Figure 1.
Dose-response curves of vtg1 and esr1 expression in zebrafish embryos.
Zebrafish embryos were treated with increasing doses of E2 continuously for 4 days and the mRNA expression levels were determined by RT-qPCR. (A) Relative vtg1 mRNA expression. (B) Relative esr1 mRNA expression. Asterisk denotes significant differences (**P<0.01; unpaired Student’s t-test compared to the controls; n=2 biological replicates; 3 technical replicates within each biological replicate). Abbreviations vtg1: vitellogenin 1 and esr1: estrogen receptor 1.
Figure 2.
Distinct sets of genes are regulated by E2 during different times of zebrafish development.
(A) Venn diagram illustrating the number of differentially expressed genes (P≤0.01, fold change ≥|±1.4|) that were regulated in common at the different time points. (B) Venn diagram of the human homologues of gene transcripts from (A).
Figure 3.
Clustering of gene expression profiles of E2 and vehicle treatment groups at different time points.
The colored cells show the mean expression level of the biological replicates at each time point (1 dpf and 2 dpf: n=3; 3 dpf and 4 dpf: n=4). Red cells represent up-regulated genes, blue cells represent down-regulated genes and black cells represent unchanged expression levels between E2 and vehicle treated groups (P<0.005).
Figure 4.
Comparison of E2 regulated genes analyzed by microarray or RT-qPCR.
(A) Relative mRNA expression of the up-regulated genes vtg1, vtg3, vtg5, esr1, cyp19a1b and f13a1a at different time points as determined by RT-qPCR and microarray analysis. (B) Relative mRNA expression of the down-regulated genes hpx, fkbp5, fabp10a, agxtb, pnp4b, nlf1, f2, klf9, zgc: 92590 and zgc: 110053 at different time points as determined by RT-qPCR and microarray analysis. (C) Relative mRNA expression of the non-changed genes esr2a, esr2b, rbp2a and dlgap1a at different time points as determined by RT-qPCR and microarray analysis. White bars represent microarray results and black bars RT-qPCR results. Asterisk denotes significant difference (*P<0.05, **P<0.01; unpaired Student’s t-test compared to the controls), n≥3 biological replicates except for genes fabp10a, agxtb and zgc:110053 which were 2 biological replicates ; each replicate consists of 30 pooled embryos. Abbreviations vtg1: vitellogenin 1; vtg3: vitellogenin 3; vtg5: vitellogenin 5; esr1: estrogen receptor 1; esr2a: estrogen receptor 2a; esr2b: estrogen receptor 2b; cyp19a1b: cytochrome P450, family 19, subfamily A, polypeptide 1b; f13a1a: coagulation factor XIII, A1 polypeptide a, tandem duplicate 1; hpx: hemopexin; fkbp5: FK506 binding protein 5; fabp10a: fatty acid binding protein 10a; agxtb: alanine-glyoxylate aminotransferase b; pnp4b: purine nucleoside phosphorylase 4b; nxf1: nuclear RNA export factor 1; klf9: krueppel-like factor 9; f2: coagulation factor II (thrombin); rbp2a: retinol binding protein 2a and dlgap1a: discs, large (Drosophila) homolog-associated protein 1a.
Figure 5.
Developmental dynamics of E2 responsive tissues in Tg(5xERE:GFP) transgenic fish.
Zebrafish larvae were treated with 1 μM E2 (in 0.1% DMSO) from 3 hpf and imaged at 1 dpf (A-C), 2 dpf (D-F), 3 dpf (G-I), 4 dpf (J-L), 5 dpf (M-R) and 6 dpf (S-U). Arrows (white) indicate the liver; arrowheads (red) indicate the pancreas. A, D, G, J, M, P and S, bright-field images; B, E, H, K, N, Q and T corresponding GFP fluorescence images; C, F, I, L, O, R and U, overlay of bright-field and GFP images. A-C and S-U, lateral view; D-O, dorsal view; P-R, ventral view; anterior to the left. Scale bars, 100 μm.
Figure 6.
Validation of co-regulated differentially expressed genes in embryos and adult males using RT-qPCR.
(A) Relative mRNA expression of the up-regulated genes vtg1, vtg3, esr1 , cyp19a1b and f13a1a in adult males upon E2 treatment. (B) Relative mRNA expression of the genes eif4e1b, cyp11a1, dazl, zp3, cpn1, sult1st3 and f2 in 3 dpf and 4 dpf larvae as well as in adult males upon E2 treatment. **P<0.01; unpaired Student’s t-test compared to the controls. Abbreviations eif4e1b: eukaryotic translation initiation factor 4e 1b, cyp11a1 (cytochrome P450, subfamily XIA, polypeptide 1), dazl: deleted in azoospermia-like, zp3: zona pellucida glycoprotein 3, cpn1(carboxypeptidase N, polypeptide 1), sult1st3: sulfotransferase family 1, cytosolic sulfotransferase 3.