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Fate map of early avian cardiac progenitor cells

¹ Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA
² Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA

View larger version (67K): [in a new window]   Fig. 1. Imaging and grid to locate position of DiI injections at ‘0’ hours. All images are ventral views of the embryo. DiI labeled cells are seen in red. (A) Fluorescence image of embryo 127 (stage 4+). (B) Bright field image of the same embryo; (C) superimposed images of the embryo shown in A,B with an overlay of the same grid used on the template at stage 4 (Fig. 2A). (D-F) Imaging of the same embryo 20 hours later: (D) fluorescence image; (E) bright field image; (F) superimposed images in D,E showing the location of DiI in the ventricle 20 hours post-injection. HN, Hensen’s node; Ht, heart; arrows indicate superimposed images of A and B in C, and D and E in F.

View larger version (95K): [in a new window]   Fig. 2. Fate map of embryos at stages 4-6. (A-C) A composite template of embryos with all injections at ‘0’ hours represented by a circle and number for each embryo, at stages 4 (A), 5 (C) and 6 (F). The color of the circle denotes the region of the heart (yellow, sinus venosus; blue, ventricle; red, atria; black, bulbus cordis or vitelline artery; and white, those that did not enter the heart) to which the DiI-labeled cells became incorporated 20 hours post-injection. The HFR is bilaterally demarcated by a black rectangle (see Materials and Methods) in A,C,F. The HFR at respective stages was superimposed on embryos stained for mRNA expression of Bmp2 at stages 4 (B), 5 (D) and 6 (G), and Nkx2.5 at stages 5 (E) and 6 (H). aip, anterior intestinal portal; hn, Hensen’s node; hp, head plate; ps, primitive streak. (I) The pre-cardiac region from Rosenquist and DeHaan (1966), as shown in Schultheiss et al., 1997. B,G,H reproduced, with permission, from Schultheiss et al., 1997; D,E reproduced, with permission, from Ehrman and Yutzey, 1999.

View larger version (152K): [in a new window]   Fig. 3. Fate map of embryos at stages 7 and 8. (A,D) A composite template of embryos with all injections at ‘0’ hours, represented by a colored circle (yellow, sinus venosus; blue, ventricle; red, atria; black, bulbus cordis or vitelline artery; and white, those that did not enter the heart) and number for each embryo at stages 7 (A) and 8 (D). The HFR at stage 7 (black rectangle, see Materials and Methods) was superimposed on a stage 7 embryo stained for Bmp2 expression (B) and Nkx2.5 expression (C). (E) The HFR at stage 8 is represented (on only one side) as a red domain on a stage 8 embryo stained for expression of Nkx2.5 mRNA. B,C reproduced, with permission, from Ehrman and Yutzey, 1999. E reproduced, with permission, from Schultheiss et al., 1995.

View larger version (71K): [in a new window]   Fig. 4. Specificity of labeling technique. (A) Superimposed bright field and fluorescence image immediately post-injection of a stage 4+ embryo, ventral side upwards, injected with DiO (green). (B-1,B-2) 150 µm section at the level shown in A (x100). Because of the thickness of the section, B-1 shows an image where the ectoderm and mesoderm layer are in focus and B-2 shows the mesoderm and endoderm in focus. (C) 150 µm section at the level shown in A (x200). The arrow denotes the location of the primitive groove. Ec, ectoderm; En, endoderm.

View larger version (179K): [in a new window]   Fig. 5. A definitive rostrocaudal pattern was not present in the HFR at stages 4-6. Stage 4+ embryo (number 758) injected with DiI (red) and DiO (green) at ‘0’ hours was photographed at 4, 8, 10, 15 and 22 hours during development. Images shown are the fluorescent DiI and DiO images superimposed on each other and on the bright field image at each time point in Adobe PhotoShop 5.5. Arrow denotes DiO-labeled cells in the ventricle and arrowhead denotes the DiO-labeled cells in the sinus venosus.

View larger version (74K): [in a new window]   Fig. 6. Nkx2.5 expression was not detected in the entire HFR. All views of whole-mount embryos are ventral except in B2, where a dorsal view is presented. (A1,B1,E,G) The fluorescent image was superimposed on the bright field image. (A1) Embryo (three pairs of somites) injected with DiI (red dot) in the HFR, based on the HFR fate map from Fig. 3. Arrow indicates level of first somite. (A2) Whole-mount in situ hybridization of the embryo in A1 with digoxigenin-labeled probe of antisense Nkx2.5 (blue). Red dot indicates position of DiI injection, as in A1. Arrow indicates level of first somite. (B1) Embryo (four pairs of somites) injected with DiI (red dot) in the HFR, based on the HFR fate map from Fig. 3. Arrow indicates level of first somite. (B2) Whole mount in situ hybridization of the embryo in B1 with digoxigenin labeled probe of antisense Nkx2.5 (blue). Red dot indicates position of DiI injection, as in B1. Arrow indicates level of first somite. (C) Control stage 11 embryo stained with digoxigenin-labeled probe of antisense Nkx2.5. Nkx2.5 was expressed specifically in the heart. (D) Stage 5 embryo (number 888) injected with DiI (red dot) in the HFR, based on the fate map from Fig. 2C. Arrow shows site of injection. (E) Same embryo as in D, 20 hours post-injection, developed to stage 10. (F) Bright field image of a 400 µm section at level shown in E (white * indicates neural tube; black *, ventricle). (G) Superimposed bright field and fluorescent image of the same section as in F.