Abstract
Erythro-myeloid progenitors (EMPs) were recently described to arise from the yolk sac endothelium, just prior to vascular remodeling, and are the source of adult/post-natal tissue resident macrophages. Questions remain, however, concerning whether EMPs differentiate directly from the endothelium or merely pass through. We provide the first evidence in vivo that EMPs can emerge directly from endothelial cells (ECs) and demonstrate a role for these cells in vascular development. We find that EMPs express most EC markers but late EMPs and EMP-derived cells do not take up acetylated low-density lipoprotein (AcLDL), as ECs do. When the endothelium is labelled with AcLDL before EMPs differentiate, EMPs and EMP-derived cells arise that are AcLDL+. If AcLDL is injected after the onset of EMP differentiation, however, the majority of EMP-derived cells are not double labelled. We find that cell division precedes entry of EMPs into circulation, and that blood flow facilitates the transition of EMPs from the endothelium into circulation in a nitric oxide-dependent manner. In gain-of-function studies, we inject the CSF1-Fc ligand in embryos and found that this increases the number of CSF1R+ cells, which localize to the venous plexus and significantly disrupt venous remodeling. This is the first study to definitively establish that EMPs arise from the endothelium in vivo and show a role for early myeloid cells in vascular development.
Highlights
In the absence of in vivo validation
In agreement with previous reports[2,4,5,6], we found that yolk sac erythro-myeloid progenitors (EMPs) can contribute to tissue resident macrophages in neonate mouse tissues (Supplemental Fig. 1)
While macrophages were classically believed to develop from progenitors in the hematopoietic blood islands[34], the recent finding that EMPs express most endothelial markers and co-reside in the endothelial lining[20] have led to a general acceptance of an endothelial cell origin to EMPs16,17
Summary
One third of EMPs arise from blood vessels outside of the blood islands. To genetically label EMPs and EMP-derived cells during early vascular development, we used two transgenic myeloid-specific Cre recombinase mouse models: Csf1riCre (constitutive) and Csf1rMeriCreMer (tamoxifen-inducible). We found that in control embryos, 5.0 ± 0.8% of cells were tdT+, compared to only 1.6 ± 0.6% in embryos cultured without blood islands These culture experiments confirm that 32% of EMPs (i.e., 1.6% divided by 5.0%) develop in the yolk sac outside the blood islands, which corroborates our results obtained using endothelial cell morphology as criteria (Fig. 1I). TdT+ cells were distributed in the vascular plexus, whereas in the absence of flow, EMPs were mostly confined to the blood islands (Supplemental Fig. 6). We investigated other features of vascular morphology, including average vessel diameter, density of branch points, average segment length and the density of avascular regions These metrics revealed no differences in the arterial and venous plexuses between control and CSF1-Fc injected embryos (Supplemental Fig. 8)
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