Fig. 4. RA regulation of p21 in vivo and in vitro. Whole-mount staining with antibodies against p21 revealed significantly reduced levels of p21 in Raldh2^-/- yolk sacs (B) compared with wild type (A). Section staining revealed p21 expression predominantly in endothelial cells in wild-type yolk sacs (C, arrows); p21 was essentially absent in Raldh2^-/- yolk sac (D). Endothelial cells were cultured for up to 72 hours in the presence or absence of 1 µM RA. Western analyses revealed that RA induced the expression of p21 in endothelial cells within 8 hours (E).