Lighting Up the Central Dogma in Embryonic Development

阐明胚胎发育的中心法则

• 批准号: 9350588
• 负责人: Hernan Gustavo Garcia
• 金额: $ 235.5万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9350588
• 关键词: Animals; Binding; Biological Phenomena; Cell Proliferation; Cells; DNA; DNA Sequence; Defect; Development; Developmental Biology; Disease; Drosophila genus; Drosophila melanogaster; Embryo; Embryonic Development; Evolution; Fluorescent Probes; Foundations; Gene Expression; Genetic Transcription; Human Genome; Image; Life; Light; Lighting; Link; Malignant Neoplasms; Measurement; Microscopy; Molecular; Nucleic Acid Regulatory Sequences; Output; Proteins; Regulation; Regulator Genes; Shapes; System; Technology; Time; Work; cell growth; experimental study; in vivo; insight; interest; movie; new technology; programs; repaired; single molecule; spatiotemporal; stem; transcription factor

Our understanding of biological phenomena is often shaped by the technology available to query our system of interest. For years, our understanding of embryonic development has been dictated by static snapshots stemming from technologies that rely on the examination of dead, fixed embryos. In this case, the spatiotemporal evolution of the gene expression programs that shape animal body plans is inferred from static images. The main hypothesis of the proposed work is that the reliance on snapshots rather than movies has hidden deep insights from view relevant to embryonic development. We propose to break free from the reign of static snapshots in the study of developmental biology by introducing new technologies that will make it possible to redo the subject of embryonic development from the standpoint of dynamics through the imaging and quantification of the central dogma in real time at the single cell level within living embryos. We will use the embryonic development of the fruit fly Drosophila melanogaster as a proof of principle in order to (i) develop new technology to quantify fast changing transcription factor concentrations and the resulting transcriptional output in development, (ii) develop new in vivo fluorescent probes to augment cutting-edge lattice light-sheet microscopy to make it possible to directly visualize how single activators and repressors bind to the DNA, and (iii) combine these input-output measurements with our single-molecule measurements in order to directly visualize the molecular mechanisms by which transcription factors perform their regulatory function in real time in living embryos. We argue that only by enabling this real-time description of gene regulatory programs in development can we reach a quantitative understanding that makes it possible to predict how DNA sequence dictates cellular commitment and how, when this regulation goes awry, developmental defects and states of unchecked cellular proliferation ensue.

我们对生物现象的理解通常是由可用于查询我们的技术来塑造的 感兴趣的系统。多年来，我们对胚胎发展的理解一直由静态决定 源于依赖死亡，固定胚胎的技术的快照。在这个 情况，塑造动物身体计划的基因表达程序的时空演化是 从静态图像推断出来。拟议工作的主要假设是对快照的依赖 而不是电影隐藏了与胚胎发展相关的深刻见解。 我们建议通过 引入新技术将使重做胚胎开发的主题成为可能 从动力学的角度通过成像和量化中央教条 在活胚胎中的单细胞水平上的时间。我们将使用果蝇的胚胎发展 果蝇Melanogaster作为原理证明，以开发新技术以快速量化 变化转录因子浓度和发育中的转录输出，（ii） 开发新的体内荧光探针，以增强尖端的晶格灯表显微镜以使 可以直接可视化单个激活因子和阻遏物与DNA的结合，并且（iii）组合 这些输入输出测量值通过我们的单分子测量值直接可视化 转录因子实时执行调节功能的分子机制 活着的胚胎。我们认为，只有通过实现对基因调节程序的实时描述 开发我们是否可以达到定量理解，这可以预测DNA 序列决定了细胞的承诺，以及当该调节出现问题时，如何发育 随之而来的缺陷和未检查的细胞增殖状态。