Embryonic mast cells arise from the Cpa3-expressing precursors but not granulocyte-monocyte progenitors

Most of the mast cells (MCs) in connective tissues, such as skin, are long-lived embryonic-derived immune cells that play important roles in host defense and in various immunological diseases, including allergies. Their embryonic origin and ontogeny remain to be fully studied since several overlapping waves of embryonic hematopoiesis have been reported to give rise to these cells. Here, combining single-cell RNA sequencing and new genetic fate mapping models, we identified a Cpa3-expressing population sequentially appearing in the yolk sac, fetal liver, and peripheral tissues which gives rise to dermal MCs during embryonic days 11.5 to 14.5. Using in vitro differentiation and in vivo transplantation assays, we identified a Ter119^-F4/80^-CD45⁺CD117⁺CD16/32⁺CD135^-CD115^-Ly6C^-CD34^lo population as potential fetal liver MC precursors (MCPs). Fate mapping with Cpa3^CreERT2 and Zbtb16^CreERT2 models, as well as the granulocyte-monocyte progenitors (GMPs) fate mapping Ms4a3^Cre and Elane^Cre models, demonstrated that MCs arise from Cpa3⁺ precursors rather than Ms4a3⁺ or Elane⁺ GMPs. A corresponding population with a similar developmental trajectory was also identified in human early yolk sac and fetal liver, suggesting a conserved MC developmental program across species. These findings reveal a distinct developmental path of skin MCs in embryos, permitting future functional studies in immunity and development.