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doi: 10.1242/10.1242/dev.00579

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Epiblast and primitive-streak origins of the endoderm in the gastrulating chick embryo

¹ Department of Neurobiology and Anatomy, and Children's Health Research Center, University of Utah School of Medicine, Salt Lake City, UT 84132-3401, USA
² University of Ghana, Accra, Ghana, West Africa

View larger version (33K): [in a new window]   Fig. 1. Schematic diagram based on details from Lopez-Sanchez et al. (Lopez-Sanchez et al., 2001) showing the two rostral (in rostral-to-caudal sequence, sites cr and c0.5 by Lopez-Sanchez et al.), two mid (in rostral-to-caudal sequence, approximately sites c1.5 and c2 by Lopez-Sanchez et al.), and two caudal primitive streak sites labeled in this study, as well as the one epiblast site (site bL by Lopez-Sanchez et al.). Scale bar: 125 µm.

View larger version (185K): [in a new window]   Fig. 2. Fate mapping the rostral primitive streak. DiI/CRSE (red) and CFSE (green) injections at sites cr and c0.5 at stages 3a/b (A-F), 3c (G-K), 3d (L-O), and 4 (P-T), respectively. (A,G,L,P) t=0 hours of reincubation. (B,C,H,M,Q) t=6 hours of reincubation. (D,I,N,R) t=9 hours of reincubation. (E,F,J,K,O,S,T) t=18-24 hours of reincubation. Lines in B,E,J,S indicate levels of sections shown in C,F,K,T, respectively. (E,F,J,K,O,S,T) Immunocytochemistry with anti-rhodamine (brown) and anti-fluorescein (purple) antibodies. In T, the more caudal injection site contributed to the somites (s; purple cells) and underlying endoderm, whereas the more rostral site contributed to the mid-dorsal endoderm and notochord (arrow), as well as the floor plate (arrowhead) of the neural tube (nt). cip, cranial intestinal portal; ep, epiblast; hn, level of Hensen's node; n, notochord; nt, neural tube, ps, primitive streak. Arrow in E indicates fluorescein-labeled (purple) cells; single arrowheads in E,J,O indicate the cranial intestinal portal; double arrowheads in E and J indicate the rostral border of the head.

View larger version (167K): [in a new window]   Fig. 3. Fate mapping the mid-primitive streak. DiI/CRSE (red) and CFSE (green) injections at mid-primitive streak level (red) and an adjacent rostral level (green) at stages 3a/b (A-G), 3c (H-L), 3d (M-P) and 4 (Q-W), respectively. (A,H,M,Q) t=0 hours of reincubation. (B,C,I,N,R) t=6 hours of reincubation. (D,J,O,S,U) t=9 hours of reincubation. (E-G,K,L,P,T,V,W) t=18-24 hours of reincubation. (Note that two stage 4 embryos are shown at 9 hours (S,U) and subsequently at 18-24 hours (T and V,W, respectively). Lines in B,E,K,V indicate levels of sections shown in C,F,G,L,W. (F,K,L,W) Immunocytochemistry with anti-rhodamine (brown) and anti-fluorescein (purple) antibodies. In C, arrow indicates the midline dorsal surface cells of the primitive streak (see Lawson and Schoenwolf, 2001a; Lawson and Schoenwolf, 2001b). cip, cranial intestinal portal; ep, epiblast; np, neural plate; nt, neural tube.

View larger version (161K): [in a new window]   Fig. 4. Fate mapping the caudal primitive streak. DiI/CRSE (red) and CFSE (green) injections at the caudal end of primitive streak at stages 3a/b (A-E), 3c (F-K) and 4 (L-N), respectively. (A,F,L) t=0 hours of reincubation. (B,G,H,M) t=6 hours of reincubation. (C,I) t=9 hours of reincubation. (D,E,J,K,N) t=18-24 hours of reincubation. Lines in D,G,J indicate levels of sections shown in E,H,K. (E,H,K) Immunocytochemistry with anti-rhodamine (brown) and anti-fluorescein (purple) antibodies. ep, epiblast; ps, primitive streak; arrow in K indicates the junction between the area pellucida (above) and area opaca (below).

View larger version (59K): [in a new window]   Fig. 5. Fate mapping the epiblast. DiI/CRSE (red) injections of epiblast adjacent to the primitive streak at stage 3c. (A,B) t=0 hours of reincubation. (C) t=3 hours of reincubation. (D) t=6 hours of reincubation. (E,F) t=22 h of reincubation. Lines in A,E represent levels of sections shown in B,F. (B,F) Immunocytochemistry with anti-rhodamine antibody (brown). Note in B that the injection is confined to the epiblast (arrow). ps, primitive streak. Note in F that the epiblast injection labeled the mesodermal cells of the somites (s) and the underlying endoderm (arrows).

View larger version (70K): [in a new window]   Fig. 6. Labeling the hypoblast and the rostral primitive streak. In situ hybridization with a Crescent RNA probe (purple), and immunocytochemistry with anti-rhodamine antibody (brown), 4 hours after injection of DiI/CRSE site cr at stage 2. (A) Crescent-labeled hypoblast in rostral half of area pellucida. (B) Crescent-labeled hypoblast as in a (purple) and rostral endoderm (brown). Line in B indicates the level of section shown in C. ep, epiblast. Note the intermixing of cells in the lower layer at the interface between the hypoblast (purple; arrowheads) and definitive endoderm (brown; arrows).

View larger version (42K): [in a new window]   Fig. 7. Prospective fate maps showing the primitive-streak origins of the definitive endoderm. The origins of the rostrocaudal subdivisions of the definitive endoderm are shown to the left of the broken vertical line in embryos at three pairs of stages: stage 3a/b to stages 7-8, stage 3c/d to stages 8-9, and stage 4 to stages 9-10. Only injections at the rostral end of the streak are shown, as more caudal injections (mid-streak and caudal streak) contributed endoderm only to the most caudal end of the embryo in a u-shaped configuration centered on the midline (more rostral injections labeled slightly more rostral cells, whereas more caudal injections labeled slightly more caudal cells). The origins of the mediolateral subdivisions of the definitive endoderm are summarized to the right of the broken vertical line, above the broken horizontal line. More rostral injections label more medial definitive endoderm, whereas more caudal injections label more lateral definitive endoderm. A comparison of the mesodermal and endodermal origins from the primitive streak is summarized in a diagrammatic cross-section to the right of the broken vertical line, below the broken horizontal line. Ectoderm is indicated in gray. Endoderm is indicated by different colors in the mediolateral plane and outlined in black; it consists of only the lower layer. Mesoderm consists of the tissue below the ectoderm and above the endoderm and is also indicated by different colors in the mediolateral plane. The comparison of the origin of the mesoderm and endoderm from the primitive streak is based on the data in the current study, as well as those published previously (Garcia-Martinez et al., 1993; Lopez-Sanchez et al., 2001). Note: (1) that for both mesoderm and endoderm, rostrocaudal position within the primitive streak translates after ingression to mediolateral position within the blastoderm (that is, more rostral sites contribute to more medial tissues and more caudal sites contribute to more lateral tissues); and (2) endoderm contributions from a particular rostrocaudal site tend to be spread more laterally than do mesodermal contributions from the same site. ao, area opaca; ap, area pellucida; ps, primitive streak; solid inverted u-shaped line, rostral border of neural plate/head; broken inverted u-shaped line, location of the cranial intestinal portal; vertical line in midline, notochord; squares, somites.