Single-cell transcriptomic dissection of two waves of endothelial-hematopoietic transition in a murine yolk sac

The yolk sac drives vertebrate embryonic hematopoiesis through primitive hematopoiesis and endothelial-to-hematopoietic transition (EHT) waves. However, dynamic cellular and molecular changes during EHT of the yolk sac remain to be elucidated. We built a comprehensive atlas of early endothelial and hematopoietic development in the yolk sac by integrating single-cell transcriptomic data from mouse embryos (E6.75-E11.0). Focusing on the yolk sac (E7.5-E9.5), we established a refined atlas capturing key cell populations of EHT in the yolk sac. This enabled the identification of distinct hemogenic endothelial cell (HEC) subpopulations and revealed 2 fundamentally distinct waves of yolk sac hemogenesis via EHT that differed in temporal emergence, cellular origin, molecular signature, and lineage bias. The first EHT wave, emerging around E8.0, originated from primordial endothelial cells and exhibited a bias toward the generation of erythromyeloid progenitors. In contrast, the second EHT wave, emerging around E8.5, originated from maturing yolk sac endothelial cells, expressed key intraembryonic HEC markers (Hlf, Nupr1, Gfi1), and showed a hematopoietic stem and progenitor cell fate bias. Furthermore, molecular dynamics analysis of the pseudo-trajectory during the 2 waves of EHT in mouse yolk sacs revealed different dynamic changes in several pathways, particularly the ribosome and metabolic pathways. The yolk sac endothelial and hematopoietic atlas is accessible from an interactive web server (https://lllab.shinyapps.io/ysshinyapp/). Collectively, this study provides novel insights into the multi-wave nature of yolk sac hematopoiesis, clarifies the fundamental principles of yolk sac EHT at a single-cell resolution, and offers potential guidance for in vitro blood cell regeneration strategies.