Abnormal and dilated blood vessels with bleeding in LPA₄-deficient embryos. (A-B) The hearts from WT (A) and LPA₄-deficient (B) embryos at E18.5 were examined macroscopically. Pericardial hemorrhage was observed only in the LPA₄-deficient embryos (B, white bold arrows). Note that a cut was made in the blood-stained pericardium in panel B. h, heart; l, lung; t, thymus. (C-D) H&E staining was performed on heart sections of WT (C) and LPA₄-deficient (D) embryos at E18.5. In LPA₄-deficient embryos, blood cells had leaked into the pericardial cavity (D, black bold arrows). a, atrium; p, pericardium; v, ventricle. Scale bars, 500 μm. (E-F) H&E staining was performed on periocular tissue sections of LPA₄-deficient embryos at E18.5 (E). The framed square area in panel E is magnified in panel F. In LPA₄-deficient embryos with subcutaneous hemorrhage, red blood cells had leaked into the mesenchyme from a blood vessel. o, oculus; b, blood vessel. Scale bars, 200 μm. (G-H) H&E staining was performed on ventral skin sections of WT embryos (G) and LPA₄-deficient embryos with subcutaneous hemorrhage (H) (as shown in Figure 3G) at E18.5. Blood vessels containing red blood cells are indicated by arrows. The bleeding LPA₄-deficient embryos (H) had dilated blood vessels compared with WT embryos (G). e, epidermis. Scale bars, 500 μm. (I and J) Immunohistochemical staining for α-SMA was performed on ventral skin sections of WT embryos (I) and LPA₄-deficient embryos with subcutaneous hemorrhage (J) at E18.5. Blood vessels containing red blood cells are indicated by arrowheads. Most WT blood vessels were covered with α-SMA⁺ cells, while some of the dilated blood vessels (asterisks) in LPA₄-deficient embryos were poorly covered with α-SMA⁺ cells. e, epidermis. Scale bars, 500 μm.