Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

Abstract

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

Fig 1. Genetic tracing of the T+ primitive steak cells.

(A) Schematics of the T^{nGPF-CreERT2/+}; R26R^tdTomato/+ allele [16]. Cre-ERT2 is expressed in cells expressing T. In the presence of tamoxifen, Cre protein is translocated to the nucleus where it recombines the R26R^tdTomato/+ reporter. As a result, the cell and its descendants are permanently labelled. (B) Diagram of the experimental approach. T-expressing cells and their descendants are labelled, from E6+21h, E7, E7+7h and E7+11h by administrating a dose of tamoxifen (Tam) to T^{nGPF-CreERT2/+}; R26R^tdTomato/+ mice (0.08 mg/body weight via oral gavage). Cell descendants in the myocardium are analysed at E12.5. (C) Representative hearts resulting from the administration of tamoxifen at different time points in T^{nGPF-CreERT2/+}; R26R^tdTomato/+ immunostained with cTnnT to reveal the cardiomyocytes (blue). Yellow arrows identify small patches of tdTomato positive cardiomyocytes in the LV (iii and v), in the RV (v), and in the outflow tract and atria (vii and viii). Views are ventral. Single epicardial cells are labelled in each condition. (D–E) Summary of all T^{nGPF-CreERT2/+}; R26R^tdTomato/+ hearts examined. The contribution of the T-expressing cells to the different compartments of the heart is quantified by measuring the proportion of tdTomato-positive myocardium. Numbers in brackets in (E) represent the number of litters assessed. Error bars are SD. The data underlying (D–E) can be found in S1 Source Data. (F) Stage variation quantified according to Downs and Davies criteria [20]. Number in brackets represents the number of litters assessed. All the timed matings were for 2-hour periods. (G) Embryos collected at E6+21h or E7+7h showing variation in stage. ((H) Representative T^{nGPF-CreERT2/+}embryos untreated or tamoxifen treated for 2 hours mice (0.08 mg/body weight via oral gavage) and immunostained with oestrogen receptor. Insets (iii) and (iv) are magnified view from (i) and (ii), respectively. (I) PCR amplicons generated from the genomic region in which Cre-mediated recombination occurs, resolved on an agarose gel. Before recombination, the PCR product is 1,145 bp (white rectangle); after recombination, it is 274 bp (black rectangle). Template gDNA was extracted from either an ear clip of an adult T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato} mouse (untreated) or T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato} embryos dissected at 2, 4, and 12 hours following oral gavage with Tamoxifen, as labelled. An increase in the proportion of the recombined band can be seen over time following Tamoxifen administration. The data underlying (I) can be found in S1 Raw image. (J) Representative T^{nGPF-CreERT2/+}embryos at the MS-LS and OB-EB stages, immunostained with. (iii) and (iv) are magnified views in (i) and (ii), respectively. (K) Quantification of T intensity in single segmented nuclei from embryo shown in (J). The data underlying (K) can be found in S2 Source Data. (L) Representative T^{nGPF-CreERT2/+}embryos at the MS-LS and OB-EB stages treated with tamoxifen for 2 hours (0.08 mg/body weight via oral gavage) and immunostained for oestrogen receptor. (iii) and (iv) are magnified views in (i) and (ii), respectively. (M) Quantification of Oestrogen receptor intensity in single segmented nuclei from the embryos shown in (L). The data underlying (M) can be found in S2 Source Data. Embryos in J (i) and (ii) and in L (i) and (ii) were immunostained together and imaged under similar conditions. ant., anterior; cTnnT, cardiac troponinin T; EB, “early bud” stage; EHF, early head fold; LA, left atria; LB, “late bud stage”; LS, late-streak; LV, left ventricle; MS, mid-streak; OB, “no bud” stage; OFT, outflow tract; post., posterior; PS, primitive streak; RA, right atria; RV, right ventricle. Scale bar: 200 μm.

Fig 2. Descendants from late E7+7h T-positive primitive streak cells contribute to posterior regions of the FHF.

(A, B) Representative embryos resulting from the administration of tamoxifen at E7+7h in T^{nGPF-CreERT2/+}; R26R^tdTomato/+ immunostained with cTnnT to reveal the cardiomyocytes of the first heart field (green) (A) and heart tube (B). Yellow arrows identify tdTomato-positive cardiomyocytes in the posterior first heart field (Aii–Aiv) and inflow tract (Bii). Blue arrows identify tdTomato-positive endodermal cells (Aii) and endocardial cells (Biii). (Aii) shows magnified view of inset in (Ai). (Ai–Aii and Bi–Bii) are z-maximum projection, (Aiii–Aiv and Biii) are single optical sections. Views are ventral. Single epicardial cells are labelled in each condition. Scale bar: 200 μm.

