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 Î¼g 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 Â°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 Âµg/Âµl 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).

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Zebrafish il-4 il-10 regulated immunity in gills

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