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

Abstract

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.

A spatiotemporal gradient of mesoderm assembly governs cell fate and morphogenesis of the early mammalian heart

“…Loss of Mesp1 disrupts the density gradient that forms after gastrulation, altering mesoderm organization To gain an understanding of cues that control the spatiotemporally-governed early cardiac progenitor behaviors, we studied gastrulation in Mesp1 mutants, where early organogenesis does not occur due to specification and/or migration defects [6,8,36,43]. Although the movement behaviors of Mesp1 knockdown cells have been studied in vitro [44], we exploited LSFM and our tracking workflow to better understand their actions in vivo.…”

A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors

“…2A). The persistence of Smarcd3-F6+ cells led us to hypothesize that these cells represent retained CPCs, suggesting that as previously described (Saga, Kitajima & Miyagawa-Tomita, 2000;Ajima et al, 2021;Saga et al, 1999), aspects of early cardiac specification may be Mesp1independent. Thus, the transcriptional and epigenomic programs regulated by and independent of Mesp1 remain to be understood during in vivo cardiogenesis.…”

“…S15). We detected linkages between Mesp1 and the characterized "EME" enhancer (Haraguchi et al, 2001;Ajima et al, 2021;Costello et al, 2011;Guo et al, 2018) with enrichment in Mesp1 KO cell types likely indicative of retained early chromatin landscape or de-repression of the locus without appropriate regulation from downstream targets (Fig. S15A).…”

“…Gain of function experiments suggest a broad and important function for Mesp1 in mesoderm differentiation, but the in vivo gene regulatory landscape controlled by Mesp1 remains unclear (Costello et al, 2011;Saga et al, 1999;Liu, 2017;Saga, Kitajima & Miyagawa-Tomita, 2000;Ajima et al, 2021;Saga et al, 1996;Kitajima et al, 2000;Lin et al, 2022).…”

“…Deletion of Mesp1 in mice variably disrupts specification and migration of cardiac progenitors (Ajima et al, 2021; Saga, Kitajima & Miyagawa-Tomita, 2000; Saga et al, 1999; Kitajima et al, 2000; Lescroart et al, 2018). During in vitro cardiac differentiation, overexpression of Mesp1 induces expression of subsequent cardiac TFs, indicating a potentially instructive role in cardiogenesis (Chiapparo et al, 2016; Bondue et al, 2008; Lindsley et al, 2008; Wu, 2008; Kelly, 2016; Bondue & Blanpain, 2010; Lin et al, 2022; Soibam et al, 2015).…”