Macrophage PAR2 regulates fetal erythropoiesis. (A) Immunohistochemistry of fetal livers collected from PAR2^flfl-LysMcre and PAR2^flfl control embryos at E15.5 and stained for the macrophage marker F4/80. Scale bar, 50 µm. (B) mRNA expression in fetal livers was analyzed for erythroid master regulators, β-globin, F4/80, macrophage markers, and regulators of apoptotic cell clearance capacity. Fetal livers were collected from PAR2^flfl-LysMcre and PAR2^flfl embryos at E15.5 (C) and from PAR2^+/− and PAR2^−/− embryos at E13.5 (D), and mRNA expression of DNase II and Hamp-1 was normalized to r18s. To compensate for reduced macrophage numbers, Hamp-1 expression was also normalized to F4/80, using the data depicted for the corresponding genotypes in panel B or Figure 3B or it was normalized to DNase II by calculating the expression relative to F4/80 or DNase II for each embryo. All data are mean ± standard deviation. (E) Livers were collected from 9- to 10-week-old adult female mice, and F4/80, DNase II, and Hamp-1 mRNA expression was normalized to r18s or F4/80. (A-D) *P < .05, **P < .01, ***P < .001, ****P < .0001, Mann-Whitney U test for Id3 and Nr1h3 (B), Hamp-1 to F4/80 and Hamp-1 to DNase II (C), and Hamp-1 and Hamp-1 to F4/80 (D); all other P values were calculated using the unpaired Student t test. (E) Mann-Whitney U test for F4/80; all other P values were calculated using the unpaired Student t test. neg, negative; pos, positive.