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First published online 5 January 2005
doi: 10.1242/dev.01602

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Four-cell stage mouse blastomeres have different developmental properties

¹ Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Genetics, Tennis Court Road, Cambridge CB2 1QR, UK
² Department of Experimental Embryology, Polish Academy of Science, Jastrzebiec 05-552, Poland
³ Développement des Vertébrés, Institut Jacques Monod, UMR7592 CNRS Universités Paris 6, 7. 2, place Jussieu, 75005 Paris, France
⁴ Department of Microbiology, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan

View larger version (121K): [in a new window]   Fig. 4. Construction of chimaeras of individual four-cell stage blastomere types. ME embryos were selected in which the later dividing two-cell blastomere had been labelled at its vegetal pole as described in the legend for Fig. 3. (A) One such embryo that has been subjected to limited digestion with pronase in order to thin the zona pellucida (red arrow) so that it continues to maintain the tetrahedral morphology of the embryo and yet does not provide any resistance for micromanipulation of the individual blastomeres. (B) The embryo is held in the left-hand pipette by a m-blastomere while the e2 blastomere is withdrawn into the pipette on the right. (C,D) The procedure is repeated to remove an e1 blastomere into the right-hand pipette. (E,F) One of the m blastomeres, which is attached to the polar body, is then withdrawn into the right hand pipette. (G) The completed dissection showing all four four-cell stage blastomeres, the second polar body is still attached to one of the m cells. Scale bar: 60 µm. (H) An aggregate of e2 blastomeres from three such dissections. (I) The same aggregate as shown in H viewed under fluorescence optics to show the fluorescent beads used to label the vegetal poles. Scale bar: 18 µm in A,H,I; 33 µm in B-F.

View larger version (25K): [in a new window]   Fig. 1. A schematic representation of the origins of blastocyst pattern in the first two cleavages of the mouse embryo. Orientation of the first cleavage, and the order and orientation of the second cleavage divisions in both two-cell stage blastomeres affect the spatial pattern of allocation of their progeny at the blastocyst stage. Two groups of embryos are presented: ME, in which the earlier second cleavage is an M-division the later an E-division; and EM, in which the earlier second cleavage is an E-division and the later one an M-division. Taken together, both of these groups account for 80% of all embryos in our studies (Piotrowska-Nitsche and Zernicka-Goetz, 2005). In both of these groups of embryos, there is a strong tendency for the one of the daughter cells to contribute most of its progeny to the future embryonic part and for the other to the future abembryonic part of the blastocyst. However, only in ME embryos does the early dividing blastomere preferentially contribute its progeny to the embryonic part. In EM embryos, the earlier dividing blastomere can equally often contribute to the embryonic or abembryonic part (hence two possible outcomes are presented).

View larger version (56K): [in a new window]   Fig. 2. Allocation to specific blastocyst regions of the progeny of blastomeres with vegetally marked membrane. (A) A lateral view of a two-cell stage embryo with one two-cell stage blastomere labelled with red dye and the other two-cell stage blastomere with a bead attached at its vegetal pole. pb, polar body. (B) Polar view of a three-cell stage embryo in which the blastomere labelled with dye has divided first through a meridional plane. (C) A four-cell stage embryo in which the dye labelled two-cell blastomere has divided meridionally and its sister two-cell blastomere undertakes an E-division. The bead remains in a `vegetal' position (on the e2 cell), indicating that membrane has not been displaced during division. The cell carrying the bead (which we term e2+) is then labelled with a blue dye (blue arrow) and allowed to develop to the blastocyst stage. (D) Blastocyst derived from the embryo in C showing contribution of the blue labelled cells to mural trophectoderm. (E) A similar four-cell stage embryo to that shown in C in which the bead is now in a position proximal to the polar body (on the e1 cell) indicating that vegetal membrane has been displaced during division. The e2 cell that does not carry the bead (that we term e2-) is then labelled with a blue dye and allowed to develop to the blastocyst stage (F) Blastocyst derived from the embryo in E, showing contribution of the blue labelled cells to the boundary zone. Scale bar: 10 µm.

View larger version (83K): [in a new window]   Fig. 3. Lineage tracing of e2+ (A-D) or e2- (E-H) four-cell blastomeres (marked in blue) to the blastocyst stage. (A-D) Series of optical sections of four blastocysts in which the e2 blastomere is labelled with blue dye. The red cells are progeny of one two-cell blastomere labelled with red dye that was first to divide to the four-cell stage. All cells have green nuclei due to the GFP-H2B transgenic marker. In all cases, the vegetal membrane of the labelled four-cell blastomere had not undergone displacement during its generation. In all four cases the blue labelled clone occupies predominantly the mural trophectoderm and does not contribute to ICM. The one exception is the embryo in B, in which a single blue cell was found in the superficial layer of the ICM. The arrowhead in B indicates a fluorescent bead that is retained in the development of this embryo. The bead, which was attached to the vegetal part of the two-cell stage blastomere still remains attached to a blue cell and so continues as a vegetal marker. (E-H) Series of optical sections of four blastocysts in which a vegetally labelled e-blastomere was found in the e1 position and the e2 cell was labelled with blue dye. In all four cases, the blue-labelled clone occupies predominantly the more superficial cells of ICM and also adjacent polar and mural trophectoderm. Scale bar: 18 µm.

