Live visualization of a functional RET‐EGFP chimeric receptor in homozygous knock‐in mice
Live visualization of a functional RET‐EGFP chimeric receptor in homozygous knock‐in mice
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纯合敲入小鼠功能性 RET-EGFP 嵌合受体的实时可视化
DOI：
10.1111/dgd.12740
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Enomoto Hideki
中科院分区：
文献类型：
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作者：
Sunardi Mukhamad;Ito Keisuke;Enomoto Hideki
GDNF家族配体（GFLs）通过激活RET受体酪氨酸激酶调节神经发育和肾脏器官发生。许多依赖RET的发育过程涉及长距离细胞间通信或细胞极性，包括细胞迁移和轴突引导。这表明，时空调控的RET蛋白亚细胞定位和RET信号在细胞内的适当传播对gfl的生理功能至关重要。然而，我们对RET蛋白在细胞中的动态知之甚少。要解决这个问题，需要开发一种系统，使活细胞中的RET可视化。在这项研究中，我们报道了一种新的敲入小鼠系，其中RET - EGFP嵌合受体在theRetpromoter下表达。与由于缺乏肠神经系统（ENS）和肾脏而在出生后死亡的eret -缺陷小鼠不同，RetRET - EGFP/RET - egfp小鼠可以存活并生长到成年，没有明显的异常，这表明RET - EGFP发挥的功能与RET相当。在神经元和ENS祖细胞中，RET - EGFP信号在细胞膜和细胞质中都被检测到，后者以点状模式出现。培养的神经细胞和胚胎的延时成像显示RET - EGFP点在神经元轴突和细胞体中的主动运输。免疫组织化学分析在早期和循环核内体中检测到RET - EGFP信号，表明RET - EGFP通过内吞途径运输。RetRET‐EGFP/RET‐EGFPmice首次实现了体内RET蛋白功能的可视化，并提供了一个独特的平台来研究RET运输的动力学和生理学。
The GDNF Family Ligands (GFLs) regulate neural development and kidney organogenesis by activating the RET receptor tyrosine kinase. Many RET‐dependent developmental processes involve long‐distance cell‐cell communications or cell polarity, which includes cell migration and axon guidance. This suggests that spatiotemporally regulated subcellular localization of RET protein and appropriate propagation of RET signaling in cells are essential for the physiological function of the GFLs. Little is known, however, about the dynamics of RET protein in cells. Addressing this issue requires development of a system that allows visualization of RET in living cells. In this study, we report generation of a novel knock‐in mouse line in which the RET‐EGFP chimeric receptor is expressed under theRetpromoter. UnlikeRet‐deficient mice that die after birth due to the absence of the enteric nervous system (ENS) and kidneys,RetRET‐EGFP/RET‐EGFPmice were viable and grew to adulthood with no overt abnormality, which indicated that RET‐EGFP exerts function comparable to RET. In neurons and ENS progenitors, RET‐EGFP signals were detected both on the cell membrane and in the cytoplasm, the latter of which appeared as a punctate pattern. Time‐lapse imaging of cultured neural cells and embryos revealed active transport of RET‐EGFP puncta in neuronal axons and cell bodies. Immunohistochemical analyses detected RET‐EGFP signals in early and recycling endosomes, indicating that RET‐EGFP is trafficked via the endocytic pathway.RetRET‐EGFP/RET‐EGFPmice enable visualization of functional RET protein in vivo for the first time and provide a unique platform to examine the dynamics and physiology of RET trafficking.