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First published online August 25, 2008
doi: 10.1242/10.1242/dev.021519

Development 135, 3081-3091 (2008)
Published by The Company of Biologists 2008
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Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst

Berenika Plusa1,2,*,{ddagger}, Anna Piliszek1,*, Stephen Frankenberg1,3,*,{dagger}, Jérôme Artus1 and Anna-Katerina Hadjantonakis1,{ddagger}

1 Developmental Biology Program, Sloan-Kettering Institute, New York, USA.
2 Faculty of Life Sciences, Manchester University, Manchester, UK.
3 Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzeebiec, Poland.


Figure 1
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  		Fig. 1. Pdgfr{alpha} is localised to the PrE layer of E4.0 blastocysts and blastocyst outgrowths. (A) Pdgfr{alpha} is co-expressed with Gata4 in the PrE of E4.0-4.5 blastocysts. (B-E) Co-expression of GFP in E4.0 and E4.5 Pdgfr{alpha}H2B-GFP/+ embryos with Pdgfr{alpha} protein (B), and with the PrE markers Gata6 (C), Gata4 (D) and Dab2 (E). (F) GFP is co-expressed with Gata4 in Pdgfr{alpha}H2B-GFP/+ blastocyst outgrowths after 72 hours of culture. Each row represents one embryo. All panels show single optical sections. bf, bright field; green, Pdgfr{alpha} (A) and GFP (B-F); red, Pdgfr{alpha} (B), Gata4 (A,D,F), Gata6 (C) and Dab2 (E); blue, Hoechst. Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. Expression of GFP in live Pdgfr{alpha}H2B-GFP/+ embryos during pre- and early postimplantation development. (A-C) Heterogeneous GFP distribution in a 16-cell morula (A), a 36-cell early blastocyst (B; arrowhead indicates GFP-expressing TE cell) and a 64-cell mid-blastocyst (C). (D) In an ~80-cell blastocyst, GFP-expressing cells are partially segregated to the prospective PrE layer. (E,F) In an ~100-cell blastocyst (E), GFP-expressing cells are restricted to the PrE layer and in a 120-cell blastocyst (F) they start to migrate along the mural TE (arrowheads). (G) At E5.5, GFP-positive cells occupy the visceral endoderm (arrowhead indicates remainder of GFP-positive parietal endoderm). The second column depicts 3D reconstructions of the z-stacks, other panels show single optical sections. bf, bright field; green, GFP; red, FM4-64. Scale bar: 20 µm.

 

Figure 3
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  		Fig. 3. Localisation of Nanog and Gata6 in embryos from 8-cell to blastocyst stage. (A) Gata6 and GFP expression in a 16-cell PdgfraH2B-GFP/+ embryo. (B) Gata6 and Nanog are co-expressed in 8-cell embryos. (C) In a 32-cell morula, Nanog and Gata6 show broad, overlapping expression. (D) In a 33-cell blastocyst, Nanog and Gata6 colocalise in some, but not all cells. (E) In a 58-cell blastocyst, Gata6 expression is mutually exclusive from that of Nanog. Each row represents a single optical section of one embryo. bf, bright field; green, GFP; white, Nanog; red, Gata6; blue, Hoechst. Scale bar: 20 µm.

 

Figure 4
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  		Fig. 4. Localisation of Nanog, Gata4 and GFP in PdgfraH2B-GFP/+ blastocysts. (A) In a 32-cell blastocyst, Nanog and GFP show broad, overlapping expression; Gata4 is undetectable. (B) Salt-and-pepper distribution of Nanog, Gata4 and GFP in a 64-cell blastocyst. Gata4 is expressed only in a subset of Nanog-negative and GFP-positive cells. (C) Partial segregation of Nanog-positive and Gata4-/GFP-positive cells in a 115-cell blastocyst. Gata4 and GFP are co-expressed in cells mostly localised to the nascent PrE layer. Nanog is expressed in the EPI layer and no longer colocalises with Gata4 or GFP. (D) In a 118-cell blastocyst, PrE markers (Gata4 and GFP) are co-expressed and mutually exclusive from the EPI marker Nanog; cells expressing PrE and EPI markers are fully restricted to their respective layers. Each row represents a single optical section of one embryo. bf, bright field; green, GFP; white, Nanog; red, Gata4 (A-D); blue, Hoechst. Scale bar: 20 µm. (E) Comparison of the number of Gata4- and Nanog-positive cells versus total cell number. An abrupt decrease in the number of Nanog-expressing cells at around the 64-cell stage coincides with the emergence of Gata4-expressing cells. Thereafter, the number of Nanog-expressing cells appears to increase only slightly, whereas the number of Gata4-expressing cells increases approximately linearly. (F) Comparison of the number of Gata4- and GFP-expressing cells with respect to total cell number.

