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常常受到现有技术的影响， 兴趣系统。多年来，我们对胚胎发育的理解一直是由静态决定的， 这些快照来自于依赖于检查死亡的、固定的胚胎的技术。在这 在这种情况下，塑造动物身体计划的基因表达程序的时空进化是 从静态图像推断。所提出的工作的主要假设是，依赖快照 而不是电影中隐藏着与胚胎发育有关的深刻见解。 我们建议在发育生物学研究中打破静态快照的统治， 引入新技术，使胚胎发育的研究重新成为可能 从动力学的观点，通过成像和量化的中心法则在真实的 在活胚胎内的单细胞水平上的时间。我们将利用果蝇的胚胎发育 果蝇作为一个证明的原则，以（一）发展新的技术，以量化快速 改变转录因子浓度和发育中产生的转录输出，（ii） 开发新的体内荧光探针，以增强尖端的晶格光片显微镜， 可以直接观察单个激活子和阻遏子如何与DNA结合，以及（iii）联合收割机 这些输入输出测量与我们的单分子测量，以便直接可视化 转录因子在真实的时间内执行其调节功能的分子机制， 活的胚胎我们认为，只有使这种实时描述基因调控程序， 我们才能达到一个定量的理解，使预测DNA如何 序列决定了细胞的承诺，以及当这种调节出错时， 随之而来的是不受控制的细胞增殖的缺陷和状态。