Fig 3. Independent Foxa2+ progenitors contribute to the ventricles and outflow tract.

(A) Schematics of the Foxa2^{nGPF-CreERT2/+}; R26R^tdTomato/+ allele [16]. (B) Pulse-labelling of the Foxa2-expressing cells and their descendants, from stage (i) E6+21h, (ii) E7+7h by administration of tamoxifen to Foxa2^{nGPF-CreERT2/+}; R26R^tdTomato/+ mice (0.08 mg/body weight via oral gavage). Cell descendants in the heart analysed at E12.5. (C) Representative hearts resulting from the administration of tamoxifen at different time points in Foxa2^{nGPF-CreERT2/+}; R26R^tdTomato/+ immunostained for cTnnT to reveal the cardiomyocytes (blue). Views are ventral. Single epicardial cells are also labelled in (iii). (D, E) Summary of all hearts analysed. The contribution of the Foxa2-expressing cells to the different compartments of the heart is quantified by measuring the proportion of tdTomato-positive myocardium. Numbers in brackets in (E) represent the number of litters assessed. Error bars are SD. Scale bar: 200 μm. The data underlying (D, E) can be found in S3 Source Data. cTnnT, cardiac troponinin T; LA, left atria; LV, left ventricle; OFT, outflow tract; RA, right atria; RV, right ventricle.

Fig 4. T and Foxa2 colocalise in primitive streak cells.

Representative embryos of E6+21h MS (A, B) and LS (C, D) and E7+7h EB (E, F). Embryos are immunostained for T (red) and Foxa2 (green). Images are z-maximum projections. Views are lateral/slightly posterior to show the whole width of the primitive streak. Insets in Bi–Biii, Di–Diii, and Fi–Fiii show magnified views and white arrows point to T+/Foxa2+ double positive cells. Scale bar: 100 μm. EB, “early bud” stage; LS, late-streak; MS, mid-streak; PS, primitive streak.

Fig 5. Foxa2 lineage-positive cells maintain Foxa2 expression at the mid-late steak stage.

(A, B) Representative embryos at the early (A) and mid-late (B) streak stages resulting from the administration of tamoxifen at E6+8h in Foxa2^{nGPF-CreERT2/+}; R26R^tdTomato/+ immunostained for Foxa2 (green) and T (blue). View is lateral in (A) and posterior in (B). (B”) Schematics of the embryo shown in (B). (C) Same embryo as in (B). Single optic sections are shown. (i–iii) are magnified views of the insets. Yellow arrows point to double Foxa2/T positive, blue arrows to Foxa2 positive T negative, and red arrows to Foxa2 negative T positive tdTomato-expressing cells. (D) Representative example of a segmented image based on Foxa2 and T signal. (E) Quantification of T and Foxa2 signal intensities along the proximal-distal axis of the embryo in single cell nuclei. All the tdTomato-positive cells are all located in the distal (anterior) portion of the primitive streak. The data underlying (E) can be found in S4 Source Data. (F) Quantification of Foxa2 and T intensities in single cell in distal (green), proximal (blue) primitive streak cells and tdTomato-positive cells. The data underlying (F) can be found in S4 Source Data. (G, H) Model. As the primitive streak extend along the proximal-distal axis, from the early to mid-late streak stages, a subset of the Foxa2-positive cells switch contribution from endoderm to cardiac (ventricles) progenitors and express T. ES, early streak; LV, left ventricle; MS, mid-streak; PS, primitive streak.

Fig 6. Ventricular precursors originate from distal regions of the primitive streak.

