a, b Brightfield image of a wild-type or ptena^−/−ptenb^−/− mutant zebrafish embryo at 35 hpf. The area from which HSPCs originate is indicated with a yellow box. A close up is indicated with a white box. c–h Four-dimensional imaging of tg(kdrl:eGFP) wild-type or ptena^−/−ptenb^−/− mutant embryos between 35 and 48 hpf. Still frames from Movie S1. Arrowheads: HSPCs undergoing EHT; asterisk: disintegrating HSPCs. Confocal image z-stacks (2 µm step size, with ×40 objective and ×2 zoom; anterior to the left; maximum projections of a representative embryo; time in hh: mm. i–l Acridine orange staining. Arrows and circles: HSPCs in VDA of 40–45 hpf embryos. Asterisks: apoptotic HSPCs. Scale bar: 50 µm. Representative embryos are shown and the number of embryos that showed this pattern/total number of embryos is indicated. DA dorsal aorta, PCV posterior cardinal vein. m–p confocal images of apoptotic endothelial cells in the VDA of fixated wild-type or ptena^−/−ptenb^−/− mutant zebrafish embryos. In green: tg(kdrl:eGFP); in red: anti-activated caspase-3 immunohistochemistry staining. Apoptotic cells are indicated with an asterisk. Representative embryos are shown and the number of embryos displaying this particular pattern/total number of embryos is indicated in the bottom right. Anterior to the left; 2 µm step size; maximum projections; scale bar: 100 µm.

a The VDA of tg(kdrl:Dendra2) was photoconverted green-to-red at 26–28 hpf. By 50–60 hpf red HSPCs derived from the photoconverted VDA had colonized the CHT in b sibling and cptena^−/−ptenb^−/− larvae. d The number of GFP^low HSPCs at 48 hpf in the CHT of tg(cd41:eGFP) siblings (sib) (n = 25) (e) and ptena^−/−ptenb^−/− mutants (mut) (n = 12) (f) is expressed as average number of cells in siblings (n = 41) or ptena^−/−ptenb^−/−. Mutants (n = 18) (g) is expressed as average number of cells in siblings (n = 33) or ptena^−/−ptenb^−/− mutants after injection with synthetic ptenb-mRNA (n = 15). Error bars indicate standard error or the mean (SEM). Shapiro–Wilk test for normal distribution and Welch’s two-tailed t-test were used for statistical analysis; ***p < 0.001. Representative embryos are shown and the number of embryos that showed this pattern/total number of embryos is indicated.

a–g Four-dimensional imaging of tg(kdrl:eGFP) transgenic embryos. a–cptena^−/−ptenb^−/− mutant embryos and d–g wild-type embryos. Imaging was done from 35 hpf onwards following treatment with 5 µM LY294002 from 32 hpf. Still frames from movie S3 (a–c) and movie S4 (d–g). arrowheads: HSPCs. Asterisks: disintegrating HSPCs. Different colors of arrowheads distinguish separate EHT events. Images were taken with ×40 objective and ×1 zoom. Time in hh:mm; DA dorsal aorta, PCV posterior cardinal vein. h, i CHTs of tg(kdrl:mCherry-CAAX/cd41:eGFP) control (n = 10) and LY294002-treated (5 µM, 32–50 hpf) (n = 16) embryos were imaged at 50 hpf. The vasculature is highlighted in red (mCherry) and some GFPlow HSPCs are indicated by arrows. j, k CHTs of tg(cd41:eGFP) control (n = 11) and LY294002-treated (5 µM, 30–60 hpf) (n = 19) embryos were imaged at 4 dpf. Anterior to the left; 2 µm step size. Representative embryos are shown and the number of embryos that showed this pattern/total number of embryos is indicated. The number of GFP^low HSPCs was determined at 50 hpf (l) and 4 dpf (m) and is expressed as average number of cells; error bars indicate standard error of the mean (SEM). Shapiro–Wilk test for normal distribution and Welch’s two-tailed t-test were used for statistical analysis; ***p < 0.001.

