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doi: 10.1242/10.1242/dev.00170

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Early patterning of the mouse embryo — contributions of sperm and egg

Karolina Piotrowska* and Magdalena Zernicka-Goetz{dagger}

Wellcome Trust/Cancer Research UK Institute, and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
* On leave from the Department of Experimental Embryology, Polish Academy of Sciences, Jastrzebiec, Poland



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  		Fig. 2. Clones derived from the 2-cell blastomeres of the parthenogenetic embryos do not respect an embryonic-abembryonic boundary in the blastocyst. Blastomeres of 2-cell embryos were labelled with different coloured dyes and the distribution of the progeny of labelled cells were analysed at the blastocyst stage. The frequencies of the four categories of blastocyst scored are indicated. Blastocysts of 4 different groups of embryos: parthenogenetic haploid eggs (A,B); parthenogenetic diploid eggs treated with cytochalasin (C,D); parthenogenetic diploid eggs in which the polar body was fused back to the embryo (E,F); and fertilised eggs (G,H) [data from Piotrowska et al. (Piotrowska et al., 2001Go)]. Blastocysts were scored ++ if 0, 1 or 2 cells crossed the boundary zone. In cases where 3 cells crossed the boundary, blastocysts were scored +. When 4-5 cells, or more than 5 cells failed to respect the boundary they were scored — and — —, respectively. In the table, the degree to which predominantly embryonic clones extend to the abembryonic part are shown in red. The degree to which abembryonic clones extend to the embryonic part are shown in blue. The micrographs represent individual optical sections mid-way through the embryo to show the cavity, which occupies the lower half of each blastocyst. The boundary zone is marked with white dashed lines and the border of the blastocoel has been traced on to a central section and is shown projected onto each of the other sections as a blue dashed line. The clonal border is marked with a yellow dashed line. The examples shown in the micrographs are all from haploid parthenogenetically activated eggs. Scale bar: 25 µm.

 


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  		Fig. 1. Experimental design: generating parthenogenetic eggs. Oocytes in metaphase of the second meiotic division (A) were activated to obtain: parthenogenetic haploid eggs (1n, B); parthenogenetic diploid eggs, by treating activated eggs with cytochalasin D to prevent extrusion of the second polar body (2n CD, D); parthenogenetic diploid eggs in which the extruded second polar body (F) was subsequently electrofused back to the egg (2n PB, G). The DIC images show these three types of parthenogenetic eggs after their generation. pb, polar body; fp, female pronucleus. The merged fluorescent and DIC images (C,E,H) show 2-cell stage embryos that developed from these activated eggs in which each blastomere was labelled with a different coloured dye. Scale bar: 25 µm.

 


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  		Fig. 3. Comparison of the boundaries between clones derived from 2-cell blastomeres in zygotes and parthenogenetically activated eggs. (A-D) Individual confocal sections of blastocysts in which the clonal border of the 2-cell stage progeny (yellow dashed line) is tilted with respect to the boundary zone between the embryonic and abembryonic parts (white dashed lines). Blastomeres of fertilised or parthenogenetically activated embryos labelled with different coloured dyes at the 2-cell stage and cultured to the blastocyst stage. The four examples are of the frequently found patterns of clonal distribution in fertilised eggs (A), haploid parthenogenetically activated eggs (B), diploid parthenogenetically activated eggs generated by cytochalasin treatment (C) or electrofusion (D). (E) Series of confocal sections of an individual blastocyst developed from parthenogenetic diploid cytochalasin-treated embryo. The boundary zone is marked with white dashed lines and the border of the blastocoel has been traced on to a central section and is shown projected onto each of the other sections as a red, blue or green dashed line. The clonal border is marked with a yellow dashed line. Panels a-j show individual optical sections at 7.5 µm intervals as a `z-series'. Panels k and l show the dissociated cells of this blastocyst observed by fluorescence or DIC optics respectively. Note that all cells are labelled but not uniformly throughout. (F) Series of confocal sections of the individual blastocyst developed from a fertilised embryo. The boundary zone is marked with red dashed lines and the border of the blastocoel was traced on to a central section and is shown projected onto each of the other sections as a white dashed line. Panels a-h show individual optical sections at 7 µm intervals as a `z-series'. Panels i and j show the dissociated cells of this blastocyst observed by fluorescence or DIC optics respectively. Scale bar: 25 µm (in A for A-D and E for E,F).

 


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  		Fig. 4. Disruption of the cortical cytoplasm associated with the position of the sperm entry disturbs spatial patterning of the blastocyst. (A) Fertilisation cone (fc) of freshly fertilised eggs was marked by attaching a fluorescent bead at the time of polar body (pb) extrusion. (B) 2-3 hours later, after the male pronucleus (mp) started to migrate toward the female pronucleus (fp) at the egg centre, the cortex and the associated cytoplasm marked by the bead was removed. (C) Fragment of excised cytoplasm. Next, the site of the operation was re-labelled with another fluorescent bead (not shown). Blastomeres at the 2-cell stage were labelled with different coloured dyes and the distribution of cells was examined by the confocal sectioning at the blastocyst stage. (D) An individual section of such a blastocyst. The clonal border of the 2-cell stage progeny (marked by yellow dashed line) is tilted with respect to the boundary zone between the embryonic and abembryonic parts (white dashed lines). A fluorescent bead (pale green) is visible. (E) A control experiment in which another region of the cortex of the zygote, approximately 90° from the fertilisation cone, was removed instead. (F) Fragment of excised cytoplasm. Resulting eggs from such a manipulation (G) were labelled at the 2-cell stage as before and cultured to the blastocyst stage. (H) An individual section of such a blastocyst showing distribution of labelled cells. Scale bar: 25 µm (in A for A,B,C,E,F,G and In D for D,H).

 

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