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Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo

Christoph Viebahn1,2,{dagger}, Christof Stortz2, Sally A. Mitchell3 and Martin Blum3,*

1 Institute of Anatomy and Cell Biology, Martin-Luther-University Halle-Wittenberg, Germany
2 Institute of Anatomy, Friedrich-Wilhelms-University Bonn, Germany
3 Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Germany
* Present address: Institute of Zoology, University of Stuttgart-Hohenheim, Germany



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  		Fig. 1. Experimental set-up for DiI injection shown in transection. (A) Whole 6.0 d.p.c. blastocyst (c) with intact zona pellucida (z) held on a metal ring (r). Embryonic disc (d) with its anterior margin (*) projects above the fluid level. The tip of the injection pipette and the injected dye lie in the space between epiblast (e) and zona pellucida (z), thereby avoiding contact with the hypoblast (h). (B) DiI injection sites just anterior to and within the PGE. Lateral injection sites were grouped symmetrically with regard to the median plane; for simplicity, they are shown only on the left of the embryonic disc and the numbers of injections carried out in corresponding injection sites are accumulated.

 


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  		Fig. 2. Early gastrulation stages of the rabbit embryo. (A) Whole blastocyst at a pre-primitive streak stage (6.0 d.p.c.); the plane of focus is on the blastocyst wall containing the embryonic disc. (B) Stage 1 with smooth anterior contour (epiblast-trophoblast junction) of the disc indicating the position of the AMC. The posterior contour (arrows) is more ragged. (C) Stage 2 with the ‘posterior gastrula extension’ (PGE), a sickle-shaped area of reduced density that lies posteriorly to the ragged posterior contour (arrows) seen at stage 1. (D) Stage 3 with primitive streak (s) in the midline of the PGE. The former posterior margin is still visible (arrows). Scale bars: 1.3 mm in A; 250 µm in B,C; 270 µm in D.

 


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  		Fig. 3. Rabbit Brachyury cDNA fragment and predicted protein sequence (A), interspecific comparison of protein sequence (B) and cladogram (C) show that rabbit cDNA and protein sequence fall within the Brachyury (T)-family. Amino acids in the T-box are shown in blue, other residues in green. Deviations from the consensus sequence are highlighted in red. Sequences were taken from the following papers: mouse (Herrmann et al., 1990Go); human (Edwards et al., 1996Go); chick (Kispert et al., 1995Go); Xenopus (Smith et al., 1991Go); zebrafish (Schulte-Merker et al., 1992Go); Amphioxus (Holland et al., 1995Go); Halocynthia roretzi (Yasuo and Satoh, 1998Go); Hydra (Technau and Bode, 1999Go); mouse tbx2 (Bollag et al., 1994Go); mouse tbx6 (Agulnik et al., 1996Go).

 


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  		Fig. 4. Brachyury expression during gastrulation (between 6.0 and 7.5 d.p.c.) as determined by in situ hybridisation; position of the AMC is indicated by asterisks. Bars and letters indicate position of sagittal sections shown in Fig. 5. (A,B) Stage 2, Brachyury-expressing cells lie singly at first (A) and a little later in a coherent patch (B) in the PGE. (C,D) Strong Brachyury expression in the emerging (C) and definitive (D) primitive streak at stage 3. (E) Early stage 4 with lack of Brachyury expression in the anterior half of the primitive streak (anterior to arrow) but distinct expression in the primordium of Hensen’s node (h). (F) Late stage 4 with node fully formed and strong Brachyury expression along the entire primitive streak. Arrow as in E. (G,H) Stage 5 and 6 with Brachyury expression also in notochordal process (n). Scale bars: 130 µm in A-D; 160 µm in E-G; 290 µm in H.

 


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  		Fig. 5. Cellular distribution of Brachyury expression as seen in sagittal (A,B) and horizontal (C,D) semi-thin sections from specimens in Fig. 4. (A,B) At stage 2, Brachyury transcripts are confined to epiblast cells (e) in the posterior third of the embryonic disc. Anterior (A) and posterior (B) margins (epiblast-trophoblast junction) are marked with single and double asterisks, respectively. (C,D) No Brachyury expression in epiblast or mesoderm of the anterior half of the primitive streak at stage 4 (arrowhead marks primitive groove in C) but strong Brachyury expression in the posterior half of the primitive streak (D) in epiblast (e) and ingressing mesoderm (i). Scale bar: 40 µm.

