microRNAs and Integrative Control on Cardiopoiesis

描述(申请人提供)：microRNAs(MiRs)自然微调信号通路的响应性，已知许多miRs在心脏发育和疾病中发挥重要作用。到目前为止，对任何生物过程中涉及的miRs的知识主要来自表达谱。相对较少的MIR被测试为实际的调节作用，因此我们目前对MIR在生物防治中的流行率、重要性和实际功能的了解仍然很少。我们的数据表明，应用高通量技术对miR寡核苷酸模拟物的全基因组文库进行基于干细胞和祖细胞的分析是可行的。使用这种方法，我们发现MIR在启动胚胎心脏形成中发挥了意想不到的作用，并通过区分心脏中胚层和前肠内胚层发挥作用。在这里，我们建议应用同样的方法来识别控制心脏发生的下一步的MIR：来自共同祖细胞的心肌细胞、平滑肌和血管内皮细胞的分化和多样化。尽管心脏造血谱的适当多样化对心脏形成、再生和疾病至关重要，但人们对这一过程是如何控制的知之甚少。为了解决这个问题，我们将应用全基因组miR文库筛选，随后是单个miR和靶点的验证和模型构建的迭代过程，以产生控制心脏生成的信号蛋白和miR的精细化网络。MiRs和靶蛋白的网络将通过在胚胎中定向获得和丧失功能的研究来评估，以直观地了解miRs如何调节信号蛋白以指导心脏生成。这项研究将产生两个结果：第一，功能筛选和随后的体内测试将揭示控制心脏生成的miRs，因此对发育、疾病和再生非常重要。其次，我们将构建和测试由miR靶标组成的信号网络，这将为深入了解心脏生成的系统水平控制提供依据。这项研究的创新之处在于，它将最先进的MIR功能筛选与MIR靶标识别和系统分析相结合，以揭示心脏中干细胞出现不同细胞类型的逻辑。

DESCRIPTION (provided by applicant): microRNAs (miRs) naturally fine-tune the responsiveness of signaling pathways, and a number of miRs are known to play important roles in heart development and disease. To date, knowledge of miRs involved in any biological process comes primarily from expression profiling. Relatively few miRs have been tested for actual regulatory roles, therefore our current knowledge of both the prevalence, importance and actual function of miRs in biological control remains rudimentary. Our data show that it is feasible to apply high throughput technology to screen stem and progenitor cell-based assays against whole genome libraries of miR oligonucleotide mimics. Using this approach, we discovered miRs that play unanticipated roles in initiating embryonic heart formation and act by distinguishing cardiac mesoderm from foregut endoderm. Here we propose to apply this same approach to identify miRs that control the next step in cardiogenesis: the differentiation and diversification cardiomyocytes, smooth muscle, and vascular endothelial cells from a common progenitor. Although the proper diversification of cardiopoietic lineages is critical to heart formation, regeneration and disease, little is known about how the process is controlled. To address this question, we will apply whole genome miR library screening, followed by an iterative process of validation of individual miRs and targets and model building to yield a refined network of signaling proteins and miRs that control cardiopoiesis. The network of miRs and target proteins will be evaluated through targeted gain and loss of function studies in embryos to visualize how miRs regulate signaling proteins in order to guide cardiopoiesis. This research will yield two outcomes: First, the functional screening and subsequent in vivo testing will reveal miRs that control cardiopoiesis and hence be important for development, disease and regeneration. Second, we will construct and test signaling networks composed of the miR targets that will offer insight into the systems-level control of cardiopoiesis. The research is innovative in that it merges state of the art functional screening of miRs with miR target identification and systems analysis in order to expose the logic underying the emergence of distinct cell types in the heart from stem cells.