Chemical toolbox for multiscale, integrative imaging: Connecting cellular gene expression to organ-scale phenotype

项目摘要 多细胞生物的生物学是在多个层次上组织的。分子丰度和相互作用 调节细胞功能;细胞与附近细胞和环境的通信进一步塑造细胞状态 和功能;细胞在器官范围内形成高度互连的功能网络。因此，功能或功能障碍 一个器官的功能是通过组成该器官的单个细胞的协调作用来表现的。到 机械地理解疾病是如何发展的，我们需要了解一个细胞中的异常变化是如何发生的。 细胞会转化为系统级功能障碍。随着测序成像和遗传学的显著进步 操纵，研究人员现在更接近于破译细胞基因型如何引起系统水平 体内表型。单细胞测序探测构成组织的单个细胞的遗传谱。 器官规模的表型分析，如细胞形态学，细胞布线，以及细胞内的蛋白质。 整个器官中细胞的空间组织。基于CRISPR的遗传扰动建立因果联系 基因和表型之间的关系。然而，这些技术大多探测 一个复杂生物系统的单一方面。这种局限性使得整合信息成为一项挑战 从不同的分子类型和规模，并提取系统级的机制基础， 表型，尤其是在体内。我们的目标是通过开发一个转型的，多尺度的， 多模态成像平台，其筛选大的组织体积以识别具有异常表型的细胞， 表征异常细胞及其邻近细胞的完整和定量的分子内容物。这 该平台将识别细胞中的异常遗传变化如何改变其表型，影响附近的细胞， 有助于疾病的发展。尽管其巨大的潜力，精简器官规模的蛋白质组 由于化学不相容性，表型和原位单细胞转录组学是不可能的， 成像要求。我们建议开发一系列化学工具，以实现多尺度，综合分析 蛋白质和RNA：在完整组织中RNA的可逆保护;多- 用于厚组织成像的基于量子点的NIR成像平台。整合这些工具，我们 将开发和实施多尺度的综合成像平台，以表征表型异常 在自闭症患者的大脑中，如异位神经元连接，并在自闭症区域的细胞转录组谱 表型缺陷这样的研究将提供一个整体的观点，病变组织破译致病 通过鉴定改变的基因，在分子水平上研究表型异常背后的机制。 异常表型附近的表达模式，导致系统- 水平表型，以及健康与患病组织中差异细胞类型的空间组织。在 除了能够进行新的生物学研究外，新开发的化学工具还将在广泛的领域推动创新。 生物医学科学的范围，包括RNA生物学，遗传学，成像和组织工程。

PROJECT SUMMARY The biology of multicellular organisms is organized on multiple levels. Molecular abundance and interactions regulate cell function; Communication of cells with nearby cells and environments further shapes cellular states and functions; Cells form highly interconnected functional networks organ-wide. Thus, a function or a dysfunction of an organ is manifested through the orchestrated action of individual cells comprising the organ. To mechanistically comprehend how a disease develops, we need to understand how the abnormal alteration in a cell is translated into system-level dysfunctions. With remarkable progress in sequencing, imaging, and genetic manipulation, researchers are now a step closer to decipher how cellular genotype gives rise to system-level phenotype in vivo. Single-cell sequencing probes the genetic profile of individual cells comprising a tissue. Organ-scale phenotyping, such as CLARITY, probes the detailed morphology of cells, cellular wiring, and the spatial organization of cells throughout an organ. CRISPR-based genetic perturbation establishes causal links between genes and phenotype in vitro at unprecedented throughput. However, these technologies mostly probe a single facet of a complex biological system. This limitation makes it challenging to integrate information obtained from different molecular types and scales, and to extract the mechanistic underpinning of system-level phenotype, especially in vivo. We aim to address this critical gap by developing a transformational, multiscale, multimodal imaging platform that screens a large tissue volume to identify cells with abnormal phenotype and characterize the complete and quantitative molecular contents or the abnormal cells as well as nearby cells. This platform will identify how the abnormal genetic change in a cell alters its phenotype, affects nearby cells, and contributes to disease development. Despite its immense potential, streamlining organ-scale proteomic phenotyping and in situ single cell transcriptomics is impossible due to the incompatibility of chemistry and imaging requirements. We propose to develop a series of chemical tools to enable multiscale, integrative profiling of proteins and RNAs: reversible protection of RNAs in an intact tissue; tissue transformation chemistry for multi- omic profiling; and quantum dot-based NIR imaging platform for thick-tissue imaging. Integrating these tools, we will develop and implement the multiscale, integrative imaging platform to characterize phenotypic abnormalities in autistic brains, such as ectopic neuronal connections, and profile cellular transcriptome at the region of phenotypic defects. Such study will provide a holistic view of diseased tissues to decipher pathogenic mechanisms behind a phenotypic abnormality at a molecular level, through the identification of altered gene expression patterns near an abnormal phenotype, intercellular communication network that leads to the system- level phenotype, and the spatial organization of differential cell types in healthy versus diseased tissues. In addition to enabling new biological studies, the newly developed chemical tools will drive innovations in a wide range of biomedical science, including RNA biology, genetics, imaging, and tissue engineering.