Gastruloids are competent to specify both cardiac and skeletal muscle lineages

Abstract

Cardiopharyngeal mesoderm contributes to the formation of the heart and head muscles. However, the mechanisms governing cardiopharyngeal mesoderm specification remain unclear. Here, we reproduce cardiopharyngeal mesoderm specification towards cardiac and skeletal muscle lineages with gastruloids from mouse embryonic stem cells. By conducting a comprehensive temporal analysis of cardiopharyngeal mesoderm development and differentiation in gastruloids compared to mouse embryos, we present the evidence for skeletal myogenesis in gastruloids. We identify different subpopulations of cardiomyocytes and skeletal muscles, the latter of which most likely correspond to different states of myogenesis with "head-like" and "trunk-like" skeletal myoblasts. In this work, we unveil the potential of gastruloids to undergo specification into both cardiac and skeletal muscle lineages, allowing the investigation of the mechanisms of cardiopharyngeal mesoderm differentiation in development and how this could be affected in congenital diseases.

Fig. 1. The culture of gastruloids for 11 days allows the transcription of markers of the CPM and their derivatives.

a Experimental scheme of the generation of gastruloids from mouse embryonic stem cells (mESCs). Gastruloids were cultured in N2B27 up to 11 days, with the addition of small molecules from day 4 to day 7 as indicated below the time line. b Representative images of gastruloids at day 3, day 4, day 5, day 6, day 7 and day 11. Arrowheads indicate beating areas. c Percentage of beating gastruloids per experiments (n = 8 independent experiments). Data are presented as mean percentage ± SEM. d Scheme of CPM specification in mouse toward the myogenic fates. Created in BioRender. Lescroart, F. (2024) https://BioRender.com/m68f097. Markers of each state are indicated in italics. e–g Expression profiles of Mesp1 (n = 9), Isl1 (n = 3) and Tbx1 (n = 6) (e), Tnnt2 (n = 9), Myh7 (n = 3) and Myl7 (n = 6) (f) and Tcf21 (n = 8), Myod (n = 7) and Myf5 (n = 5 independent experiments) (g) throughout the culture of gastruloids as measured by quantitative RT-PCR. Results are normalized to the expression of Tbp. Fold changes are represented relative to expression on day 0. Data are presented as mean values ± SEM.

Fig. 2. Gastruloids faithfully recapitulate CPM specification and differentiation toward its myogenic fates.

a–d Representative maximum intensity projection (MIP) of mouse embryos at E7.5 (a), E7.75 (b), gastruloids on day 4 (c) and day 5 (d) after RNAscope with Mesp1 (cyan), Isl1 (yellow) and Tbx1 (purple) probes. a′, b′, c′, and d′: Optical sections of the area indicated with dotted lines. Arrowheads indicate overlapping expression of Mesp1 and Isl1 (white) or of Tbx1 and Isl1 with no expression of Mesp1 (empty). n = 4 embryos and n > 5 gastruloids. e–h Representative MIP of embryos at E7.75 (e), E8.5 (f), gastruloids on day 5 (g) and day 6 (h) after RNAscope with Nkx2–5 (cyan), Ebf3 (yellow) and Tbx1 (purple) probes. e′, f′, g′, and h′: Optical sections of the area indicated with dotted lines. White arrowheads indicate overlapping expression of Ebf3 and Tbx1. n = 4 embryos and n > 5 gastruloids. i–l Embryos at E8.5 (a), E9.5 (b), gastruloids on day 6 (c), and day 7 (d) after RNAscope with Isl1 (cyan), Tcf21 (yellow) and Tbx1 (purple). i′, j′, k′, and l′: Optical sections of the area indicated with dotted lines. Arrowheads indicate overlapping expression of Tbx1 and Tcf21 (white) or of Isl1 and Tcf21 (white). n = 4 embryos and n > 5 gastruloids. m, n Representative MIP of the pharyngeal region of embryo at E9.5 (m) and gastruloid on day 11 (n) after RNAscope with Tnnt2 (green) and Tbx1 (purple). o Percentage of gastruloids expressing Tnnt2 on day 7 (left - n = 14) and day 11 (right – n = 23). p, q Representative MIP of an embryo at E9.5 (p) and gastruloid on day 11 (q) after RNAscope with Tcf21 (green) and Myf5 (purple). r Percentage of gastruloids expressing Myf5 on day 11 (n = 45). s, t Representative MIP of the pharyngeal region of an embryo at E10.5 (s) and gastruloid on day 11 (t) after HCR with Myl7 (green) and Myh3 (purple). u Percentage of gastruloids expressing Myl7 on day 11 (left - n = 34) or expressing Myh3 on day 11 (right – n = 14). a, anterior, p, posterior, PS, primitive streak, PhA, pharyngeal arch. Scale bars: 100 µm. All data derived from at least 2 independent experiments.

