Sonic Hedgehog signaling regulates the optimal differentiation pace from early‐stage mesoderm to cardiogenic mesoderm in mice

Sonic Hedgehog (Shh), encoding an extracellular signaling molecule, is vital for heart development. Shh null mutants show congenital heart disease due to left-right asymmetry defects stemming from functional anomaly in the midline structure in mice. Shh signaling is also known to affect cardiomyocyte differentiation, endocardium development, and heart morphogenesis, particularly in second heart field (SHF) cardiac progenitor cells that contribute to the right ventricle, outflow tract, and parts of the atrium. Despite extensive studies, our understanding remains incomplete. Notably, Shh signaling is suggested to promote cardiac differentiation, while paradoxically preventing premature differentiation of SHF progenitors. In this study, we elucidate the role of Shh signaling in the earliest phase of cardiac differentiation. Our meta-analysis of single-cell RNA sequencing suggests that cardiogenic nascent mesoderm cells expressing the bHLH transcription factor Mesp1 interact with axial mesoderm via Hh signaling. Activation of Hh signaling using a Smoothened agonist delayed or suppressed the differentiation of primitive streak cells expressing T-box transcription factor T to Mesp1⁺ nascent mesoderm cells both in vitro and ex vivo. Conversely, inhibition of Hh signaling by cyclopamine facilitated cardiac differentiation. The reduction of Eomes, an inducer of Mesp1, by Hh signaling appears to be the underlying mechanism of this phenomenon. Our data suggest that SHH secreted from axial mesoderm inhibits premature differentiation of T⁺ cells to Mesp1⁺ nascent mesoderm cells, thereby regulating the pace of cardiac differentiation. These findings enhance our comprehension of Shh signaling in cardiac development, underscoring its crucial role in early cardiac differentiation.