DOI: 10.1126/science.1178577 , 294 (2009); 326Science et al.Shane P. Herbert,FormationSprouting: An Alternative Mode of Blood Vessel Arterial-Venous Segregation by Selective CellThis copy is for your personal, non-commercial use only.. clicking herecolleagues, clients, or customers by , you can order high-quality copies for yourIf you wish to distribute this article to others. herefollowing the guidelines can be obtained byPermission to republish or repurpose articles or portions of articles (this information is current as of March 29, 2010 ):The following resources related to this article are available online at www.sciencemag.org http://www.sciencemag.org/cgi/content/full/326/5950/294version of this article at: including high-resolution figures, can be found in the onlineUpdated information and services, http://www.sciencemag.org/cgi/content/full/326/5950/294/DC1 can be found at: Supporting Online Materialfound at: can berelated to this articleA list of selected additional articles on the Science Web sites http://www.sciencemag.org/cgi/content/full/326/5950/294#related-content http://www.sciencemag.org/cgi/content/full/326/5950/294#otherarticles, 11 of which can be accessed for free: cites 26 articlesThis article http://www.sciencemag.org/cgi/content/full/326/5950/294#otherarticles 1 articles hosted by HighWire Press; see: cited byThis article has been http://www.sciencemag.org/cgi/collection/developmentDevelopment : subject collectionsThis article appears in the following registered trademark of AAAS. is aScience2009 by the American Association for the Advancement of Science; all rights reserved. The title CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience on March 29, 2010 www.sciencemag.orgDownloaded fromArterial-Venous Segregation bySelective Cell Sprouting: An AlternativeMode of Blood Vessel FormationShane P. Herbert,1,2Jan Huisken,1Tyson N. Kim,3Morri E. Feldman,4Benjamin T. Houseman,5Rong A. Wang,3Kevan M. Shokat,5Didier Y. R. Stainier1*Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexistingvessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, weuncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, twounconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formedby selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation.These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which areexpressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient thedirection of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, aprocess essential for vascular development.During early stag es of vertebrate embryo-genesis, coordinated sorting and seg-regation of art erial- and venous-fatedangioblasts into distinct networks of arteries andveins is essential to establish a functional vas-culature. Recent studies in mouse and zebrafishhave elucidated key roles for a number of sig-naling pathways and transcriptional regulators inarterial-venous specification (1, 2). However , westill lack a basic mechanistic understanding ofhow mixed populations of specified arterial-venous cells coordinate their behavior to segre-gate and form distinct vessels.During zebrafish vascular development, an-gioblasts migrate from the lateral plate meso-derm to the midline (3, 4)andeventuallygiverise to the first embryonic artery (dorsal aorta,DA) and vein (caudal vein, CV). Notochord -derived Sonic hedgehog, which induces the ex-1Department of Biochemistry and Biophysics, Programs inDevelopmental Biology, Genetics and Human Genetics,Cardiovascular Research Institute, University of California,San Francisco, CA 94158, USA.2Multidisciplinary Cardiovas-cular Research Centre and Institute of Molecular and CellularBiology, Faculty of Biological Sciences, University of Leeds,Leeds LS2 9JT, UK.3Laboratory for Accelerated VascularResearch, Division of Vascular Surgery, Department of Surgery,University of California, San Francisco, CA 94143, USA.4Graduate Group in Biophysics, University of California, SanFrancisco, CA 94158, USA.5Howard Hughes Medical Instituteand Department of Cellular and Molecular Pharmacology,University of California, San Francisco, CA 94158, USA.*To whom correspondence should be addressed. E-mail:[email protected]. 1. The CV forms by selective angioblast sprouting. (A to E)Mid-trunktransverse sections [(A) a nd (D)], lateral views [(B) and (E)], or lateral time-lapse [(C) and movie S1] of Tg(kdrl:GFP)s843[(A) to (C)] or Tg(kdrl:GFP)s843;Tg(gata1:dsRed)sd2[(D) and (E)] embryos. Angioblasts coalesce and remodelto form the DA by 22 hpf [white and red brackets; (A) to (C)]. Between 21and 24 hpf, venous angioblasts sprout ventrally from the DA [arrowheadsand dotted lines, (A) to (C)] and contribute to the CV primordium [yellow andblue brackets, (A) to (C)]. ISVs sprout dorsally from 23 hpf [arrows, (B)]. By25 hpf, venous angioblasts surround Tg(gata1:dsRed)sd2-positive erythro-cytes [asterisks, (A) and (D); red c ells, (D) and (E)]. Erythrocyte displacementtypically clears the CV lumen by 26 hpf [(A), (D), and (E)] but not in tnnt2MO–injected embryos (E). Scale bars indicate 35 mm.9 OCTOBER 2009 VOL 326 SCIENCE www.sciencemag.org294REPORTS on March 29, 2010 www.sciencemag.orgDownloaded frompression of vascular endothelial growth factora(Vegfa)intheventralsomites,isessentialforangioblast differentiation (5). Vegfa-induced ac-tivatio n of Notch signalin g (5, 6), as well as otherfactors (2, 7–9), subsequently promotes arterialspecification in a subset of angioblasts (4), beforearterial-venous segregation. To investigate mech-anisms of arterial-venous angioblast sorting andsegregat ion, we analyzed vascular developmentwith high temporal resolution in Tg(kdrl:GFP)s843(4)embryos(10). These transgenic embryo s ex-press green fluorescent protein (GFP) in theendothelial lineage, enabling the tracking ofangioblasts during early stages of vascular de-velopment. GFP-positive angioblasts coalescedat the midline to form a single vascular cord bythe 21-somite