Fig. 3. Transcriptomic analysis reveals cardiopharyngeal mesoderm subpopulations in gastruloids.

UMAP representation of Leiden clustering of gastruloids at day 4 (a), day 5 (c), day 6 (e) and day 11 (g). FeaturePlots of key markers in gastruloids at day 4 (b), day 5 (d), day 6 (f) and day 11 (h). Scale bars represent expression levels. Mes., mesoderm; CardioPharyng., cardiopharyngeal; Intermed., intermediate; Visc., visceral; Som. Somitic; Diff., differentiating; PS, primitive streak.

Fig. 4. Different subtypes of cardiomyocytes differentiate in gastruloids.

a, c, e UMAP representation of day 11 cardiomyocytes. Red dots represent cells of cluster 6 (a), cluster 9 (c) or cluster 8 (e). The most differentially expressed genes of the cluster are shown in green. b, d, f Feature and violin plots of key differentially expressed genes in the cardiomyocyte cluster 6 (b), cluster 9 (d) and cluster 8 (f) at day 11. g Representative maximum intensity projection image of a mouse embryonic heart at E10.5 after HCR experiment with Myl2 (green) and Myl7 (purple) probes. n = 2 hearts. h–h′ Representative maximum intensity projection images of gastruloids at day 11 after HCR experiment with Myl2 (green) and Myl7 (purple). n = 15 gastruloids in 4 independent experiments. i Representative FACS analysis of the combined expression of Myl7 and Myl2 expression in all of the living gastruloids’ cells at day 11 after HCR experiment. Negative controls with no probe are shown in left. n = 4 independent experiments. j Scheme of the experimental design with the use of the Mef2c-AHF-enhaner-Cre; Rosa-tdTomato (Mef2c-Cre; Rosa-tdTomato) line that label the right ventricle and outflow tract. Created in BioRender. Lescroart, F. (2024) https://BioRender.com/b93x660k. Representative FACS analysis of the combined expression of tdTomato and cTnT expression in all of the living gastruloids’ cells at day 11. l, m Graphs showing the proportion of tdTomato+ (Tom + ) cells in cardiomyocytes (cTnT+ cells) derived from day 11 gastruloids (l) and embryonic cells at E10.5/E11.5 (m) (in n = 4 and n = 5 independent experiment (mean with standard error of mean (SEM)). n–n” Confocal images (maximum intensity projection) of 3 independent Mef2c-Cre; Rosa-tdTomato gastruloids at day 11 after HCR with Myl2 (yellow) tdTomato (purple) and Myl7 (blue) probes. n = 16 gastruloids in 3 independent experiments. o–o” Optical sections from the same gastruloids shown in (n–n”) to specifically observe the co-expression of Myl2 (green) and tdTomato (purple) marked by white arrowheads. Empty arrowheads indicate Myl2+ tdTomato- cells. Scale bars: 100 µm. LA, left atrium; RA, right atrium; LV, left ventricle; OFT, outflow tract; a, anterior; p, posterior.

