KEGG pathway analysis of cold-regulated genes.

<p>A. Up-regulated genes. B. Down-regulated genes. Up- and down-regulated genes were submitted for KEGG enrichment analysis using the SAS online analysis system. Color scales represent <i>P</i> values of enrichment tests and gray cells indicate an empty value or <i>P</i>>0.05.</p

Analyzing Cold Tolerance Mechanism in Transgenic Zebrafish (<i>Danio rerio</i>)

<div><p>Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish <i>Tg</i> (<i>smyd1:m3ck</i>) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter <i>Smyd1</i> was used to express the carp muscle form III of creatine kinase (<i>M3</i>-<i>CK</i>), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. <i>In situ</i> hybridization showed that <i>M3</i>-<i>CK</i> was expressed strongly in the skeletal muscle. When exposed to 13°C, <i>Tg</i> (<i>smyd1:m3ck</i>) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in <i>Tg</i> (<i>smyd1:m3ck</i>) versus wild-type fish at 28°C. After 2 h at 13°C, ATP concentrations were 2.16-fold higher in <i>Tg</i> (<i>smyd1:m3ck</i>) than in wild-type (<i>P</i><0.05). At 13°C, the ATP concentration in <i>Tg</i> (<i>smyd1:m3ck</i>) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28°C. Microarray analysis revealed differential expression of 1249 transcripts in <i>Tg</i> (<i>smyd1:m3ck</i>) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish.</p></div

Relative abundance of up- and down-regulated transcripts in the experimental groups.

<p>The numbers of differentially expressed genes are shown. Transcripts with fold change ≥2, <i>P</i> value <0.05, were considered to be differentially expressed.</p

GO enrichment analysis of up-regulated transcripts in transgenic and wild-type fish under cold stress.

<p>A. Biological processes of wt-13°C vs. wt-28°C up-regulated genes. B. Molecular functions of wt-13°C vs. wt-28°C up-regulated genes. C. Biological processes of m3ck-13°C vs. m3ck-28°C up-regulated genes. D. Molecular functions of m3ck-13°C vs. m3ck-28°C up-regulated genes. The horizontal axis represents transcript count in each GO term. Up-regulated transcripts were submitted for GO enrichment analysis using the SAS online analysis system.</p

GO enrichment analysis of down-regulated transcripts between transgenic and wild-type fish under cold stress or normal temperature.

<p>A. m3ck-28°C vs. wt-28°C down-regulated transcripts. B. m3ck-13°C vs. wt-13°C down-regulated transcripts. The horizontal axis represents transcript count in each GO term. Up- and down-regulated transcripts were submitted for GO enrichment analysis using the SAS online analysis system.</p

GO enrichment analysis of up-regulated transcripts between transgenic fish and wild-type fish under cold stress or normal temperature.

<p>A. m3ck-28°C vs. wt-28°C up-regulated transcripts. B. m3ck-13°C vs. wt-13°C up-regulated transcripts. The horizontal axis represents transcript count in each GO term. Up-regulated transcripts were submitted for GO enrichment analysis using the SAS online analysis system.</p

Expression of <i>m3ck</i> in transgenic fish by <i>in situ</i> hybridization.

<p>Whole-mount <i>in situ</i> hybridization was used to detect <i>m3ck</i> expression in wild-type (A) and <i>Tg</i> (<i>smyd1:m3ck</i>) fish (B) at 72 hpf. The signal was evaluated by microscopy for color detection of immunoreactive signals. Scale bars = µm.</p

Status of transgenic and non-transgenic fish at 13°C.

<p>The percentages smooth swimming, occasional swing, response to external stimulation, no movement, inversion, and dead fish were recorded. Data were analyzed by one-way ANOVA and <i>t-test</i> to compare data between fish lines. Data represent mean ± S.E.M. (<i>n</i> = 30). “*”indicates <i>P</i><0.05.</p

ATP concentration in transgenic and non-transgenic fish at 28°C or 13°C.

<p>The concentration of ATP in <i>Tg</i> (<i>smyd1:m3ck</i>) and wild-type fish was measured at the indicated times. Data were analyzed by one-way ANOVA and <i>t-test</i>. Data represent mean ± S.E.M. (<i>n</i> = 5). “*”indicates <i>P</i><0.05.</p

Overlapping expressed transcripts under cold stress.

<p>Venn diagrams represent the number of overlapping and differentially expressed genes between A. Up-regulated transcripts under cold stress (2 h) of <i>Tg</i> (<i>smyd1:m3ck</i>) and wild-type fish. B. Down-regulated transcripts under cold stress of <i>Tg</i> (<i>smyd1:m3ck</i>) and wild-type fish. C. Down-regulated transcripts under cold stress of <i>Tg</i> (<i>smyd1:m3ck</i>) and up-regulated transcripts in wild-type fish. D. Up-regulated transcripts under cold stress of <i>Tg</i> (<i>smyd1:m3ck</i>) and down-regulated transcripts in wild-type fish.</p