TISSUE IN SITU METABOLITE PROFILING IN ZEBRAFISH

斑马鱼组织原位代谢物分析

• 批准号: 8749733
• 负责人: STEPHEN L JOHNSON
• 金额: $ 19.06万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8749733
• 关键词: Amputation; Anabolism; Animals; Antibiotic Resistance; Antibiotics; Architecture; Benign; Biogenesis; Biological Availability; Cell division; Cells; Complex; Coupling; Detection; Development; Fishes; Gene Transfer Techniques; Genes; Genetic; Glucose; Goals; Growth; Image; Image Analysis; In Situ; Label; Malignant Neoplasms; Mass Spectrum Analysis; Metabolism; Methods; Modeling; Natural regeneration; Organ; Physiological; Pigments; Positioning Attribute; Proliferating; Resolution; Role; Sampling; Signal Transduction; Signaling Molecule; Staging; Stem cells; Surveys; Tissues; Transgenes; Transgenic Organisms; Warburg Effect; Work; Zebrafish; bone; cancer cell; cell type; melanocyte; metabolomics; method development; public health relevance; small molecule

DESCRIPTION (provided by applicant): Understanding how cells develop and perform their physiological roles in complex tissues will require understanding the small molecule metabolites that are used as building blocks for biosynthesis, as signaling molecules to regulate the activity of the cell, or used to signal between cells. Visualizing these metabolites in intact tissue that preserves the architecture of the tissue will be required to get a better understanding of how the tissue works or develops. The problem is compounded by the need to survey 10,000s of metabolites, or the entire metabolome, in unbiased approaches to discover underlying mechanisms of the tissue. Mass spectrometry (MS) methods to visualize entire metabolomes in tissue sections, Metabolomic In Situ Imaging (MISI), have been developed, but remain poorly utilized. This is largely due to the inability to relate specific cell types to metabolite profilesin the MISI image. We propose to develop methods that will allow unambiguous identification of distinct cell types. We will identify or generate labels that uniquely mark different cells. These labels will be ionized and taken into the MS together with the sampled metabolome, thus coupling cell type identifiers to each metabolome profile in the section. We will explore three approaches to identifying cell type specific labeling for MISI (a) use of endogenous metabolites specific to cell types, (b) detection of cell-type transgenic expression of GFP, and (c) development of cell type-specific antibiotic conversion by transgenically expressed antibiotic resistance genes. We will use cell type-specific labeling to explore different metabolisms of growing or quiescent cells in the regenerating zebrafish fin. Development of these methods will open the doors on our ability to see how cells of a lineage behave differently at different positions within an organ, or how different cell types next to each other coordinate their functions. Such analysis will push forward our understanding of how animals develop properly, or how cancer cells do it wrong.

描述（由申请人提供）：了解细胞如何在复杂组织中发育和发挥其生理作用将需要了解小分子代谢物，这些小分子代谢物用作生物合成的构建块，用作调节细胞活性的信号分子，或用于在细胞之间发出信号。为了更好地了解组织如何工作或发育，需要在保留组织结构的完整组织中可视化这些代谢物。由于需要以公正的方法调查数以万计的代谢物或整个代谢组，以发现组织的潜在机制，因此问题变得更加复杂。已经开发出用于可视化组织切片中整个代谢组的质谱（MS）方法，即代谢组原位成像（MISI），但仍然没有得到很好的利用。这主要是由于无法将特定细胞类型与 MISI 图像中的代谢物谱联系起来。我们建议开发能够明确识别不同细胞类型的方法。我们将识别或生成唯一标记不同细胞的标签。这些标记将被电离并与采样的代谢组一起进入 MS，从而将细胞类型标识符与切片中的每个代谢组图谱耦合。我们将探索三种方法来识别 MISI 的细胞类型特异性标记（a）使用细胞类型特异性的内源代谢物，（b）检测 GFP 的细胞类型转基因表达，以及（c）通过转基因表达的抗生素抗性基因开发细胞类型特异性抗生素转化。我们将使用细胞类型特异性标记来探索再生斑马鱼鳍中生长或静止细胞的不同代谢。这些方法的发展将为我们了解一个谱系的细胞在器官内不同位置如何表现不同，或者彼此相邻的不同细胞类型如何协调其功能打开大门。这样的分析将促进我们对动物如何正常发育或癌细胞如何错误发育的理解。