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First published online March 23, 2006
doi: 10.1242/10.1242/dev.02317

Development 133, 1597-1607 (2006)
Published by The Company of Biologists 2006
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Developmental abnormalities of NT mouse embryos appear early after implantation

Alice Jouneau1,*,{dagger}, Qi Zhou1,*, Anne Camus2, Vincent Brochard1, Linda Maulny1, Jérôme Collignon2 and Jean-Paul Renard1,{dagger}

1 Unité de Biologie du Développement et de la Reproduction, UMR INRA-ENVA, Institut National de la Recherche Agronomique (INRA), Jouy-en-Josas 78352, France.
2 Laboratoire de Développement des Vertébrés, Institut Jacques Monod, UMR 7592 CNRS, Université Paris 6 et 7, 2 place Jussieu, 75251 Paris, France.


Figure 1
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  		Fig. 1. Expression of Oct4 and outgrowth of NT blastocysts. (A,C,E) In vivo fertilised control embryos. (B,D,F) ES-cell NT blastocysts. (A,B) Whole-mount in situ hybridisation with Oct4 mRNA probe. (C,D) Immunofluorescent staining using an Oct4 antibody. Overlapping of chromatin staining (red) and Oct4 expression (green) is revealed by a yellow stain. Both Oct4 mRNA and proteins are correctly expressed in ICM of NT embryos. (E,F) Examples of 3-day cultures of control and NT blastocysts. (G) Mean surface occupied by the ICM-derived clusters of cells (arbitrary units). No statistical difference could be detected between outgrowths.

 

Figure 2
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  		Fig. 2. Growth of the conceptus during foetal stages. (A) Control conceptus at E13. NT conceptus with a living foetus (B), or consisting of only a placenta with no foetus or membrane present (C). Scale bar: 1 mm. Weight curves of the foetus (D) and the placenta (E) for control and NT conceptus from E13 to term (E19). *P<0.01; **P<0.05.

 

Figure 3
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  		Fig. 3. Two morphological abnormalities observed in E7 NT embryos. (A,C) Control embryos at early-streak (A) and mid-streak (C) stages. (B) Two examples of NT embryos with abnormal rounded shape at pre-streak (left) and early-streak stage (right). (D) The normal proportion of the embryonic and extra-embryonic regions is perturbed in some NT conceptuses (Exe phenotype). (E) Mean ratios of the epiblast (=number of cells in epiblast/total cell number, in %) in controls (black), in NT displaying the Exe phenotype (white), and in other NT embryos (grey). Scale bars: 100 µm. *P<0.01.

 

Figure 4
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  		Fig. 4. Linear regression (P<0.01) of the number of mitotic cells and the total cell number in the epiblast of both NT and control embryos. Forty-one percent (19/47) of the NT embryos are outliers (black diamonds), with either too high or too low a number of mitotic cells. By contrast, only 10% of the controls (3/29) are outliers (open triangles). The coefficients of determination R2 were statistically significant (P<0.01).

 

Figure 5
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  		Fig. 5. Molecular characterisation of NT embryos with abnormal morphologies. Whole-mount in situ hybridisation showing the expression of brachyury (A-D), Nodal (E,F), Bmp4 (G,H), Eomes (I,J) and Cdx2 (K,L) in primitive-streak stage (A,B,E-L) and neural-plate stage (C,D) embryos. (A,C,E,G,I,K) Controls; (B,F,J) NT embryos with abnormal shape; (D,H) NT embryos displaying the `large Exe' phenotype. Scale bars: in A, 100 µm for A,B, 50 µm for C,D; in E, 100 µm for E-L.

 

Figure 6
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  		Fig. 6. Developmental defects at E7 are not rescued by incorporation of normal cells into the epiblast and the visceral endoderm. (A,D) Chimaeras made by injection of normal lacZ-expressing ES cells into blastocysts. Normal NT chimaeras at early-streak (A) and headfold (C) stages. (B) NT chimaera with an abnormal shape; (D) NT chimaera with a `large Exe' phenotype. (E,F) Early-streak stage chimaeras made by injection of normal GFP-expressing ICM cells into NT blastocysts. The chimaera in F displays a `large Exe' phenotype (as well as an abnormal shape), in comparison with the one in E. The nuclei are stained in red by propidium iodide. Cytoplasmic GFP appears green. Scale bars: 100 µm.

 

Figure 7
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  		Fig. 7. Tetraploid cells can rescue abnormal morphologies of NT embryos at E7. (A,B) Normal chimaeras made by aggregation of a NT embryo and a tetraploid, GFP-expressing, embryo developed from a fertilised zygote. (C) Tetraploid embryo developed after disappearance of NT blastomeres. The embryo in A is at early streak stage; the embryo in B is at late-streak stage. (Inset) Higher magnification of C showing that all epiblast cells express GFP. Scale bars: 100 µm.

 

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© The Company of Biologists Ltd 2006