 


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  		Fig. 6. Cellular migration in the posterior half of 6.2 d.p.c. embryonic discs as determined by DiI labelling and culturing for 8 (A-C), 12 (D-H) and 16 hours (I-M). Dark-field photographs taken immediately after the injection (A,D) and after culture (B,E,I). DiI deposits are numbered in the order of injection in each embryo and appear with a shadow image under asymmetrical dark field illumination (A,B,I) and ring-like under circular illumination (D,E). Direction and distance of dye spread (white bars in B,E,I), as determined in fluorescence micrographs of embryonic discs mounted on microscope slides (C,F,G,J-L). (C) Dye spread in posteromedial direction (white line) following injection of bolus #1 (position indicated by white ring) placed mediolateral and anterior to the PGE of embryo shown in A,B. The DiI bolus was lost during mounting after removing the zona pellucida but left a mark of reduced cellular density seen in DAPI labelling (see inset). Translocated DiI appears as minute spots in the area of the PGE; these lie in one plane except for some spots that produce typically larger out-of-focus images (arrowhead). (F,G) Dye spread in posteromedial (F) and anteromedial direction (G) after injection anteromedially (#1) or posteromedially (#2) in the PGE in the embryo shown in D,E. (H) DAPI-labelled frozen section (position marked with broken line in F) showing epiblast (arrowheads pointing downwards) and not hypoblast cells (arrowheads pointing upwards) being labelled with DiI. (J,K) Dye spread after injections near the lateral margin anterior to the PGE (#1), posteromedially in the PGE (#2) and in the anterior centre of the PGE (#3) in the embryo shown in I. Injection #3 was inadvertently positioned between epiblast and hypoblast and gave rise – in addition to the typical spot-like labelling (compare C,F,G) – to complete cells being labelled (e.g. arrowhead). (M) Nomarski image of semi-thin section (position marked with broken line in K) of the specimen in K after photoconversion. DAB precipitates are found in the strongly labelled (hypoblast) cells only (arrowheads as in H; m, mesoderm). (L) Posterior and some anterior dye spread after injection similar to #3 in I and after development to late stage 3 (dark-field view of embryo not shown). Scale bars: 300 µm in A,B,D,E,I; 55 µm in C (inset 80 µm); 75 µm in F,G; 20 µm in H; 70 µm in J-L; 35 µm in M.

 


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  		Fig. 7. Cellular proliferation after pulse BrdU labelling in vitro. (A) Stage 1 with uniform labelling of the embryonic disc and background labelling in the bordering extra-embryonic tissues. A' and A'' anterior and posterior border, respectively, of embryo in A to show unlabelled (horizontal arrowheads) and labelled (vertical arrowheads) nuclei. (B,C) Early and late stage 2, respectively, with PGE (anterior border marked with broken line) less densely labelled than remainder of embryonic disc. Vertical bar indicates position of section shown in F. (D,E) Early and late stage 3, respectively, with intense labelling of AMC (compare with C) and primitive streak. Vertical bar indicates position of section shown in G. (F,G) Posterior sagittal sections from embryo in C,D, respectively, confirm the absence of mesoderm cells at stage 2 (C,F) and vigorous mesoderm ingression at early stage 3 embryo (D,G). At stage 2 (F), labelled nuclei are almost exclusively found in the epiblast (e) and at stage 3 (G), the hypoblast (h) is still unlabelled, while many nuclei are labelled in epiblast (e), in ingressing (i) and definite (m) mesoderm. Double asterisks mark posterior embryonic border at the junction with the extra-embryonic trophoblast. Scale bars: 250 µm in A-E; 60 µm in A' and A''; 30 µm in F,G.

 


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  		Fig. 8. Morphometric analysis of BrdU labelling at stage 2. (A) Position of grid boxes used for counting in anterior (red), central (yellow) and posterior (green) regions of the embryonic discs. (B) Graphical comparison and confidence values between average labelling indices obtained in these three regions.

 


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  		Fig. 9. Integration of the migratory, proliferative and differentiation behaviour of epiblast cells at the late pre-primitive streak stage (stage 2) of mammalian gastrulation: differentiation towards a mesodermal fate as revealed by Brachyury expression in the epiblast of the PGE (compare with Fig. 4A) occurs under the condition of a reduced proliferative activity (light green) in the PGE, when compared with high proliferative activity (yellow-red) in the remainder of the embryonic disc. This is accompanied by cellular rearrangement (grey arrows) between anterior and posterior areas of the embryonic disc and within the PGE to form the primitive streak. Each arrow represents the summary of results obtained for the individual sites chosen for injection anterior to and within the PGE (compare with Fig. 1B). Extra-embryonic tissues (chorion) are depicted as largely non-proliferative (light blue).

 

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© The Company of Biologists Ltd 2002