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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要在脊椎动物的发育过程中，体节——预示着骨骼和肌肉的形态片段成体——在胚胎伸长的体轴的后端有节奏地、顺序地形成。这种节奏规范是由高度保守的生物钟决定的，该生物钟由振荡基因组成在人类中，该网络无法在单个细胞中剧烈振荡或同步。邻近细胞之间的组织范围振荡与发育缺陷有关，例如脊柱侧凸。尽管经过多年的研究确定了这种振荡网络的分子组成部分，但我们仍然不知道至关重要的是，了解这些成分中重要的致病突变的影响机制。迄今为止，关于这一高度动态的发展过程的绝大多数信息都来自来自固定组织或荧光生产者的推论，其成熟时间太慢而无法揭示这些突变在转录动力学水平上的机制基础。为了克服这一主要障碍，我们建立了 MS2 系统来测量转录动态在活的、发育中的斑马鱼胚胎的单个细胞中，该网络内的基因启动，这是一种广泛存在的现象这种新能力使我们能够重新审视分子过程。从个体细胞动力学的角度来看，潜在的脊椎动物分割。技术，我们发现表征体细胞发生的平滑蛋白质振荡是在这里，我们建议描述斑马鱼体细胞发生的特征。健康条件下并揭示（i）体细胞发生中转录爆发的分子起源，以及（ii）这些突发如何通过信号通路在相邻细胞之间协调，以产生我们预计，我们的工作将确定健康发展所必需的组织范围内的连贯振荡。精确诊断脊椎动物发育中疾病表型的分子基础的阶段。此外，这里开发的实验和计算技术将为生物学界提供支持对脊椎动物发育动力学的单细胞性质进行探索，与现有的研究相媲美最终，这些知识与创新技术相结合，目前是可以在无脊椎动物中实现的。这里提出的技术和分析——将使合理修改这些转录成为可能用于生物工程或治疗目的的随意动力学。