a–h Control and LY294002-treated (from 32–60 hpf) embryos were fixed at 4 dpf. Markers for definitive blood lineages were assessed by in situ hybridization in the CHT: c-myb (HSPCs; a, b), globin (erythrocyte lineage; c, d), ikaros (lymphocyte lineage; e, f), l-plastin (leukocytes; g, h). Representative embryos are shown, with anterior to the left. The number of embryos that showed a particular pattern/total number of embryos is indicated in the bottom right corner of each panel. i, j GFP^high thrombocytes were imaged at 5 dpf in tg(cd41:eGFP) embryos. Scale bar: 100 µm. k–n High-resolution imaging at 12 dpf of kidney (k, l, dorsal view) (control, n = 6; LY294002-treated, n = 8; 4 µm step size) and thymus (m, n, lateral view) (control, n = 6; LY294002-treated, n = 7; 2 µm step size). Anterior to the left; maximum projections of representative larvae. Scale bar: 100 µm.

Tissue from control and LY294002-treated embryos (~2000 each) was dissected, the AGM regions pooled, dissociated and FACS sorted, after which the SORT-seq protocol was performed. a–e t-SNE maps highlighting the expression of marker genes for each of the different cell types found. Transcript counts are given in a linear scale. a HSPCs I, b HSPCs II, c EHT progenitor, d Myeloid/monocyte progenitors, e Myeloid/neutrophil progenitors. f t-SNE map highlighting the distribution of cells from LY294002-treated embryos and their controls (g). Visualization of k-medoid clustering and cell-to-cell distances using t-SNEs. Each dot represents a single cell. Colors and numbers indicate cluster and correspond to colors in i. The distribution of in total 2512 cells over the five clusters are shown as percentage of total for control and LY294002-treated embryos. Fisher’s exact test with multiple testing correction (Fdr) were used for statistical analysis. ***p < 0.001. cl1: Myeloid/neutrophil progenitor, cl2: HSPC II, cl3: myeloid/monocyte progenitor, cl4: HSPC I, cl5: EHT progenitor. h Normalized expression level of ENSDARG00000080337_ACO24175.4 for HSPC I and HSPC II cluster using violin plots. Normalized expression is plotted on a log10 scale.

CHTs of control and LY294002-treated embryos (5 dpf, ~100 embryos each) were dissected, pooled, dissociated, FACS sorted and submitted to SORT-seq. a Visualization of k-medoid clustering and cell-to-cell distances using t-SNEs. Each dot represents a single cell. Colors and numbers indicate cluster and correspond to colors in h. In total, 684 cells are shown. b–f t-SNEs maps highlighting the expression of marker genes for each of the different cell types found. Transcript counts are given in a linear scale. b Erythrocyte progenitors, c Thrombocyte/erythrocyte progenitors, d HSPCs, e Myeloid progenitors, f Neutrophil progenitors. g t-SNE map highlighting the distribution of LY294002-treated embryos and their controls (h). The percentage of cells from LY294002-treated embryos and their controls in the different clusters. Fisher’s exact test with multiple testing correction (Fdr) were used for statistical analysis. **p < 0.01, ***p < 0.001.

CHTs of control and ptena^−/−ptenb^−/− mutant embryos (5 dpf, ~100 embryos each) were dissected, pooled, dissociated, FACS sorted and submitted to SORT-seq. a Visualization of k-medoid clustering and cell-to-cells distances using t-SNEs. Each dot represents a single cell. Colors and numbers indicate cluster and correspond to colors in h. In total, 614 cells are shown. b–f t-SNEs maps highlighting the expression of marker genes for each of the different cell types found. Transcript counts are given in a linear scale. b Erythrocyte progenitors, c Thrombocyte/erythrocyte progenitor, d HSPCs, e Myeloid progenitors, f Neutrophil progenitors. g t-SNE map highlighting the distribution of ptena^−/−ptenb^−/− mutant embryos and their siblings. h The percentages of cells from ptena^−/−ptenb^−/− mutant embryos and their siblings in the different clusters. Fisher’s exact test with multiple testing correction (Fdr) were used for statistical analysis. *p < 0.05, ***p < 0.001.