Fig 1: Contribution of Lyl-1-expressing MF-progenitors to the fetal liver and brain.A All MF-progenitors from E10-Brain expressed Lyl-1, contrary to the corresponding YS (Fig. 1b) which harbor both FDG+/Lyl-1+ and FDG-/Lyl-1- subsets. MF-progenitors from E10 Lyl-1LacZ/LacZ FL harbored both FDG+/Lyl-1+ and FDG-/Lyl-1 MF-progenitor subsets. in E10-brain, mature MFs (CD11b+F4/80+ gate) were all FDG+/Lyl-1+. The contour plots in WT samples indicate the level of non-specific background ß-Gal activity/FDG labeling in WT samples. Representative profiles of 3 independent samples, each consisting of 3-4 E10-Brain or 8-12 E10-FL. B Quantification of Kit+CD45+CD11b+ MF-progenitors in E10-FL (plots show mean ± s.e.m.; Unpaired, two-tailed t-Test). C Lyl-1 marks the entire F4/80 + microglia/BAM population from the onset of brain colonization. FDG/Lyl-1 expression in F4/80+ microglia/BAM from the brain of Lyl-1WT/LacZ and Lyl-1LacZ/LacZ embryos at E9 to E11. The rare CD11b+ F4/80low-neg cells present in the brain at E9 are FDG+/Lyl-1+. Gray histograms indicate non-specific background ß-Gal activity/FDG levels in WT samples. Representative profiles of 3–5 independent samples, each consisting of 3-5 brains. D MF-progenitors from E10-brain express Myb levels similar to E9-YS MFPrim progenitors. RT-qPCR quantification of Myb expression levels in Kit+CD45+CD11b+ MF-progenitors sorted from WT E9-YS and from WT and Lyl-1WT/LacZ brain at E10. Lin-Sca+cKit+ (LSK) progenitors from WT E12-FL were used as positive control. Myb expression levels, shown on a Log2 scale, were normalized to the mean expression value obtained for WT E10-YS, considered as 1 (error bars show mean ± s.e.m.; Unpaired, two-tailed t-Test). E RT-qPCR analyses of Lyl-1 expression in A1 to A3 MF subsets isolated from Cx3cr1WT/GFP brain at E10. Lyl-1 is expressed by the 3 subsets, with levels decreasing with differentiation. Expression levels were normalized to the mean value obtained for Cx3cr1WT/GFP YS A1 progenitors (n = 2; Error bars show mean ± s.e.m). F Defective differentiation of brain MF-progenitor in Lyl-1 mutant embryos. Distribution of A1-A2 and A3 MF subsets in E10 brain from Cx3cr1WT/GFP:Lyl-1WT/WT, Cx3cr1WT/GFP:Lyl-1WT/LacZ and Cx3cr1WT/GFP:Lyl-1LacZ/LacZ embryos. The size of the whole MF population was similar in the three genotypes, but Lyl-1 deficiency modified the distribution of the MF subsets with an increased size of the A1 subset and a reduced A3 pool (5-12 independent analyses, 6–8 brains per sample. Error bars show mean ± s.e.m.; Unpaired, two-tailed t Test). G Heatmap expression profile of the genes that mark the development of tissue resident-MFs in WT E9 and E10 MF-progenitors (Heatmap displays transformed log2-expression values; Unpaired, two-tailed t-test).
Fig 2: Characterization of the Ly6 antigen series on brain-infiltrating leukocytes (BILs) and peripheral blood leukocytes (PBLs). Flow cytometric analysis of PBLs (A, C, E, G, I, K, M, O) and BILs (B, D, F, H, J, L, N, P) collected from the same mouse 18 h after infection with Theiler's murine encephalomyelitis virus (TMEV) revealed the presence of readily distinguishable neutrophil populations in both cell preparations. However, inflammatory monocytes were only detected in the BILs. Consistent with the analyses performed above, the CD45hiCD11b++F4/80+ population of inflammatory monocytes was positive for Gr1 (Ly6C/G), 7/4 (Ly6B), and AL-21 (Ly6C) but negative for 1A8 (Ly6G). The CD45hiCD11b+++F4/80- neutrophil population was positive for all four Ly6 series antigens. Of note, CD45hiCD11b++F4/80+ inflammatory monocytes in the BILs expressed higher levels of surface Ly6C/G (B, D) and Ly6B (J, L) as compared to CD45hiCD11b+++F4/80- neutrophils and expressed 10-fold higher levels of the AL-21 antigen (N, P). The CD45+CD11b+ macrophage population in the blood was negative for all Ly6 series antigens. Results are representative of five mice.
