Regulation of early cardiopharyngeal fates specification

 描述（由申请人提供）：遗传性和获得性心血管疾病的现代诊断和治疗需要深入了解胚胎发育期间控制心脏细胞身份的机制。在哺乳动物中，面部和下颌肌肉-统称为咽肌-与心咽中胚层中的心脏祖细胞有共同的起源。这种关系反映在DiGeorge综合征中，其中Tbx 1功能改变导致心血管和颅面畸形。在早期哺乳动物胚胎中很难研究心脏与咽肌的命运选择，细胞环境也是如此。niche，决定心咽祖细胞是否保持多能性或特异性进入心肌或咽肌。海鞘玻璃海鞘是一种与脊椎动物关系最近的海洋无脊椎动物，它的幼虫具有简化的心咽细胞谱系，可以以简单和刻板的方式做出连续的心脏肌肉与咽肌肉的选择。这可以使用靶向分子扰动、共聚焦显微镜和谱系特异性转录谱分析以高时空分辨率进行研究，所述谱系特异性转录谱分析结合了荧光激活细胞分选（FACS）和下一代RNA测序（RNA-seq）。海鞘心咽中胚层起源于两个祖细胞，它们产生心脏和控制呼气开口的心房虹吸肌（ASM）。发现双能心咽祖细胞经历定向不对称细胞分裂，产生不同的第一和第二心脏前体和ASM前体。在分子上，心咽祖细胞显示多谱系转录启动，即它们激活早期心脏和ASM程序。这些然后分离到其相应的前体由于监管交叉拮抗作用：早期ASM监管机构抑制心脏程序在ASM前体，而ASM程序被抑制在心脏前体。在这里，监管机制进行性ASM命运规范将通过检验前馈调控电路控制顺序基因激活的假设进行分析。接下来，将探讨不对称细胞分裂的方向决定特定小生境与ASM与心脏前体之间的差异相互作用的假设。最后，定义的组织特异性分子扰动、FACS和RNA-seq测定（包括来自单细胞样品的测定）将定义多能心咽祖细胞、第一和第二心脏前体和早期ASM前体的转录特征。这些结果将表征定义心咽多能性的调节特性，并揭示在脊索动物中调节保守的心脏与咽肌命运选择的机制。

 DESCRIPTION (provided by applicant): Modern diagnostics and treatments for inherited and acquired cardiovascular diseases require in-depth knowledge of the mechanisms that control heart cell identity during embryonic development. In mammals, the facial and lower jaw muscles - collectively referred to as pharyngeal muscles - share a common origin with heart progenitors in the cardiopharyngeal mesoderm. This relationship is reflected in the DiGeorge syndrome, where altered Tbx1 function causes cardiovascular and craniofacial malformations. The heart vs. pharyngeal muscle fate choice is difficult to study in the early mammalian embryos, as is the cellular environment, a.k.a. niche, which determines whether cardiopharyngeal progenitor cells remain multipotent or become specified into either cardiac or pharyngeal muscles. Larvae of the ascidian Ciona intestinalis, a marine invertebrate among the closest relatives to the vertebrates, possess a simplified cardiopharyngeal lineage of cells that make successive heart vs. pharyngeal muscles choices in a simple and stereotyped manner. This can be studied with high spatiotemporal resolution using targeted molecular perturbations, confocal microscopy and lineage- specific transcription profiling that combines fluorescence activated cell sorting (FACS) and next generation RNA sequencing (RNA-seq). The ascidian cardiopharyngeal mesoderm arises from two progenitors, which produce the heart and the atrial siphon muscles (ASM) that control the exhalant opening. It was found that bipotent cardiopharyngeal progenitors undergo oriented asymmetrical cell divisions that produce distinct first and second heart precursors and ASM precursors. Molecularly, the cardiopharyngeal progenitors display multilineage transcriptional priming, i.e. they activate both early cardiac and ASM programs. These then segregate to their corresponding precursors due to regulatory cross-antagonisms: early ASM regulators inhibit the heart program in ASM precursors, while the ASM program is inhibited in the heart precursors. Here, regulatory mechanisms governing progressive ASM fate specification will be analyzed by testing the hypothesis that feedforward regulatory circuits control sequential gene activation. Next, the hypothesis that the orientation of asymmetric cell division determines differential interaction between a specific niche and the ASM vs. heart precursors will be explored. Finally, defined tissue-specific molecular perturbations, FACS and RNA-seq assays, including from single-cell samples, will define transcriptional signatures for multipotent cardiopharyngeal progenitors, first and second heart precursors and early ASM precursors. These results will characterize the regulatory properties that define cardiopharyngeal multipotency and uncover mechanisms that regulate conserved heart vs. pharyngeal muscle fate choices in chordates.