Mesoderm

Pluripotency, PGC regulator, mesoderm, endoderm, and ectoderm gene sets were curated in the literature (Ding et al. 2015 (link); Hackett et al. 2018 ) and in the R&D database (Table S15). First, for each gene in a specific category, such as pluripotency, the log₂FC of the gene was calculated. Then the mean log₂FC of all genes in a category was considered as the overall activity value.

To generate the Sox2-Flp line, we obtained the original plasmid used for the generation of the Sox2-Cre line from Dr. A. McMahon’s lab, which contains a ~13.7 kb genomic fragment encompassing the Sox2 promoter that drives expression of the downstream gene in an epiblast-specific manner^{25 (link)}. We replaced Cre with a FlpO coding sequence using standard recombinant DNA techniques. A PmeI-excised Sox2-FlpO insert was used for pronuclear injection into fertilized C57BL/6 x (DBA x C57BL/6) oocytes. 5 original transgenic founders were obtained that were propagated as hemizygous lines by mating to C57BL/6-Elite (Charles River) mice. Cell lineage-specific activity of the newly established Sox2-Flp line was tested as follows: 1. Sox2-FlpO transgenic males were crossed to the RCE:FRT reporter strain (MMRRC Strain #032038-JAX) which harbors the R26R CAG-boosted EGFP (RCE) reporter allele with a FRT-flanked STOP cassette upstream of an enhanced green fluorescent protein (EGFP) gene. After removal of the FRT-flanked STOP cassette by FLP-mediated recombination, EGFP reporter expression is directed to the cells/tissues in which FLP is expressed. 2. In addition, Sox2-Flp transgenic males were crossed with heterozygous Ssr2 tm1a females in which FLP activity is identified by the loss of LacZ expression. 3. Detailed genotyping was performed on fine-dissected tissue to establish full allele conversions. These strategies collectively illustrated FLP activity in all epiblast-derived tissues, resulting in non-mosaic EGFP expression (on mating with the RCE:FRT strain) and complete loss of LacZ expression (on mating to Ssr2 tm1a/+ females) in all cells of the embryo proper and of the extra-embryonic mesoderm (Fig. 3b, Supplementary Figs. 3, 4). Epiblast-restricted activity of Sox2-Flp was given in most instances, with only rare occasions of ectopic expression in trophoblast cells of the junctional zone in one placenta, with <1% of junctional zone cells affected. This was observed with the RCE:FRT reporter but not with the Ssr2 tm1a reporter alleles. Thus, the Sox2-Flp line was established as a novel tool to revert knockout-first alleles into functional alleles in all cells of the embryo proper while leaving the gene knocked out in all, or the vast majority of, trophoblast cells. Two Sox2-Flp founder lines (1.27 and 4.2.28) matched these criteria and were used in further study.