Queen's Medical Research Institute, Cardiovascular Science Centre, University of Edinburgh, Edinburgh, UK.
Queen's Medical Research Institute, Cardiovascular Science Centre, University of Edinburgh, Edinburgh, UK,
Cell Physiol Biochem. 2021 May 5;55(S4):35-47. doi: 10.33594/000000366.
BACKGROUND/AIMS: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage.
We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line.
We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes.
We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair.
背景/目的：从果蝇到硬骨鱼再到哺乳动物，足细胞在物种间的作用高度保守。鉴定出能主动维持足细胞完整性的分子标记物，将有助于我们更好地理解导致损伤的变化。
我们生成了转(transgenic)基因斑马鱼，其足细胞蛋白启动子驱动荧光报告基因表达，用于足细胞的可视化和分离。我们对来自斑马鱼报告系的分离足细胞进行了单细胞 RNA 测序(scRNA-seq)。
我们证明了 LifeAct-TagRFP-T 荧光报告基因在体内能真实复制足细胞蛋白的表达。我们还能够使用光片(single plane illumination)显微镜显示自发的 GCaMP6s 荧光。我们在与人类和小鼠成熟足细胞共有的基因中鉴定出许多与钙结合和肌动蛋白丝组装相关的足细胞转录本。
我们描述了新型转基因斑马鱼的建立，并利用它们来鉴定和分离正常足细胞，用于制备 scRNA-seq 文库。scRNA-seq 数据鉴定了不同的细胞群，并可能在簇间发生基因转换。这些数据为未来的比较研究以及利用斑马鱼作为肾脏发育、疾病、损伤和修复的模型提供了基础。
