Sixteen inner or outer blastomeres from 16-cell embryos and 32 inner or outer blastomeres from 32-cell embryos (nascent blastocysts) were reaggregated and cultured in vitro. In 24 h old blastocysts developed from blastomeres derived from 16-cell embryos the expression of Cdx2 protein was upregulated in outer cells (new trophectoderm) of the inner cells-derived aggregates and downregulated in inner cells (new inner cell mass) of the external cells-derived aggregates. After transfer to pseudopregnant recipients blastocysts originating from both inner and outer blastomeres of 16-cell embryo developed into normal, fertile mice, but the implantation rate of embryos formed from inner cell aggregates was lower. The aggregates of external blastomeres derived from 32 cell embryo usually formed trophoblastic vesicles accompanied by vacuolated cells. In contrast, the aggregates of inner blastomeres quickly compacted but cavitation was delayed. Although in the latter embryos the Cdx2 protein appeared in the new trophectoderm within 24 h of in vitro culture, these embryos formed only very small outgrowths of Troma1-positive giant trophoblastic cells and none of these embryos was able to implant in recipient females. In separate experiment we have produced normal and fertile mice from 16- and 32-cell embryos that were first disaggregated, and then the sister outer and inner blastomeres were reaggregated at random. In blastocysts developed from aggregates, within 24 h of in vitro culture, the majority of inner and outer blastomeres located themselves in their original position (internally and externally), which implies that in these embryos development was regulated mainly by cell sorting.

The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8→16 (primary inner cells) and 16→32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.

In order to ensure successful development, cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or inner cell mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential cell fate decisions. We revealed that TE-deprived ICMs derived from 32-cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage.

During preimplantation mouse development stages, emerging pluripotent epiblast (Epi) and extraembryonic primitive endoderm (PrE) cells are first distributed in the blastocyst in a “salt-and-pepper” manner before they segregate into separate layers. As a result of segregation, PrE cells become localised on the surface of the inner cell mass (ICM), and the Epi is enclosed by the PrE on one side and by the trophectoderm on the other. During later development, a subpopulation of PrE cells migrates away from the ICM and forms the parietal endoderm (PE), while cells remaining in contact with the Epi form the visceral endoderm (VE). Here, we asked: what are the mechanisms mediating Epi and PrE cell segregation and the subsequent VE vs PE specification? Differences in cell adhesion have been proposed; however, we demonstrate that the levels of plasma membrane-bound E-cadherin (CDH1, cadherin 1) in Epi and PrE cells only differ after the segregation of these lineages within the ICM. Moreover, manipulating E-cadherin levels did not affect lineage specification or segregation, thus failing to confirm its role during these processes. Rather, we report changes in E-cadherin localisation during later PrE-to-PE transition which are accompanied by the presence of Vimentin and Twist, supporting the hypothesis that an epithelial-to-mesenchymal transition process occurs in the mouse peri-implantation blastocyst.

The integration of extracellular signals and lineage-specific transcription factors allows cells to react flexibly to their environment, thus endowing the mammalian embryo with the capacity of regulative development. The combination of genetic and pharmacological tools allowing disruption of the fibroblast growth factor / extracellular signal-regulated kinase (FGF/ERK) pathway, together with animal models expressing lineage-specific reporters provided new insights into the role of this signaling cascade during mammalian development, as well as in embryo-derived stem cells. Here, we combine current knowledge acquired from different mammalian models to consider the universality of this cascade in specifying cellular fate across mammalian species.

In order to examine interactions between cells originating from different species during embryonic development we constructed interspecific mouse↔rat chimaeras by aggregation of 8-cell embryos. Embryos of both species expressed different fluorescent markers (eGFP and DsRed), which enabled us to follow the fate of both components from the moment of aggregation until adulthood. We revealed that in majority of embryos the blastocyst cavity appeared inside the group of rat cells, while the mouse component was allocated to the deeper layer of the inner cell mass and to the polar trophectoderm. However, due to rearrangement of all cells and selective elimination of rat cells, shortly before implantation all primary lineages became chimaeric. Moreover, despite the fact that rat cells were always present in the mural trophectoderm, majority of mouse↔rat chimaeric blastocysts implanted in mouse uterus, and out of those 46% developed into foetuses and pups, half of which were chimaeric. In contrast to mural trophectoderm, polar trophectoderm derivatives, i.e. the placentae of all chimaeras were exclusively of mouse origin. This strongly suggests that the successful postimplantation development of chimaeras is enabled by gradual elimination of xenogeneic cells from the nascent placenta. The size of chimaeric newborns was within the limits of control mouse neonates. The rat component located preferentially in the anterior part of the body, where it contributed mainly to the neural tube. Our observations indicate that although chimaeric animals were able to reach adulthood, high contribution of rat cells tended to diminish their viability.

Spontaneous diploid-triploid chimaeras occur sporadically in various mammalian species including man, but so far have never been produced experimentally. In order to get a deeper insight into the developmental consequences of this anomaly, we have developed two procedures that enabled for the first time to produce routinely diploid-triploid embryos, foetuses, and animals in the mouse. These procedures are: (1) aggregation of cleaving diploid embryos with triploid embryos produced by suppression of the second polar body in zygotes, and (2) fusion of a haploid karyoplast with one blastomere of the two-cell diploid embryos. The first procedure yielded 23 living and 6 dead postimplantation embryos and foetuses (age: 8th-19th day) out of which 22 were chimaeric. In addition, three chimaeric neonates reached adulthood. Two animals were fertile, and one--an overt chimaera--was an infertile male. The rate of postimplantation development of aggregation chimaeras was normal or only slightly retarded, and with one exception the foetuses were morphologically normal. Generally, the highest contribution of the 3n component in extra-embryonic structures was noted in the yolk sac, and usually it was higher than its contribution to the organs of the body. Chimaerism was most often noted in the liver, the heart, the intestine, and the lungs. Participation of triploid cells to all tissues studied, both in the body and in extra-embryonic structures, appeared to decrease slightly as development progressed. The second procedure yielded 10 foetuses and 6 adults. Three foetuses were chimaeric. Six fertile adults were probably non-chimaeras: the triploid component was absent in the coat and in the blood.