Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

RESEARCH ARTICLE Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo Ioannis Kokkinopoulos1, Hidekazu Ishida1, Rie Saba1, Prashant Ruchaya1,2, Claudia Cabrera3,4,5, Monika Struebig4, Michael Barnes4, Anna Terry4, Masahiro Kaneko1, Yasunori Shintani1, Steven Coppen1, Hidetaka Shiratori6, Torath Ameen1, Charles Mein4, Hiroshi Hamada6, Ken Suzuki1, Kenta Yashiro1* a11111 OPEN ACCESS Citation: Kokkinopoulos I, Ishida H, Saba R, Ruchaya P, Cabrera C, Struebig M, et al. (2015) Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo. PLoS ONE 10(10): e0140831. doi:10.1371/ journal.pone.0140831 Editor: Li Chen, University of Houston, UNITED STATES Received: June 1, 2015 Accepted: September 29, 2015 Published: October 15, 2015 Copyright: © 2015 Kokkinopoulos 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. Data Availability Statement: All relevant data except for the raw data of deep sequencing are within the paper and its Supporting Information files. The raw data of deep sequencing are available from NCBI Gene Expression Omnibus (GEO, http://www.ncbi. nlm.nih.gov/geo) under the accession number GSE63796. 1 Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 2 Physiology and Pathology, University of São Paulo State – UNESP, Araraquara School of Dentistry, Araraquara, São Paulo, Brazil, 3 Clinical Pharmacology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 4 Genome Centre, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 5 NIHR Barts Cardiovascular Biomedical Research Unit, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 6 Department of Developmental Genetics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan * Abstract In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study. Funding: This work was supported by the Medical Research Council (MRC) New Investigator Research Grant (G0900105) and the MRC Research Grant (MR/J007625/1 to KY) (http://www.mrc.ac.uk/). The funders had no role in study design, data collection PLOS ONE | DOI:10.1371/journal.pone.0140831 October 15, 2015 1 / 25 Single-Cell Expression Profiling of the Cardiac Crescent Cells and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction The heart is one of the first organs formed during vertebrate embryogenesis. Cardiac mesoderm cells emerge from the anterior portion of the primitive streak between the Early and Mid —Primitive Streak stages in the mouse embryo [1–4]. These cells migrate to the most anterior part of the lateral plate mesoderm (LPM), where cardiac progenitor/precursor cells (CPs) populate the heart field that will form the heart tube upon the Neural Plate stage [3, 5]. Subsequent morphogenetic events include the formation and looping of the heart tube, expansion of the ventricular and atrial chambers, and septation of the ventricles, atria, and outflow tract. Lineage tracing experiments have led to the identification of the first heart field (FHF) and second heart field (SHF), from which the SHF CPs have been well characterised to date [1, 2, 6–8]. The SHF derives from cells of the subpharyngeal mesoderm [6, 9]. This population is localized initially in the mediodorsal region neighboring the FHF at E7.5 in the mouse embryo. Continuous addition of cells from CPs of the SHF to the arterial and venous poles of the heart tube as well as to the atrial septum occur until the separated systemic and pulmonary circulation is completed, underling their contribution to the right ventricle, outflow tract, and parts of the atria. The multipotency of SHF CPs gives rise to cardiomyocytes, electric conduction system, smooth muscle and endocardial/endothelial cells [10]. In contrast, the FHF gives rise to the first differentiated cardiomyocytes in the anterior splanchnopleuric layer of the LPM and directly contributes to the linear primitive heart tube [3, 11–15]. Although the detailed mechanisms regulating the segregation of the two heart fields remain unknown, it has been indicated that the FHF’s specification precedes that of the SHF in the primitive streak at Primitive Streak stage [4, 13, 14, 16]. The expression of the transcription factor Tbx5 and potassium ion channel Hcn4 at E7.5 were shown to be specific to the FHF, although the expression pattern of both genes are dynamically shifted in later stages of embryo development [11, 12, 17, 18]. Tbx5 expression is also suggested to start at the Primitive Streak stage [14], whereas Hcn4 likely starts after the Late Headfold stage [4, 11, 12]. Recent lineage tracing experiments indicate that the FHF contributes mainly to the left ventricle and portions of the atria [12–14]. In addition, different from the SHF, the FHF CPs marked by Hcn4 and the FHF progenitor derived from the bHLH transcription factor Mesp1+ cardiac mesoderm cells were shown to b (...truncated)