Fig 2: Ifi30 overexpression increases the number of HSPCs in the CHT and rescues the loss of HSPCs after ROS challenge.a Experimental outline to generate double-transgenic kdrl:Gal4;UAS:ifi30. b WISH for ifi30 at 24 hpf in kdrl:Gal4+ embryos that were either UAS:ifi30 negative (upper) or positive (lower). c WISH for cmyb at 4 dpf in kdrl:Gal4+ embryos that were either UAS:ifi30 negative (upper) or positive (lower). d WISH against cmyb at 48 hpf in kdrl:Gal4+ embryos that were either UAS:ifi30- (upper) or UAS:ifi30+ (lower), NT and H2O2-treated. e Quantification of cmyb-expressing cells. Statistical analysis: one-way ANOVA with Tukey–Kramer post hoc tests, adjusted for multiple comparisons, **P = 0.013; ****P < 0.0001. Center values of all statistical analyses denote the mean, and error values denote s.e.m. Scale bars: 200 µm (b, c), 100 µm (d).

Fig 3: HSPCs interact with IFI30+ cells in human fetal liver.a Double immunostaining against GILT/IFI30 and CD117 on sections of human fetal liver at different stages of gestation. b Distribution of GILT/IFI30+ cells (red line), CD117+ cells (brown line), and IFI30+ cells in contact with CD117+ cells (green line). For each developmental stage, only one human fetal liver sample was used (N = 1), but three different sections were analyzed. c High magnification of double immunostaining against GILT/IFI30 (red) CD117 (brown) and nuclei (blue), on sections of human fetal liver at 13 weeks of gestation. Scale bars: 10 µm. d Double immunostaining against GILT/IFI30 (black), CD68 (cyan), and nuclei (blue) in sections of human fetal liver at 13 weeks of gestation. All red arrows point to double-stained cells. Scale bars: 100 µm (a); 25 µm (c); 200 µm. Right panel shows a magnification of the region delimited by the red dotted line (d).

Fig 4: ifi30 is expressed in CHT endothelial cells.a WISH against ifi30 at 24, 33, and 48 hpf. b Double WISH at 48 hpf against ifi30 (purple) and cdh5 (red), black arrows indicate the expression of cdh5 in dorsal aorta (DA) and the overlapping expression with ifi30 in the posterior cardinal vein (PCV). c qPCR data examining ifi30 expression (fold change relative to expression in kdrl:GFP+ head subset) in FACS-sorted cells from kdrl:GFP, ikaros:GFP, and mpeg1:GFP embryos. GFP+ cells from dissected heads and tails were sorted from 48 hpf embryos (for each transgenic line have been pooled n = 20 embryos), and each experiment was repeated independently three times. Statistical analysis was performed using a one-way ANOVA with Tukey–Kramer post hoc tests, adjusted for multiple comparisons, *P = 0.013; **P = 0.001; ****P < 0.0001. Center values denote the mean, and error values denote s.e.m. d Imaged area in the tail at 48 hpf, as indicated by the black dotted line. Confocal imaging in the CHT of kdrl:mCherry/cmyb:GFP embryos in non-injected and ifi30-full mRNA injected embryos. The white arrows indicate kdrl and cmyb double-positive cells. e Quantification of HSPCs associated to ECs. Statistical analysis: unpaired two-tailed t test ****P < 0.0001. Center values denote the mean, and error values denote s.e.m. Scale bars: 200 µm (a, b); 50 µm (d).

Fig 5: ifi30-deficiency increases ROS levels in ECs and induces a defect in HSPC proliferation.a cmyb expression at 48 hpf in wild type and ifi30-/- embryos, after the injection of control or ifi30-morpholino. b Quantification of cmyb-expressing cells. Statistical analysis: one-way ANOVA, with Tukey–Kramer post hoc tests, adjusted for multiple comparisons, ****P < 0.0001, (n.s.) non-significant P = 0.84; P = 0.32. Center values of all statistical analysis denote the mean, and error values denote s.e.m. c Confocal imaging in the CHT of kdrl:GFP+ cells (green), CellROX fluorescent probe (red) and merge (yellow). d Representative FACS plots showing dissociated zebrafish embryos after injection of control or (e) ifi30-morpholinos. FACS plots are gated on live cells. f Histogram plot showing the overlap of both samples, gated on kdrl+ cells. g Quantification of the percentage of kdrl:GFP+ cells affected by oxidative stress (ROS+) in ifi30-morphants (ifi30-MO) and controls (ctrl-MO) at 48 hpf. Data points are the mean of n = 4 biological replicates ± SD. Statistical analysis: unpaired two-tailed t test, **P = 0.007. Center values of all statistical analysis denote the mean, and error values denote s.e.m. h Fluorescence imaging in the CHT of kdrl:mCherry;cmyb:GFP embryos injected with control- and ifi30-MOs and treated with GSH. i Quantification of HSPCs associated with ECs. Statistical analysis: one-way ANOVA, with Tukey–Kramer post hoc tests, adjusted for multiple comparisons, *P = 0.028; **P = 0.008; ****P < 0.0001. The center values of all statistical analyses denote the mean, and error values denote s.e.m. Scale bars: 200 µm (a), 100 µm (c, d).