Signalling pathway dysregulation in human FGR samples. Quantitative RT-PCR analysis performed on umbilical cords from FGR cases and AGA controls, focusing on ten signalling pathways, identified significant upregulation in seven pathways (Hif/Hypoxia, Wnt/β-catenin, Jak/Stat3, Notch, Bmp, TGFβ, Hippo/YAP-TAZ), downregulation in two (FGF, Shh) and no modification in one (CREB). Sample size: n = 8. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001, Log2 FC stands for Log2 Fold Change. Test: Unpaired t-test.

Long-term hypoxia treatment leads to growth restriction in zebrafish embryos. (A–E) Morphological analysis of 3-dpf zebrafish embryos after a long-term (2-days long) treatment in 5% hypoxia; hypoxia-treated embryos display reduced body length (A–C) and ocular size (D), poorly absorbed yolk and a pericardial effusion (arrows in B′) leading to a significant enlargement of the cardiac region size (E). All embryos are in lateral view, anterior to the left. Sample size: n = 45; **** = p < 0.0001; R.R. = Relative Ratio. Test: Unpaired t-test.

Long-term hypoxia induces cardiovascular modifications in zebrafish embryos/larvae. (A–D) After a 5-days incubation in 5% hypoxia, 6-dpf zebrafish larvae display enlarged heart and inflated myocardial layer, as evidenced by the fluorescence of the myocardial transgene Tg(tg:EGFP-myl7:EGFP)^ia300. (E,F) After a 2-days incubation in 5% hypoxia, 3-dpf zebrafish embryos display disorganized intersegmental vessels (arrows), identified by the endothelial transgene Tg(fli1:EGFP)^y1. 6-dpf larvae and 3-dpf embryos are in lateral view, anterior to the left. Sample size: n = 10; ** = p < 0.01; *** = p < 0.001; R.I. = Relative Intensity; R.D. = Relative Diameter. Test: Unpaired t-test.

Hyper-activation of Hif/Hypoxia, Wnt/β-catenin and Jak/Stat3 signalling in the cardiovascular and brain regions of hypoxia-treated zebrafish. The GFP-based (green) reporters for Hif/Hypoxia (A–C), Wnt/β-catenin (D–F) and Jak/Stat3 (G–I) signalling show upregulation of the three pathways in all considered anatomical regions. All embryos are at 3 dpf and displayed in lateral view, anterior to the top. br = brain; he = heart; ph = pharynx. Sample size: n = 6 per condition; ns = not significant; * = p < 0.05; ** = p < 0.01; R.I. = Relative Intensity. Test: Unpaired t-test.

Stat3 signalling modulation does not rescue FGR anomalies. Downregulation of Stat3 signalling with 50 μM AG490 inhibitor does not rescue body length, eye size and cardiac morphology under hypoxia. Upregulation of Stat3 signalling with 20 μM LIF agonist does not rescue body length, eye size and cardiac morphology under hypoxia. All embryos, at 3 dpf, are displayed in lateral view, anterior to the left. Sample size: n = 15; **** = p < 0.0001; ns = not significant; R.R. = Relative Ratio. Test: One-way ANOVA followed by Tukey’s test.

Wnt/β-catenin signalling upregulation can rescue FGR anomalies. Downregulation of Wnt/β-catenin signalling with 5 µM XAV939 (XAV) inhibitor does not rescue body length or eye size, and worsens the pericardial effusion under hypoxia. Upregulation of Wnt/β-catenin signalling with 40 µM SB216763 (SB) agonist can rescue the body length and the pericardial effusion under hypoxia. All embryos, at 3 dpf, are displayed in lateral view, anterior to the left. For display purposes, images of control embryos under normoxia and hypoxia correspond to control images of Figure 5, as the same control conditions were used for both Stat3 and Wnt experiments. Sample size: n = 15; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant; R.R. = Relative Ratio. Test: One-way ANOVA followed by Tukey’s test.

Wnt/β-catenin upregulation can rescue hypoxia-induced vascular defects. Upregulation of Wnt/β-catenin signalling using 40 µM SB216763 (SB) can rescue the vascular organization (reciprocal distance and vessel diameter) in hypoxia-treated embryos. Conversely, downregulation of Wnt/β-catenin signalling using 5 µM XAV939 leads to a worsened phenotype. Intersegmental vessels (arrows) were inspected in 3 dpf embryos, after 2-days long incubation in normoxia (control) or 5% hypoxia, exploiting the endothelial-specific transgene Tg(fli1:EGFP)^y1. All pictures display trunk regions in lateral view, anterior to the left. Sample size: n = 15; ns = not significant; * = p < 0.05; ** = p < 0.01; **** = p < 0.0001; R.D. = Relative Distance. Test: One-way ANOVA followed by Tukey’s test.