Generation and validation of the cecr1b-LoF zebrafish models.

a Representative brightfield images of the CHT of 3 dpf embryos stained with the Leucognost-Pox colorimetric assay for neutrophils detection. Scale bar: 300 microns. b Quantification of neutrophils number in the CHT. Each dot represents the count of a single embryo (mean ± SD). Statistical significance was assessed by two-tailed, unpaired t-student test and Ordinary one-way ANOVA test with Tukey’s correction for cecr1b-MOs embryos (ctrl, n = 38 embryos; cecr1b-MOs n = 37 embryos) and cecr1a/cecr1b-sgRNA embryos (Cas9, n = 35 embryos; cecr1a-sgRNA, n = 28 embryos; cecr1b-sgRNA, n = 29 embryos), respectively; ***, p < 0.001; *, p < 0.05; ns, not significant (c) Representative confocal images of Tnfα expressing cells in the trunk-tail region of 24–26 hpf embryos. Magnification: 20X. Scale bar: 100 microns. d Count of Tnfα+ cells in the trunk region. Each dot in the graph represents the count of a single embryo (mean ± SD). Statistical significance was assessed by two-tailed, unpaired t-student test; **p < 0.002 (ctrl, n = 10 embryos; cecr1b-LoF, n = 13 embryos) (e) RT-qPCR results of pro-inflammatory (il1β, tnfα, il6) and anti-inflammatory (il10) cytokines in 3 dpf embryos (mean ± SEM). n = 3 biologically independent experiments. Statistical significance was assessed by a two-tailed unpaired t-student test; *p < 0.05, ns not significant.

Analysis and modulation of the A_2bR pathway, which is dysregulated in the endothelium of cecr1b-LoF embryos.

a Schematic representation of the A_2bR-dependent eAdo signaling pathway. A_2bR activation in the endothelium leads to the expression of HSPC-specific genes (runx1 and cmyb) through activation of the cAMP/Pka. Expression levels of (b) cxcl8 and (c) cmyb measured by RT-qPCR in endothelial cells sorted (ECs) from 30–32 hpf Tg(fli1a:GFP)^y1. Data are presented as mean ± SEM; n = 3 biologically independent experiments. Statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; ***p < 0.001; **p < 0.002; ns, not significant. d Representative images of WISH staining for the HE markers runx1 and scl in the trunk region of 24–26 hpf embryos and for the specifying HSPCs marker cmyb in 30-32 hpf embryos. DA dorsal aorta, PCV posterior cardinal vein, s-HSPCs specifying HSPCs, HE hemogenic endothelium. Numbers indicate the embryos belonging to the representative phenotype of each category, shown in the image. Scale bar = 100 microns. e WISH staining of the arterial (efnb2) and venous (efnb4) markers in the trunk region of 28–30 hpf embryos. DA dorsal aorta, PCV posterior cardinal vein. Red arrowheads indicate the region of the DA, blue arrowheads indicate the region of the PCV. Scale bar = 100 microns.

Defective HSPC emergence from HE and altered CHT colonization.

a Sequence of images from time-lapse experiments on Tg(kdrl:GFP) ctrl and cecr1b-LoF embryos from 30 hpf. Yellow arrowheads indicate HSPCs emerging from the ventral wall of the dorsal aorta in the trunk region. DA dorsal aorta, PCV posterior cardinal vein. Magnification 20X, scale bar = 50 microns. b Representative fluorescence images of the CHT region of 3 dpf Tg(CD41:GFP) ctrl and cecr1b-LoF embryos. The red dotted line divides the CHT into the anterior and posterior parts. Scale bar = 150 microns. c Quantification graph of the CD41+ HSPC number (mean ± SD) in the CHT region of ctrl and cecr1b-LoF embryos at the stage of 3 dpf. Each dot represents the count of a single embryo. Statistical significance was assessed by a two-tailed, unpaired t-student test. ***p < 0.001. (ctrl, n = 29 embryos; cecr1b-LoF, n = 41 embryos). d Histograms showing the percentage of embryos with completely colonized (white) or partially colonized (gray) CHT. Data are presented as the mean ± SD of independent experiments. n = 3 biologically independent experiments. Statistical significance was assessed with the Chi-square test (Fisher’s exact test, confidence interval 95%). ***p < 0.001. e Representative fluorescence images of the CVP region of 3 dpf Tg(kdrl:GFP). The red dotted line divides the CVP region into the anterior and posterior parts. Quantification graph of the GFP+ area in the (f) anterior and (g) posterior part of the CVP. Each dot represents the count of a single embryo. Data are presented as mean ± SD. Statistical significance was assessed with an unpaired t-student-test. ***p < 0.001; **p < 0.002 (ctrl, n = 18 embryos; cecr1b-LoF, n = 28 embryos).

