Epiblast-derived CX3CR1+ progenitors generate cardiovascular cells during cardiogenesis

CX3CR1+ cells generate tissue macrophages in the developing heart and play cardioprotective roles in response to ischemic injuries in the adult heart. However, the origin and fate of CX3CR1+ cells during cardiogenesis remain unclear. Here, we performed genetic lineage tracing of CX3CR1+ cells and their progeny (termed Cx3cr1 lineage cells) in the mouse and demonstrated that they emerge from a subset of epiblast cells at embryonic day E6.5 and contribute to the parietal endoderm cells at E7.0. At E8.0-9.5 of development, Cx3cr1 lineage cells produced cardiomyocytes and endothelial cells via both de novo differentiation and fusion with pre-existing cardiomyocytes or endothelial cells, respectively. Cx3cr1 lineage cells persisted in the adult heart, comprising ~13% of cardiomyocytes and ~31% of endothelial cells. Additionally, CX3CR1+ cells differentiated from mouse embryonic stem cells generated cardiomyocytes, endothelial cells, and macrophages in vitro, ex vivo, and in vivo. Single-cell RNA sequencing revealed that Cx3cr1+ cells represent an intermediate cell population transitioning from embryonic stem cells to mesoderm. Taken together, embryonic CX3CR1+ cells constitute a multipotent epiblast-derived progenitor population that contributes not only to the formation of macrophages, but also of cardiomyocytes and endothelial cells.