SUMMARYVascular endothelial cells in vivo are exquisitely regulated by their local environment, which is absent or disrupted when using methods such as FACS or in vitro cell culture to study native signaling pathways. 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 endothelially expressed genes. Additionally, we generated a ‘UAS:RiboTag’ transgenic line to allow for the study of a wider array of zebrafish cell and tissue types using TRAP-RNAseq methods. This new tool offers an unparalleled resource to study cause and effect relationships in the context of gene loss or gain of function in vivo.HIGHLIGHTSAn ‘AngioTag’ transgenic line permits in vivo endothelial expression profilingThe AngioTag line is used for Translating Ribosome Affinity Purification - RNAseqA ‘UAS:RiboTag’ line enables profiling of any zebrafish cell and tissue type