Profiling the endothelial translatome in vivo using 'AngioTag' zebrafish

Abstract

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

Tissue Regeneration Requires Edema Fluid Clearance by Compensatory Lymphangiogenesis in Zebrafish

“…This technique bypasses the cell dissociation steps that disrupt cell identity and alter gene expression, giving a true “ in vivo snapshot” of molecular changes in highly specific cell populations 38–40 . To this end, we generated a novel Tg(mrc1a:egfp-2a-rpl10a3xHA) transgenic line using the mrc1a promoter 18 to drive the expression of an eGFP-2A-rpl10a-3xHA transgene cassette 37 . This expression cassette consists of an eGFP coexpressed via a viral 2A peptide linkage with a 60S ribosomal protein l 10a (rpl10a) tagged with triplicate hemagglutinin epitope sequences (3xHA) to permit simultaneous visualization of transgene expression and translatome collection ( Supplementary Fig.…”

“…To probe the in vivo molecular changes in lymphatic endothelial cells in response to tissue damage and repair, we carried out transgene-assisted Translating Ribosome Affinity Purification (TRAP), which has been used to isolate ribosome-bound mRNAs undergoing active translation from specific cell types or tissues in vivo 36, 37 . This technique bypasses the cell dissociation steps that disrupt cell identity and alter gene expression, giving a true “ in vivo snapshot” of molecular changes in highly specific cell populations 38–40 .…”

“…The RiboTag construct was generated using Tol2kit components with Gateway Technology 61 . To generate mrc1a:eGFP-2A-rpl10a-3xHA DNA construct, LR clonase II (Thermo Fisher Scientific) was used to recombine p5E-mrc1a promoter 18 , pME-eGFP-2A-rp110a-3xHA 37 , and p3E-polyA 61 into pDestTol2pA2 61 . The DNA construct and Tol2 transposase RNA were microinjected into the blastomere of one-cell stage zebrafish embryos.…”

“…TRAP was performed following a modified protocol 37 . For each untreated or edematous sample, 500 4dpf mrc1a RiboTag zebrafish were dechorionated, deyolked, and dounce-homogenized in homogenization buffer consisting of 1 mM Tris pH 7.4, 100 mM KCl, 12 mM MgCl 2 , 1% NP-40, 1 mM DTT, 1x Protease inhibitors (Sigma), 200 units/mL RNAsin (Promega), 100ug/mL cycloheximide (Sigma) and 1mg/mL heparin (Sigma).…”

CXCR3-CXCL11 signaling restricts angiogenesis and promotes pericyte recruitment

“…Zebrafish transgenic lines-Tg(fli1a:eGFP) y1 ; Tg(kdrl:mCherry-CAAX) y171 ; TgBAC(pdgfrb:Gal4FF) ncv24 ; Tg(5xUAS:RFP) zf83 ; Tg(kdrl:rpl10a-3xHA-2a-eGFP) y530 -are previously published 21,[53][54][55][56] .…”