TISSUE IN SITU METABOLITE PROFILING IN ZEBRAFISH

斑马鱼组织原位代谢物分析

• 批准号: 8892222
• 负责人: STEPHEN L JOHNSON
• 金额: $ 22.3万
• 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 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8892222
• 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; Genetic; Glucose; Goals; Growth; Health; 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; resistance gene; 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(申请人提供)：要了解细胞如何在复杂的组织中发展和发挥其生理作用，需要了解小分子代谢物，这些代谢物被用作生物合成的基石，作为调节细胞活动的信号分子，或用于细胞之间的信号传递。在保存组织结构的完整组织中可视化这些代谢物将需要更好地了解组织是如何工作或发育的。为了发现组织的潜在机制，需要以无偏见的方法调查1万个代谢物或整个代谢组，这使问题变得更加复杂。在组织切片中显示整个代谢物的质谱学(MS)方法--代谢原位成像(MISI)已经被开发出来，但仍然没有得到很好的利用。这在很大程度上是因为无法将特定的细胞类型与MISI图像中的代谢物图谱联系起来。我们建议开发能够明确识别不同细胞类型的方法。我们将标识或生成唯一标记不同单元格的标签。这些标记将被电离，并与采样的代谢组一起进入MS，从而将细胞类型识别符与区段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)使用针对细胞类型的内源性代谢物，(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耐药基因发展细胞类型特异性抗生素转换。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打开大门，让我们了解同一谱系的细胞在器官内不同位置的不同表现，或者不同类型的细胞如何相互协调其功能。这样的分析将推动我们理解动物是如何正常发育的，或者癌细胞是如何做错的。