Fig 2: Cell cycle defects in fetal liver erythroblasts devoid of Cdh1. (A) Fetal liver cells from E17.5 control (Cdh1-WT) and Cdh1-null (Cdh1-KO) embryos were stained for Ter119 and CD71 surface markers and DNA content was analyzed in the indicated populations. The percentage of cells with 2N DNA content (G1) or with higher-than-2N DNA content (S/G2-M) was measured and plotted (right panel; n = 5). Representative cell cycle profiles are shown (left panels). (B) Quantitation of pH3 positive cells in the different erythroid populations gated based on Ter119 and CD71 expression, from control and Cdh1-deficient E17.5 fetal livers (n = 5). Examples of flow cytometry plots showing the quantified region are included on the left. (C) The percentage of EdU positive cells was assessed in sorted Ter119+ CD71+and Ter119+ CD71low/- erythroblast populations from control (Cdh1-WT) and Cdh1-null (Cdh1-KO) E17.5 fetal livers (n = 4). **P < 0.01, ns (not significant) P > 0.05.

Fig 3: Cdh1 deficiency leads to inefficient fetal erythropoiesis and anemia at birth. (A) Red cell parameters from peripheral blood of control (Cdh1 WT) and Cdh1-deficient (Cdh1 KO) pups collected 12 to 18 h after birth. The scatter dot plots show mean ± s.e.m of the corresponding data (n = 15 for WT and 12 for KO). (B) Erythroid maturation was assessed based on expression of Ter119 and CD71 cell surface markers in fetal livers of the indicated embryonic stage and genotype. The ratios of pro-erythroblasts (Ter119- CD71+), immature erythroblasts (Ter119+ CD71+), and mature erythroblasts and reticulocytes (Ter119+ CD71low/-) were determined in 4–7 samples for each stage and genotype (bar graphs, right). Representative flow cytometry plots are included on the left. (C) Quantification of the percentage of enucleated erythroid cells (Ter119+ Draq5-) in fetal livers from the indicated embryonic stage and genotype. The top panels show representative flow cytometry profiles. n = 10 for E13.5; n = 6 for E17.5; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Signs of anemia and impaired terminal erythroid differentiation in adult mice lacking Cdh1 expression in hematopoietic cells. (A) Red cell parameters from peripheral blood of 8–12 week old mice of the indicated genotype. The scatter dot plots show mean ± s.e.m of the corresponding data. (n = 51 for Vav-Cdh1 WT and 46 for Vav-Cdh1 KO). (B) Relative weight of the spleen in control (Vav-Cdh1 WT) and mutant (Vav-Cdh1 KO) adult mice (n = 10). A representative picture is included (left). (C, D) Terminal erythroid maturation was assessed based on CD71 expression in Ter119+ gated cells in the bone marrow (C) and spleen (D) from adult mice of the indicated genotype. The relative abundance of populations showing high, low, or no CD71 expression, representing increasingly higher maturation stages, were quantified (bottom graphs). Representative flow cytometry plots are included (top panels). (n = 6). *P < 0.05, **P < 0.01, *** P < 0.001.

Fig 5: Effects of exercise training, aging, obesity, and pressure overload on cardiac endothelial cell (EC) number and vascular density.(A and B) Fluorescence-activated cell sorting (FACS) analysis and quantification of mean fluorescence intensity (MFI) of the cardiac ECs (CD31+CD140a-CD45-Ter119-DAPI-) in various mouse models. (C and D) Representative immunofluorescence images and quantification of CD31+ blood vessel area (%) in the heart. Scale bar 100 µm. Data is presented as mean ± SEM. Student’s t-test was used, *p<0.05, **p<0.01, ***p<0.001. In panel (B), each color-coded circle indicates an individual biological sample. In panel (D), the number of mice in each experimental group is indicated in the respective graph, N = 3–5 male mice/group.Figure 1—source data 1.Source data for Figure 1B and D.Figure 1—source data 2.Echocardiography measurements of cardiac function and ventricular dimensions in the indicated experimental group.