In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters
In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters
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DOI：
10.1152/ajpgi.90513.2008
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发表时间：
2009-02-01
影响因子：
4.5
通讯作者：
Farber, Steven A.
中科院分区：
文献类型：
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作者：
Hama, Kotaro;Provost, Elayne;Farber, Steven A.
Hama K，Provost E，Baranowski TC，Rubinstein AL，安德森JL，利奇SD，法伯SA.使用多重淬灭荧光报告子对斑马鱼消化器官功能的体内成像。美国生理学杂志胃肠和肝脏生理学296：G445-G453，2009年。首次发表于2008年12月4日; doi：10.1152/ajpgi.90513.2008。斑马鱼幼虫的光学清晰度使其成为实时分析脊椎动物器官功能的理想工具，通过使用酶活性的荧光报告。消化器官的一个关键功能是将酶的产生与机械过程结合起来，使营养物质的可用性和吸收成为可能。然而，直接观察活脊椎动物的消化过程是极其困难的，在许多情况下是不可能的。在这里，我们描述了一种新的方法来可视化肠道蛋白质和脂质加工同时在活斑马鱼幼虫使用淬灭的荧光蛋白（EnzChek）和磷脂（PED 6）。通过采用这些试剂，我们发现，野生型幼虫表现出显着的变化，肠磷脂酶和蛋白酶的活动在一组内，但显示出很强的相关性，在个人的活动。此外，我们发现胰腺功能是必不可少的幼虫消化蛋白酶活性，但不是幼虫肠道磷脂酶活性。虽然无脂（ffr）突变幼虫先前被描述为表现出受损的脂质过程，我们发现他们也有显着降低蛋白酶活性。最后，我们选择并评估了以前被认为具有改变的磷脂酶活性并且已知或怀疑在肠道中具有炎症作用的化合物，包括非甾体抗炎药，并鉴定了一种显著增加肠道磷脂加工的化合物。因此，基于多重荧光标记的方法有助于快速分析活斑马鱼幼体的消化器官功能。
Hama K, Provost E, Baranowski TC, Rubinstein AL, Anderson JL, Leach SD, Farber SA. In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters. Am J Physiol Gastrointest Liver Physiol 296: G445-G453, 2009. First published December 4, 2008; doi:10.1152/ajpgi.90513.2008.-Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.