TISSUE IN SITU METABOLITE PROFILING IN ZEBRAFISH

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用作生物合成构建块的小分子代谢物，作为调节细胞活性的信号分子，或用于细胞间信号传递。为了更好地了解组织是如何工作或发育的，需要在保存组织结构的完整组织中可视化这些代谢物。为了发现组织的潜在机制，需要以无偏倚的方法调查10,000种代谢物或整个代谢物组，这使得问题更加复杂。质谱（MS）方法在组织切片中显示整个代谢组，代谢组原位成像（MISI），已经开发出来，但仍然很少使用。这主要是由于无法将MISI图像中的特定细胞类型与代谢物谱联系起来。我们建议开发方法，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。这些标签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耦合到切片中的每个代谢组谱。我们将探索三种方法来鉴定MISI的细胞类型特异性标记(a)使用细胞类型特异性的内源性代谢物，(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，使我们能够看到一个谱系的细胞在器官内不同位置的不同行为，或者不同类型的细胞如何彼此相邻协调其功能。这样的分析将推动我们理解动物是如何正常发育的，或者癌细胞是如何出错的。

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.