Regulation of early cardiopharyngeal fates specification

 描述(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特性的机制。在哺乳动物中，面部和下颌肌--统称为咽肌--与心咽中胚层的心脏祖细胞有着共同的起源。这种关系反映在DiGeorge综合征中，其中TBX1功能改变会导致心血管和颅面畸形。心脏和咽肌的命运选择在早期哺乳动物胚胎中很难研究，细胞环境也是如此。它决定了心咽祖细胞是保持多潜能，还是成为特定的心脏或咽肌细胞。海鞘幼虫是一种海洋无脊椎动物，是与脊椎动物最近的近亲之一，拥有简化的心咽细胞谱系，以简单和刻板的方式选择连续的心脏和咽部肌肉。这可以利用靶向分子扰动、共聚焦显微镜和结合荧光激活细胞分类(FACS)和下一代RNA测序(RNA-SEQ)的谱系特异性转录图谱以高时空分辨率进行研究。腹水状的心咽中胚层来自两个祖细胞，它们产生心脏和控制呼出口的心房虹吸肌(ASM)。研究发现，双能心咽前体细胞经历定向不对称的细胞分裂，产生不同的第一和第二心脏前体和ASM前体。在分子上，心咽前体细胞表现出多谱系转录启动，即它们既激活早期的心脏程序，也激活ASM程序。由于调节的交叉拮抗，这些细胞然后分离到它们相应的前体：早期的ASM调节器在ASM前体中抑制心脏程序，而ASM程序在心脏前体中被抑制。在这里，将通过测试前馈调控电路控制顺序基因激活的假设来分析调控渐进ASM命运规范的调控机制。接下来，我们将探索这样的假设，即细胞不对称分裂的方向决定了特定的利基和ASM与心脏前体之间的不同相互作用。最后，明确的组织特异性分子扰动、FACS和RNA-SEQ分析，包括来自单细胞样本的分析，将确定多潜能心咽前体细胞、第一和第二心脏前体细胞以及早期ASM前体细胞的转录特征。这些结果将描述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和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.