The overall goal of this proposal is to bridge current in vitro models of human embryonic development with the latest progress in developmental hematopoiesis studied in vivo. This project will establish a new framework for defining the prenatal transformation events leading to childhood leukemia. Background and objectives: During embryonic development, multilineage hematopoietic stem and progenitor cells derive from the endothelium, and there is a remarkable transcriptional heterogeneity in the endothelial populations of the mouse embryo. Currently little is known about the impact of these specific endothelial population origins on their hematopoietic fates. In the present proposal, we will test if this heterogeneity is conserved in an in vitro model of human development using human pluripotent stem cell (hPSC) differentiation. We will then determine if it plays a role in developmental disorders and prenatal transformation. To address this, we will analyze publicly available and freshly generated single-cell RNA/ATAC sequencing data through the following specific research axes: Axis 1: Identification of transcriptional signatures for specific embryonic endothelial cell populations from publicly available single cell sequencing data of mouse embryos [https://doi.org/10.1242/dev.201867]. This axis has an overall goal of understanding the endothelial diversity in the context of blood emergence in the mouse embryo, and to describe the trajectories and fates of the different endothelial cell populations. Axis 2: Describe the dynamics of epigenetic and transcriptional landscapes occurring during in vitro differentiation of hPSCs towards the hematopoietic by using single cell sequencing technologies. We'll explore transcriptional and epigenetic trajectories leading to different endothelial and hematopoietic lineages and we will compare with signatures and trajectories previously identified in publications using mouse embryos (Axis 1). Axis 3: Establish an in vitro strategy to identify developmental populations susceptible to transformation leading to childhood myeloid leukemia. For this we will implement our differentiation protocol (characterized in Axis 1) using established human pluripotent stem cell lines harboring cytogenetic abnormalities relevant for childhood leukemia. Samples will be collected at multiple time-points for single-cell RNA and ATAC sequencing. Data analysis will involve integration with dataset established in Axis 1 to identify transcriptional states affected by the presence of cytogenetic abnormalities. This project will not use sensitive data. Human data are derived from well-established commercial cell lines.