Abstract
During pre-implantation, the totipotent 1-cell embryo undergoes cleavages to derive a blastocyst. Here, cells break symmetry during mitosis, and daughter cells diverge towards two distinct functional paths with the formation of the inner cell mass and trophectoderm. This work was motivated by our need to understand which, and how, genes are involved in the decision-making process leading to the commitment of differentiated cell types. We produced mice embryos heterozygotes for the Gt(ROSA)26Sortm1(CAG-Brainbow2.1)Cle/J construct and heterozygotes for the Tg(CAG-cre/Esr1*)5Amc/J construct. At the 2-cell stage, embryos were treated with 4-Hydroxytamoxifen in culture for one hour to promote the translocation of the CRE protein to the nuclei. CRE proteins recombined the Brainbow2.1 construct to allow for the production of one of the four possible fluorescent proteins (red, green, yellow or cyan). The embryos were then in vitro cultured and harvested at 4-, 8-cell or blastocyst stages. Blastocysts were evaluated for the binary distribution of colored cells in either the inner cell mass or trophectoderm. We identified a significant bias of sister cells towards one of the cell types in blastocysts. This result demonstrates that the cell fate could be traced back to 2-cell stage embryos. Embryos were then evaluated at 4-, 8-cell stages for the presence of fluorescence proteins; and those that showed markedly expression of one or two fluorescent proteins were split for single-cell RNA-sequencing. Using the expression of the transcript correspondent to fluorescent proteins, we assigned each blastomere to a group of cells that could be traced back to either one of the cells in the 2-cell embryo. Using these two groups we determined that few genes showed differences in expression levels that resembled the lineages. Remarkable differences were observed when we analyzed the transcriptome as a whole. With Rainbow-seq we connected transcriptome profiles with lineages at the single-cell resolution.