Figure 1. EMPs and EMP-derived cells increase with developmental stage but lose their endothelial cell characteristics at later stages.

Csf1r^iCreRosa26^tdT yolk sacs were harvested at E8.5 (10 somite pairs, A) E9.0 (15 somite pairs, B) and E9.5 (25 somite pairs, C) to visualize tdT⁺ cells. The number of total of tdT+ cells increased based on immunofluorescence of Csf1r^iCreRosa26^tdT embryos (D, n = 4–5 per stage) and of CSF1R⁺ cells based on flow cytometry of wild type embryos (E, n = 6 at E8.5 and n = 9 at E9.5). Blood vessels were counterstained with CD31 (F–H, green). Endothelial-like tdT⁺ cells (F and H, arrowheads) were blindly quantified based on 3 phenotypic criteria (CD31 co-expression, cell shape, and vessel integration) by immunofluoresence. Analysis was limited to yolk sac vessels proximal to the embryo proper. The number of tdT⁺ cells with an endothelial phenotype was initially 32% but decreased in older embryos (I, n = 4–5 embryos per stage). The percentage of CSF1R⁺ cells that co-stained for VE-Cadherin was also measured by flow cytometry and confirmed the decrease in endothelial staining in older embryos (J). Scale bars: 250 μm (A–C), 50 μm. (F–H) All values are mean ± SEM. *p < 0.05; two-tailed Student’s t-test.