TISSUE IN SITU METABOLITE PROFILING IN ZEBRAFISH

描述（由申请人提供）：了解细胞如何发育并在复杂组织中发挥其生理作用将需要了解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细胞之间信号传递的小分子代谢物。在保存组织结构的完整组织中观察这些代谢物将需要更好地了解组织如何工作或发育。这个问题是复杂的，需要调查10,000代谢物，或整个代谢组，在公正的方法，以发现组织的潜在机制。已经开发了用于可视化组织切片中的整个代谢组的质谱（MS）方法，即代谢组学原位成像（MISI），但仍然利用不多。这在很大程度上是由于无法将特定的细胞类型与MISI图像中的代谢产物谱相关联。我们建议开发的方法，将允许明确识别不同的细胞类型。我们将识别或生成唯一标记不同细胞的标签。这些标记将被离子化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偶联。我们将探索三种方法来确定MISI的细胞类型特异性标记（a）使用特定于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打开大门，让我们能够看到一个谱系的细胞在器官内的不同位置如何表现出不同的行为，或者不同的细胞类型如何相互协调它们的功能。这样的分析将推动我们理解动物如何正常发育，或者癌细胞如何出错。

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.