Effects of hypothermia on gene expression in zebrafish gills
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE7853 | GEO | 2008/09/01
SECONDARY ACCESSION(S): PRJNA99991
REPOSITORIES: GEO
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Project description:Ectothermic vertebrates are different from mammals that are sensitive to hypothermia and they have to maintain core temperature for survival. Why and how ectothermic animals can survive, grow and reproduce in low temperature have been for a long time a scientifically challenging and important inquiry to biologists. We used a microarray to profile the gill transcriptome in zebrafish (Danio rerio) after exposure to low temperature. Adult zebrafish were acclimated to a low temperature of 12 ï°C for 1 (1-d) and 30 d (30-d), and the gill transcriptome was compared to wild types by oligonucleotide microarray hybridization. Results showed 11 and 22 transcripts were found to be upregulated by low-temperature treatment for 1-d and 30-d respectively, while 56 and 70 transcrips were downregulated. The gill transcriptome profiles revealed that ionoregulation-related gene was highly upregulated in cold-acclimated zebrafish. This observation encouraged us to investigate the role of ionoregulatory genes in zebrafish gills during cold acclimation. Cold acclimation caused upregulation of genes that are essential for ionocyte specification, differentiation, ionoregulation, and acid/base balance, and also increased the numbers of cells expressing these genes. mRNA expression of epithelial Ca2+ channel (ECaC), one of these genes, was increased in parallel with the level of Ca2+ influx, revealing a functional compensation after long-term acclimation to cold. Phospho-histone H3 and TUNEL staining showed that the cell turnover rate was retarded in cold-acclimated gills. These results suggest that gills may sustain their functions by yielding mature ionocytes from preexisting undifferentiated progenitors in low-temperature environments. The AB strain of zebrafish (D. rerio) was originally obtained from the University of Oregon, and were kept in the zebrafish stock center at Academia Sinica, Taipei, Taiwan. Fish were reared in local tap water at 28 ï°C and a photoperiod regime of 14-h L/10-h D. Adult zebrafish were acclimated to 12 ï°C with a gradually reduced temperature at a gradient of 4 ï°C/h in order to prevent temperature shock and reduce mortality. After 30 d of acclimation, surviving (over an 80% survival rate) fish appeared to be feeding and behaving normally compared with control fish. The experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (approval no. RFiZOOHP2006083). After the low-temperature treatment, gill tissues dissected from 6 individuals were pooled as a sample and then homogenized in 5 ml Trizol reagent (Invitrogen, Carlsbad, CA). Thirty six individuals (18 for low-temperature treatment and 18 for control) were sacrificed for each microarray hybridization experiment, and another 60 individuals were used for quantitative reverse-transcription polymerase chain reaction. After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama). The quantity and quality of total RNA were assessed by spectrophotometry and agarose gel electrophoresis, respectively. The commercial zebrafish 14K oligonucleotides set (MWG Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufactureâs instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %. The detailed description of the oligonucleotides information can be obtained on the Ocimun Biosolution website (http://www.ocimumbio.com/web/default.asp). cDNA probes were synthesized by reverse transcription of 20 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and were labeled with Cy5 (cold treatment groups) and Cy3 (control groups)(Amersham Bioscience, Buckinghamshire, UK) , respectively. The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 °C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 µg/µl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (5 min), 1x SSC and 0.1% SDS (5 min), 0.5x SSC (5 min), and twice with 0.1x SSC (2 min each). Scanning was performed with a Genepix scanner (Molecular Devices, Sunnyvale, CA). The acquired images were analyzed using ScanArray Express 3.0 (PerkinElmer, Waltham, MA) and Genespring (Aglient Technologies, Foster City, CA) software. The measurements of spots were filtered by flags, and the lowest normalization was performed after subtraction of the median background. To assess the differential expressions of genes, we identified the ratio of Cy5/Cy3 intensity > 2 or < 0.5. Each experiment contained 3 biological replicates (including 1 dye swap) with different samples, and the differentially expressed genes were selected from those with at least 2 of 3 significant signals (ratio > 2 or < 0.5).
