Fig. 2

The endocardium is molecularly distinct from the vascular endothelium. (A,C) Live images of Gt(endocard:egfp) and Tg(fli1a:egfp) embryos at the 15 somite-stage (16.5 hpf) showing GFP fluorescence. GFP fluorescence can be seen in the endocardial progenitors (white arrowhead) of Gt(endocard:egfp) embryos (A), some auto-fluorescence is seen in the yolk (asterisk). In Tg(fli1a:egfp) embryos (C), GFP fluorescence can be seen in the entire vascular endothelium of the trunk (black arrows) and head (white arrow) as well as the endocardium (white arrowhead). Scale bars: 50 μm. (B,D) FACS plots from dissociated Gt(endocard:egfp) (B) and Tg(fli1a:egfp) (D) embryos show the gating strategy to collect GFP⁺ cells: two gates were used to collect high and low GFP⁺ populations for Gt(endocard:egfp) embryos, and all analysis was performed on cells collected above the red line (B), whereas a single population was collected for Tg(fli1a:egfp) embryos (D). (E) Hierarchical clustering of the top 200 most variable genes across all samples shows that samples segregate according to transgene. (F) Volcano plot showing the spread of differentially expressed genes with a subset of genes labelled, the dotted lines show a log₂ fold change of 1 or −1. (G) Validation of the subset of genes highlighted in F by qPCR (G). Log₂ fold change is represented, error bars indicate 95% confidence intervals. (H) GO term analysis using a PANTHER overrepresentation test was performed on TREAT lists of genes upregulated in the Gt(endocard:egfp)^High samples relative to Tg(fli1a:egfp). The 25 most significant GO terms for genes enriched in the endocardium are shown.