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First published online 27 August 2003
doi: 10.1242/dev.00725

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Analysis of cell lineage in two- and four-cell mouse embryos

Toshihiko Fujimori^1,*,, Yoko Kurotaki¹, Jun-ichi Miyazaki² and Yo-ichi Nabeshima¹

¹ Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe Cho, Sakyo-ku, Kyoto 606-8501, Japan
² Division of Stem Cell Regulation Research, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

View larger version (97K): [in a new window]   Fig. 1. Expression of ß-galactosidase in embryos injected at the one-cell stage. Two representative embryos, 1-5 (A-D) and 1-4 (E-H) are shown. (A,E) Whole mounts and (B-D,F-H) Hematoxylin and Eosin-stained sections. Almost all parts of the embryos were positive for X-gal staining (A,B,E-F). (C,D,G,H) Higher magnifications of B and F respectively. (H) Derivatives of primitive endoderm (arrows) did not show X-gal staining. Scale bars: 300 µm (A,E), 150 µm (B,F), 30 µm (C,G).

View larger version (69K): [in a new window]   Fig. 2. Distribution of X-gal-positive cells in an embryo derived from a mating with a F1 male lacking the floxed CAT gene. (A) Whole-mount embryo in the deciduum. All embryonic cells expressed ß-gal except for the layers of primitive endoderm origin. (B,C) Sections stained with Hematoxylin and Eosin. The staining pattern was similar to that of embryos after injection at the one-cell stage. Almost all cells were stained except for the parietal endoderm (arrowhead) and visceral yolk sac endoderm (arrow). Scale bars, 300 µm (A), 150 µm (B), 30 µm (C).

View larger version (89K): [in a new window]   Fig. 3. Embryos injected at the two-cell stage showed random distribution of labeled cells. Embryos showed spotted distribution of X-gal-positive cells, and this pattern was seen evenly throughout the body axis (A,B) without regard to any embryonic axes. C and D are higher magnifications of B. In all cell layers, X-gal-positive and -negative cells were randomly intermingled. (E) Quantitative analysis. Classification of cell layers, and the result from one embryo, 2-8, are shown. Left panels indicate the origin of the each layer at 3.5 days and 4.5 days based on classic studies, and the name and abbreviation of the each tissue is also shown. The actual numbers of positive and negative cells are given and also represented by the histograms. F,G,H and I,J,K show the results of embryos 2-2 and 2-34, respectively. In the amnion of embryo 2-2, all the cells were positive for X-gal, however, this was an exceptional case because positive cells were intermingled with negative cells in other embryos. Scale bars: 300 µm (A) 150 µm (B), 30 µm (C).

View larger version (83K): [in a new window]   Fig. 4. Embryos with one type of X-gal distribution after activation of Cre recombinase in a blastomere at the four-cell stage. (A-G) The results of embryo 4-7. In this embryo, X-gal-positive cells were distributed randomly following labeling at the four-cell stage. (A) Whole mount, (B-F) sections showing that the stained cells were in all cell layers. The numbers of positive cells were less than in those embryos labeled at the two-cell stage. (C) In the extra-embryonic ectoderm of this embryo, clusters of relatively large numbers of X-gal-positive cells were observed. (G) Quantitative analysis. X-gal-positive cells accounted for between 5% and 31% of the total cells depending on the tissue. (H-O) The results of another example, embryo 4-6, which showed a similar pattern of distribution of labeled cells. (L,N) Sections at different positions of the same embryo. (I,J,K) High magnifications of L. Histograms show that the contribution of labeled cells is between 5% and 41%, however localization to any specific tissue or position in the body was not observed. Scale bars: 300 µm (A), 150 µm (B), 30 µm (C).

View larger version (89K): [in a new window]   Fig. 5. Two other types of labeled cell distribution after injection at the four-cell stage. (A-D) In the second type of distribution, only extra-embryonic ectoderm contained X-gal-positive cells. (A,C) Whole mounts; (B,D) sections of the embryos in A and C, respectively. In both embryos, a portion of trophoblast giant cells (arrows) and a part of the ectoplacental cone is for X-gal-positive. Note that embryo 4-59 (C,D, arrows) has labeled cells localized to the trophoblast giant cell layer. (E,F) The last type of cell distribution. In this embryo, there were no X-gal-positive cells in the extra-embryonic ectoderm region. In other regions, X-gal-positive and -negative cells were intermingled. Scale bars: 300 µm (A), 150 µm (B).

View larger version (157K): [in a new window]   Fig. 6. Distribution of labeled cells at the blastocyst stage. Embryos were cultured in vitro following injection of Cre recombinase into a blastomere at the two-cell (A-F) or at the 4-cell (G-I) stage. Labeled cells were seen as clusters in these embryos. The boundary between the embryonic and abembryonic parts is indicated by the dashed line.

View larger version (59K): [in a new window]   Fig. 7. Analysis of alginate-embedded embryos. Normal embryos without injection or embryos labeled with Cre recombinase were cultured in alginate gel to maintain their position. (A) The angle (a) between the first cleavage plane (P) and the embryonic-abembryonic axis (E-Ab) of the blastocyst was measured by comparing photographs. Only the embryos in which the first cleavage plane was orthogonal to the bottom of culture plate were used for the analysis. (B) The angle was found to be 80° in 28 out of 123 of the normal uninjected embryos. The angle was greater than 50° in 90 embryos (73%) (columns are colored in dark red). (C) In the case of DNA-injected embryos, similar distribution in angles were observed after analyzing 71 embryos. (D-I) Examples of uninjected embryos used for analysis. In the embryo in D-F, the angle was 70°, and the first blastomere to divide contributed mainly to the embryonic part of the embryo. In another normal embryo shown in G-I the angle was 80°, and the early-dividing blastomere contributed to the abembryonic part of the embryo. (J-Q) Two examples of labeled embryos. (J-M) The injected blastomere divided later than the other blastomere and contributed to the embryonic part in the embryo as can be seen from the X-gal staining in this region (M). In the embryo shown in N-Q, the injected blastomere also divided late. The labeled cells were seen in the trophectoderm of the abembryonic part and around the boundary zone. Scale bar: 30 µm.