study of acoustic overexposure and functional reproductive condition-dependent plasticity within the auditory periphery, the mechanisms underlying acoustic experience-mediated central nervous system plasticity in fishes are generally poorly characterized. Recent research has highlighted neurochemical and transcriptional flexibility within the central nervous systems of fishes in response to prolonged exposure to music. However, the contributions of the acoustic characteristics of musical stimulation to central nervous system plasticity remain unclear. To evaluate the contributions of sound stimulus frequency to brain plasticity, I employed a targeted transcriptional analysis of neuroplasticity-associated genes within the brain of zebrafish (Danio rerio) exposed to 100 Hz and 800 Hz continuous pure tones at a sound pressure level of 140 dB (re 1 μPa) for 1-week intervals across a 4-week period. The transcription of genes involved in mediating connective plasticity fluctuated as a function of duration and frequency of sound exposure, while cellular proliferation did not show variation with sound treatment; suggesting prolonged tonal stimulation may facilitate connective plasticity within the zebrafish brain. These results provide evidence of central nervous system plasticity in response to pure tone exposure and implicate sound-induced behaviour and multisensory inputs in the mediation of sound-induced transcriptional flexibility within the zebrafish brain. Collectively, this thesis highlights the complexity of auditory system plasticity and emphasizes the value of investigating acoustic experience-mediated nervous system plasticity beyond the auditory periphery in fishes

The Function of Hedgehog and Wnt Signaling Pathways in Otic Development

The inner ear is a complex sensory organ essential for hearing and balance. During embryonic development, the inner ear depends on signaling information originating from the embryonic hindbrain to establish dorsoventral and anteroposterior identity. The Hedgehog (Hh) and Wnt signaling pathways are active in the hindbrain and implicated in otic development, but their exact mechanisms of action remained unclear. We investigated the function of Hh in ear development using a mouse model where we conditionally inactivated Hh signaling in the otic vesicle, a transient embryonic structure that gives rise to the inner ear, while leaving nearby Hh dependent tissues unaffected. We found Hh signaling within the otic vesicle functions to establish ventral otic identity and drive the proliferation of cochlear-vestibular ganglion (cvg) neuroblasts that will innervate the ear. We identified presumptive Hh target genes in the developing inner ear using microarrays. Several of these presumptive Hh targets are known to function in ear development or hearing. We also identified many novel targets that have not been characterized in the ear. Many of these novel presumptive Hh target genes are expressed in the ventral otic vesicle, a region that will give rise to the cochlear duct. To interrogate the function of Wnt signaling in ear development, we used a Wnt responsive inducible Cre recombinase (TopCreERT2) to genetically label cells at different stages of ear development. We found cells that make up dorsal, vestibular, structures and cvg neurons are Wnt responsive for prolonged periods of ear development. In the cochlear duct, we found both sensory and support cells originate from a Wnt responsive population. Surprisingly, we found the Wnt responsive population of cochlear progenitors was also labeled using a cre recombinase expressed from the Gbx2 locus. TopCreERT2 and Gbx2 expression overlap in the dorsomedial wall of the otic vesicle, suggesting this region is a likely source for auditory cells