Fig 2: Subepicardial location of embryonic macrophages. Cross sections at the level of mid-ventricle of E16 (a1–a4) and E12 (b1–b5) hearts. The epicardium is labeled with WGA lectin (a2–a4) and with WT1 antigen (b3–b5), and less intense staining with WGA is visible around cardiac cells (a2–a4). The CD206-positive (a1, a3, a4) and the Lyve-1+/CD64+ macrophages are located under the epicardial cells (b1–b5); asterisks in panels a4 and b5 indicate the magnified area in a1–a3 and b1–b4, respectively. WGA wheat germ agglutinin, WT1 Wilms’ tumor protein, ep epicardium, my myocardium, en—endocardium; scale bars—100 µm

Fig 3: PCR analysis reveals differences among selected embryonic cTM subpopulation at two developmental stages (E14.0 and E17.0). The panel presenting expression levels of mRNAs for selected genes at two developmental stages: E14.0 and E17.0 within sorted cTMs subpopulations. P1: CD45+CD11b+F4/80+CD64low; P2: CD45+CD11b+F4/80+CD64high CD206-; P3: CD45+CD11b+F4/80+CD64 high CD206+. Statistically significant differences (p value = 0.05) are marked (*)

Fig 4: Flow cytometry analysis with identification of embryonic cTM subpopulations: CD64low; CD64highCD206-, and CD64highCD206+. a Gating strategy of E14 mouse cardiac tissue macrophages from single cell suspension. Firstly, dead cells were excluded. Within living cells [gate P2], the CD45-CD11b-double-positive population was selected [gate P3]. These cells were also identified as CD64+ with the significant differentiation on CD64low [gate P4] and CD64high [gate P5] subpopulations. b Representative histograms for CX3CR1, F4/80 and CD206 detected in CD45+CD11b+CD64low population (yellow) and CD45+CD11b+CD64high population (blue). Control staining is shown in gray. c Representative dot-plot analysis of pregated CD45+CD11b+CD64low population (yellow) and CD45+CD11b+CD64high population (blue) for CD206 and CX3CR3 expression. There is a clear differentiation on CD206+ and CD206- within CD64high subpopulation in comparison with CD64low subpopulation, which is CD206-. The red dashed lines indicate three selected cTM subpopulations: CD45+CD11b+CX3CR1+CD64low, CD45+CD11b+CX3CR1+CD64highCD206- and CD45+CD11b+CX3CR1+CD64high CD206+.

Fig 5: mRNAs for Retlna/FIZZ1, Ym1/Chil3 and bFGF are detectable only at the later developmental stage (E17.0). The panel presents expression levels of mRNAs for Retlna/FIZZ1, Ym1/Chil3 and bFGF at E17. These mRNAs were mainly expressed in the P1 subpopulation being undetectable at E14. P1: CD45+CD11b+F4/80+CD64+low; P2: CD45+CD11b+F4/80+CD64 + high CD206−; P3: CD45+CD11b+F4/80+CD64+high CD206+. Statistically significant differences (p value ≤ 0.05) are marked (*)