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.

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.

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.

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.

E2 up-regulates expression of vtg4 in the liver of 4 dpf DZ zebrafish embryos. Whole-mount ISH was performed with anti-sense vtg4 RNA probes on 4 dpf E2-treated embryos (A) and DMSO-treated embryos (B). ISH of sense vtg4 RNA probes on E2-treated embryos (C) and DMSO-treated embryos were performed as controls. Lateral view; anterior to the left. Arrow (red) indicates expression location of vtg4 in the liver. Scale bars, 200 μm.

Maternal effect of Tg(5xERE:GFP) transgenic fish at 5 hpf in the absence of E2. (A, B) Tg(5xERE:GFP) transgenic fish embryos. (C, D) Embryos from cross of female Tg(5xERE:GFP) transgenic fish and male wild type DZ fish. (E, F) Embryos from cross of male Tg(5xERE:GFP) transgenic fish and female wild type DZ fish. (G, H) Wild type DZ fish embryos. A, C, E and G, bright-field images; B, D, F and H corresponding GFP fluorescence images; Scale bars, 500 μm.

Endocrine pancreas is a novel E2 responsive tissue in embryonic zebrafish. Double transgenic Tg(5xERE:GFP)/Tg(ins:mCherry) embryos (4dpf) showing co-localization of GFP and mCherry signals in pancreatic islets upon E2 treatment. (A) GFP fluorescence image; (B) mCherry fluorescence image; (C) merged image of GFP and mCherry; (D) merged image of bright field (BF), GFP and mCherry. Arrows indicate the liver; arrowheads indicate the pancreatic islets. Dorsal view; anterior to the left. Scale bars, 100 μm.