Regulation of muscle fate specification and cell migration in cardiogenic lineage

描述（由申请人提供）：了解指导心脏细胞从多能心血管祖细胞进行性特化的机制对于使用诱导多能干细胞（iPS）开发再生疗法至关重要。一个简单的脊索动物模型系统，被囊玻璃海鞘，将被用来分析细胞和分子机制，确定在心源性谱系的肌肉型规格。在玻璃海鞘胚胎中，两侧成对的心前细胞，称为躯干腹侧细胞（TVC），经历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。后者然后向背外侧心房虹吸基板迁移。在TVC的不对称分裂之后，编码转录因子COE和Islet的基因在ASM中特异性上调。此外，COE是必要的，足以阻止心脏特化和促进ASM的命运，包括表达的ASM特异性胰岛增强子和细胞迁移到背侧的幼虫。最后，使用TVC特异性增强子靶向表达组成型活性Notch胞内结构域足以抑制COE、胰岛和细胞迁移的ASM特异性表达。这些观察结果导致了这样的假设，即初始不对称分裂导致心脏特异性Notch信号传导，其可能通过抑制COE的表达来阻断ASM命运特化。为了检验这一假设，将分离和表征控制COE的ASM特异性表达的顺式调节序列，并确定COE上游Notch信号传导的功能。Notch信号传导的内源性调节因子和效应因子的表达和定位模式将被记录，以深入了解在不对称TVC分裂期间抑制Notch信号的机制。将使用先前建立的试验分析Notch信号传导、COE和胰岛对心脏与ASM命运特化和细胞迁移的影响，以便开始表征这些调节剂之间的上位关系。最后，将通过使用荧光激活细胞分选和微阵列获得心脏和ASM特异性转录谱，记录心脏与ASM命运规范相关的全基因组基因表达变化。该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规范的负调节剂，并阐明了控制心肌命运规范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.