 

Figure 5
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  		Fig. 5. Changes in EPI and PrE marker expression correlate with GFP-positive cell behaviour during in vitro culture of Pdgfr{alpha}H2B-GFP/+ blastocysts. (A-D) Immunolocalisation of Gata4 and Nanog in the ICM of embryos of 64 cells or more. (A) Salt-and-pepper distribution of Gata4 and Nanog in a 65-cell embryo. Some cells expressing Gata4 are also Nanog positive. (B) Salt-and-pepper distribution of Gata4 and Nanog in a 72-cell embryo. (C) Partial segregation of Gata4- and Nanog-positive cells in a 108-cell embryo. (D) Gata4- and Nanog-positive cells are completely segregated in a 115-cell embryo. (E-H) Changes in the GFP-positive cell distribution within the ICM of a PdgfraH2B-GFP/+ embryo dissected ~90 hpc, visualized by 3D time-lapse microscopy. From 20 minutes (E) to 235 minutes (F) of culture, GFP-positive cells are randomly distributed in the ICM. Then, after around 370 minutes, partial segregation of the GFP-positive cells to the layer of cells lining the cavity can be observed (G). Complete segregation of GFP-positive cells to the PrE layer is achieved by 575 minutes (H). (E'-H') High-magnification views of ICMs of embryos at successive time-points E-H. (H') PrE layer can be distinguished by different refractive properties on a phase contrast (bright field) image from EPI cells (arrowheads). Each row represents single section time-lapse images of the same embryo (E-H, GFP fluorescence overlaid on a bright-field image; E'-H', bright-field image only). Green, GFP; red, Gata4; white, Nanog; blue, Hoechst. Scale bar: 20 µm. (I) Quantification of embryos exhibiting salt-and-pepper, partially sorted, or sorted distribution of PrE precursors relative to cell number.

 

Figure 6
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  		Fig. 6. Diverse behaviour of GFP-positive cells contributing to the PrE layer in PdgfraH2B-GFP/+ embryos. (A) 3D reconstructions of z-stacks taken during fluorescence time-lapse imaging of a single Pdgfr{alpha}H2B-GFP/+ blastocyst during PrE formation. GFP-positive cells are initially randomly distributed throughout the ICM, but then segregate progressively to the surface of the ICM in contact with the blastocyst cavity. Some GFP-positive cells stay on the cavity throughout the movie (dark blue dot). Note the intercalation of GFP-positive cells highlighted by magenta and red dots. During PrE layer formation, GFP-positive cells remaining in the EPI can undergo apoptosis (arrowhead) or downregulate GFP expression, in contrast to cells in the PrE, which upregulate GFP expression (pale blue dot). A weakly GFP-positive cell is shown migrating away from the cavity (yellow dot). Scale bar: 20 µm. (B) Relative contribution of different types of cell behaviour to PrE segregation.

 

Figure 7
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  		Fig. 7. Multi-step model of EPI/PrE lineage formation. Initial (16- to 32-cell stage) overlapping expression of lineage-specific transcription factors is followed by a progression towards a stabilised salt-and-pepper pattern of PrE (Gata6) and EPI (Nanog) expression (32- to 64-cell stage). Subsequently, stabilised Nanog or Gata6 expression biases cells towards a particular fate (~64-cell stage) and cell sorting proceeds, although positional signals (arrows) are still required to complete specification.

 

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