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First published online 18 January 2005
doi: 10.1242/jcs.01666

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Downregulation of Par3 and aPKC function directs cells towards the ICM in the preimplantation mouse embryo

Berenika Plusa^1,3,*,, Stephen Frankenberg^1,3,*, Andrew Chalmers^1,5, Anna-Katerina Hadjantonakis^4,, Catherine A. Moore^1,3, Nancy Papalopulu^1,5, Virginia E. Papaioannou⁴, David M. Glover^2,3 and Magdalena Zernicka-Goetz^1,3,¶

¹ Wellcome Trust and Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QR, UK
² Cancer Research UK Cell Cycle Genetics Research Group, Downing Street, Cambridge CB2 3EH, UK
³ Department of Genetics, Downing Street, Cambridge CB2 3EH, UK
⁴ Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA
⁵ Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK

View larger version (76K): [in a new window]   Fig. 1. Localization of Par3 (A-D) and aPKC (E-H) in preimplantation mouse embryos. (A) Par3 (red) localizes to apical cell surfaces (arrow) at the 8-cell stage. (B-D) Three optical sections of the same blastocyst at 5 µm, 24 µm and 42 µm showing concentration of Par3 at apico-lateral surfaces of cells (arrowheads). DNA is stained blue. (E,F) aPKC is concentrated at apical cell surfaces (arrow on this glancing optical section) and nuclei (arrowhead) at the 8-cell stage. (G,H) Confocal sections of the same blastocyst at 14 µm and 26 µm showing concentration at outer apico-lateral cell borders (arrowheads). The inset to panel H shows a mitotic spindle in a dividing blastomere revealed by staining of aPKC (red), Par3 (green) and DNA (blue). Bar, 10 µm.

View larger version (70K): [in a new window]   Fig. 2. Representative series of confocal sections of blastocysts injected at the 4-cell stage with DsRed mRNA, dsPar3 RNA and dominant negative aPKC mRNA. Nuclei fluoresce green, reflecting expression of GFP-tagged histone H2B. The clone formed by the progeny of the injected cells is marked in red. The mean proportions of labelled ICM cells to total labelled cells was 0.34 for DsRed-injected embryos (n=38); 0.53 for dsPar3 RNA-injected embryos (n=16); and 0.71 for dominant negative aPKC-injected embryos (n=25). Bar, 10 µm.

View larger version (16K): [in a new window]   Fig. 3. Proportions of labelled and unlabelled ICM and trophectoderm cells in experimental and control groups of embryos. Note that the relative proportions of ICM versus trophectoderm cells remain almost constant, whereas the relative numbers of labelled cells in ICM or trophectoderm differ between groups.

View larger version (23K): [in a new window]   Fig. 4. Proportional contribution of labelled cells to mural trophectoderm (M) against relative contribution of labelled cells to trophectoderm versus ICM (F). The derivation of the values M and F is described in the text. (A) Values of M and F for a group of 16 experimental embryos injected at the 4-cell stage with dsPar3 RNA (pale blue). A group of 14 control embryos were injected with dsGFP RNA (orange). (B) Values of M and F for a group of 25 experimental embryos injected at the 4-cell stage with mRNA for dominant negative aPKC (blue). A group of 26 embryos were injected with mRNA for wild-type aPKC (purple). (C) Combined groups of control embryos injected with DsRed mRNA at either the 4- or 8-cell stage (red and pink, respectively) or dsGFP RNA at the 4-cell stage (orange). (C, left panel) A control embryo (corresponding to the indicated data point) representing a clone of cells making a major contribution to the ICM and also the polar trophectoderm. (C, right panel) A control embryo (corresponding to the indicated data point) representing a clone of cells contributing predominantly to mural trophectoderm. Bar, 10 µm. Positive rank (Spearman) correlation coefficients were obtained for the values of F and M in both the dsGFP and DsRed control groups (r=0.44, 0.10<P< 0.20; and r=0.37, P<0.05, respectively). Control blastomeres injected with DsRed at the 8-cell stage, or with wild-type aPKC at the 4-cell stage, also gave respective positive correlation coefficients of r=0.52 (0.10<P< 0.20) and r=0.35 (0.05<P< 0.10). When all control groups were combined (panel C), the correlation was highly significant (r=0.51, P<0.001).

View larger version (130K): [in a new window]   Fig. 5. Dominant negative aPKC changes cleavage orientations and leads to cells `falling' from the outside cell layer into the inside part of the embryo (A',B'). Examples of the conservative (A') and differentiative (B') divisions in embryos followed by time-lapse microscopy through five focal planes after injection of a single 4-cell-stage blastomere with mRNA for DsRed and dominant negative aPKC. Images A-D represent stages of development of a single embryo at a central focal plane. The parental cells being followed are outlined with red and the daughter cells with blue and yellow dashed lines. (C') Example of the internalization of a cell followed by time-lapse microscopy through five focal planes in a pre-cavitation embryo injected with mRNA for DsRed and dominant negative aPKC into a single 4-cell-stage blastomere. Images A-D represent stages of development of a single embryo at a central focal plane. Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 6. Recruitment of tight junction proteins following expression of dominant negative aPKC. (A-D) Localization of the tight junction protein ZO1 at the late 8-cell stage in embryos injected with mRNA for dominant negative aPKC and DsRed into one 4-cell-stage blastomere. ZO1 (green and monochrome) localizes to apposing cell surfaces (arrows) between both injected (A,B; injected cells are red and marked with an asterisk) and non-injected (C,D) progeny; 11 embryos were analysed. (E,F) Similar embryo at the blastocyst stage. Occludin (green and monochrome) localizes to the tight junction (arrow) in both non-injected and injected progeny; 9 stained embryos were analysed. The latter are red and marked with an asterisk. Bar, 10 µm.

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