Fig. 1

Tregs in the maternal serum promote CM proliferation. a Rat neonatal CMs were exposed to the serum collected from neonatal (NS), adult (AS), pregnant (PS) and Treg-depleted pregnant (TDPS) animals and proliferation measured by EdU incorporation. Nuclei are stained blue with 4’-6-diamidino-2-phenylindole (DAPI) and CMs green by anti-α-actinin antibodies. Red nuclei indicate EdU incorporation. Scale bar, 100 μm. Insets shows a higher magnification of the area defined by the white dotted line. b Quantification of EdU⁺ CM nuclei (% of total CMs) after exposure to the different sera. c Outline of the experimental procedure followed for Treg depletion in vivo in pregnant animal. The Treg-depleting agent, either PC61 antibodies in CD1 mice or Dyphteria Toxin (DT) in DEREG mice, was administered i.p. to the pregnant mother at embryonic days 10 (E10) and 15 (E15). EdU was administered i.p. every day from E10 to E18, when embryos were removed for histological analysis. d Quantification of EdU⁺/ α-actinin CM nuclei (% of total CMs) in the heart of embryos harvested from either control (white bars) or Treg-depleted (gray bars) mothers, in the two models of Treg depletion described in panel (c). e Longitudinal section of a whole E18 heart, in which nuclei are stained blue with DAPI, CMs green by anti-α-actinin antibodies and EdU incorporation is shown in red. Scale bar, 1 mm. f High magnification images of the indicated regions of embryonic hearts from control (C) and Treg-depleted (TD) mothers, in which nuclei are stained blue with DAPI, CMs green by either anti-α-actinin or PCM1 antibodies and EdU incorporation is shown in red. RV: right ventricle, LV: left ventricle. White numbers indicate the percentage of EdU⁺ CMs in each cardiac region. Scale bar, 20 μm. Values in (b) and (d) are mean ± s.e.m., n ≥ 3 biological replicates. One-way analysis of variance and Bonferroni/Dunn’s post hoc tests were used to compare multiple groups (b). Pairwise comparison was performed with the Student’s t-test (d). **P < 0.01, ***P < 0.001 relative to control