Fig. 1. Morphology and immunostaining in 20-hour allantoic regenerates. (A-C) Schematic drawings illustrate the formation of allantoic regenerates between the neural plate/early allantoic bud (EB) through 6-somite pair stages (lower left). (A-B) The allantois (al) overlies the primitive streak (ps) and was removed. (B) Epiblast (curved red arrow) continues to ingress into the primitive streak, so that, during whole embryo culture, operated conceptuses regenerate a new allantois (al-r) (C). (D,E) H/E-stained low- (D) and high- (E) magnification views of the posterior primitive streak immediately after removal of the allantois. The arrow in D indicates the anterior point of microcapillary entry into the yolk sac. Arrowheads in D and E indicate the site of removal of the allantois, just above the primitive streak. (F) H/E-stained 20-hour allantoic regenerate (al-r) contains mesothelium (m) and nascent endothelial channels (asterisks). (G-L) Immunostaining of allantoic regenerates and cultured unoperated controls. Histological sections immunostained with one antibody (G,H,J,K) were counterstained in hematoxylin. Doubly immunostained histological sections (I,L) were not counterstained. (G) FLK1 (brown color) in the endothelial plexus (e.g. asterisks) throughout the allantoic regenerate. (H) VCAM1 (brown color) in the mesothelium (m) of the distal allantoic two-thirds and a few distal core cells (arrowhead) of the regenerate. (I) FLK1 (blue color)/VCAM1 (brown color) double immunostaining reveals closely associated but separate populations of FLK1- and VCAM1-containing cells within the distal core, as previously reported (Downs et al., 2001). (J-L) Control unoperated host allantoises immunostained for (J) FLK1, (K) VCAM1 and (L) FLK1 (blue color)/VCAM1 (brown color). Other abbreviations: ac, amniotic cavity; am, amnion; ch, chorion; ec, ectoplacental cavity; x, exocoelomic cavity; ys, yolk sac. In F-L, embryo stages before and after culture are separated by `/'. Scale bars in L: 50 µm (E); 100 µm (F,G,I); 200 µm (D,H,J-L).