(A) Localisation of the tdTomato+ cells from the Foxa2 lineage are assessed (i, ii) at E7+7h (OB-EB stage). nGFP signal from the transgene is shown in (i). The embryos are immunostained for T (blue) and Foxa2 (green) (ii). Image is a z-maximum projection. (iii) The same embryo (i, ii), but with Foxa2+ cells masked to reveal the tdTomato+/Foxa2− cells. Insets (iv) and (v) show magnified views in single optical sections of (ii). Red arrows point to tdTomato+/Foxa2−/T− mesodermal cells. Yellow arrows point to tdFtomato+/Foxa2−/T+ primitive streak cells. Green arrows point to tdTomato+/Foxa2+/T− endodermal cells. No tdTomato+/Foxa2+/T+ cells are identifiable. (B) Localisation of the tdTomato+ cells from the Foxa2 lineage are assessed in Bre-cerulean (BMP reporter) embryos at (i, ii) E7+12h (EHF stage). nGFP signal from the transgene is shown in (i). The embryos are immunostained for Foxa2 (blue) (ii). Image is a z-maximum projection. (iii) The same embryo (i, ii), but with Foxa2+ cells masked to reveal the tdTomato+/Foxa2− cells. Insets (iv) and (v) show magnified views in single optical sections of (ii). Red arrows point to tdTomato+/Foxa2−/Bre-cerulean+ mesodermal cells. Blue arrow points to tdTomato+/Foxa2−/Bre-cerulean− cell. (C) (i) A common progenitor between the RV and the outflow tract in the distal primitive streak. Foxa2 is down-regulated as it switches its contribution towards outflow tract myocardium. (ii) Proximal PS contribute to the atria and are descended from cells that had not expressed Foxa2 in their past. Ant, anterior; EB, early bud; LS, late-streak; nGFP, nuclear localised GFP; OB, no bud; Post, posterior; OFT, outflow tract; PS, primitive streak; RV, right ventricle. Scale bar: 100 μm.

Fig 7. Ventricular, outflow tract, and atrial progenitors are located in distinct regions of the mesoderm.

(A, B) tdTomato localisation in T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato}, Bre-Cerulean (BMP reporter) embryos at the EHF stage and following tamoxifen administration at E6+21h (A) and at E7+7h (B, C). Insets Aii, Bii–Biv, and Ci show magnified views of Ai, Bi, and Ci, respectively. Yellow arrows in Ai, Bii–Biv, and Cii point to double tdTomato/Bre-cerulean-positive cells and red arrows in Bii–Biv and Cii to tdTomato+ bre-cerulean− cells. (D) tdTomato localisation in T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato} embryos at the EHF stage, following tamoxifen administration at E7+7h and immunostained for Raldh2. Insets in ii–iv show magnified view of i. Images are z-maximum projection of 37 sections acquired every 5 μm and covering 185 μm. Interval between frames: 6 mn and 30 s. cc, cardiac crescent; EHF, early head fold; ml, midline; n, node; PS, primitive streak. Scale bar: 100 μm.

Fig 8. Subclusters corresponding to the left and right ventricle, outflow tract, and atrial progenitors can be defined within the FHF, AHF, and pSHF.

(A) The 4 T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato} embryos (dissected at E7+14 h EHF stage) analysed in the single-cell transcriptomic assay. (B) UMAP plot showing the cell populations coloured by cluster identity (i). (ii, iii) Magnified view of (i) showing the cardiomyocytes, pharyngeal mesoderm, anterior paraxial and paraxial mesoderm clusters (ii) and subclusters (iii). The cardiomyocytes, pharyngeal mesoderm, and paraxial mesoderm clusters are renamed FHF, AHF, and pSHF based on the marker genes shown in (C). (C) UMAP showing the log normalised counts of the selected genes. (D) UMAP showing the log normalised counts of the tdTomato gene (i). (ii) Magnified view of (i) showing the log normalised counts of the tdTomato gene in the FHF, AHF, and pSHF clusters. (iii) Table showing the percentage of tdTomato+ cells in each mesodermal cluster. The data underlying (iii) can be found in S6 Source Data. (E) Diagram showing the repartition of the tdTomato-positive cells from the FHF, AHF, and pSHF clusters into 6 subclusters, each with different proportion of tdTomato-positive cells (see also S13A Fig). We hypothesis that cells in clusters enriched in tdTomato reads contribute to the OFT and Atria. (F) Expression heat map of marker genes and tdTomato. Scale indicates z-scored expression values. The full heat map with all the genes display is shown S13B Fig. The data underlying (F) can be found in S5 Source Data. (G) UMAP showing the log normalised counts of selected genes. AHF, anterior second heart field; aParaxial meso, anterior paraxial mesoderm; AVC, atrioventricular canal; Caudal meso, caudal mesoderm; EHF, early head fold; Exe endoderm, extra-embryonic endoderm; FHF, first heart field; GFP, green fluorescence protein; Intermediate meso, intermediate mesoderm; misc, miscellaneous; n., node; OFT, outflow tract; Pharyngeal mes, Pharyngeal mesoderm; Presomitic meso, presomitic mesoderm; PS, Primitive streak; pSHF, posterior second heart field; Tam, tamoxifen; UMAP, Uniform Manifold Approximation Projection.

