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

项目总结 多细胞生物的生物学是在多个层次上组织起来的。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沟通进一步塑造细胞状态 和功能；细胞在整个器官范围内形成高度相互连接的功能网络。因此，一种功能或功能障碍 器官的功能是通过构成器官的单个细胞的协调动作来体现的。至 从机械上理解疾病是如何发展的，我们需要理解一种疾病的异常变化是如何发生的 细胞被转化为系统级的功能障碍。在测序、成像和遗传学方面取得了显著进展 通过操纵，研究人员现在更接近于破译细胞基因型是如何引起系统水平的 活体表型。单细胞测序探测组成组织的单个细胞的基因图谱。 器官尺度的表型分析，如Clarity，探测细胞的详细形态，细胞连接，以及 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在体外以前所未有的吞吐量。然而，这些技术主要是探查 复杂生物系统的一个方面。这一限制使集成信息变得具有挑战性 从不同的分子类型和尺度获得，并提取系统级的机理支撑 表型，尤指活体内的。我们的目标是通过开发变革性的、多尺度的、 多模式成像平台，筛选大组织体积以识别表型异常的细胞和 表征完整和定量的分子含量或异常细胞以及附近的细胞。这 Platform将确定细胞中的异常基因变化如何改变其表型，影响附近的细胞，以及 有助于疾病的发展。尽管它具有巨大的潜力，但简化器官规模的蛋白质组 表型鉴定和原位单细胞转录切割是不可能的，因为化学和 成像要求。我们建议开发一系列化学工具，以实现多尺度、综合的分析 蛋白质和RNA：完整组织中RNA的可逆保护；组织转化化学 基因组成像；以及用于厚组织成像的基于量子点的近红外成像平台。集成这些工具，我们 将开发和实施多尺度、综合成像平台来表征表型异常 在自闭症患者的大脑中，例如异位神经元连接，以及在 表型缺陷。这项研究将提供对疾病组织的整体看法，以破译致病原因 通过鉴定改变的基因，在分子水平上的表型异常背后的机制 异常表型、细胞间通讯网络附近的表达模式导致系统- 水平表型，以及健康和疾病组织中不同细胞类型的空间组织。在……里面 除了能够进行新的生物学研究外，新开发的化学工具还将推动广泛的创新 生物医学的范围，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.