Additional Supporting Information may be found in the online version of this article.

sc-12-0250_sm_SupplFigure1.pdf241KSupplemental Figure 1. Expression pattern of Sox17GFPCre/+ at E7.5 A) 3D reconstruction of an E7.5 Sox17GFPCre/+ embryo. Intense GFP fluorescence was observed in the definitive endoderm area (arrow) and weaker expression seen in visceral endoderm area (arrowhead). Anterior (A), posterior (P), proximal (Pr), distal (Di). Scale bar = 100 μm.
sc-12-0250_sm_SupplFigure2.pdf481KSupplemental Figure 2. Cre recombinase efficiency assessment A-D) β -D-Galactosidase staining of E9.5 Sox17GFPCre/+;R26RLacZ/+ embryo sections showed LacZ activity in all endoderm-derived cells such as (A) the first branchial pouch (BP1) (B) the second branchial pouch (BP2) (C) the foregut (Fg), the hepatic primordium (HP) and (D) in the hindgut (Hg). It was also present in endothelia as illustrated for the dorsal aorta endothelium (DA). E-F) β -D-Galactosidase staining was performed on E12.5 Sox17GFPCre/+;R26RLacZ/+ embryo sections. The recombination of the Rosa26 locus was detected in (E) the endocardium (heart, H) as well as in the endothelia, such as in the aorta (A). The enzyme activity was also present in all endoderm-derived organs, such as the liver (Li), the stomach (St), the dorsal and ventral pancreas (DP and VP), the small intestine (It) (F), as well as the colon and the caecum (G). Scale bar = 100 μ m (A-D, F-G), 200 μ m (E). Magnification insert scale bar = 20 μ m.
sc-12-0250_sm_SupplFigure3.pdf625KSupplemental Figure 3. Fate tracing of Sox17-expressing cells at multiple stages A) Immunolabeling of E9.5 R26ReYFP ;Sox17GFPCre embryos revealed YFP and Foxa2 colocalization in the gut tube (GT), ventral pancreas (VP), and liver (Li). However, Foxa2 is not detected in YFP-positive endothelial cells (arrows). Scale bar = 50 μm. B) Immunolabeling of E11.5 and E15.5 R26ReYFP;Sox17GFPCre pancreata revealed YFP co-localization with pancreatic markers, such as Ptf1a, Ngn3, Ins, and the ductal stain, Dolichos biflorus agglutinin (DBA). Scale bar = 50 μm.
sc-12-0250_sm_SupplFigure4.pdf726KSupplemental Figure 4. Fate tracing of Sox17-expressing cells in different organs A) Immunolabeling of E15.5 R26ReYFP;Sox17GFPCre embryos revealed YFP and PECAM colocalization in the vein (V), liver (Li), pancreas (P), and hepatic vein (HV). B) Immunolabeling of E9.5 R26ReYFP;Sox17GFPCre embryos revealed YFP and pre-HSC markers co-localization in the blood island of yolk sac.
sc-12-0250_sm_SupplFigure5.pdf474KSupplemental Figure 5. Analysis of the two Sox17-expressing cell populations A) EpCAM immunolabeling was used to analyze the number of EpCAM+ and EpCAM- cells present in E9.5 Sox17GFPCre/+ whole mouse embryos. GFP/EpCAM co-positive cells represent ventral pancreatic cells (EpCAM+), and GFP-positive/EpCAM-negative cells represent hemogenic endothelial cells (EpCAM-). Percentages indicate the mean percentage of EpCAM+ or EpCAM- cells which are GFP-positive (n=4). B) Quantification of EpCAM+ and EpCAM- cells analyzed by FACS from all GFP-positive cells identified in E9.5 Sox17GFPCre/+ whole mouse embryos. C) The mid region including ventral pancreas (red box) was dissected from whole embryos to increase sorting efficiency. Immunohistochemistry on E9.5 Sox17GFPCre/+ whole mount embryo cryo-sections showed that GFPCre was expressed in the ventral pancreatic bud (VP) with weaker expression in the dorsal aorta (DA) in P-Sp area. Image was acquired using AZ100 multizoom microscope, Nikon. Zoom = 5.7x. Scale bar = 100 μm.
sc-12-0250_sm_SupplFigure6.tif1270KSupplemental Figure 6. Comparison of coverage plots on mouse Sox17 gene locus Coverage plot of uniquely mapped sequence reads (red) from EpCAM- and EpCAM+ samples following RNA-Seq. Predicted Sox17 transcripts taken from either the UCSC database (Sox17 (UCSC)) or RefSeq and EMBL/GenBank databases (Sox17 (TROMER)) are shown below mapped reads. Exons 1 – 5 are noted, and a CpG island spanning exons 4 – 5 is indicated. Blue box indicates exon 2 region which contains mapped reads for EpCAM- sample.
sc-12-0250_sm_SupplTable1.tif842KSupplemental Table 1.
sc-12-0250_sm_SupplTable2.tif995KSupplemental Table 2.
sc-12-0250_sm_SupplTable3.tif2117KSupplemental Table 3.
sc-12-0250_sm_SupplTable4.xls10431KSupplemental Table 4.
sc-12-0250_sm_SupplTable5.xls94KSupplemental Table 5.

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