Figure 3

Dual non-invasive reporter visualization in chimeric embryos generated by tetraploid <-> diploid aggregation. In all panels the tetraploid compartment is yellow/green fluorescent (EYFP+) and the diploid is blue/cyan fluorescent (ECFP+). (a) schematic representation of the tetraploid procedure. Electrofusion of E1.5 embryos to render the blastomeres tetraploid, in vitro culture, and subsequent aggregation schemes using double-tagged compartments. Sandwich aggregations were made using either ECFP+ (diploid) morulae or ES cells. (b-d) anterior view of an E7.5 double-tagged polarized chimera dissected free of its deciduum and therefore missing primary giant cells and parietal endoderm. Here the epiblast (lower half of embryo) and its derivatives are ECFP+ whereas the extraembryonic ectoderm, the visceral endoderm and trophoblast are EYFP+ (upper half or embryo). (b) dark field image taken through an EYFP filter. (c) dark field image taken through an ECFP filter. (d) dark field double exposure image acquired by consecutively using ECFP and EYFP filters. (e) dark field double filter exposure of an E9 chimera dissected free of all extraembryonic membranes except for a small piece of yolk sac (lower left). The embryo itself is ECFP+ whereas the yolk sac both ECFP+ and EYFP+ in the mesoderm and endoderm respectively. A small number of EYFP+ cells are also observed in the ventral midline of the embryo (arrowhead). This observation has been noted previously in tetraploid chimeras, and is presumed to represent a small population of visceral endoderm cells that fail to be displaced by the definitive endoderm at earlier stages. (f) dark field double filter exposure of an E10 chimera. The embryo is exclusively ECFP+ whereas its placenta (to the left of the embryo) is predominantly EYFP+. The labyrinthine trophoblast layer of the placenta comprises both ECFP+ and EYFP+ cells.