Transcriptomic analysis of the zebrafish inner ear points to growth hormone mediated regeneration following acoustic trauma
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ABSTRACT:
ORGANISM(S): Danio rerio
SUBMITTER: Michael Smith
PROVIDER: E-GEOD-29669 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Considerable interest and controversy has arisen over the potential effects of seismic surveys carried out during exploration for oil and gas deposits. Regarding fish, there is a concern that intense sound sources, such as seismic airguns, may injure their auditory system. In this study, salmonid cDNA microarrays, reciprocal suppression subtractive hybridization (SSH) cDNA libraries and quantitative reverse transcription – polymerase chain reaction (QPCR) were used to identify and study a responsive gene set in the inner ear of Atlantic salmon (Salmo salar) following seismic airgun exposure. Microarray analyses on pooled seismic exposed inner ear RNA versus pooled control inner ear RNA revealed 79 unique transcripts (passing background threshold) that were greater than 1.75-fold differentially-regulated by acoustic stress on at least 3 of the 4 slides in the study (including at least one dye-swap). QPCR analyses of 8 microarray-identified transcripts of interest revealed a significant up-regulation (P<0.05) of transcripts encoding nicotinamide riboside kinase 2 (1.89-fold) and hemoglobin subunit alpha-4 (3.78-fold), and a significant down-regulation (P<0.05) of a transcript encoding C14orf159 protein (1.35-fold). QPCR analyses also confirmed an overall up-regulation of transcripts encoding growth hormone I (7.78-fold), c-type lectin receptor A (2.20-fold) and retinol binding protein I (1.24-fold), however these differences were not considered to be statistically significant (P<0.05) due to the high biological variability in the seismic exposed group for these transcripts of interest. A total of 683 expressed sequence tags (ESTs) generated from SSH cDNA salmon ear libraries enriched for genes responsive to seismic airgun noise have been deposited in the GenBank dbEST. Targeted gene discovery in salmon ear allowed for the identification of novel transcripts, including some with sensory-relevant functional annotations, and represents a significant contribution to salmonid hearing research. Initial results demonstrate that genomics has the potential to greatly enhance our understanding of the impact of seismic airguns on gene and molecular pathways involved in hearing, and provide valuable molecular biomarkers that can act as an early warning sensor to acoustic stress. Juvenile Atlantic salmon (Salmo salar) smolt were obtained from North Water Products Ltd., Daniel’s Harbour, NL and held at ambient seawater temperature, in a flow-through system supplied with air at the Northwest Atlantic Fisheries Centre, St. John’s, NL. Two weeks prior to seismic airgun exposures, fish were divided into two 1m3 cages, one each for control (non-exposed) and exposed groups, in a 15,000L aquarium at ambient seawater temperature (0.2°C). Each cage was placed the same distance from the water intake and airstones were placed next to each cage. Fish were fasted for two days prior to exposure. Sixteen control (non-exposed) fish from one cage were sampled prior to seismic activity. Immediately following sampling of control fish, seventeen fish in the remaining cage were placed 2m from a 10in3 Texas Instruments airgun. Fish were subjected to 50 exposures, 1 exposure every 10 seconds, at an average sound pressure level of 204 dB peak-to-peak relative to 1µPa; considered to be a worse case scenario within a few hundred meters of a survey vessel. Seventeen seismic exposed fish were sampled 16 h following exposure. The only aquarium available that was suitable for seismic airgun exposures was not designed to have a regulated photoperiod. For this reason the fish were held under a constant daylight regime. Fish were collected by dip-net and euthanized by severing the spinal cord. The inner ears from each salmon were removed, placed immediately in RNase-free 2 ml tubes, and then flash frozen in liquid nitrogen. RNA isolated from the right inner ear of the 12 seismic exposed and 12 control individuals that gave the highest total RNA yields were used to generate 2 mRNA pools (a “seismic” pool and a “control” pool). Each sample contributed 4.0 µg column purified total RNA to each pool. Comparisons were made for the seismic exposed mRNA pool compared to the control (non-exposed) mRNA pool using the consortium for Genomic Research on All Salmonids Project (cGRASP) 16K (salmonid) cDNA array and the 3DNA Array 900 Detection Kit and instructions (Genisphere). Technical quadruplicate slides including 2 dye-swaps were run for the comparison. Slide GG003_011: Cy5-labeled control ear, Cy3-labeled seismic Slide GG003_012: Cy5-labeled control ear, Cy3-labeled seismic Slide GG003_013: Cy3-labeled control ear, Cy5-labeled seismic Slide GG003_014: Cy3-labeled control ear, Cy5-labeled seismic
2012-02-28 | E-GEOD-33257 | biostudies-arrayexpress
Project description:Through its activity in stereocilia, MYO7A (myosin VIIA) is essential for hair cell function in the inner ear. Utilizing multiple stages of immunoaffinity enrichment, we have developed a strategy that allows us to partially purify stereocilia membranes from thousands of chick inner ears and isolate low-abundance MYO7A protein complexes from those membranes. The D10 stereocilia membrane enrichment protocol involves density centrifugation steps and immuno-enrichment of stereocilia using the D10 antibody, directed against the major stereocilia transmembrane protein PTPRQ. The data in this submission document the effectiveness of the enrichment procedure.
