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To access, purchase, authenticate, or subscribe to the full-text of this article, please visit this link: http://dx.doi.org/10.1016/j.ydbio.2017.03.017 Byline: Robyn A. Umans (a,b), Hannah E. Henson (a,b), Fangzhou Mu (c), Chaithanyarani Parupalli (a), Bensheng Ju (a), Jennifer L. Peters (d), Kevin A. Lanham (c), Jessica S. Plavicki (c), Michael R. Taylor [michael.taylor@wisc.edu] (a,c,*) Keywords Blood-brain barrier (BBB); Brain endothelial cell (BEC); Barriergenesis; Angiogenesis; Glucose transporter 1 (Glut1); Plasmalemma vesicle-associated protein (Plvap) Highlights * Zebrafish glut1b:mCherry serves as an in vivo reporter of blood-brain barriergenesis. * Live imaging reveals that CNS angiogenesis and barriergenesis occur simultaneously. * Forward genetic screen using glut1b:mCherry identifies a zebrafish gpr124 mutant. * Zebrafish plvap:EGFP is an in vivo indicator of blood-brain barrier maturation. Abstract The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously. Author Affiliation: (a) Department of Chemical Biology & Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA (b) Integrated Program in Biomedical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA (c) Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, WI, USA (d) Cell and Tissue Imaging, St. Jude Children's Research Hospital, Memphis, TN, USA * Corresponding author at: Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, WI, USA. Article History: Received 27 October 2016; Revised 24 February 2017; Accepted 18 March 2017