Fig 1. Genetic tracing of the T+ primitive steak cells.

(A) Schematics of the T^{nGPF-CreERT2/+}; R26R^tdTomato/+ allele [16]. Cre-ERT2 is expressed in cells expressing T. In the presence of tamoxifen, Cre protein is translocated to the nucleus where it recombines the R26R^tdTomato/+ reporter. As a result, the cell and its descendants are permanently labelled. (B) Diagram of the experimental approach. T-expressing cells and their descendants are labelled, from E6+21h, E7, E7+7h and E7+11h by administrating a dose of tamoxifen (Tam) to T^{nGPF-CreERT2/+}; R26R^tdTomato/+ mice (0.08 mg/body weight via oral gavage). Cell descendants in the myocardium are analysed at E12.5. (C) Representative hearts resulting from the administration of tamoxifen at different time points in T^{nGPF-CreERT2/+}; R26R^tdTomato/+ immunostained with cTnnT to reveal the cardiomyocytes (blue). Yellow arrows identify small patches of tdTomato positive cardiomyocytes in the LV (iii and v), in the RV (v), and in the outflow tract and atria (vii and viii). Views are ventral. Single epicardial cells are labelled in each condition. (D–E) Summary of all T^{nGPF-CreERT2/+}; R26R^tdTomato/+ hearts examined. The contribution of the T-expressing cells to the different compartments of the heart is quantified by measuring the proportion of tdTomato-positive myocardium. Numbers in brackets in (E) represent the number of litters assessed. Error bars are SD. The data underlying (D–E) can be found in S1 Source Data. (F) Stage variation quantified according to Downs and Davies criteria [20]. Number in brackets represents the number of litters assessed. All the timed matings were for 2-hour periods. (G) Embryos collected at E6+21h or E7+7h showing variation in stage. ((H) Representative T^{nGPF-CreERT2/+}embryos untreated or tamoxifen treated for 2 hours mice (0.08 mg/body weight via oral gavage) and immunostained with oestrogen receptor. Insets (iii) and (iv) are magnified view from (i) and (ii), respectively. (I) PCR amplicons generated from the genomic region in which Cre-mediated recombination occurs, resolved on an agarose gel. Before recombination, the PCR product is 1,145 bp (white rectangle); after recombination, it is 274 bp (black rectangle). Template gDNA was extracted from either an ear clip of an adult T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato} mouse (untreated) or T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato} embryos dissected at 2, 4, and 12 hours following oral gavage with Tamoxifen, as labelled. An increase in the proportion of the recombined band can be seen over time following Tamoxifen administration. The data underlying (I) can be found in S1 Raw image. (J) Representative T^{nGPF-CreERT2/+}embryos at the MS-LS and OB-EB stages, immunostained with. (iii) and (iv) are magnified views in (i) and (ii), respectively. (K) Quantification of T intensity in single segmented nuclei from embryo shown in (J). The data underlying (K) can be found in S2 Source Data. (L) Representative T^{nGPF-CreERT2/+}embryos at the MS-LS and OB-EB stages treated with tamoxifen for 2 hours (0.08 mg/body weight via oral gavage) and immunostained for oestrogen receptor. (iii) and (iv) are magnified views in (i) and (ii), respectively. (M) Quantification of Oestrogen receptor intensity in single segmented nuclei from the embryos shown in (L). The data underlying (M) can be found in S2 Source Data. Embryos in J (i) and (ii) and in L (i) and (ii) were immunostained together and imaged under similar conditions. ant., anterior; cTnnT, cardiac troponinin T; EB, “early bud” stage; EHF, early head fold; LA, left atria; LB, “late bud stage”; LS, late-streak; LV, left ventricle; MS, mid-streak; OB, “no bud” stage; OFT, outflow tract; post., posterior; PS, primitive streak; RA, right atria; RV, right ventricle. Scale bar: 200 μm.