Table 1.
Observed endpoints at specific time points during the zebrafish early life stage test.
Fig 1.
mRNA expression signature in head, abdomen and tail of deiodinase knockdown larvae at 72 hpf.
The results of unsupervised hierarchical clustering (Pearson correlation, average linkage) generated on the total set of significantly differentially expressed transcripts in D1D2MO and D3MO in the head (H), abdomen (A) and tail (T) regions are shown at the top. This analysis shows (dis)similarities among expression patterns in the different body parts of the deiodinase knockdowns. The transcriptional response in the head was similar in both deiodinase knockdowns, and distinct from that in other body parts. In contrast, the response in abdomen and tail was knockdown-specific but similar among these two body parts. Bootstrap support values (in %) are denoted at each node, showing the level of confidence in each step of the clustering. The legend on the right shows the Pearson correlation coefficient. The bar chart gives an overview of the number of up- and downregulated transcripts for each knockdown in each body part. Pie charts summarize the most important GO classes (gene ontology: biological process) affected by each knockdown in each body part. GO classes were manually grouped into broad biological categories. GO classes received a score calculated in Blast2GO based on the number of sequences they contained and the level of their specificity. GPCR = G-protein coupled receptor signalling pathway. D1D2MO: combination of deiodinase type 1 and type 2 morpholino-injected, D3MO: deiodinase type 3 morpholino-injected, and SCMO: standard control morpholino-injected.
Fig 2.
Overview of transcriptional effects on purine metabolism and vision.
The figure shows the affected part of the KEGG pathway ‘purine metabolism’, and its link to vision. Small heat maps show logFC values of differentially expressed transcripts coding for the enzymes involved in the pathway. Green indicates downregulated (Log2FC ≤ -0.585, FDR ≤ 0.05), and red indicates upregulated (Log2FC ≥ 0.585, FDR ≤ 0.05) transcripts. White boxes indicate that transcripts were not differentially expressed. D1D2: combination of deiodinase type 1 and type 2 morpholino-injected, D3: deiodinase type 3 morpholino-injected, I: inhibitory subunit of PDE. Numbers refer to S2 Table listing the details of the differentially expressed transcripts.
Fig 3.
Overview of transcriptional effects on carbohydrate metabolism.
The figure shows carbohydrate metabolism starting with glycogen breakdown, and including glycolysis/gluconeogenesis, the citric acid cycle and oxidative phosphorylation. Small heat maps show logFC values of differentially expressed transcripts coding for the enzymes involved in the pathway. Green indicates downregulated (Log2FC ≤ -0.585, FDR ≤ 0.05), and red indicates upregulated (Log2FC ≥ 0.585, FDR ≤ 0.05) transcripts. White boxes indicate that transcripts were not differentially expressed. D1D2: combination of deiodinase type 1 and type 2 morpholino-injected, D3: deiodinase type 3 morpholino-injected. Numbers refer to S2 Table listing the details of the differentially expressed transcripts.
Fig 4.
Heat map of differentially expressed transcripts involved in protein metabolism in abdomen and tail region.
Green indicates downregulated (Log2FC ≤ -0.585, FDR ≤ 0.05), and red indicates upregulated (Log2FC ≥ 0.585, FDR ≤ 0.05) transcripts. White boxes indicate that transcripts were not differentially expressed. Grey boxes indicate that transcripts were removed from analysis in a specific body part because FG < BG + 2SD (See Materials and Methods). Abd: abdomen, D1D2MO: combination of deiodinase type 1 and type 2 morpholino-injected, D3MO: deiodinase type 3 morpholino-injected. All differentially expressed transcripts are listed with their details in S2 Table.
Fig 5.
Heat map of differentially expressed transcripts involved in developmental processes in abdomen and tail region.
Green indicates downregulated (Log2FC ≤ -0.585, FDR ≤ 0.05), and red indicates upregulated (Log2FC ≥ 0.585, FDR ≤ 0.05) transcripts. White boxes indicate that transcripts were not differentially expressed. Grey boxes indicate that transcripts were removed from analysis in a specific body part because FG < BG + 2SD (See Materials and Methods). Abd: abdomen, D1D2MO: combination of deiodinase type 1 and type 2 morpholino-injected, D3MO: deiodinase type 3 morpholino-injected. All differentially expressed transcripts are listed with their details in S2 Table.
Fig 6.
Phenotypic effects observed in D1D2MO and D3MO zebrafish larvae.
(A) heart rate at 24 and 48 hpf, (B) larval length at 96 hpf, (C) swim bladder inflation success at 96 hpf, (D) frequency of pericardial oedema at 96 hpf, (E) hatching success as a function of time, and (F) larval motility at 96hpf: boxplot with median, 10 and 90 percentile of the travelled distance during 45 minutes. In (A) and (B), data are shown as average ± SD and SD was calculated over all individual embryos. Knockdowns were initially compared to SCMO, and * indicates a significant difference from SCMO. If further differences were observed, additional comparisons were made, and different letters indicate significant differences among experimental groups. In (E), letters indicate significant differences among hatching curves taking all time points into account. D1D2MO: combination of deiodinase type 1 and type 2 morpholino-injected, D3MO: deiodinase type 3 morpholino-injected, UC: uninjected control. SCMO: standard control morpholino-injected.
Fig 7.
Effect of deiodinase knockdown on energy status of zebrafish larvae.
(A) carbohydrate content, (B) protein content, (C) lipid content, and (D) total energy content in zebrafish larvae at 72 hpf. Data are shown as average ± SD and SD was calculated over four biological replicate samples. Error bars in (D) show SD of total energy content. * indicates a significant difference from SCMO. X indicates a significant difference from the total energy content of UC. D1D2MO: combination of deiodinase type 1 and type 2 morpholino-injected, D3MO: deiodinase type 3 morpholino-injected, UC: uninjected control. SCMO: standard control morpholino-injected.