Hes1 Is Expressed in the Second Heart Field and Is Required for Outflow Tract Development Francesca Rochais1., Mathieu Dandonneau1., Karim Mesbah1, The´ re` se Jarry1, Marie-Genevie` ve Mattei2, Robert G. Kelly1* 1 Developmental Biology Institute of Marseilles-Luminy, UMR 6216 CNRS-Universite´ de la Me´diterrane´e, Campus de Luminy, Marseille, France, 2 Inserm UMR 910, Faculte´ de Me´decine, Hoˆ pital d’Enfants de la Timone, Marseille, France Abstract Background: Rapid growth of the embryonic heart occurs by addition of progenitor cells of the second heart field to the poles of the elongating heart tube. Failure or perturbation of this process leads to congenital heart defects. In order to provide further insight into second heart field development we characterized the insertion site of a transgene expressed in the second heart field and outflow tract as the result of an integration site position effect. Results: Here we show that the integration site of the A17-Myf5-nlacZ-T55 transgene lies upstream of Hes1, encoding a basic helix-loop-helix containing transcriptional repressor required for the maintenance of diverse progenitor cell populations during embryonic development. Transgene expression in a subset of Hes1 expression sites, including the CNS, pharyngeal epithelia, pericardium, limb bud and lung endoderm suggests that Hes1 is the endogenous target of regulatory elements trapped by the transgene. Hes1 is expressed in pharyngeal endoderm and mesoderm including the second heart field. Analysis of Hes1 mutant hearts at embryonic day 15.5 reveals outflow tract alignment defects including ventricular septal defects and overriding aorta. At earlier developmental stages, Hes1 mutant embryos display defects in second heart field proliferation, a reduction in cardiac neural crest cells and failure to completely extend the outflow tract. Conclusions: Hes1 is expressed in cardiac progenitor cells in the early embryo and is required for development of the arterial pole of the heart. Citation: Rochais F, Dandonneau M, Mesbah K, Jarry T, Mattei M-G, et al. (2009) Hes1 Is Expressed in the Second Heart Field and Is Required for Outflow Tract Development. PLoS ONE 4(7): e6267. doi:10.1371/journal.pone.0006267 Editor: Mai Har Sham, The University of Hong Kong, China Received February 3, 2009; Accepted June 12, 2009; Published July 17, 2009 Copyright: 2009 Rochais et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Inserm Avenir program, the Fondation de France, the European Community’s Sixth Framework Program contract (Heart Repair) LSHM-CT-2005-018630, the Agence National pour la Recherche and the 2008 ISHR-ES/Servier Research Fellowship (FR). FR is an Inserm postdoctoral fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kelly@ibdml.univ-mrs.fr . These authors contributed equally to this work. Introduction Cardiac progenitor cells of the second heart field (SHF) contribute to the rapid growth of the embryonic heart by adding cells to the poles of the heart tube [1]. At the arterial pole, SHF cells give rise to right ventricular and outflow tract (OFT) myocardium in addition to smooth muscle at the base of the great arteries [2]. The SHF originates in splanchnic mesoderm medial to the cardiac crescent from which the linear heart tube is derived and is characterized by the expression of Fgf10, Isl1 and Tbx1 [3–5]. The contribution of SHF derived cells to the heart is coordinated with that of cardiac neural crest cells, which are essential for dividing the embryonic OFT into the ascending aorta and pulmonary trunk [6]. Interactions between these two cell types in the pharyngeal region prior to their addition to the heart are critical for normal OFT development; perturbations in SHF or neural crest development results in failure to correctly elongate and divide the OFT, resulting in congenital heart defects including overriding aorta, double outlet right ventricle and common arterial trunk [6–8]. Despite recent advances in our understanding of OFT development, the molecular mechanisms maintaining SHF cells in a progenitor state and regulating their contribution to the elongating OFT remain poorly defined. Notch intercellular signaling has been implicated in multiple aspects of heart morphogenesis including cushion development, trabecular growth, atrioventricular patterning, neural crest differentiation and arterial pole development [9–12] (reviewed in [13]). Mutations in the genes encoding the Notch ligand JAGGED1 or the NOTCH2 receptor have been identified in Alagille syndrome, associated with arterial pole defects including tetralogy of Fallot, and mutations in Notch1 are associated with aortic valve disease [14–16]. The Hey (Hairy and Enhancer of Split related) family of basic-helix-loop-helix transcription factors is thought to mediate Notch signaling in the developing cardiovascular system [13]. Hey2 mutant mice display arterial pole alignment and ventricular septal defects, associated with activation of the atrial program in ventricular myocardium [17– 20]. Members of the related Hes (Hairy and Enhancer of Split) family of transcription factors are also activated by Notch signaling and play critical roles in development including maintaining PLoS ONE | www.plosone.org 1 July 2009 | Volume 4 | Issue 7 | e6267