Unveiling Complexity and Multipotentiality of Early Heart Fields

Abstract

[Figure: see text].

Keywords: congenital heart defects; embryonic development; genomics; myocardium; stem cells.

Figure 1.. Mesp1-Cre single-cell maps reveal diverse cell types participating in early mouse mesoderm development.

(A), Mesp1-Cre scRNA-seq experimental design. Mesp1-Cre; Rosa26-tdT embryos were harvested for scRNA-seq at E7.25 (no bud stage); E7.5 (early bud stage); E7.75 (early head fold stage); and E8.25 (somite stage) as shown in representative bright-field and Mesp1-Cre; tdT+ (Mesp1 lineage) micrographs. Illustration below these micrographs shows tissues genetically labeled by Mesp1-Cre in embryos, and workflow for capturing these labeled single cells for RNA sequencing. Scale bars, 150 μm. (B), scRNA-seq data is displayed by tSNE plots at each developmental stage. Cells are colored according to their cell identities in D, E, F. (C, D), tSNE plot of scRNA-seq data across all examined stages displays individual cells (single dots) by (C) developmental stages or (D) cell types. (E), Dot plot shows distribution of each cell type across different embryonic stages. (F), Dot plot of key marker genes identifies each cell cluster. Al, Allantois; Bl, Blood; MM, Mixed Mesoderm; CrPh, Cranial-pharyngeal mesoderm; CM, Cardiomyocytes; DC, Developing Cardiomyocytes; En, Endothelium; Ep, Epithelium; EEM, Early Extraembryonic Mesoderm; Hem, Hemangiogenic mesoderm; HT, Heart tube; LEM, Late Extraembryonic Mesoderm; LPM, Lateral plate mesoderm; NM, Nascent Mesoderm; PSM, Pre-somitic mesoderm; PS, Primitive streak, SM, Somite mesoderm.

Figure 2.. Mesp1-Cre scRNA-seq trajectory analysis reconstructs developmental cell lineage trees during mesoderm/heart organogenesis.

(A, B), URD inferred lineage tree, as displayed by (A) dendogram or (B) force-directed layout, reveals the developmental history of Mesp1 mesoderm-derived organs. Red dashed box in A, B outlines cardiomyocyte branch, which is further magnified in B. The magnified cardiomyocyte branch shows that cardiomyocytes may derive from both late extraembryonic mesoderm (LEM) and lateral plate mesoderm (LPM) progenitor cells. (C), tSNE layout of cells from only the cardiomyocyte branch (boxed area in A, B) reveals seven cardiac subclusters composing the cardiomyocyte branch including three distinct cardiomyocyte populations (CM1–3) and four specific cardiac progenitor cell-types (CP4–7). (D), Heatmap of differentially expressed marker genes identifies each cardiac subcluster.

Figure 3.. Distinct cardiomyocyte lineages derive from intra- and extra-embryonic related developmental origins.

(A, B), Reconstructed URD developmental cell lineage trees using the three distinct subclustered cardiomyocyte populations predict that CM1/CM2 and CM3 cardiomyocytes derive respectively from intra- and extra-embryonic related progenitor sources, as displayed by (A) cell type and (B) developmental stages. The cardiomyocyte-related branches of the URD developmental tree are outlined with box. (C), Marker genes differentially expressed among the lineages for each cardiomyocyte subcluster are plotted on the URD cardiomyocyte-related branches. Hand1 and Mab21l2 mark early and late regions of the CM3 lineage, respectively. Mesp1, Tbx5, Isl1, Irx4 and Tdgf1 label different regions of the CM1 and CM2 lineage branches. (D, E), RNAscope in situ hybridization (ISH) of Mesp1 and Hand1 was performed in (D) E7.25 and (E) E7.5 Mesp1-Cre; Rosa26-tdT embryos. The diagram illustrates both the gene expression pattern of Hand1 and Mesp1 and Mesp1-Cre lineage-traced cells in these embryos. (F, G), RNAscope ISH of Hand1, Tbx5, and Isl1 was performed in E7.75 embryos. The diagram illustrates the expression pattern of Hand1, Tbx5, and Isl1 in these embryos. n = 3 per panel. Scale bars, 100 μm. EXE, Extraembryonic Ectoderm; YS, Yolk Sac.

Figure 4.. Lineage tracing studies reveal that early gastrulating Hand1+ cells contribute to not only a distinct subpopulation of first heart lineage cardiomyocytes but also serosal mesothelial lineages (pericardial, epicardial cells) in the heart.

