Extended Data Fig. 6: Blood development.

a, Diagram illustrating the two waves of embryonic blood development. At E6.5, gastrulation begins. Previous work using transplantation assays has shown that the proximo-posterior epiblast cells closest to the primitive streak at this stage (red) mainly give rise to primitive erythroid cells in the yolk sac, whereas the epiblast cells located in the middle of the embryo at E6.5 but closer to the primitive streak at a later stage are enriched for endothelial progenitors⁵⁶. At E7.5, blood islands are apparent (zoomed box of primitive blood wave), where primitive erythroid cells are surrounded by endothelium. At around E8.25, some endothelial cells (haemogenic endothelium) undergo an endothelial-to-haematopoietic transition and become EMPs, which migrate to the fetal liver and give rise to definitive erythrocytes. Adapted from a previous study³⁸. b, Force directed layout of Fig. 3a coloured by original clusters from Fig. 1c. c, Gene expression levels of Cdh5 and Pecam1, overlaid on the graph abstraction visualization from Fig. 3b. d, Experimental design to isolate FLK1⁺ cells from yolk sac, allantois, and embryo proper for Smart-seq2 scRNA-seq. e, Representative image of an embryo collected for the transcriptional analysis of endothelial cells from the yolk sac, allantois, and embryo proper. f, Sorting strategy of FLK1⁺ cells from the yolk sac, embryo proper, and allantois on live cells (DAPI). x axis: FLK1 intensity. y axis: DAPI intensity. g, Evidence to support myeloid annotation of the myeloid cell cluster in Fig. 3. Haemato-endothelial cells from Fig. 3a were mapped to a published dataset⁵⁴ that profiled haematopoietic cells collected at E9.5, E10.5, and E11.5 from different organs. Bar charts show the fraction of atlas cells in the myeloid cell cluster mapped to the clusters defined in figure 8 of the previous study⁵⁴. h, Representative images of the dissected regions collected to study the location of CSF1R⁺CD16/32⁺ cells. Scale bars, 0.25 mm. i, Flow cytometry plots indicating the frequency of CSF1R⁺CD16/32⁺ cells in each embryonic region. Two biological replicates were performed for this experiment: with pools of 12 and 13 embryos, respectively. Plots illustrate one biological replicate.