Fig. 3: A population of embryonic macrophages in the bone marrow.

a Dot plots show gating for myeloid cells in the bone marrow of Rank^cre/+;R26.eYFP^fl/+ mice. For macrophages, dapi-negative singlets were gated on CD45⁺ cells. B220, CD3, and CD19 postitive cells were further subdivided by Ly6C and Gr1. Gr1^low cells were further gated on CD11b vs F4/80. Monocytes and Eosinophils were identified as CD11b⁺ cells separated by scatter characteristics as shown. Macrophages were gated on F4/80⁺ cells and further subdived by CD206⁺ and VCAM1⁺. Double-positive population were identified as macrophages and analyzed for eYFP expression. Bar graph shows the quantification of eYFP frequencies within indicated myeloid cells in the bone marrow. Mp macrophages, Eo eosinophils, mono monocytes, PMN polymorph nuclear neutrophils. b Quantification of F4/80^hi CD206⁺ VCAM1⁺ cells per 2 femura (left). Contribution of eYFP⁺ cells to F4/80^hi CD206⁺ VCAM1⁺ cells (right). c Histograms show the frequency of eYFP expression in 3-week-old (young, green) and 83-week-old (old, grey) macrophages of Rank^cre/+;R26.eYFP^fl/+ bone marrow. Mp=macrophages. d Repopulation of indicated myeloid cell populations with donor-derived cells in the bone marrow of mice that have received congenic bone marrow cells 32 weeks before. A two-sided unpaired Student’s t-test was used. (n = 10, 3 biological replicates). e Giemsa staining of tdTomato⁺ and tdTomato^- bone marrow macrophages cytospins, from 6–9-week-old mice, isolated as depicted in a. (n = 3, 2 biological replicates). f Plots show the number of leukocytes (CD45), monocytes (mono), and macrophages (Mp) in the bone marrow of 3-week-old mice of indicated genotypes (left). A two-sided unpaired Student’s t-test was used. (n = 24, 5 biological replicates for Rank^cre/+;Csf1r^fl/−, n = 14, 4 biological replicates for Csf1r^−/−, n = 30, 5 biological replicates for Vav^cre/+;Csf1r^fl/−). Fold-change comparisons between indicated bone marrow cell populations of wild type and Rank^cre/+;Csf1r^fl/− or Csf1r^−/− or Vav^cre/+;Csf1r^fl/− mice (right). A Mann–Whitney U test was performed for statistical analysis. (n = 14, 6 biological replicates) g Gene set enrichment analysis (GSEA) of genes differentially expressed by eYFP⁺ embryonic and eYFP^- adult bone marrow macrophages using gene sets from MSigDB v2023.2Mm database. Normalized enrichment score (NES) and by Benjamini-Hochberg method adjusted p-values are shown for GO terms myeloid leukocyte migration and myeloid leukocyte activation. h Fold-change of mean fluorescence intensity (MFI) of Lysotracker-positive cells within embryonic (eYFP⁺) and adult-derived (eYFP^-) bone marrow macrophages from 4–7-week-old mice. Fold-change was calculated by dividing individual MFI values of eYFP⁺ macrophages to the experimental average of the eYFP^- MFI values in each experiment. A Mann–Whitney U test was performed for statistical analysis. (n = 7, 3 biological replicates) i Frequency of pHrodo positive cells within embryonic (eYFP⁺) and adult-derived (eYFP^-) bone marrow macrophages after a 2 h ex vivo incubation with E.coli. bioparticles (left). A two-sided unpaired Student’s t-test was used. (n = 7, 3 biological replicates). Fold-change of pHrodo mean fluorescence intensity (MFI) values of eYFP⁺ and eYFP^- macrophages (right) Fold-change was calculated by dividing individual MFI values of eYFP⁺ macrophages to the experimental average of the eYFP^- MFI values in each experiment. A Mann–Whitney U test was performed for statistical analysis. (n = 7, 3 biological replicates). 4–7-week-old mice were used. j Quantification of MSC cell numbers after culturing with or without bone marrow myeloid cells for 8 days. A two-sided unpaired Student’s t-test was used. (n = 5, 3 biological replicates). Source data are provided as a Source Data file.