A high-resolution in situ proteomics atlas of salivary gland development

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和特定挑战主题，06- de -102颅面发育的结构和分子地图集。在系统水平上理解器官发育的一个关键问题是了解整个发育过程中基因和蛋白质表达的时空模式。我们开发了一种新的基于荧光的多路复用技术，用于同时跟踪单个福尔马林固定的石蜡包埋组织切片中的数十种蛋白质，以及用于在亚细胞水平上对这些标记物的蛋白质定位模式进行量化和分类的新型软件算法。该方法包括直接免疫组织化学（IHC）和自动成像，然后直接结合到抗体探针的荧光团完全失活。与经典的间接免疫组化相比，这种直接免疫组化方法允许用多种抗体对同一组织进行顺序多路探测，从而显著增加单个样品中可以同时可视化的标记物数量。这种方法消除了对多个光谱兼容的荧光团和连续组织切片的需要，从而允许从少量材料生成大量数据。染料失活多路复用程序已经通过100轮循环进行了彻底的表征和测试，没有损失组织形态或抗原性。在本项目中，我们将首次将这种创新的多路复用技术应用于发育系统，在发育性唾液腺组织阵列中分析小鼠下颌下和舌下唾液腺形态发生和分化过程中信号蛋白的表达模式。最终产品将包括一个高分辨率的原位蛋白质组学图谱，该图谱将在所有关键的发育时间点对特定细胞类型和亚细胞区室中的活性信号蛋白表达水平进行分类和量化。这种高分辨率的解剖系统级分析是不可能用传统的基因组学方法创建的，这是在mRNA水平上完成的，或者用传统的蛋白质组学方法，这破坏了组织环境。该数据集将补充通过校内NIDCR项目（唾液腺图谱项目）开发的基因表达图谱。K. Yamada和M. Hoffman)，并将与FaceBase中的其他数据库集成，并为系统生物学提供真正独特的数据组件。形态发生相关数据集还将为唾液腺发育的数学模型提供信息（RO1DE0192444-01， M. Larsen,），分化相关数据集将为人工唾液腺的智能工程（R21DE0192444-01， M. Larsen）识别新的分化标记和途径成分。本研究将为今后在其他正常和患病小鼠及人类颅面组织的研究提供基础的发育生物学基础。最后，拟议的研究将通过直接材料成本（即显微镜，流体，计算机和控制器，抗体和耗材，这些都将从美国供应商购买）促进经济复苏，并提供两个新职位来完成抗体标记，并成为Larsen博士实验室的关键操作员，以便该技术可以应用于她未来的研究。应用于地图集的技术；然而，目前还没有商业上提供给公共研究界，但这个项目将促进这种强大的多路复用方法的适应和过渡到主流的研究应用。提出的研究将创建一个图谱的蛋白质表达模式在发展中唾液腺使用新的高含量的方法。这些方法允许在同一样品中检查多个标记，允许在发育阶段对多个蛋白质进行真正的共定位。这些研究将提供唾液腺发育和分化过程中发生的基本信号事件的见解。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-DE-102 Structural and Molecular Atlases of Craniofacial Development. A critical issue for understanding organ development at a systems level is knowledge of temporal and spatial patterns of gene and protein expression throughout development. We have developed a novel fluorescence-based multiplexing technology for simultaneously tracking dozens of proteins within single formalin-fixed, paraffin-embedded tissue sections and novel software algorithms for quantifying and categorizing protein localization patterns for these markers at the subcellular level. This method involves direct immunohistochemistry (IHC) and automated imaging followed by complete inactivation of fluorophores that are directly conjugated to antibody probes. This direct IHC approach allows sequential multiplexed probing of the same tissue with multiple antibodies to dramatically increase the number of markers that can be simultaneously visualized in a single sample when compared with classical indirect IHC. This method eliminates the need for multiple spectrally compatible fluorophores and for serial tissue sections, thus allowing large amounts of data to be generated from a small amount of material. The dye-inactivation multiplexing procedure has been thoroughly characterized and tested through 100 rounds of cycling with no loss in tissue morphology or antigenicity. In this project, we will apply this innovative multiplexing technology for the first time to a developmental system to profile expression patterns of signaling proteins during mouse submandibular and sublingual salivary gland morphogenesis and differentiation in a developmental salivary gland tissue array. The end product will include a high- resolution in situ proteomics-based atlas, which categorizes and quantifies active signaling protein expression levels within specific cell types and subcellular compartments, throughout all key developmental time-points. This high-resolution anatomical systems-level analysis would be impossible to create using traditional genomics methods, which are done at the level of mRNA, or by traditional proteomic methods, which destroy the tissue context. This data set will complement the gene expression atlases developed through the intramural NIDCR program (Salivary Gland Atlas project, Drs. K. Yamada and M. Hoffman) and will integrate with other databases in FaceBase, and provide a truly unique data component for systems biology. The morphogenesis-related dataset will also inform a mathematical model of the developing salivary gland, (RO1DE0192444-01, M. Larsen,) and the differentiation-related dataset will identify new differentiation markers and pathway components for intelligent engineering of an artificial salivary gland (R21DE0192444-01, M. Larsen). This study will provide a foundation of basic developmental biology knowledge needed to interpret future studies in other normal and diseased mouse and human craniofacial tissues. Finally, the proposed studies will facilitate economic recovery through direct materials costs (i.e. microscope, fluidics, computers and controllers, and antibodies and supplies, which will all purchased from US vendors) and by providing two new positions to complete the antibody labeling and to become a key operator in Dr. Larsen's lab so that the technology can be applied to her future studies. The technology being applied to the atlas; however, is not currently commercially available to the public research community, but this project will facilitate the adaptation and transition of this powerful multiplexing method to mainstream research applications. The proposed studies will create an atlas of protein expression patterns in developing salivary gland using novel high-content methods. These methods allow multiple markers to be examined in the same sample, allowing true co-localization of multiple proteins through developmental stages. These studies will provide insight into the fundamental signaling events occurring during the process of salivary gland development and differentiation.