2016-08-16 | PXD004222 | Pride
Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. There are 8 control samples and 9 samples trans-tympanically injected with H flu 10e9 for 6 hours. Each sample is from a single animal.
2013-07-23 | E-GEOD-49128 | biostudies-arrayexpress
Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. There are 6 control samples and 8 samples trans-tympanically injected with H flu 10e9 for 6 hours. Each sample is a pool of 4 animals
2013-07-23 | E-GEOD-49122 | biostudies-arrayexpress
Project description:Considerable interest and controversy has arisen over the potential effects of seismic surveys carried out during exploration for oil and gas deposits. Regarding fish, there is a concern that intense sound sources, such as seismic airguns, may injure their auditory system. In this study, salmonid cDNA microarrays and quantitative reverse transcription – polymerase chain reaction (QPCR) were used to identify and study a responsive gene set in the inner ear of Atlantic salmon (Salmo salar) following seismic airgun exposure. Microarray analyses on pooled seismic exposed inner ear RNA versus pooled control inner ear RNA revealed 79 unique transcripts (passing background threshold) that were greater than 1.75-fold differentially-regulated by acoustic stress on at least 3 of the 4 slides in the study (including at least one dye-swap). QPCR analyses of 8 microarray-identified transcripts of interest revealed a significant up-regulation (P<0.05) of transcripts encoding nicotinamide riboside kinase 2 (1.89-fold) and hemoglobin subunit alpha-4 (3.78-fold), and a significant down-regulation (P<0.05) of a transcript encoding C14orf159 protein (1.35-fold). QPCR analyses also confirmed an overall up-regulation of transcripts encoding growth hormone I (7.78-fold), c-type lectin receptor A (2.20-fold) and retinol binding protein I (1.24-fold), however these differences were not considered to be statistically significant (P<0.05) due to the high biological variability in the seismic exposed group for these transcripts of interest. Initial results demonstrate that genomics has the potential to greatly enhance our understanding of the impact of seismic airguns on gene and molecular pathways involved in hearing, and provide valuable molecular biomarkers that can act as an early warning sensor to acoustic stress.
2012-02-28 | GSE33257 | GEO
Project description: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.
2011-06-02 | GSE29669 | GEO
Project description:Mesenchymal stromal cells (MSCs) are an adult derived stem cell-like population that has been shown to mediate repair in a wide range of degenerative disorders. The protective effects of MSCs are mainly mediated by the release of growth factors and cytokines thereby modulating the diseased environment and the immune system. Within the inner ear, MSCs have been shown protective against tissue damage induced by sound and a variety of ototoxins. To better understand the mechanism of action of MSCs in the inner ear, mice were exposed to narrow band noise. After exposure, MSCs derived from human umbilical cord Wharton’s jelly were injected into the perilymph. RNAseq analysis of gene expression changes induced by MSCs was compared to sound trauma only treated animals.
2021-12-15 | GSE187029 | GEO
Project description:Capping protein controls stereocilia length and width during hair bundle development. To determine what other proteins are involved in capping protein regulation, we carried out immunoaffinity purifications targeted at either CAPZA or CAPZB2. The starting material for immunopurification was crude stereocilia membranes isolated from mouse inner ear.
2017-08-25 | PXD006077 | Pride
Project description:Eukaryotic cells are equipped with multiple mechanosensing systems and perceive wide range of mechanical stimuli from the environment. However, cell-level responses against acoustic waves, which transmits feeble but highly frequent physical perturbation, largely remains uninvestigated, especially with regard to audible range of sound. Here we investigate the effect of acoustic stimulation on gene expression profiles of mammalian cultured cells. A direct sound emission system was set up using a vibrational transducer to directly generate acoustic waves in culture medium. A custom-made vibrating plate made of PEEK (poly ether ether ketone) plastic was used as a diaphragm. A set of sound patterns including single-frequency sound and white noise were generated by NCH Tone Generator software. Sound intensity was directly measured by recording it in water using a hydrophone and the pressure level was calculated. C2C12 myoblasts cultured in a plastic dish with approximately 50% confluency were subjected to acoustic stimulation. 440 Hz and 14k Hz single-frequency sine wave sound were selected as representatives of low and high audible frequencies, and white noise was selected as a random noise pattern. 2 and 24 hours after continuous emission of these sound at 100 Pa, total RNA was extracted and subjected to the gene expression profiling analysis by RNA-sequencing technique. Total 42 early- and 145 late-response genes were identified as sound-sensitive genes in 2 and 24 hours stimulation, respectively. Gene annotation analyses revealed that in addition to the known mechanosensitive activities such as fluid shear stress response, cell migration, cell adhesion and blood vessel development, variety of pathways and processes were identified to be affected by acoustic stimulation.
2025-03-12 | GSE247726 | GEO
Project description:Through its activity in stereocilia, MYO7A (myosin VIIA) is essential for hair cell function in the inner ear. Utilizing multiple stages of immunoaffinity enrichment, we have developed a strategy that allows us to partially purify stereocilia membranes from thousands of chick inner ears and isolate low-abundance MYO7A protein complexes from those membranes. By analysis of MYO7A and co-purifying molecules with shotgun and targeted mass spectrometry, we demonstrated that MYO7A forms a complex with PDZD7, a paralog of USH1C and DFNB31, which has been shown to localize to the ankle-link region of stereocilia.
2016-08-16 | PXD004221 | Pride