2010-06-20 | E-GEOD-7853 | biostudies-arrayexpress
Project description:Ambient temperature affects organisms comprehensively, however cold responses are different among tissues. Here, we adopt a transcript screening approach to explore and compare the cold responses in zebrafish gills and brain. Zebrafish were exposed to cold and the oligonucleotide-based microarray was used to identify cold-induced genes. Principle component analysis (PCA) of the gene expression profiles indicated that gills develop different strategies for the increasing of exposure period while brain relatively remained stable. Combining statistic and clustering methods, we found that gills showed higher protein metabolism and cell activity while brain showed higher stress responses and detoxification during cold acclimation. According to the microarray data sets, we extended the study on ionocyte- and isotocin neuron-related genes in gills and brain, respectively, and found these genes were broadly stimulated by cold. These data suggest that cold activates specific physiological functions in different tissues. Taken together, our results provide molecular evidences to elucidate the cold acclimation in zebrafish gills and brain. Keywords: Time course, Tissue types
2016-07-01 | GSE10958 | GEO
Project description:The fish gill is a multifunctional organ containing a variety of specialized cells including respiratory chemoreceptors, neuroepithelial cells (NECs). Although the structure, function and development of the gill have been studied extensively, transcriptomic profiling of individual gill cells is lacking. Using the 10x Genomics Chromium technology, we conducted a single transcriptomic study of cells from distal gill filament of ETvmat2:GFP zebrafish, acclimated to 14 days of normoxia and hypoxia. Overall, approximately 13,000 cells were sequenced with an average depth of 27,000 reads per cell. We identified 16 cell clusters in the gill, including NECs, neurons, pavement cells, endothelial cells and mitochondrion-rich cells. NECs were identified through expression of vmat2, encoding vesicular monoamine transporter, and showed highly differential expressions of tph1a, sv2, and mitochondrial proteins implicated in O2 sensing. Differential gene expression analysis showed a shift in transcriptome in NECs following 14 days of acclimation to hypoxia. This study presents a comprehensive cell atlas for the zebrafish gill and provides a framework for future investigations of molecular biology and physiology in gills.
2022-05-26 | GSE198044 | GEO
Project description:Ambient temperature affects organisms comprehensively, however cold responses are different among tissues. Here, we adopt a transcript screening approach to explore and compare the cold responses in zebrafish gills and brain. Zebrafish were exposed to cold and the oligonucleotide-based microarray was used to identify cold-induced genes. Principle component analysis (PCA) of the gene expression profiles indicated that gills develop different strategies for the increasing of exposure period while brain relatively remained stable. Combining statistic and clustering methods, we found that gills showed higher protein metabolism and cell activity while brain showed higher stress responses and detoxification during cold acclimation. According to the microarray data sets, we extended the study on ionocyte- and isotocin neuron-related genes in gills and brain, respectively, and found these genes were broadly stimulated by cold. These data suggest that cold activates specific physiological functions in different tissues. Taken together, our results provide molecular evidences to elucidate the cold acclimation in zebrafish gills and brain. Keywords: Time course, Tissue types After the low-temperature treatment, brain and gill tissues dissected from 10 individuals were pooled as a sample and then homogenized in 0.8 ml Trizol reagent (Invitrogen, Carlsbad, CA). 120 individuals (60 for low-temperature treatment and 60 for control) were sacrificed for each microarray hybridization experiment, and another 200 individuals were used for quantitative reverse-transcription polymerase chain reaction. After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama). The quantity and quality of total RNA were assessed by spectrophotometry and Bioanalyzer (Aglient Technologies, Foster City, CA). The commercial zebrafish 14K oligonucleotides set (MWG Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufactureâ??s instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %. The detailed description of the oligonucleotides information can be obtained on the Ocimun Biosolution website. cDNA probes were synthesized and purified by reverse transcription of 20 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and MinElute PCR purification kit (Qiagen) and were labeled with Alexa 647 dye (cold treatment groups) and Alexa 555 dye (control groups)(Invitrogen), respectively. The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 °C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 µg/µl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (5 min), 1x SSC and 0.1% SDS (5 min), 0.5x SSC (5 min), and twice with 0.1x SSC (2 min each). Scanning was performed with a Genepix scanner (Molecular Devices, Sunnyvale, CA). The acquired images were analyzed using Genepix and Genespring software (Aglient Technologies, Foster City, CA). The measurements of spots were filtered by flags, and the Lowess normalization was performed after subtraction of the median background. Each experiment contained 3 biological replicates (including 1 dye swap) with different samples. The differentially expressed genes were selected from those with at least 2 of 3 significant signals (ratio > 2 or < 0.5), and then the Significant Analysis of Microarray method (SAM 3.02) was used to determine statistical significances.
2016-07-01 | E-GEOD-10958 | biostudies-arrayexpress
Project description:Two azide mutagenized lines Freeze Resistance (FR, 75% survival) and Freeze Susceptible (FS, 30% survival) were compared with and without 4°C ± 1.5 cold acclimation of crown tissue to identify genes responsible for the difference in freeze resistance. Keywords: Wheat cold acclimation, stress response, cold, low temperature
2013-07-01 | GSE14697 | GEO
Project description:MicroRNAs (miRNAs) play vital roles in various biological processes by leading to mRNA cleavage or translational repression, and are also involved in multiple stress conditions. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, dre-miR-100-3p inhibitor or dre-miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. In summary, the present data show that miRNAs are closely involved in cold acclimation in fish and provide information for further understanding of the roles of miRNAs in cold acclimation.
