Formal proof of the requirement of MESP1 and MESP2 in mesoderm specification and their transcriptional control via specific enhancers in mice.

MESP1 and MESP2 are transcriptional factors involved in mesoderm specification, somite boundary formation and somite polarity regulation. However, Mesp quadruple mutant zebrafish displayed only abnormal somite polarity without mesoderm specification defects. In order to re-evaluate Mesp1/Mesp2 mutants in mice, Mesp1 and Mesp2 single knockouts (KOs), and a Mesp1/Mesp2 double KO were established using genome-editing techniques without introducing selection markers commonly used before. The Mesp1/Mesp2 double KO embryos exhibited markedly severe mesoderm formation defects that were similar to the previously reported Mesp1/Mesp2 double KO embryos, indicating species differences in the function of MESP family proteins. However, the Mesp1 KO did not display any phenotype, including heart formation defects, which have been reported previously. We noted upregulation of Mesp2 in the Mesp1 KO embryos, suggesting that MESP2 rescues the loss of MESP1 in mesoderm specification. We also found that Mesp1 and Mesp2 expression in the early mesoderm is regulated by the cooperation of two independent enhancers containing T-box- and TCF/Lef-binding sites. Deletion of both enhancers caused the downregulation of both genes, resulting in heart formation defects. This study suggests dose-dependent roles of MESP1 and MESP2 in early mesoderm formation.