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First published online 31 October 2007
doi: 10.1242/dev.003798

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Stochastic patterning in the mouse pre-implantation embryo

Jens-Erik Dietrich^* and Takashi Hiiragi^*,

Max-Planck Institute of Immunobiology, Department of Developmental Biology, Freiburg i. Br., Germany.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Strategy for synchronizing pre-implantation embryos at compaction and the timing of morphogenetic events during the analyzed period. Embryos were selected at the onset of compaction (about 60 to 70 hp-hCG; 0 hp-compaction). Groups of embryos were fixed every 5 hours, through morula and blastocyst stages bI to III, until 55 hp-compaction, corresponding to embryonic days (E) 2.5 to 4.5.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Protein expression in blastomeres post-compaction. Average number of blastomeres that were positive for Oct4 (light blue), Cdx2 (pink) and Nanog (green) in relation to the total cell number (black) and the number of the inside population (dark blue) as a function of time after compaction. As a reference, the average number of blastomeres of in vivo developed embryos is provided (crosses) at 10, 30 and 55 hp-c. Error bars indicate s.d.

View larger version (91K): [in this window] [in a new window]   Fig. 3. Patterning of the pre-implantation embryo progresses through an initial phase of high variability. Oct4 (A), Cdx2 (B) and Nanog (C) distribution at defined stages of development. For every embryo, the counterstained DNA is shown in the row below. White and green arrowheads indicate nuclei of interest of inside and outside cells, respectively. Actin (red) marks the cell membranes. Asterisks indicate blastomeres with only minimal contact to the outside. Images are single optical sections, except for those at 0 hp-c, being maximum intensity projections. Scale bar: 20 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Cdx2 levels are variable and initially without correlation to Oct4. (A) Cdx2 and Oct4 distribution within the same embryo at defined stages of development. For every embryo the counterstained DNA is shown in the row below. White and green arrowheads indicate nuclei of interest of inside and outside cells, respectively. Actin (red) marks the cell membranes. All images are single optical sections. Scale bar is 20 µm. (B) Scatter plots showing the normalized mean fluorescence intensity of Cdx2 relative to Oct4 for individual blastomeres. Each dot represents one blastomere; different colors represent different embryos. Red lines mark the background fluorescence level.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Cdx2 and Nanog variability are not correlated. (A) Cdx2 and Nanog at defined stages of development. For every embryo the counterstained DNA is shown in the row below. White and green arrowheads indicate nuclei of interest of inside and outside cells, respectively. Actin (red) marks the cell membranes. Asterisks indicate blastomeres with only minimal contact to the outside. All images are single optical sections. Scale bar: 20 µm. (B) Scatter plots showing the normalized mean fluorescence intensity of Cdx2 relative to Nanog in individual blastomeres. Each dot represents one blastomere; different colors represent different embryos. Red lines mark the background fluorescence level.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Variability of Cdx2, but not Nanog, may be generated by asymmetric cell divisions at the 8-cell stage. (A,B) Cdx2 and Nanog patterning of blastomeres isolated at the 8-cell stage and analyzed at 70 (A) and 78 (B) hp-hCG. (C) Scatter plot illustrating the normalized mean fluorescence intensity of Cdx2 (left panels) and Nanog (right panels) in sister blastomeres at 70 (C1) and 78 (C2) hp-hCG. The `smaller' (at 70 hp-hCG) or `inside' (at 78 hp-hCG) cell is plotted on the x-axis, the `bigger' or `outside' one on the y-axis. A filled circle represents a doublet with clear asymmetry; an empty circle represents a doublet with slight asymmetry; an empty rhombus represents symmetric doublets. Each symbol represents a 2/16 doublet; different colors represent different embryos. Red lines mark the background fluorescence level. (D) Cdx2 and Nanog patterning at 90 hp-hCG in blastomeres isolated at the 8-cell stage. (E) Cdx2 and Nanog patterning of blastomeres isolated at 8-cell and re-isolated at the 16-cell stage. A cross marks an apolar cell; black triangle, a polar cell; asterisk, eccentric localization of the nucleus; arrowhead, the pole of intense actin staining in polar cells. DNA staining provides a reference value. Actin (red) marks the cell membranes. All images are single optical sections. Scale bar: 20 µm. (F) Scatter plot illustrating the Cdx2 variance in relation to Nanog levels of polar (filled triangles) and apolar (crosses) cells. Each symbol represents one blastomere. Black lines and dotted lines represent the average protein levels of polar cells and apolar cells, respectively. Error bars indicate s.d.

View larger version (45K): [in this window] [in a new window]   Fig. 7. A summary scheme of the temporal and spatial expression patterns and the proposed model for autonomous establishment of asymmetries in the pre-implantation mouse embryo. Oct4 (red) is present at high levels in all blastomeres until 45 hp-c, gradually declining in the trophectoderm (orange gradient represents the degree of ICM restriction, with darker being more restricted). Cdx2 and Nanog reflect two patterning phases, including initiation of variability among blastomeres after compaction and subsequent sorting (dark green represents strong expression, light green weak expression and empty circles absence of nuclear Cdx2 or Nanog; blue gradient represents the degree of restriction, with darker being more restricted; see text for details).

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