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

SC_11-0922_sm_supplFigure1.pdf258KSupplemental Figure 1. (A, B) FACS analysis of the E7.5 Etv2 WT (top) and mutant (bottom). Mutant embryos showed a decreased number of cells associated with the hematopoietic (CD41) and endothelial (Flk1) lineages in the Etv2 mutant (**: p<0.01, *: p<0.05).
SC_11-0922_sm_supplFigure2.pdf636KSupplemental Figure 2 (A) Illustration of the parasagittal sections used in this study. Mesoderm is shaded in gray. The direction of mesoderm migration within the planes of sections is indicated by red arrows 42. Note that primitive streak (PS) and node are not in a parasagittal section. (B-C) Immunohistochemistry of late streak (B) and early bud (C) stage embryos. Parasagittal sections were stained with antibodies against EYFP (green) and Pdgfra (red). Distally located EYFP+ cells coexpress Pdgfra (B, inset; arrowheads), whereas proximally located cells are negative for Pdgfra (A, inset; arrows). At the late streak stage, all EYFP+ cells are localized in developing blood islands in the yolk sac and do not colabel with Pdgfra (Am, amnion: BI, blood island: EC, exocoelomic cavity: Ecto, embryonic ectoderm: EEE, extraembryonic ectoderm: EEM, extraembryonic mesoderm: Meso, embryonic mesoderm: VE, visceral endoderm. Boxed areas are enlarged in successive panels and insets. Bars: 100 micron). (D) Blood islands in the Tie2-lacZ transgenic yolk sac in the Etv2 WT background. The section is stained for β-galactosidase activity. (E) A blood island of the Etv2 WT yolk sac stained with the Tie2 antibody. Note Tie2 signal in the endothelial and hematopoietic lineages. Arrowheads indicate the endothelial layer and asterisks denote blood.
SC_11-0922_sm_supplFigure3.pdf184KSupplemental Figure 3 qRT-PCR analysis of the E8.0 yolk sac cells. Y-axis represents relative expression level between mutant and wildtype yolk sacs (the expression of a gene in the mutant yolk sac is divided by the expression in the wildtype yolk sac).
SC_11-0922_sm_supplFigure4.pdf195KSupplemental Figure 4 Quantification of the CD41 and Tie2 positive cells after Etv2 induction from Day 3 to Day 9 in differentiated EBs [data represent an average of 3 independent experiments (bars represent SEM)].
SC_11-0922_sm_supplTable1.tif2754KSupplemental Table 1. Sequences of oligonucleotides used in the molecular analysis. The primer sequences utilized in the Lmo2 regulation studies are outlined as follows. (A) The PCR primers used for ChIP analysis where the locus is the distance from the transcriptional start site. (B) The primers for the Lmo2 +1K region that was subcloned into the luciferase reporter construct are indicated. (C) The primers for the mutagenesis of the four EBS sites in the +1K region are indicated. (D) The oligonucleotide sequences harboring the EBS3 utilized in the EMSA is provided.
SC_11-0922_sm_supplTable2.tif568KSupplemental Table 2. List of genes upregulated after 12 h of Etv2 induction in EBs at 3 days of differentiation. Genes that showed significant changes between Etv2 uninduced and induced conditions (p<0.005) are listed. Microarray data analysis was performed using three independent cultures of Etv2 inducible ES cells as described in the Methods section of the main text. False discovery rate (FDR) control was attempted using Benjamini-Hochberg method (see the last column of the table) 61, but unusual distribution of unadjusted p values resulted in extremely high FDR values estimated. An unadjusted p value cut-off of 0.005 was selected for significant differential gene expression.

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