Fig 9. The molecular signature of the primitive streak cells contributing to the ventricles or outflow/atria are distinct.

(A) Mid-late streak and OB/EB T^{nGPF-CreERT2/+}; R26R^{tdTomato/tdTomato} embryos analysed in the scRNA-seq. Embryos were selected at the mid-late streak stages (dissected at E6+21h and resulting from tamoxifen administration at E6+5h) and at the OB-EB stage (dissected at E7+3h and resulting from tamoxifen administration at E6+21h). (B, C) Expression heat map of marker genes. Scale indicates z-scored expression values. The data can be found in S7 Source Data. (D) UMAP plot showing the integrated data from the 2 scRNA-seq mid-late streak and OB/EB datasets (see also S11 Fig). Colour codes correspond to the embryonic stage of collection or population identity. The data underlying (D) can be found in S7 Source Data. (E) Expression heat map of marker genes comparing the aPS and primitive streak cells at the mid-late streak stages and primitive streak cells at the OB-EB stages (S7 Source Data). Scale indicates z-scored expression values. (F) UMAP plot of the aPS, MS-LS primitive streak, and OB-EB primitive streak cells colour coded. (G, H) Violin plots showing the normalised log2 expression value of Eomes and T (G) and Foxa2, Lhx1, Sp8 Hes1, Ccnd2, and Notch2 (H) in the aPS and primitive streak at MS-LS and OB-EB stages. The data underlying (G, H) can be found in S7 Source Data. aPS, anterior primitive streak; DE, definitive endoderm; EB, early bud; LPM/Ex-meso, lateral plate mesoderm and extraembryonic mesoderm, mesenchyme; LS, late streak; MS, mid-streak; Nascent meso, nascent mesoderm; OB, no bud; PGC, primordial germs cells; PS, primitive streak; scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation Projection.

Fig 10. Live imaging of the mesodermal cells reveal their trajectories during gastrulation.

(A) Image sequence from time-lapse video (S1 Movie) of an T^{nGPF-CreERT2/+}; R26^mgpf/+ embryo resulting from the administration of tamoxifen at around E6.5 (overnight timed matings). Yellow arrows point to progenitor initially located in proximity to the node/distal regions and migrating along the midline in medial regions of the prospective cardiac crescent. Red arrows point to progenitors initially located proximally and migrated towards posterior regions of the prospective cardiac crescent and posterior second heart field. Blue arrow points to a progenitor located at the embryonic-extraembryonic border. Images are z-maximum projection of 37 sections acquired every 5 μm and covering 185 μm. Interval between frames: 6 mn and 30 s. cc, cardiac crescent; EHF, early head fold; ml, midline; n, node; OB, no bud; pCC, posterior cardiac crescent; pSHF, posterior second heart field. The movie can be found in S1 Movie.

Fig 11. Model of early cardiac development.

(A) Cells located in distal regions of the primitive streak contribute first to the left ventricle (mid-streak stage), then to the right ventricle (late-streak stage), and finally to the outflow tract (OB-EB stages). Although the outflow and atria leave the primitive streak at similar stages, they arise from different regions. The outflow tract originates from distal locations in the primitive streak while atrial progenitors are positioned more proximally. The distal primitive streak cells express Foxa2 when they contribute to the ventricles. They stop expressing Foxa2 when they contribute to the outflow tract. (B) Proposed location of the FHF, AHF, and pSHF in comparison to the hypothetical fate of these different cardiac regions at the pre-headfold stage in (A). (C) Lineage tree of the primitive streak. Within the distal end of the primitive streak, independent primitive streak cells contribute to the left ventricle, the right ventricle, the (at the mid-late streak stage) and to the outflow tract (OB-EB stages). A common progenitor between the right ventricle and the outflow tract exists. By contrast, atrial progenitors are located in the proximal primitive streak and a common progenitor between the left ventricle and atrium may exist in the primitive streak. AHF, anterior heart field; aPS, anterior primitive streak; AVC, atrioventricular canal; cranial pxm, cranial paraxial mesoderm; EB, early bud; epi, epicardium; FHF, first heart field; LV, left ventricle; OB, no bud; OFT, outflow tract; Pha, Pharyngeal arches; PS, primitive streak; pSHF, posterior second heart field; RV, right ventricle.