View larger version (112K): [in a new window]   Fig. 5. Defects in preimplantation and postimplantation development of chimaeras derived from e1-, e1+, e2- and e2+ blastomeres. (A-L) The rows of vegetally labelled e1 and e2 (A-F) or non-vegetally labelled (e1 and e2) (G-L) chimaeras display embryos in which the marked vegetal membrane had either not undergone displacement or had been displaced during their generation in the second cleavage. A similar range of defects were seen in all groups. Many chimaeras developed to a late morula-like stage (A,B,D,G,J). Some embryos appeared to have begun to cavitate (D). Other embryos developed to form trophoblastic vesicles (F). When chimaeras developed to the blastocyst stage, they could contain very little ICM (E,H) or have a normal appearance (C,I,K,L). Scale bar: 20 µm. (M-P) In situ hybridisation showing Oct4 expression in ICM cells or their precursors in such chimaeras. Blastocysts derived from e1- or e2- (M,N) and e2+ or e1+ (O,P) chimaeras. (Q-W') Chimaeras constructed as described in Fig. 4 were allowed to develop in culture to the equivalent of the late blastocyst stage (see Materials and methods) and transferred to foster mothers together with carrier embryos. The chimaeras, which are recognised by their fluorescent nuclei, were recovered at E6.5, equivalent to the onset of gastrulation. (Q-Q') m chimaera: a gastrulating embryo of normal appearance. The embryo is also shown under fluorescent optics (Q') to reveal GFP-H2B expression and a fluorescent bead (arrow in Q') applied in subset of experiments also in m chimaeras. This shows that the bead did not interfere with embryo development. The GFP marker is more strongly fluorescent in epiblast cells. Scale bar: 100 µm. (R-T) e2+ chimaeras: embryos either appear almost normal morphologically, although delayed in their development in comparison with m chimaeras (S,T) or are abnormal (R). The characteristic thickening of the visceral endoderm (presumably anterior visceral endoderm) is still at the distal tip of the egg cylinder (arrow in T) as found in E5.5, but not in E6.5 wild-type embryos. (R'-T') Fluorescent images of embryos in R-T. Arrows indicate epiblast. None of the e2 cells used to make these chimaeras had undergone displacement of the vegetal membrane at the time of transition to the four-cell stage. Scale bar: 80 µm. (U-W) e1- and e2- chimaeras: two embryos that show significant levels of tissue disorganisation (U,V). (U) Asymmetric outgrowth of extra-embryonic tissue occurs (arrow in U). This is a e1- chimaera in which there was no displacement of the vegetally marked membrane. (V) e2- chimaera lacking or with reduced extra-embryonic structures (arrow in V). (W) Embryo (e1-) of apparently normal morphology. (U'-W') Fluorescent images of embryos in U-W. Arrows indicate epiblast. (X-Z') In situ hybridisation showing Fgf8, Cer1 or Bmp4 expression in m chimaera (X), e1+ chimaeras (X',Y), e2+ (Y') e1- chimaeras (Z,Z'). (X') This embryo shows a thick Cer1-positive AVE (red arrow) that is reminiscent of wild-type E5.75 embryos. However, although the embryo is small, the expression of Bmp4 appears normally localised (green arrow). (Y) An embryo that shows normal expression of Cer1 and Bmp4 (red and green arrows, respectively). (Y') Small e2+ chimaera showing no expression of Fgf8 and Cer1, indicating a delay or a complete arrest in development. Nevertheless, all three germ layers could be distinguished morphologically. (Z) An example of an e1- chimaera with a visceral endoderm layer as well as an inner epithelial layer, but with an abnormally placed ectoplacental cone (on one side of the embryo rather than proximally). The same embryo is shown in U. Bmp4 expression is relatively normal in a ring of cells in the inner epithelium (green arrow), presumably marking the boundary between the epiblast and the extra-embryonic ectoderm. Cer1 (red arrow) is expressed in a group of visceral endoderm cells presumably marking the distal tip of the embryo. (Z') An e1- chimaera showing expression of Cer1 (red arrow) and Fgf8 (black arrow) as observed in control m chimaeras (X).

View larger version (86K): [in a new window]   Fig. 6. Developmental potential of vegetally marked four-cell blastomeres when surrounded in chimaeras by four-cell blastomeres from random positions. Three chimaeras recovered at E5.5 to observe the distribution of H2B-GFP marked cells in comparison to progeny of not expressing GFP. (A,B) Two sides of the embryo in which the e2+-derived progeny contributed mainly to epiblast and extra-embryonic ectoderm. The contribution of labelled cells appears to be stronger on one side of the embryo (A). (C) Chimaera of e2+ four-cell stage blastomere (visible bead) and four wild-type blastomeres of F1 (wild-type) strain. (D,E) Two sides of embryo in which e2+ blastomere contributed to epiblast, extra-embryonic ectoderm and some visceral endoderm. (F,G) Two sides of chimaera in which e2+ contributed predominantly to visceral endoderm. Scale bar: in A, 50 µm for A,B,D-G; in C, 30 µm for C.