Figure 1: Mesp1-lineage cells are a CPC-enriched population, which contain endoderm components.

(A) Schematic characterization of embryonic stem cells (Mesp1^Cre/+/Rosa26^EYFP/+) in this study; in vitro Mesp1-CPC characterization and in vivo Mesp1-CPC functional characterization. The genome-wide identification of Mesp1 targets, and the establishment of the reporter ES cell line, was previously published¹⁹. (B) Mesp1-EYFP+ signals were located in the mesoderm in E7.5 and E9.5 embryos. E7.5 panel:an embryonic sagettal section showing EYFP signal mainly in the mesoderm (Mes), and less in endoderm (En) and primitive streak (PS). E9.5 panel(left): an embryonic heart showing EYFP signal in left ventricle (LV), right ventricle (RV) and out flow tract (OFT). E9.5 panel (right): an embryonic heart section showing EYFP signal in atrium (A), ventricle (V) and out flow tract (OFT). (C) Enrichment of cardiac-related genes in the EYFP+ population. Gene expression levels were measured by real-time RT-PCR. Mesp1 was measured at day 5 of EB culture. Nkx2-5, αMHC and Ryr2 were measured at day 8 of EB culture. N ≥ 3; *p < 0.05 versus control cells. (D) Mesp1-lineage cells are a CPC-enriched population, which contain endoderm components. Co-localization of EYFP and lineage markers in differentiating ES cells. Day 5 EBs were stained by EYFP, Foxa2, Gata4, CD31, and α-SMA antibodies. (E,F) Expression of cardiac and hematopoietic transcription factors in EYFP+ cells during differentiation. (E) Transcription factors Wnt3, Eomes, Mixl1, Foxa2, Mesp1 and Cxcr4 showed strong induction between days 4–6. (F) Cardiac transcription factors Nkx2-5, Mef2c and Tbx5 showed much stronger induction over the time course than hematopoietic transcription factors Tal1, Lmo2 and Gata1.