2019-12-17 | GSE134399 | GEO
Project description:Long non-coding RNAs (lncRNAs) are increasingly regarded as a key role in regulating diverse biological processes in various tissues and species. So far, no data is available on screening and functional prediction of lncRNAs in cold acclimation in fishes. Here we compared expression profiles of lncRNAs in zebrafish embryonic fibroblast cells (ZF4) cultured at 28°C as control with that of cold acclimated cells cultured at 18°C for 30 days by high-throughput sequencing. There are 8363 novel lncRNAs were identified. Including known and novel lncRNAs, there are 347 lncRNAs up-regulated and 342 lncRNAs down-regulated in cold acclimated cells, compared with that of control cells at 28°C. Among differentially expressed lncRNAs, 74 and 61 were found only in control cells or in cold-acclimated cells, respectively. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses of genes adjacent to differentially expressed lncRNAs showed that enriched genes were involved in electron transport, cell adhesion, oxidation-reduction process, and so on. We predicted target genes of differentially expressed lncRNAs by looking for interactions between lncRNAs and mRNA, and constructed a complex interaction network. In summary, our genome-wide systematic identification and function prediction of lncRNAs in zebrafish cells, lays the foundation for future studies to elucidate crucial roles of lncRNAs in cold acclimation in fishes.
2018-04-18 | GSE107369 | GEO
Project description:Plants in temperate regions have evolved mechanisms to survive sudden temperature drops. Previous reports have indicated that the cold acclimation mechanism is light-dependent and does not fully operate under a low light intensity. In these studies, plants were grown under a long-day photoperiod and were more sensitive to freezing stress. However, winter annuals like Arabidopsis thaliana Col-0 germinate in the fall, overwinter as rosettes, and therefore must acclimate under short photoperiods and low irradiance. The role of light intensity was analysed in plants grown under a short-day photoperiod at the growth stage 1.14. Plants were acclimated at 4 °C for seven days under 100 and 20 μmol m-2s-1 PPFD for control and limited-light conditions, respectively. All cold acclimated plants accumulated molecular markers reportedly associated with acquired freezing tolerance, including proline, sucrose, CBFs, and COR gene protein products dehydrins and low-temperature-responsive proteins LTIs. Observed changes indicated that low PPFD did not inhibit the cold acclimation process, and the freezing stress experiment confirmed similar survival rates. The molecular analysis found distinct PPFD-specific adaptation mechanisms that were manifested in contrasting content of anthocyanins, cytokinin conjugates, abundances of proteins forming photosystems, and enzymes of protein, energy, and ROS metabolism pathways. Finally, this study led to the identification of putative proteins and metabolite markers correlating with susceptibility to freezing stress of non-acclimated plants grown under low PPFD. Our data show that Arabidopsis plants grown under short-day photoperiod can be fully cold-acclimated under limited light conditions, employing standard and PPFD-specific pathways.
2021-07-08 | PXD025234 | Pride
Project description:Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known. Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater. We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation. Thermal acclimation for as little as 6 hours at 8˚C enhanced crab tolerance during a 1h exposure to -2°C relative to crabs acclimated to 18˚C. Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm and cold acclimated crabs during the first day of thermal acclimation. No changes in gene expression were detected until 12h of thermal acclimation. Genes strongly upregulated in warm acclimated crabs represented immune response and extracellular / intercellular processes, suggesting that warm acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes. Genes strongly upregulated in cold acclimated crabs included many that are involved in glucose production suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant. Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold acclimated crabs. There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition. Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene expression-related changes in homeostasis begin within 12 hours – the length of a tidal cycle. all array data and raw images archived at the Porcelain Crab Array Database (http://array.sfsu.edu)
2012-03-01 | GSE35307 | GEO
Project description:Two azide mutagenized lines Freeze Resistance (FR, 75% survival) and Freeze Susceptible (FS, 30% survival) were compared with and without 4°C ± 1.5 cold acclimation of crown tissue to identify genes responsible for the difference in freeze resistance. Keywords: Wheat cold acclimation, stress response, cold, low temperature Experiment design (8 hybridizations): Genotype: SD16029 (FR) or SD16169 (FS) Temperature: 25°C or 4°C
2013-07-01 | E-GEOD-14697 | biostudies-arrayexpress