Background: Mesp1 is critical for early cardiomyocyte differentiation and heart development. We previously observed downregulation of Mesp1 expression in YY1-ablated mouse embryonic hearts. However, how Mesp1 expression is mediated by YY1 is not well understood. Results: We excised YY1 in the murine embryos using Sox2-cre and found that Mesp1 was downregulated in the embryonic day (E) 7.5 mutant embryos. Also, YY1 activated the 6 kb Mesp1 regulatory element fused to a luciferase reporter. We identified two putative YY1 binding sites in the proximal promoter region of Mesp1 gene, and found that mutation of these sites significantly reduced YY1-induced activation of the Mesp1 promoter. We also uncovered one cognitive site for SP1, one of the earliest binding partners of YY1 identified. Mutation of this SP1 site repressed SP1-induced activation of the Mesp1 promoter. Moreover, YY1 and SP1 synergistically activated the Mesp1 promoter. Consistently, while Lacz expression driven by the wild-type 6 kb regulatory element of Mesp1 gene was robust in E7.5 mouse embryos, the mutation of these binding sites in the context of this 6 kb sequence substantially reduced the LacZ expression during embryogenesis. Conclusions: YY1 and SP1 independently and cooperatively govern the Mesp1 expression during embryogenesis. Developmental Dynamics 245:379-387, 2016. V C 2015 Wiley Periodicals, Inc.

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The mesendoderm is thought to be a population of the intermediate and transient common progenitors prior to the emergence of the me soderm (Me) and the en doderm (En) in vitro ; however, it is unclear whether it exists in vivo , and if so, what are the molecular mechanisms that establish the subsequent Me or En fate of the mesendoderm. Here we show that Mesp1 transiently marks a subset of the epiblast at the p rimitive s treak (PS) initiation site at gastrulation in vivo . It is also evidenced by the cell fate mapping that the progeny of Mesp1 + progenitors gives rise to both the Me and the definitive En, but neither the primitive En nor the ectoderm, suggesting that Mesp1 + cells are the bio-potent mesendodermal progenitors. Mesp1 -fated dorsal foregut En (derived from the definitive En) subsequently contributes exclusively to the Pdx1 + /Foxa2 + progenitors in the p ancreatic b ud (PB), and ultimately to the endocrine cells in the pancreas. RNA-seq and ch romatin- i mmuno p recipitation of Mesp1 -fated cells show that Mesp1 directly binds to the endogenous regulatory elements of mesendodermal and endodermal modulators (e.g. Foxa2 ). Conditional ablation of Foxa2 in the Mesp1 -fated cells results in aberrant PB formation and postnatal death. In situ hybridization shows that ablation of Mesp1 abolishes the majority of mesendodermal modulators while it leads to ectopic expression of endodermal transcription factors in the distal portion of the PS, indicating that transient Mesp1 expression maintains bi-potency of the mesendoderm by inhibiting the endodermal transcription program. We generated chimera embryos by injecting into WT blastocysts the Mesp1 Cre/+ ; Rosa26-EYFP (control) or the Mesp1 Cre/Cre ; Rosa26-EYFP (mutant) ES cell lines (n=3/group). We found that, the mutant ESCs fail to contribute to the cardiac mesoderm, but instead they adopt the endodermal fate evidenced by their increased contribution to the pancreatic endocrine cells, indicating that the inhibition of the endodermal transcription program by Mesp1 in the mesendoderm progenitors is a cell-autonomous effect. Our results demonstrate that Mesp1 marks the bi-potent mesendoderm in vivo and functions as a critical molecular switch between the Me and the En formation via an orchestrating dual molecular mechanism.