ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Molecular characteristics of zebrafish Tmem39b. (A) Predicted structure of zebrafish Tmem39b protein, which contains 8 transmembrane domains. (B) A phylogenetic tree of TMEM39 proteins from the representative species. (C) Sequence identity between the TMEM39 proteins of several vertebrate species. (D) GFP-tagged zebrafish Tmem39b localizes to the endoplasmic reticulum. (E) Distribution of tmem39b transcripts in the tissues of adult zebrafish.

Knockout of the tmem39b gene sensitized zebrafish larvae to lethal cold stress. (A) Genotypes of the mutant lines. The mutations result in truncated proteins containing only 2 transmembrane domains. The genotypes are confirmed by DNA sequencing. The red box indicates the target sequence and the red arrows demonstrate the mutation sites. (B) Photos of the WT and zko3151b mutant larvae following exposure to cold-warming stress. The larvae were exposed to 10 °C for 24 h and photos were taken following the return to 28 °C for 12h. The red arrows point to dead fish. (C) Survival rates of WT and zko3151b mutant larvae following exposure to 10 °C for the indicated time points followed by recovery at 28 °C for 24 h. *, p < 0.05 (n = 4).

Dysfunction of the tmem39b gene decreases the cold resistance of adult zebrafish. (A) Survival rates of WT and zko3151b adults upon exposure to lethal cold stress. (B) Photos of the hematoxylin and eosin (H&E)-stained tissue sections. The scale bar represents 60 μm. (C) Fluorescence intensity of the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)-stained tissue sections. *, p < 0.05; **, p < 0.01 (n = 3). (D) Fluorescence images showing more severe cell apoptosis in tissues of the zko3151b mutants in comparison to the WT. The scale bar represents 150 μm.

Effects of Tmem39b dysfunction on gene transcriptional expression of zebrafish larvae during cold exposure and rewarming. (A) Results of principal component analysis (PCA) illustrate the overall change in gene expression between different treatments and fish lines. The dashed arrows show the trajectory of gene expression alteration during cold exposure and rewarming. The red double-headed arrow demonstrates the augmented distance between the WT and zko3151b mutants during the recovery phase. (B) Numbers of the genes differentially expressed between different treatments and fish lines. The up- and down-regulated gene sets (U and D) are numbered sequentially according to the comparisons. (C) Venn diagrams demonstrate the number of genes affected by tmem39b mutation under different circumstances. (D) A volcano plot indicates the genes affected by Tmem39b deficiency during rewarming. Representative genes induced by rewarming and negatively affected by tmem39b mutation are shown. NC, not changed; U6 and D6, the same as in (B); GS3 and GS4, the same as in (C).

Representative genes affected by Tmem39b dysfunction upon cold exposure and rewarming. Heatmaps demonstrate expressions of the representative up-regulated genes upon cold exposure (A) and rewarming (B). The up-regulation of these genes is attenuated in the tmem39b mutants in comparison to the corresponding genes in WT. (C) Transcriptional expression of the representative genes determined using qPCR. *, p ≤ 0.05; **, p ≤ 0.01 (n = 4).

Tmem39b protects the organism from cold-warm stress-induced DNA damage. The upper panel contains Western blots for the WT and tmem39b mutant samples collected at the indicated time points. The membranes were immunostained with antibodies against γH2AX and histone H3. The line charts illustrate the γH2AX/H3 ratio of optical density obtained by analyzing the corresponding protein bands.

Effects of Tmem39b dysfunction on endoplasmic reticulum stress response and autophagy. (A) A complex heatmap indicating the transcriptional expression of genes involved in endoplasmic reticulum stress response (red) and autophagy (blue). The genes were up-regulated at certain time points upon cold exposure or recovery under a normal temperature. The left part of the heatmap indicates the results of a previous study and the right part displays the data of this study. The rows of the heatmap represent genes and the columns show the averaged expression of the genes during different conditions. The row Z scores are calculated separately for the two experiments. Expression of representative genes involved in ER stress response (B) and autophagy (C) are determined by qPCR. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; #, p ≤ 0.05 (n = 4). “#” indicates significant difference in gene expression between WT and zko3151b under the same condition.

Biological processes and pathways influenced by tmem39b mutation. (A) A heatmap demonstrating the expression of DEGs across all the samples. The DEGs are classified into 15 clusters according to their abundance using the k-means clustering algorithm. The cluster names are shown on the right of the heatmap. The clusters regulated by both cold-warm stress and tmem39b mutation are shown in red, while those only influenced by a Tmem39b deficiency are shown in blue. The clusters associated with the functions of Tmem39b in determining cold resistance and damage repair are highlighted with red arrow heads. (B) Eigengenes of the gene clusters associated with the functions of Tmem39b. GO terms (C) and KEGG pathways (D) were enriched for the gene clusters affected by tmem39b mutation. FDR, false discovery rate; ratio, percentage of the identified genes to all the genes associated with a certain term.

Zebrafish Tmem39b regulates the production of C-reactive protein (CRP) during rewarming and a working model for Tmem39b’s function in determining cold resilience. (A) Deficiency of tmem39b attenuates the up-regulation of CRP in zebrafish larvae during the recovery phase following cold exposure. CRP concentration is normalized as μg/g protein. All the data points are shown. *, p ≤ 0.05. (B) A working model for zebrafish Tmem39b’s function.