A Regulatory Pathway Involving Notch1/β-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate. Mus musculus

The regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle, or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/β-Catenin signaling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated β-Catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and β-Catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs prior to their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involve unanticipated functions of β-Catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs. Overall design: YFP+ cells marking precardiac mesoderm (Isl1-cre domain) were FACS-sorted (w/wo stabilized Beta-catenin). Their total RNA was amplified with the WT-Ovation Pico RNA Amplification System, fragmented and labeled with the FL-Ovation cDNA Biotin Module V2 (Nugen). The hybridization, staining and scanning of the Affymetrix GeneChips were performed in the Gladstone Genomics Core Lab. Raw data generated from at least three independent experiments were further analyzed by the group of Dr. Ru-Fang Yeh at the Center for Informatics and Molecular Biostatistics, UCSF.

多能心脏祖细胞（multipotent cardiac progenitor cell，CPC）的增殖以及后续分化为心肌细胞、平滑肌细胞或内皮细胞的调控过程，是心血管基础生物学与心脏再生医学的核心基础之一。然而，调控这些细胞命运抉择的分子机制仍未明确。本研究证实，促进CPC增殖的Wnt/β-连环蛋白（Wnt/β-Catenin）信号通路，可通过Notch1介导的CPC内磷酸化β-连环蛋白积累产生负向调控；而CPC中的Notch1活性对于其分化过程不可或缺。Notch1正向调控、β-连环蛋白负向调控心脏转录因子Isl1、Myocd及Smyd1的表达。令人意外的是，通常在CPC分化前短暂表达的Isl1基因敲除，会在体内及胚胎干细胞（embryonic stem，ES）细胞系统中导致CPC的异常增殖。进一步研究发现，Isl1对于CPC分化为心肌细胞和平滑肌细胞是必需的，但对于其向内皮细胞分化则非必需。上述研究结果揭示了一套调控CPC增殖与细胞命运的网络，其中涉及β-连环蛋白、Notch1及Isl1的未被认知的功能，该调控网络有望应用于基于CPC的再生医学策略。
整体实验设计：通过荧光报告蛋白YFP标记心前中胚层（对应Isl1-cre重组酶表达区域）的细胞，经荧光激活细胞分选（FACS）分离（辅以/不辅以稳定化β-连环蛋白处理）。采用WT-Ovation Pico RNA扩增系统对总RNA进行扩增，随后使用FL-Ovation cDNA生物素标记模块V2（Nugen）进行片段化与标记。Affymetrix基因芯片的杂交、染色与扫描实验均在格拉德斯通基因组学核心实验室（Gladstone Genomics Core Lab）完成。至少三次独立实验产生的原始数据，由加州大学旧金山分校（UCSF）信息学与分子生物统计学中心的Ru-Fang Yeh博士课题组进行后续分析。