Regulation of muscle fate specification and cell migration in cardiogenic lineage

描述(由申请人提供)：了解从多能心血管前体细胞中定向渐进指定心脏细胞的机制对于开发使用诱导多能干细胞(IPS)的再生疗法至关重要。一个简单的脊索模型系统，被囊状肠囊，将被用来分析决定心源性谱系中肌肉类型规格的细胞和分子机制。在Ciona胚胎中，两侧的心脏前细胞对，称为主干腹侧细胞(TVCs)，经历了刻板的不对称细胞分裂，将心脏与心房虹吸肌(ASM)前体区分开来。然后，后者向背外侧心房虹吸管移行。随着TVCs的不对称分裂，编码转录因子CoE和Islet的基因在ASM中特异性上调。此外，COE是阻断心脏规范和促进ASM命运的必要条件和充分条件，包括ASM特异性胰岛增强子的表达和细胞向幼虫背侧的迁移。最后，使用TVC特异性增强子靶向表达具有结构性活性的Notch胞内区，足以抑制COE、胰岛和细胞迁移的ASM特异性表达。这些观察结果导致了一种假设，即最初的不对称分裂导致心脏特异的Notch信号，该信号可能通过抑制COE的表达来阻止ASM命运的指定。为了验证这一假说，我们将分离和鉴定控制COE ASM特异性表达的顺式调控序列，并确定COE上游Notch信号的功能。为了深入了解Notch信号在不对称TVC分裂过程中极化的机制，将记录Notch信号的内源性调节因子和效应器的表达和定位模式。Notch信号、COE和Islet对心脏与ASM命运规范和细胞迁移的影响将使用先前建立的分析方法进行分析，以便开始表征这些调节因子之间的上位关系。最后，通过使用荧光激活细胞分类和微阵列获得心脏和ASM特定的转录图谱，将记录下心脏和ASM命运规范下的全基因组基因表达变化。本项目完成后获得的结果将表征COE的调节和功能，COE是一种新的心脏命运指定的负调控因子，并阐明在心源性谱系中控制肌肉命运指定和细胞迁移的细胞和分子机制。

DESCRIPTION (provided by applicant): Understanding the mechanisms directing progressive specification of heart cells from multipotent cardiovascular progenitors is essential for the development of regenerative therapies using induced pluripotent stem (iPS) cells. A simple chordate model system, the tunicate Ciona intestinalis, will be used to analyze the cellular and molecular mechanisms that determine muscle-type specification in the cardiogenic lineage. In Ciona embryos, the bilateral pairs of precardiac cells, called trunk ventral cells (TVCs), undergo stereotyped asymmetric cell divisions that distinguish the heart from the atrial siphon muscle (ASM) precursors. The latter then migrate toward the dorso-lateral atrial siphon placode. Following asymmetric divisions of the TVCs, the genes encoding the transcription factors COE and Islet are specifically up- regulated in the ASMs. In addition, COE is necessary and sufficient to block heart specification and promote the ASM fate, including expression of an ASM-specific Islet enhancer and cell migration toward the dorsal side of the larva. Finally, targeted expression of the constitutively active Notch intracellular domain using a TVC-specific enhancer is sufficient to inhibit ASM- specific expression of COE, Islet and cell migration. These observations led to the hypothesis that the initial asymmetric divisions result in heart-specific Notch signaling, which blocks ASM fate specification, possibly by inhibiting the expression of COE. In order to test this hypothesis, the cis-regulatory sequences that control ASM-specific expression of COE will be isolated and characterized, and the function of Notch signaling upstream of COE will be determined. The expression and localization patterns of endogenous regulators and effectors of Notch signaling will be documented in order to gain insight into the mechanisms that polarize the Notch signal during asymmetric TVC divisions. The effects of Notch signaling, COE and Islet on heart vs. ASM fate specification and cell migration will be analyzed using previously established assays in order to begin to characterize the epistatic relationships between these regulators. Finally, whole genome gene expression changes underlying heart vs. ASM fate specification will be documented by obtaining heart and ASM-specific transcription profiles using fluorescence activated cell sorting and microarrays. The results obtained upon completion of this project will characterize the regulation and function of COE, a novel negative regulator of heart fate specification, and illuminate the cellular and molecular mechanisms controlling muscle fate specification and cell migration in the cardiogenic lineage.