Numb family proteins are essential for cardiac morphogenesis and progenitor differentiation

Abstract

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 doubleknockout (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.

Adaptor proteins NUMB and NUMBL promote cell cycle withdrawal by targeting ERBB2 for degradation

“…Immunostaining for NUMB confirmed cardiomyocyte-specific loss of NUMB at E10.5 (Supplemental Figure 1B). Observed cardiac phenotypes with double cKOs were consistent with previous studies utilizing myocardial Cres to ablate both Nb and Nbl (17,18) (Figure 1, A and B). We also observed similar, but less severe, cardiac phenotypes in Nb cKO alone ( Figure 1, A and B).…”

CDC42 is required for epicardial and pro-epicardial development by mediating FGF receptor trafficking to the plasma membrane

“…EC proliferation was measured by BrdU pulse labeling as described (Zhao et al, 2014). Additional details of lineage tracing and BrdU labeling are provided in the supplementary Materials and Methods.…”

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

“…Using tweezers and scissors, collect the female mouse and embryo tail samples for genotyping as previously reported 22 . Using a plastic pipette, transfer each embryo to a well of a 48 well plate containing 1 ml of 1x PBS.…”

“…The fourth population consists of epicardial cells derived from the pro-epicardial organ (PEO), and contributes to fibroblasts, smooth muscle cells and potentially other cardiac cell types 31 . The epicardium regulates coronary vascular development, cardiac growth and morphogenesis The most important concept about cardiac morphogenesis is that during heart development, abnormal cell migration/differentiation of the four progenitor cell populations will result in malformations or congenital heart defects 4,22,32 , which is the number one cause of birth defects in the world. Determining the mechanisms of cardiac morphogenesis at the cellular and molecular level is an essential step toward improving diagnosis and potential treatments for CHDs.…”

“…Cardiac morphogenesis depends on cardiac lineage differentiation and individual cell organization at the whole heart level 22 , and thus, it is essential to image single cardiac progenitor cell differentiation to different cardiac cell types and to also image cell morphology at single cell resolution. To achieve that, Rosa26Cre ERT2 15 was crossed to R26R-Confetti 16 , and the pregnant female was gavaged with Tamoxifen at a low concentration.…”