Numb family proteins are essential for cardiac morphogenesis and progenitor differentiation.

Numb family proteins (NFPs), including Numb and numb-like (Numbl), are cell fate determinants for multiple progenitor cell types. Their functions in cardiac progenitor differentiation and cardiac morphogenesis are unknown. To avoid early embryonic lethality and study NFP function in later cardiac development, Numb and Numbl were deleted specifically in heart to generate myocardial double-knockout (MDKO) mice. MDKOs were embryonic lethal and displayed a variety of defects in cardiac progenitor differentiation, cardiomyocyte proliferation, outflow tract (OFT) and atrioventricular septation, and OFT alignment. By ablating NFPs in different cardiac populations followed by lineage tracing, we determined that NFPs in the second heart field (SHF) are required for OFT and atrioventricular septation and OFT alignment. MDKOs displayed an SHF progenitor cell differentiation defect, as revealed by a variety of methods including mRNA deep sequencing. Numb regulated cardiac progenitor cell differentiation in an endocytosis-dependent manner. Studies including the use of a transgenic Notch reporter line showed that Notch signaling was upregulated in the MDKO. Suppression of Notch1 signaling in MDKOs rescued defects in p57 expression, proliferation and trabecular thickness. Further studies showed that Numb inhibits Notch1 signaling by promoting the degradation of the Notch1 intracellular domain in cardiomyocytes. This study reveals that NFPs regulate trabecular thickness by inhibiting Notch1 signaling, control cardiac morphogenesis in a Notch1-independent manner, and regulate cardiac progenitor cell differentiation in an endocytosis-dependent manner. The function of NFPs in cardiac progenitor differentiation and cardiac morphogenesis suggests that NFPs might be potential therapeutic candidates for cardiac regeneration and congenital heart diseases.