Profiling the endothelial translatome in vivo using 'AngioTag' zebrafish

Vascular endothelial cells in vivo are exquisitely regulated by their local environment, which is disrupted or absent when using methods such as FACS sorting of cells isolated from animals or in vitro cell culture. Here, we profile the gene expression patterns of undisturbed endothelial cells in living animals using a novel “AngioTag” zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. By performing TRAP-RNAseq on AngioTag animals, we demonstrate strong enrichment of endothelial specific genes and uncover novel endothelial genes and unique endothelial gene expression signatures for different adult organs. Finally, we generated a versatile “UAS:RiboTag” transgenic line to allow a widerarray of different zebrafish cell and tissue types to be examined using TRAP-RNAseq methods. These new tools offer an unparalleled resource to study the molecular identity of cells in their normal in vivo context. Overall design: We profiled the endothelial translatome of different vascular beds within organs of the adult zebrafish as well as the whole fish to determine endothelial genes shared among all organs and those unique to each vascular bed.