This is a free sample of content from The Biology of Heart Disease. Click here for more information on how to buy the book. Cardiac Cell Lineages that Form the Heart Sigole` ne M. Meilhac1, Fabienne Lescroart2,Ce´ dric Blanpain2,3, and Margaret E. Buckingham1 1Institut Pasteur, Department of Developmental and Stem Cell Biology, CNRS URA2578, 75015 Paris, France 2Universite´ Libre de Bruxelles, IRIBHM, Brussels B-1070, Belgium 3WELBIO, Universite´ Libre de Bruxelles, Brussels B-1070, Belgium Correspondence: [email protected]; [email protected] Myocardial cells ensure the contractility of the heart, which also depends on other meso- dermal cell types for its function. Embryological experiments had identified the sources of cardiac precursorcells. With the advent of genetic engineering, novel tools have been used to reconstruct the lineage tree of cardiac cells that contribute to different parts of the heart, map the development of cardiac regions, and characterize their genetic signature. Such knowl- edge is of fundamental importance for our understanding of cardiogenesis and also for the diagnosis and treatment of heart malformations. he sources of cells that form the different tracing experiments based on the engineered Tparts of the heart and their relationships to temporal and spatial expression of a reporter each other are of major fundamental interest gene in different cardiac progenitors and their for understanding cardiogenesis. They are also descendants, with the mouse as the principal of biomedical significance in the context of model system. congenital heart malformations and for future therapeutic approaches to cardiac malfunction SOURCES OF CARDIAC CELLS IN THE based on stem cell therapies. In this review we EARLY EMBRYO mainly focus on myocardial cell lineages, with reference to the origin of the inner endocardial At the epiblast stage of embryonic development and outer epicardial cell layers of the heart. All (about E6.5 in the mouse), the cardiac fate of these are derived from mesoderm. Neural crest individual cells labeled with horseradish perox- cells, which play an important role in the mat- idase was determined and different cardiac pro- uration of the arterial pole of the heart are of genitor cells were shown to be clonally related to neuroectodermal origin and under different paraxial mesoderm and extraembryonic meso- genetic regulation, not treated here. Wewill dis- derm, as well as neurectoderm and endoderm cuss the current view emerging from a combi- (Lawson and Pedersen 1987; Buckingham et al. nation of approaches: cell lineage analyses that 1997). These challenging experiments depend- define the derivatives of a single mesodermal ed on embryo culture and did not permit anal- progenitor cell, cell labeling of groups of pro- ysis of cell contributions to the compartments genitors that reflects cell movement, and genetic of the maturing heart, mainly because of dilu- Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a013888 1 © 2015 by Cold Spring Harbor Laboratory Press. All rights reserved. This is a free sample of content from The Biology of Heart Disease. Click here for more information on how to buy the book. S.M. Meilhac et al. tion of the marker. More recent retrospective ley et al. 2008). Although Mesp1 loss of function clonal analysis also indicated these early lineage did not lead to an absence of cardiomyocyte relationships (Tzouanacou et al. 2009). Grafting differentiation during embryonic development, of regions of the epiblast showed that progeni- possibly owing to redundancy with Mesp2 (Ki- tors for the endocardium and the pericardium tajima et al. 2000), Mesp1 plays a key role as an are located in the same region as those for the upstream regulator of myocardial cell fate, as myocardium (Tam et al. 1997). These experi- indicated by the major increase in cardiomyo- ments also showed that cells are not committed cyte differentiation following Mesp1 overexpres- to a cardiac fate at this stage, but will adopt the sion in ES cells (Bondue et al. 2008, 2011; David fate dictated by their location. This continues et al. 2008; Lindsleyet al. 2008). In the absence of to be the case during gastrulation, when cells that Mesp1 and Mesp2, no mesodermal cells leave will form the mesoderm ingress through the the primitive streak, demonstrating the essential primitive streak. Fate mapping has shown that role of Mesp1/2 in the delamination of cardiac cardiac progenitors ingress early, at the mid- mesoderm (Kitajima et al. 2000). streak stage, to become located in the anterior region of the primitive streak, which comprises newly forming mesoderm, in close proximity to MYOCARDIAL CELL LINEAGES: progenitors of cranial (head) mesoderm (Kin- REGIONALIZATION OF THE MYOCARDIUM der et al. 1999). Distinct progenitors of the en- Two Myocardial Cell Lineages that docardium or myocardium have been identified Segregate Early in the primitive streak by retroviral labeling in the chick embryo (Weiand Mikawa 2000), how- Retrospective clonal analysis in the mouse em- ever, the timing of segregation of these cell types bryo (see Buckingham and Meilhac 2011) indi- in the mouse remains controversial, as reviewed cated that two major lineages contribute to the in Harris and Black 2010. Mesp1 (Saga et al. myocardium of the heart. The first lineage con- 1999) is expressed in the nascent mesoderm in tributes left ventricular myocardium, whereas the primitive streak, including cardiac progen- the second lineage is the source of outflow tract itors, as well as in cells that will contribute to and most right ventricular myocardium, with the anterior paraxial mesoderm. Genetic trac- both lineages contributing to the atria and other ing with a Mesp1-Cre and Rosa26 conditional parts of the heart (Meilhac et al. 2004). The size reporter shows that almost all cardiac cells in of first or second lineage clones gives an indica- the heart are labeled, so that Mesp1 marks all tion of when segregation of the two lineages cardiac progenitors (Fig. 1A,C) (Saga et al. took place, leading to the conclusion that pro- 1999, 2000; Y Saga, unpubl.). genitors with these distinct contributions to the Once cardiac progenitors have progressed heart segregate early during embryonic devel- through the streak they delaminate and move opment, probably at the onset of gastrulation. anteriorly to take up a position on either side This genetic approach, which depends on a ran- of the midline under the head folds, at about dom recombination event that results in a func- E7.5 in the mouse embryo. It is at this stage tional reporter gene in cardiac progenitors and that the first differentiated myocardial cells are their progeny, does not involve any precon- detected in an epithelial crescent-shaped struc- ceived idea about the progenitor cell. In combi- ture, which will subsequently fuse at the midline nation with DiI labeling to identify the left/ to form the early heart tube. Mesp1 may play a right derivatives of the posterior heart field, ret- role in the delamination process, because it ac- rospective clonal analysis showed that right and tivates epithelio-mesenchymal transition (EMT) left progenitors diverge before first and second in the embryonic stem (ES) cell system, with heart lineages (Dominguez et al. 2012). For pre- genes like Snail, Twist1, Zeb1, and Zeb2 as down- cise information on the timing of segregation stream targets, that mediate the repression of and also on the location of first and second E-cadherinexpression(Bondueetal.2008;Linds- lineage progenitors, it will be necessary to de- 2 Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a013888 © 2015 by Cold Spring Harbor Laboratory Press. All rights reserved. This is a free sample of content from The Biology of Heart Disease. Click here for more information on how to buy the book. Cardiac Cell Lineages that Form the Heart + A + LV B Nkx2.5 CMs Nkx2.5 + + RV Isl1 -low Isl1 -low + RA HCN4 Conductiveductive CMsC LA E6.5-E7.5 E7.75 + HCN4HCN4 LV + Brachyury + Nkx2.5Nkx2. CMsCMs + + Mesp1 Flk1+ ckitckit Mef2c-AHF-enhMef2c-AH + SMs OFT RV Tbx1Tbx1+ CMsCMs OFT CMs CMs Nkx2.5Nkx2. + + + Isl1Isl1 Nkx2.5Nkx2. SMs + SMsSMs SSMsMs Multipotentultipotent ? Isl1Isl1 Nkx2.55+ RA ECs MMesp1esp1+ ECsECs ECECss Isl1Isl1+ LA Tbx18Tbx18+ CMsCMs Wt1+ WWnt2nt2+ SMs Isl1+ PEO ECs In vivo PericytesPericyte Wnt2Wnt2 + PV Nkx2.5-Nkx2.5- Isl1-Isl1- Tbx18Tbx18 + LSCV RSCV C E6.5 E7.5 E14.5 AA PA SCV PV Myocardium HCN4 + Ao Nkx2.5 + RA LA Isl1– PS Nkx2.5 + + ICV Mesp1 Isl1 + Endocardium LV RV Nkx2.5– Isl1– Coronaries Valves Tbx18 + Epicardium Cardiac fibroblasts IVS Ventricular cardiac conduction system Figure 1. Genetic signature of cardiac precursor cells. (A) Genetic origin of cardiac components, summarizing results from genetic tracing at the population level. (B) In vitro differentiation potential of cardiac precursors, summarizing results from clonal differentiation assays of ES cells or precardiac mesoderm, after cell sorting. (C) Schematic representation of Mesp1-positive cells leaving the primitive streak (PS) (E6.5) contributing to the heart fields (E7.5) and to the tissues of the mature heart (E14.5), with color coding as in A and B. AA, aortic arch arteries; Ao, aorta; CMs, cardiomyocytes; ECs, endothelial or endocardial cells; ICV, inferior caval vein; IVS, interventricular septum; LA, left atrium; LSCV,left superior caval vein; LV,left ventricle; OFT, outflow tract; PA, pulmonary arteries; PEO, proepicardial organ; PV,pulmonary vein; RA; right atrium; RSCV,right superior caval vein; RV, right ventricle; SCV, superior caval vein; SMs, smooth muscle cells. velop inducible genetic tools for clonal analysis tracing with an HCN4-Cre shows labeling of the (see Buckingham and Meilhac 2011). embryonic left ventricle, mainly in the myocar- The cardiac crescent and early heart tube dial lineage (Fig. 1A) (Liang et al. 2013; Spater mainly contribute to the left ventricular myocar- et al.