Uncovering single-cell transcriptional dynamics in somitogenesis in live zebrafish embryos

揭示活体斑马鱼胚胎体节发生中的单细胞转录动力学

• 批准号: 10742431
• 负责人: Hernan Gustavo Garcia
• 金额: $ 44.14万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10742431
• 关键词: Adult; Affect; Animals; Biological Clocks; Biological Models; Biology; Biomedical Engineering; Cell Communication; Cells; Communities; Congenital Scoliosis; Data; Defect; Development; Developmental Biology; Developmental Process; Disease; Embryo; Embryonic Development; Ensure; Failure; Feedback; Gene Expression; Genes; Genetic Transcription; Human; Individual; Invertebrates; Investigation; Knowledge; Measurement; Measures; Mesoderm; Modeling; Molecular; Molecular Diagnosis; Morphology; Muscle; Mutation; Nature; Noise; Notch Signaling Pathway; Outcome; Pattern; Periodicals; Periodicity; Process; Proteins; Reporter; Repression; Research; Research Personnel; Signal Pathway; Signal Transduction; Somites; Specific qualifier value; System; Technology; Theoretical model; Therapeutic; Thinking; Time; Tissues; Transcription Initiation; Transcriptional Regulation; Visit; Work; Zebrafish; bone; disease phenotype; disease-causing mutation; empowerment; experimental study; gene regulatory network; genetic manipulation; knock-down; new technology; notch protein; predictive modeling; programs; response; scoliosis; small molecular inhibitor; somitogenesis; stem; success; technological innovation; therapy development; tissue fixing; vertebrate embryos

Project Summary/Abstract In vertebrate development, somites—morphological segments that prefigure the bones and muscles of the adult—are formed rhythmically and sequentially at the posterior end of the elongating body axis of the embryo. This rhythmic specification is dictated by a highly conserved biological clock that consists of an oscillatory gene regulatory network. In humans, failure of this network to robustly oscillate in individual cells or to synchronize tissue-wide oscillations across neighboring cells has been associated with developmental defects such as scoliosis. Despite years of research identifying the molecular components of this oscillatory network, we still do not understand the mechanisms affected by important disease-causing mutations in these components. Crucially, to date, the vast majority of information about this highly dynamic developmental process stems from inferences from fixed tissue or from fluorescent reporters whose maturation time is too slow to reveal the mechanistic basis of these mutations at the level of their transcriptional dynamics. To overcome this major obstacle, we established the MS2 system to measure the dynamics of transcriptional initiation of genes within this network in individual cells of living, developing zebrafish embryos, a widespread model of somitogenesis and scoliosis. This new ability empowers us to revisit the molecular processes underlying vertebrate segmentation from the standpoint of the dynamics of individual cells. Using this technology, we have discovered that the smooth protein oscillations that characterize somitogenesis are produced by bursts of transcriptional initiation. Here, we propose to characterize zebrafish somitogenesis in healthy conditions and to uncover (i) the molecular origins of transcriptional bursting in somitogenesis and (ii) how these bursts are coordinated between neighboring cells through signaling pathways in order to produce the coherent tissue-wide oscillations necessary for healthy development. We envision that our work will set the stage for precisely diagnosing the molecular underpinnings of disease phenotypes in vertebrate development. Further, the experimental and computational technologies developed here will empower the biology community to launch explorations into the single-cell nature of vertebrate development dynamics on par with what is currently attainable in invertebrate animals. Ultimately, this knowledge—combined with the innovated technologies and analyses proposed here—will make it possible to rationally modify these transcriptional dynamics at will for bioengineering or therapeutic purposes.

项目摘要/摘要 在脊椎动物的发展中，concomes-形态段的骨骼和肌肉 成人 - 在胚胎的伸长体轴的后端有节奏和依次形成。 这种节奏规范由由振荡基因组成的高度保守的生物钟决定 监管网络。在人类中，该网络无法在单个细胞中稳健振荡或同步 跨邻近细胞的整个组织振荡与发育缺陷有关 脊柱侧弯。 尽管多年的研究确定了该振荡网络的分子成分，但我们仍然没有 了解这些成分中受重要疾病突变影响的机制。十字 迄今为止，大多数有关此高度动态发展过程步骤的信息 固定组织或荧光记者的推论，其成熟时间太慢而无法揭示 这些突变在其转录动力学水平上的机械基础。 为了克服这一主要障碍，我们建立了MS2系统以衡量转录的动态 在这个网络中启动基因在生存的单个细胞中，开发斑马鱼胚胎，宽度 体体发生和脊柱侧弯的模型。这种新能力使我们能够重新审视分子过程 从单个细胞动力学的角度来看，脊椎动物的基础分割。使用此 技术，我们发现表征体重生成的光滑蛋白质振荡是 由转录计划爆发产生。在这里，我们建议在 健康条件并揭示（i）转录爆发的分子起源，（ii） 这些爆发如何通过信号通路之间在相邻单元之间协调以产生 健康发育所需的一致性组织的相干振荡。我们设想我们的工作将设定 精确诊断的阶段脊椎动物发育中疾病表型的分子基础。 此外，这里开发的实验和计算技术将增强生物学社区的能力 与什么相当 目前可以在无脊椎动物中获得。最终，这种知识与被感染的 这里提出的技术和分析将使合理地修改这些转录 随意出于生物工程或治疗目的。