ISSN: 1524-4571 Copyright © 2008 American Heart Association. All rights reserved. Print ISSN: 0009-7330. OnlineTX 72514Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas,DOI: 10.1161/CIRCRESAHA.108.181818 2008;103;784-795 Circ. Res.Arie Horowitz and Michael Simons Branching Morphogenesis http://circres.ahajournals.org/cgi/content/full/103/8/784located on the World Wide Web at: The online version of this article, along with updated information and services, is http://www.lww.com/reprintsReprints: Information about reprints can be found online at [email protected]. E-mail: Fax:Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters http://circres.ahajournals.org/subscriptions/Subscriptions: Information about subscribing to Circulation Research is online at at UNIV OF NORTH CAROLINA on November 11, 2008 circres.ahajournals.orgDownloaded fromThis Review is part of a thematic series on Arterial Specification: A Finishing School for the Endothelium, whichincludes the following articles:Role of Crosstalk Between Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase/Mitogen-ActivatedProtein Kinase Pathways in Artery–Vein SpecificationBranching MorphogenesisBrothers and Sisters: Molecular Insights into Arterial–Venous HeterogeneityFibroblast Growth Factor–Hedgehog Signaling in Coronary Arterial CirculationArterial GuidanceArterial–Venous Specification in Development Michael Simons, Guest EditorBranching MorphogenesisArie Horowitz, Michael SimonsAbstract—Tubular structures are a fundamental anatomic theme recurring in a wide range of animal species. In mammals,tubulogenesis underscores the development of several systems and organs, including the vascular system, the lungs, andthe kidneys. All tubular systems are hierarchical, branching into segments of gradually diminishing diameter. There areonly 2 cell types that form the lumen of tubular systems: either endothelial cells in the vascular system or epithelial cellsin all other organs. The most important feature in determining the morphology of the tubular systems is the frequencyand geometry of branching. Hence, deciphering the molecular mechanisms underlying the sprouting of new branchesfrom preexisting ones is the key to understanding the formation of tubular systems. The morphological similaritybetween the various tubular systems is underscored by similarities between the signaling pathways which control theirbranching. A prominent feature common to these pathways is their duality—an agonist counterbalanced by an inhibitor.The formation of the tracheal system in Drosophila melanogaster is driven by fibroblast growth factor and inhibited bySprouty/Notch. In vertebrates, the analogous pathways are fibroblast growth factor and transforming growth factor-!inepithelial tubular systems or vascular endothelial growth factor and Notch in the vascular system. (Circ Res.2008;103:784-795.)Key Words: branching!tubulogenesis!vascular system!tracheal system!ureteric systemHierarchical tubular networks are a recurrent anatomicform in numerous systems and organs. The vascularsystem is probably the most prominent instance of such anetwork, but other organs incorporate similar patterns, in-cluding the bronchial network and the urinary collectingsystem. A theme common to all tubular systems is theirbranched and hierarchal nature. Starting from a single majorconduit (eg, the aorta in the case of the arterial system),tubular systems elaborate into gradually thinner conduits,down to the level of tubes whose walls are made up of a singlecell layer. The lumen of tubular systems is continuous andformed throughout by the same cell type. These cells are of nomore than 2 types: endothelial or epithelial. Endothelial cellscoat the lumen of the vascular and lymphatic systems, whereasepithelial cells coat the lumen of all other tubular systems.The architecture of all tubular systems is determined to alarge extent by a single topological feature— branching—namely the sprouting of new tubes from preexisting ones. Thefrequency of branching determines the density of the tubularsystem and the length of its segments. The angle betweenOriginal received June 18, 2008; revision received August 20, 2008; accepted August 25, 2008.From the Angiogenesis Research Center and Section of Cardiology (A.H.), Dartmouth Medical School, Lebanon, NH; and Section of CardiovascularMedicine (M.S.), Department of Internal Medicine, Yale University School of Medicine, New Haven, Conn.Correspondence to Arie Horowitz, PhD, Angiogenesis Research Center, Dartmouth Medical School, One Medical Center Dr, Borwell 554W, Lebanon,NH 03756. E-mail [email protected]© 2008 American Heart Association, Inc.Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.181818784Review at UNIV OF NORTH CAROLINA on November 11, 2008 circres.ahajournals.orgDownloaded frombranches determines the shape of the tubular system. There-fore, finding out how the decision to sprout is made at themolecular level, and how sprout location and direction arespecified is the key to understanding the morphogenic prin-ciples of branched tubular networks.Although the focus of this review is branching in thevascular system, we discuss branching mechanisms in 3tubular systems composed of epithelial cells. The resultingbroader scope provides basis for comparison between theepithelial and endothelial-based systems and facilitates iden-tification of general principles shared by all tubular systems.As vascular biologists, our focus is on vertebrate organisms,particularly the zebrafish and the mouse. However, some ofthe most fundamental and detailed knowledge of branchingmechanisms was derived from studies on the development ofthe tracheal system of the fruit fly Drosophila melanogaster.Types of Branched StructuresThe Drosophila melanogaster Tracheal System: APrototypical Model of BranchingStructureThe relatively simple structure of the respiratory system ofDrosophila melanogaster and the ease of generating muta-tions in this model system afforded some of the majorinsights into the molecular mechanisms governing tubularnetwork development. The Drosophila larva is oxygenated bya tracheal system comprising more than 10 000 intercon-nected tubular segments.1Each