Extended Data Fig. 2: Foxa2-Venus populations.
(a) Single cell FACS strategy. The sorted cells were consecutively gated in order to remove cell debris, to select singlet from multiplet cells and to select live cells. A general gate was created in the FSC-A vs SSC-A plot to include the population of interest and minimize low FSC and high SSC events. The events in this gate were then gated for single cells based on FSC-W to exclude doublets and aggregates. The single cells were then gated for DAPI exclusion. Single-color controls were used to determine the gates for the different fluorophores. The FOXA2POS cells were mixed with pre-labelled carrier cells (labelled with CellVue Maroon) and the FOXA2POS cells (negative for CellVue Maroon staining) were used for the final index single cell sort. Each cell specifications (such as forward scatter - cell size and FOXA2-Venus protein levels) were recorded and linked to a specific sorted cell. (b) Bar plot representation of samples per cluster. Each colour represents a time point between E6.5 and E9.5. (c) Images of E6.5 - E9.5 FOXA2POS embryos, the E8.5 and E9.5 embryos were dissected as illustrated. (d) Re-clustered UMAP dimensional embedding of gastrulation stage lineages. Here, the Node segregates into two clusters. (e) FACS and cell cycle chart for gastrulation stage lineages representing the cell size (radius of circles), Foxa2 mRNA level (X-axis), FOXA2 protein level (Y-axis) and cell cycle phase (colour of pie charts, G1-phase in red, G2M-phase in green, and S-phase in blue) for specific clusters. The Node is represented by a proliferative and non-proliferative cell populations. AVE – anterior visceral endoderm, DE1- early definitive endoderm, DE2 – later definitive endoderm, EmVE – embryonic visceral endoderm, ExVE – extra-embryonic endoderm, InterVE – intermediate visceral endoderm, PS1 – early primitive streak, PS2 – later primitive streak.