(A) RNAscope ISH in Hand1-CreERT2 embryos shows that expression of CreERT2 precisely recapitulates the expression of Hand1. (B), Lineage tracing studies using Hand1-CreERT2 and Rosa26-tdT mice (shown in left) map the fate of early gastrulating Hand1+ cells. Schematic outlines the experimental strategy for Hand1-CreERT2 genetic fate mapping studies shown in right. Tamoxifen was given at E5.75, and embryos were examined for Hand1-CreERT2 genetically-labeled tdT+ cells at E7.75, E8.25, E9.5 and E12.5. (C-F), RNAscope ISH and immunohistochemistry of whole mount and cross sections of these embryos reveal the contribution of Hand1-CreERT2 genetically-labeled tdT+ cells at (C) E7.75, (D) E8.25, (E) E9.5 and (F) E12.5. (C’, D’, E’, E’’, E’’’), Insets show transverse sections of C, D, E at indicated dashed lines, respectively. (F’), Inset shows coronal section of F. (C’’, F’’), Insets are magnification of C’, F’ boxed area. Arrowheads point to tdT+ cells expressing (C, C’’) Hcn4, (D, D’) Myl7, (E’’) Erg1, (E’’’) Wt1 and (F’’) α-Actinin. (C’’’, D’’’, E’’’’, F’’’), Diagrams summarize the anatomical location of Hand1-CreERT2 genetically-labeled tdT+ cells at the embryonic stages analyzed. n = 3 embryos for each stage. Scale bars, 100 μm. AM, Amnion; AVC, Atrioventricular Canal; BA, Base of the Atrium; CC, Cardiac Crescent; Epi, Epicardium; HT, Heart tube; LA, Left Atrium; LV, Left Ventricle; OFT, Outflow Tract; Peri, Pericardium; Pro, Proepicardium; RA, Right Atrium; RV, Right Ventricle; SV, Sinus Venosus; ST, Septum transversum; YS, Yolk Sac.

Figure 5.. Clonal analysis reveals multipotentiality in early Hand1+ progenitors.

(A), Schematic outlines experimental strategy for Hand1-CreERT2; Rosa26-Confetti clonal analyses. (B), Bar graph reveals the percentage of E9.5 embryos that displayed fluorescence at titrated doses of tamoxifen. The numerator is the number of fluorescence-positive embryos, and the denominator is the number of total embryos examined. (C), Clonal analyses of uni-color E9.5 embryos reveal that individual Hand1-CreERT2; Rosa26-Confetti clones labeled at E6.75 (0.0025 mg/g Tamoxifen) have the capacity to generate multiple cell types that can contribute to the yolk sac and/or heart. (D), Diagram summarizes the contribution of Hand1-CreERT2; Rosa26-Confetti genetically-labeled clones in the heart and yolk sac at E9.5. (E), Clonal analyses of uni-color E12.5 hearts reveal that individual Hand1-CreERT2; Rosa26-Confetti clones labeled at E6.75 or E7.25 (0.005 mg/g Tamoxifen) have the capacity to generate multiple cell types within the heart. (F), Diagram summarizes the contribution of Hand1-CreERT2; Rosa26-Confetti genetically-labeled clones in the heart at E12.5. (G), Representative E12.5 uni-color hearts show individual Hand1-CreERT2; Rosa26-Confetti genetically-labeled clones contributing to different combinations of tissues and cell types: AVC/SV and RA (clone # 237-RFP); LV and Epi (clone # 329-RFP); the AVC/SV and Epi (clone # 319-YFP). Scale bars, 200 μm. (H), Bar graph displays the number of uni-color E12.5 hearts with clones contributing to cardiomyocytes only, epicardial cells and cardiomyocytes, or only epicardial cells. (I), Model summarizes the multipotentiality of Hand1+ cardiac progenitor cells (CPC) between E6.75 - E7.25 in relation to the contribution of reported FHF/SHF progenitors. AVC, Atrioventricular Canal; BA, Base of the Atrium; CM, cardiomyocytes; Epi, Epicardium; LA, Left Atrium; LV, Left Ventricle; OFT, Outflow Tract; Peri, Pericardium; Pro, Proepicardium; RA, Right Atrium; RV, Right Ventricle; SV, Sinus Venosus; ST, Septum Transversum; YS, Yolk Sac.