Transcriptomic characterization of cold acclimation in larval zebrafish

Abstract

Background: Temperature is one of key environmental parameters that affect the whole life of fishes and an increasing number of studies have been directed towards understanding the mechanisms of cold acclimation in fish. However, the adaptation of larvae to cold stress and the cold-specific transcriptional alterations in fish larvae remain largely unknown. In this study, we characterized the development of cold-tolerance in zebrafish larvae and investigated the transcriptional profiles under cold stress using RNA-seq.

Results: Pre-exposure of 96 hpf zebrafish larvae to cold stress (16°C) for 24 h significantly increased their survival rates under severe cold stress (12°C). RNA-seq generated 272 million raw reads from six sequencing libraries and about 92% of the processed reads were mapped to the reference genome of zebrafish. Differential expression analysis identified 1,431 up- and 399 down-regulated genes. Gene ontology enrichment analysis of cold-induced genes revealed that RNA splicing, ribosome biogenesis and protein catabolic process were the most highly overrepresented biological processes. Spliceosome, proteasome, eukaryotic ribosome biogenesis and RNA transport were the most highly enriched pathways for genes up-regulated by cold stress. Moreover, alternative splicing of 197 genes and promoter switching of 64 genes were found to be regulated by cold stress. A shorter isoform of stk16 that lacks 67 amino acids at the N-terminus was specifically generated by skipping the second exon in cold-treated larvae. Alternative promoter usage was detected for per3 gene under cold stress, which leading to a highly up-regulated transcript encoding a truncated protein lacking the C-terminal domains.

Conclusions: These findings indicate that zebrafish larvae possess the ability to build cold-tolerance under mild low temperature and transcriptional and post-transcriptional regulations are extensively involved in this acclimation process.

Figure 1

Establishment of cold resistance in zebrafish larvae after pre-exposure to cold stress. (A) Flowchart of cold exposure. Zebrafish embryos were incubated at 28°C from fertilization to 96 hpf. Larvae at 96 hpf were exposed to 16°C for 24 h and the controls were maintained at 28°C. Samples for RNA-seq were collected at 120 hpf. The pre-treated and control larvae were further exposed to 12°C for 6, 12, 24, 36 and 48 h. The time scales were shown on the left. (B) Death rates of pre-treated and control (ck) larvae exposed to 12°C for different times. Data was shown as mean ± standard deviation (n = 4). “**” above error bars indicate p < 0.01. (C) Images of pre-treated and control larvae exposed to 12°C for 36 h. Images were taken under a stereomicroscope from Zeiss with a color CCD camera. Red arrowheads indicate representative dead larvae.

Figure 2

Bioinformatic analysis of RNA-seq data. (A and B) Distribution of FPKM values for genes expressed in the control (A) and cold-treated (B) zebrafish larvae. The red interpolation line denotes a bimodal distribution of the frequency of FPKM. The blue dashed line indicates the FPKM value for the second peak of frequency. (C) Principle component analysis of gene expression in the control (ck) and cold-treated samples. (D) Correlation of gene expression between the control and cold-treated group. The up- and down-regulated genes were shown in red and blue, respectively. Genes not regulated by cold treatment were shown in green.

Figure 3

Validation of RNA-seq data using qPCR. Fold changes of gene expression detected by RNA-seq were plotted against the data of qPCR. The reference line indicates the linear relationship between the results of RNA-seq and qPCR.

Figure 4

Alternative promoter usage of per3 under cold stress. (A) Read coverage at per3 locus. The top panel shows the read coverage of each sample at per3 locus and the bottom panel indicates the structure of per3 transcripts. (B) The relative abundance of per3 isoforms under cold stress. (C) Promoter switching of per3 under cold stress. (D) qPCR validation of the alternative promoter usage for per3. Error bars indicate standard deviation (n = 3).

Figure 5

GO enrichment analysis for genes up-regulated by cold stress. The size of circles is proportional to the number of genes associated with the GO term. The arrows represent the relationship between parent–child terms. The color scale indicates corrected p-value of enrichment analysis.

Figure 6

GO enrichment analysis for genes down-regulated by cold stress. The size of circles is proportional to the number of genes associated with the GO term. The arrows represent the relationship between parent–child terms. The color scale indicates the corrected p-value of enrichment analysis.

Figure 7

Cold-induced genes associated with the spliceosome pathway. Gene expression value was mapped to the reference pathway using the KegArray. Up-regulated genes are shown in yellow.

Figure 8

Cold-induced genes associated with the ribosome biogenesis in eukaryotes pathway. Gene expression value was mapped to the reference pathway using the KegArray. Up-regulated genes are shown in yellow.

Figure 9

Differential splicing of stk16 under cold stress. (A) Read coverage at stk16 locus. The top panel shows the read coverage of each sample at sk16 locus and the bottom panel indicates the structure of stk16 transcripts. The red arrowheads represent relative positions of primers for RT-PCR. The dashed red box indicates the unchanged coverage of exon 2 under cold stress. (B) RT-PCR analysis to reveal the cold-specificity of stk16-J2. Stk16-J2-F and stk16-J2-R primers were displayed in (A). (C) The relative abundance of stk16 isoforms under cold stress. (D) Partial peptide sequence alignment of Stk16-J1 and Stk16-J2. The identical amino acids are shown in yellow.