Analysis of the defective HSPC population in the CHT of cecr1b-LoF embryos and its correction through A_2bR modulation.

a Representative fluorescence images of the CHT region of 3 dpf Tg(CD41:GFP) embryos. Scale bar: 150 microns. b Quantification of CD41+ HSPCs in the CHT in cecr1b-LoF embryos treated with H89 from early or late time. Each dot represents the count of a single embryo. Data are presented as mean ± SD. Statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; ***, p < 0.001; **, p < 0.002; *, p < 0.05; ns not significant (ctrl, n = 96 embryos; cecr1b-LoF, n = 82 embryos; cecr1b-LoF+H89 (early), n = 52 embryos; cecr1b-LoF+H89 (late), n = 15 embryos). c Histogram showing the proportion of embryos showing complete or defective CHT colonization in the different experimental conditions (mean of biological experimental replicates±SD). n = 3 biologically independent experiments. Statistical significance was assessed by multiple Chi-Square analyses (Fisher’s exact test, confidence interval 99%); ***p < 0.001; **p < 0.002; *p < 0.05; ns not significant.

Analysis of the defective HSPC population in the CHT of cecr1b-LoF embryos and its correction trough tnfα modulation.

RT-qPCR expression levels of (a) tnfα and (b) il1β measured in endothelial cells sorted from 30-32 hpf Tg(fli1a:GFP) ctrl and cecr1b-LoF embryos untreated or treated with CGS. Data are presented as mean ± SEM. n = 3 biologically independent experiments. Statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; **p < 0.002; ns, not significant. c Representative fluorescence images of the CHT region of 3 dpf Tg(CD41:GFP) embryos belonging to the different experimental categories. Scale bar: 150 microns. The red dotted line divides the CHT region into the anterior and posterior parts. d Quantification graph of CD41+ HSPCs in the CHT of ctrl 3 dpf embryos belonging to the different experimental categories. Each dot represents the count of a single embryo. Data are presented as mean ± SD. Statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; ***, p < 0.001; **, p < 0.002; (ctrl, n = 62 embryos; cecr1b-LoF, n = 48 embryos; cecr1b-LoF+tnfα-ATGMO, n = 58 embryos); e Histogram showing the proportion of embryos with complete or defective CHT colonization in the different experimental conditions (mean ± SD). n = 3 biologically independent experiments. Statistical significance was assessed by multiple two-sided Chi-Square analysis (confidence interval 99%); ***p < 0.001; *p < 0.05. Representative bright-field images (f) and quantification graph (g) of neutrophils stained with the Leucognost-Pox colorimetric assay in the CHT of 3 dpf embryos. Scale bar: 300 microns. Each dot represents the count of a single embryo. Data are presented as mean ± SD. Statistical significance statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; ***p < 0.001, ns, not significant (ctrl, n = 100 embryos; cecr1b-LoF, n = 107 embryos; cecr1b-LoF+tnfα-ATGMO, n = 104 embryos).

Recovery of the cecr1b-dependent hematopoietic phenotypes through supplementation of the rhADA2.

a Representative fluorescence images of the CHT region of 3 dpf Tg(CD41:GFP) embryos belonging to the different experimental conditions. The red dotted line divides the CHT region into the anterior and posterior parts. Scale bar: 150 microns. b Quantification graphs of CD41+HSPC number in the CHT region of the embryos. Each dot represents the count of a single embryo (mean ± SD). Statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; ***p < 0.001; *p < 0.05 (ctrl, n = 109 embryos; cecr1b-LoF, n = 109 embryos; cecr1b-LoF+rhADA2, n = 107 embryos). c Histogram showing the proportions of embryos showing complete or defective CHT colonization (mean ± SD). n = 5 biologically independent experiments. Statistical significance was assessed by multiple two-sided Chi-Square analysis (Fisher’s exact test, confidence interval 99%); ***p < 0.001; *p < 0.05. d Representative confocal images of the CHT region of 3 dpf embryos stained for CD41/GFP and the proliferation marker pH3 with double immuno-fluorescence. Yellow arrowheads indicate double CD41-GFP+/pH3+ cells. Magnification: 20X. Scale bar: 100 microns. e The proliferation rate was estimated as the number of double CD41-GFP+/pH3+ cells normalized on the total number of CD41-GFP+ in the CHT of each embryo. Statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; *p < 0.05, ns not significant (ctrl, n = 6 embryos; cecr1b-LoF, n = 6 embryos; cecr1b-LoF+rhADA2, n = 5 embryos). f Representative confocal images of the CHT region of 2.5 dpf embryos stained with the apoptotic TUNEL assay. Magnification: 20×. Scale bar: 100 microns (ctrl, n = 4 embryos; cecr1b-LoF, n = 4 embryos; cecr1b-LoF, n = 4 embryos). g, h Representative images and quantification of neutrophils stained with the Leucognost-Pox colorimetric assay in the CHT of 3 dpf embryos. Scale bar: 300 microns. Each dot in the graph represents the count of a single embryo (mean ± SD). Statistical significance was assessed by Ordinary one-way ANOVA test with Tukey’s correction; ***p < 0.001 (ctrl, n = 24 embryos; cecr1b-LoF, n = 34 embryos; cecr1b-LoF+rhADA2, n = 36 embryos).

ADA2 has a role in regulating inflammation and HSPC emergence via the A_2bR pathway in the cecr1b-LoF zebrafish model.

Schematic representation of the function of ADA2 validated in the cecr1b-LoF zebrafish model.