Fig 3: Lyl-1 expression marks MFPrim progenitors in the early YS.a Lyl-1-deficiency leads to an increased production of MF-progenitors in the early YS: Clonogenic potential of E8 OrgD1-YS cells: production of MF-progenitors (MFP) in WT, Lyl-1WT/LacZ and Lyl-1LacZ/LacZ OrgD1-YS. Distribution of other progenitors with a myeloid potential (EMP and GM) in WT, Lyl-1WT/LacZ and Lyl-1LacZ/LacZ E8 OrgD1-YS. (n = 3–5, 3–6 YS per sample; mean ± s.e.m.; Unpaired, two-tailed t-test). b Lyl-1 expression in MF-progenitors: FACS-Gal assay, using the ß-Gal fluorescent substrate FDG, was used as a reporter for Lyl-1 expression. While all MF-progenitors in E9-YS expressed FDG/Lyl-1, E9.5 and E10-YS harbored two MF-progenitor subsets discriminated by their FDG/Lyl-1 expression. FDG+/Lyl-1+ and FDG-/Lyl-1- mature MFs (CD11b+F4/80+) also coexisted in E10-YS. The contour plots in WT samples indicate the level of non-specific background ß-Gal activity/FDG labeling in WT samples. Representative profiles of 3 independent samples, each consisting of 3–4 YS (see gating strategy in Supplementary Fig. 1a). c In clonogenic assays, the number of MF-progenitors obtained from E8-YS (0-3 Somites) was increased in Lyl-1WT/LacZ and Lyl-1LacZ/LacZ compared to WT. The majority of the 25–30 progenitors per YS were EryP (60 to 80% in each 3 genotypes). Other progenitors were occasionally and randomly detected in WT and mutant samples (less than one EMP (0.81% ± 0.66; n = 3) and/or GM progenitor per E8-YS), confirming that the assay was performed at a time when MFT-Def progenitors were absent. (n = 3–4, 5–10 YS per sample; error bars show mean ± s.e.m.; Unpaired, two-tailed t-test). d MFPrim progenitors express Lyl-1. Flow cytometry profiles of WT and Lyl-1WT/LacZ E8-YS (0-3 S). CD11b+CD31- MFs correspond to maternal MFs present in E8-YS11. All CD11b+CD31+ MF-progenitors displayed FDG/Lyl-1 expression (Red line). The contour plots in WT samples indicate the level of non-specific background ß-Gal activity/FDG labeling in WT samples. Shown are representative profiles of 4 independent experiments. e RT-qPCR quantification of Myb expression levels: Kit+CD45+CD11b+ progenitors were sorted from WT E9-YS, WT and Lyl-1WT/LacZ E10-YS, and from FDG/Lyl-1 positive and negative fractions of MF-progenitors from Lyl-1WT/LacZ E10-YS. Lin-Sca+Kit+ (LSK) progenitors from WT E12-FL were used as positive control. FDG+/Lyl-1+ MF-progenitors from E10-YS expressed MybLow/Neg levels similar to MFPrim progenitors from E9-YS. The FDG-/Lyl-1- fraction expressed significantly higher Myb levels, similar to LSK cells from E12-FL. Myb expression levels, shown on a Log2 scale, were normalized to the mean expression value obtained for WT E10-YS, considered as 1 (Each dot represent an independent experiment; Error bars show means ± s.e.m; Unpaired, two-tailed t-test). f FDG/Lyl-1 positive and negative myeloid progenitors produce a distinct progeny: Clonogenic assays characterization of the type of progenitors produced by myeloid progenitors (Ter119-Kit+CD45+CD11b+) sorted from WT and Lyl-1WT/LacZ E9-YS ( < 18 S; n = 7) and E10-YS (n = 15) in 3 independent experiments. At E10, myeloid progenitors from Lyl-1WT/LacZ YS were subdivided into FDG/Lyl-1 negative (n = 15) and positive (n = 12) fractions (5 independent experiments). Samples were biological replicates comprising 6–8 YS. 100 to 150 Kit+CD45+CD11b+ cells per condition were platted in triplicate. All samples produced few non-myeloid contaminants, such as Erythro-Megakaryocytic progenitors and EMP in similar, non-significant amounts. FDG+/Lyl-1+ progenitors essentially produced MF-progenitors (MFP), while FDG-/Lyl-1- progenitors produced also granulo-monocytic- (GMP) and granulocyte- (GP) progenitors. Error bar shows means ± s.e.m.
Fig 4: Flow cytometric assessment of the infiltrate present in the brain of mice acutely infected with Theiler's murine encephalomyelitis virus (TMEV). TMEV-infected (B-G) or sham-infected (A) mice were killed at 18 h post infection (hpi). Brain was collected fresh and processed for the isolation of brain-infiltrating leukocytes (BILs). BILs were stained with antibodies against CD45, CD11b, F4/80, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. No CD45hi cells were observed in sham-infected mice (A). In contrast, TMEV-infected mice exhibited a large population of CD45hi cells at 18 hpi. Analysis of the CD45hi cells in BILs from infected mice revealed the presence of CD11b++ cells that were also strongly positive for the Gr1 antigen (C) and CD11b+++ cells that were positive for both the Gr1 antigen (C) and the 1A8 antigen (D). In addition, a population of the Gr1+ cells was also positive for F4/80 (E); no 1A8+ cells were F4/80+ (F). Combined gating revealed that CD45hiCD11b++Gr1+ cells were also F4/80+ while CD45hiCD11b+++Gr1+ cells were F4/80-. Quantitation of neutrophils (CD45hi1A8+Gr1+CD11b+++F4/80-) and inflammatory monocytes (CD45hi1A8-Gr1+CD11b++F4/80+) in BILs from 10 individual mice revealed that about 80% of the CD45hi cells belonged to one of these populations (G). Error bars in (G) represent 95% confidence intervals; black circles represent individual animals; 10 mice per group were analyzed and the flow plots in (A-F) are representative.
Fig 5: Gating strategy for the isolation of various populations of 4T1 tumor-infiltrating TAMs, including total TAMs (single, viable, CD11b+, F4/80+), PoEMs (single, viable, CD11b+, F4/80+, PDPN+) and non-PoEMs (single, viable, CD11b+, F4/80+, PDPN-).
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