A myocardial lineage derives from Tbx18 epicardial cells

Abstract

Understanding the origins and roles of cardiac progenitor cells is important for elucidating the pathogenesis of congenital and acquired heart diseases. Moreover, manipulation of cardiac myocyte progenitors has potential for cell-based repair strategies for various myocardial disorders. Here we report the identification in mouse of a previously unknown cardiac myocyte lineage that derives from the proepicardial organ. These progenitor cells, which express the T-box transcription factor Tbx18, migrate onto the outer cardiac surface to form the epicardium, and then make a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Tbx18-expressing cardiac progenitors also give rise to cardiac fibroblasts and coronary smooth muscle cells. The pluripotency of Tbx18 proepicardial cells provides a theoretical framework for applying these progenitors to effect cardiac repair and regeneration.

Figure 1. LacZ expression in Tbx18:nlacZ knock-in mice recapitulates endogenousTbx18 expression

a–d, Section RNA in situ hybridization (ISH) of Tbx18 in mouse embryos (E9.0–E11.5). Tbx18 is expressed very early in the proepicardium (a) and epicardium (b–d). For parallel whole-mount RNA ISH, see Supplementary Fig. 2. e–g, X-gal staining on cardiac sections from Tbx18:nlacZ embryos at E9.5–E11.5. Tbx18:nlacZ cells are detected in the early epicardium at E9.5 (e) and in all epicardial cells covering the heart after E10.5 (f, g, data not shown for E12.5–E13.5). X-gal staining on Tbx18:nlacZ mouse tissues is consistent with Tbx18 mRNA ISH. For the Tbx18:nlacZ targeting strategy, see Supplementary Fig. 3. Black arrows indicate Tbx18 mRNA expression (a–d) and Tbx18:nlacZ cells (e–g); red arrows in c and d indicate that Tbx18 is not expressed in heart between E9.5 and E11.5. For e–g, lower panels are high-magnification views of the upper panels in the heart (black arrow regions). LA/RA, left/right atrium; LV/RV, left/right ventricle; OT, outflow tract; and SV, sinus venosus.

Figure 2. Cells derived fromTbx18-expressing cells are observed within the heart by E10.5, and exhibit a cardiomyocyte identity

Tbx18:Cre mice were crossed to R26R^lacZ indicator mice (Tbx18:Cre/R26R^lacZ). a–c, X-gal staining on whole-mount embryos (E9.5–E11.5) shows Tbx18-lineage-traced cells in the proepicardium and epicardium (arrows in a–c). d–f, X-gal staining on cryosections from E10.5 to E12.5. Tbx18-lineage-traced cells are observed in the epicardium (arrows in the upper panels of d–f) and within the heart, particularly within the apical region in the developing ventricular septum (arrows in the lower panels of d–f). The lower panels of d–f show high-magnification views of the ventricles in the upper panel micrographs. For Tbx18-lineage-traced cells at E13.5, see Supplementary Fig. 7. g, β-galactosidase antibody staining on Tbx18-lineage-traced tissue at E11.5. h, Cardiac troponin T antibody staining. i, Overlay of g and h revealed that Tbx18-lineage-traced cells in heart were cardiac-troponin-T-positive cells. In g–i, lower panels are high-magnification views of the upper panels in the ventricular septum region. Arrows in d–i indicate Tbx18-lineage-traced cells in the ventricular septum region. LA/RA, left/right atrium; LV/ RV, left/right ventricle; and VS, ventricular septum.

Figure 3. Tbx18 lineage tracing in the adult heart

a, b, Whole-mount X-gal staining of Tbx18:Cre/R26R^lacZ adult mouse heart (6 weeks). Coronary vasculature (green arrows) is derived from Tbx18 lineages. Dense staining in the septum is visible (black arrow) c, X-gal staining of tissues from Tbx18:Cre/R26R^lacZ adult mouse heart (6 weeks). Tbx18 can give rise to: cardiomyocytes within atria (arrows in c, inset 1 and Supplementary Fig. 8), the ventricular septum (black arrow in c, inset 2) and the ventricular wall (black arrow in c, inset 3); coronary vascular support cells (green arrows in c, insets 2 and 3); and atrioventricular valves (c, inset 4, high magnification for bicuspid valve). Most Tbx18-lineage-traced cells within the ventricular septum co-stained with cardiac troponin T (arrows in d–f; f is a high magnification of the lower panel of e), demonstrating they are cardiomyocytes. g, A subset of cardiomyocytes isolated from adult Tbx18-lineage-traced hearts exhibited X-gal-positive staining. h, Tbx18 lineages give rise to coronary vascular smooth muscle cells (co-localized with smooth muscle myosin heavy chain). i, Tbx18 lineages give rise to coronary artery smooth muscle cells (co-localized with NP-1, an artery marker). j, k, X-gal staining on Tbx18:nLacZ knock-in mice shows that Tbx18 expression is maintained in some coronary vascular smooth muscle cells from embryonic stages to adulthood. l–o, Tbx18 lineages do not give rise to coronary vascular endothelial cells (not co-localized with Pecam1, l). m, Coronary vascular endothelial cells were isolated from the hearts (green fraction) and react with Pecam1 antibody (n, right panel), whereas Pecam1⁻ fraction (purple) cells do not (n, left panel). o, Purified Pecam1⁺ cells are Xgal⁻ (o, right panel), confirming that Tbx18-expressing lineages do not give rise to coronary vascular endothelial cells. A portion of cells in Pecam1⁻ fraction are Xgal⁺ (o, left panel). p, Cardiac fibroblasts isolated from Tbx18-lineage-traced hearts demonstrated that approximately 30% of these cells derive from Tbx18 lineages. Abbreviations are as in Fig. 2.

Figure 4. Tbx18-expressing progenitors within proepicardium are distinct, and retain the capacity to differentiate into cardiomyocytes and smooth muscle cells in vitro

a, b, Tbx18-expressing progenitors within the proepicardium are not co-stained with antibodies to Flk-1 (a) or Nkx2.5 (b) (E9.0, data not shown for E9.5), suggesting that Flk-1 endothelial progenitors within the proepicardium are a distinct population from Tbx18-expressing cells. Tbx18 progenitors with myocyte potential do not yet express Nkx2.5 within the proepicardium, although Tbx18-derived myocytes do express Nkx2.5 within the heart. The second panels in a and b are high-magnification images of the top panels of a and b, respectively. c, Tbx18-lineage-traced proepicardial cells (Tbx18:Cre/R26R^EYFP) were disassociated and single cells were expanded in proepicardial culture medium. A significant percentage of clones (34%) derived from single proepicardial cells adopt cardiomyocyte fate after incubating in the differentiation medium (co-stained with cardiac troponin T, bottom panel of c). d, Every clone (40 out of 40) derived from single Tbx18 proepicardial cells displayed staining for smooth muscle myosin heavy chain (SM-MHC) antibody after incubating in smooth muscle culture medium. e–g, Real-time Ca²⁺ transients of cells derived from single Tbx18-lineage-traced proepicardial cells after myocytic differentiation. e, Left panel: monitored cell is YFP⁺, suggesting it derived from the Tbx18:Cre/R26R^EYFP proepicardium. e, Middle panel: differentiated cells were monitored under an inverted confocal microscope after they were preloaded with Ca²⁺ indicator Rhod-2-AM. e, Right panel, transmitted light image of the monitored cell. f, Line-scanning images and time course (g) of spontaneous Ca²⁺ transients obtained from differentiated YFP⁺ cells. PE, proepicardium; SV, sinus venosus; and V, ventricle.