Fig. 5. Skeletal myogenesis takes place in gastruloids from the CPM and somitic mesoderm.

a UMAP representation of day 11 myoblast clusters highlighted in purple (cluster 16) and in pink (cluster 18). b UMAP representation of day 11 cells with their cell cycle status, G1 (red), G2/M (green) and S (blue). The numbers of cells in each phase of the cell cycle are indicated for the myoblasts cluster 16 and 18. c Scheme of the genetic regulation of skeletal myogenesis from the CPM (red) or somitic mesoderm (green). Created in BioRender. Lescroart, F. (2024) https://BioRender.com/o57x120. d Violin plots showing the level of expression of Pax7, Myf5, Met, MyoD, Myog and Myh3 specifically in the myoblast clusters of gastruloids at day 11. e Violin plots showing the level of expression of Tcf21, Isl1, Pitx2, Tbx1, MyoR and Pax3 specifically in the myoblast clusters at day 11. f, g UMAP representation of day 11 cluster 16. Blue and red dots represent Tbx1 and Pax3 expressing cells, respectively. Black circles represent Myf5 (f) or Myod (g) expressing cells. h Representative FACS analysis of the combined expression of Myh3 and Myog expression in gastruloid cells at day 11 after HCR experiment. Negative controls with no probe are shown on the left. n = 4 independent experiments. i Representative maximum intensity projection (MIP) images of two gastruloids at day 11 after immuno-fluorescent experiment with MyoG antibody (purple). n = 13 gastruloids in 3 independent experiments. a, anterior; p, posterior. Scale bars: 100 µm. j Representative MIP of a gastruloid at day 11 after RNAscope experiment with Tcf21 (yellow), Pax3 (blue) and Myf5 (purple). n = 10 gastruloids in 3 independent experiments. Scale bar: 100 µm. k Representative MIP of a gastruloid at day 11 after RNAscope experiment with Tbx1 (yellow), Pax3 (blue) and Myf5 (purple). n = 9 gastruloids in 3 independent experiments. Scale bar: 100 µm. Gray dotted lines delinate the gastruloid. Optical sections of the area indicated with dotted lines are shown in (k’–k”). White arrowheads indicate overlapping expression of Myf5 with Pax3 (k’) or with Tbx1 (k”). Scale bars for k’ and k”: 25 µm. a, anterior; p, posterior.

Fig. 6. Myogenic trajectories are found in gastruloids over time.

a UMAP representation of all gastruloid cells merged from day 4 (red), day 5 (green), day 6 (blue) and day 11 (purple). Numbers represent identified clusters of day 11 (as defined in Fig. 3g). b, c Fate probabilities towards the cardiomyocyte macrostate (b) or towards the skeletal myoblast macrostate (c). Scale bars represent absorption probabilities. Numbers represent key cardiomyocytes and myoblast clusters of day 11. d URD trajectories showing transition from cells at day 4 (root) towards cells at day 11 (tips). Framed letters represent branches along the tree. The dotted box represents the lineage branch detailed in (f–o). e–i Expression of Isl1 (e), Tcf21 (f), Myl7 (g), Myl2 (h) and Myog (i) across URD trajectory. Scale bars represent expression levels. j–n Representation of clusters’ cells across URD trajectory. Red dots represent cells of the cardiopharyngeal mesoderm (CardioPharyng. mes.) cluster 1 of day 5 (in j, Fig. 3c, cluster 1), of the cardiopharyngeal mesoderm (CardioPharyng. mes.) cluster 2 of day 6 (in k, Fig. 3e, cluster 5), of the cardiopharyngeal mesoderm (CardioPharyng. mes.) cluster 3 of day 6 (in l, Fig. 3e, cluster 8), of the myoblast cluster 1 of day 11 (in m, Fig. 3g, cluster 16) and of the myoblast cluster 2 of day 11 (in n, Fig. 3g, cluster 18).

Fig. 7. Schematic representation of the developmental timing of CPM specification and differentiation in gastruloids in comparison with the mouse model.

The scheme of the embryo has been created in BioRender. Lescroart, F. (2024) https://BioRender.com/m68f097.