利用胚胎斑马鱼前脑抗体摄取试验研究体内神经祖细胞中的Notch信号动力学
Antibody Uptake Assay in the Embryonic Zebrafish Forebrain to Study Notch Signaling Dynamics in Neural Progenitor Cells In Vivo.
作者信息
Tong Kai, Wagle Mahendra, Guo Su
机构信息
State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences, ELi and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA, USA.
出版信息
Methods Mol Biol. 2019;1576:273-281. doi: 10.1007/7651_2017_22.
Stem cells can generate cell fate heterogeneity through asymmetric cell division (ACD). ACD derives from the asymmetric segregation of fate-determining molecules and/or organelles in the dividing cell. Radial glia in the embryonic zebrafish forebrain are an excellent model for studying the molecular mechanisms regulating ACD of stem cells in vertebrates, especially for live imaging concerning in vivo molecular and cellular dynamics. Due to the current difficulty in expressing fluorescent reporter-tagged proteins at physiological levels in zebrafish for live imaging, we have developed an antibody uptake assay to label proteins in live embryonic zebrafish forebrain with high specificity. DeltaD is a transmembrane ligand in Notch signaling pathway in the context of ACD of radial glia in zebrafish. By using this assay, we have successfully observed the in vivo dynamics of DeltaD for studying ACD of radial glia in the embryonic zebrafish forebrain.
干细胞可通过不对称细胞分裂（ACD）产生细胞命运异质性。ACD源于分裂细胞中命运决定分子和/或细胞器的不对称分离。斑马鱼胚胎前脑的放射状胶质细胞是研究脊椎动物干细胞ACD调控分子机制的优秀模型，尤其适用于体内分子和细胞动力学的活体成像。由于目前在斑马鱼中以生理水平表达荧光报告标签蛋白用于活体成像存在困难，我们开发了一种抗体摄取测定法，以高特异性标记活斑马鱼胚胎前脑中的蛋白质。DeltaD是斑马鱼放射状胶质细胞ACD背景下Notch信号通路中的跨膜配体。通过使用该测定法，我们成功观察到DeltaD的体内动力学，以研究斑马鱼胚胎前脑放射状胶质细胞的ACD。
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