Stem Cell

Gastruloids are competent to specify both cardiac and skeletal muscle lineages

Time Stamp: November 22, 2024 7:00 PM
Source Node: 755877

CPM markers are expressed in gastruloids

To generate gastruloids that form CPM and characterize their differentiation to cardiac and skeletal myogenic fates, we extended a previous protocol described by Rossi et al.³⁵ to culture gastruloids for an prolonged time, until day 11 (Fig. 1a) (see Methods). In brief, mESCs were aggregated at day 0, following centrifugation, and treated with a Wnt agonist (Chiron) for 24 h from day 2. Cardiogenic factors (bFGF, VEGF and ascorbic acid) were added to the culture media at 96 h (day 4) for 3 days. After day 7, gastruloids were cultured in N2B27 culture media. Shaking (80–100 rpm) was continuous from day 4 to the end of the culture. As previously reported^32,35,38, we observed efficient and robust elongation starting on day 4, with beating areas appearing by day 7. On average, about 86.79% ( ± 7.4 SEM) of gastruloids in each experiment showed beating areas, primarily in the anterior part of the gastruloid (Fig. 1b, c and Supplementary Video 1). On day 7, gastruloids express cardiac troponin T (cTnT), VEGFR2 and E-cadherin, as previously reported³⁵, indicating their ability to form cardiomyocytes, endothelial cells and endoderm (Supplementary Fig. 1a, b). Interestingly, gastruloids on day 11 continue to express cTnT, VEGFR2 and E-cadherin (Supplementary Fig. 1c, d).

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

To investigate whether the anterior-posterior axis is maintained at day 11, we used multiplex fluorescent in situ hybridization or RNAscope to analyze the expression of Hoxc4, a posterior marker³⁹. We found Hoxc4 expression at one pole of the gastruloids at both day 7 and day 11 (Supplementary Fig. 1e, f). However, we observed an anti-correlation between the expression of Hoxc4 and troponin (Tnnt2), a marker of cardiomyocytes, where the expression of Hoxc4 decreased as Tnnt2 expression expanded. This suggests that as gastruloids develop more cardiomyocytes, they lose their posterior region, with highly beating gastruloids becoming predominantly anterior. With the extension of the protocol, gastruloid morphology is affected from day 7.

To investigate whether key markers of CPM and its derivatives (Fig. 1d) were expressed during the time-course culture, we used quantitative RT-PCR. We observed the transient expression of the bHLH transcription factor Mesp1 and the expression of the CPM transcription factors Islet1 (Isl1) and Tbx1, as previously described³⁵ (Fig. 1e). We also observed an increasing expression of transcripts encoding the cardiac specific myosin (Myl7 and Myh7) and Tnnt2 at day 5 (Fig. 1f). Interestingly, the CPM marker, Tcf21^20,25, began to be expressed at day 3, while the myogenic transcription factors Myf5 and MyoD were expressed at day 7 (Fig. 1g). Importantly, similar kinetics of expression were also observed for Mesp1, Isl1 and Tcf21 with another wild-type mESC line (see Methods for details on the lines and Supplementary Fig. 2). These data demonstrate that CPM and downstream myogenic transcriptional programs are robustly activated in gastruloids under these extended culture conditions.

Similar spatio-temporal gene expression with mouse embryo

To explore whether gastruloids faithfully mimic mouse CPM development, we compared gastruloids with mouse embryos at equivalent developmental stages. Our goal was to investigate the expression of markers of the CPM, cardiomyocytes, and the robustness of the CPM specification. We used multiplex fluorescent in situ hybridization (RNAscope or HCR) to explore gene expression. We first compared early day 4, spheric, gastruloids with E7.5 (Early bud stage) embryos. It showed a small overlapping expression of Mesp1 with Isl1 and in a smaller proportion with Tbx1 (Fig. 2a–a′ and c–c′). After symmetry breaking, comparison of day 5 gastruloids with E7.75 cardiac crescent embryos showed that Mesp1 expression is in non-overlapping domains with Isl1 and Tbx1 (Fig. 2b–d and Supplementary Fig. 3). In the embryo, Mesp1 was observed in the posterior mesoderm (likely corresponding to the somitic mesoderm), while Tbx1 and Isl1 expression overlapped in the anterior region of the embryo (Fig. 2b). Similarly, Tbx1 and Isl1 expression overlapped in gastruloids (Fig. 2d–d’).

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

We then investigated the expression of additional markers of the CPM such as Ebf3, Tcf21 and Nkx2−5, together with Isl1 or Tbx1, at later time points in E7.75 to E9.5 embryos and day 5 to day 7 gastruloids (Fig. 2e–l and Supplementary Fig. 3c, d). In the embryo, Nkx2–5 expression labeled the heart tube while Isl1 and Tbx1 were expressed in second heart field (SHF) progenitors, located in pharyngeal mesoderm behind the differentiated heart tube (Fig. 2e–j and Supplementary Fig. S3c). Ebf3 and Tcf21 are expressed more broadly, but they are also found in the pharyngeal region of the mouse embryo, where their expression overlaps with Tbx1 and Isl1 ((Fig. 2e–j). Nkx2–5 was also expressed in the anterior part of the gastruloid, marking cardiomyocytes (Fig. 2g, h and Supplementary Fig. 3d). On days 5 and 6, gastruloids expressed Tbx1 and Isl1 in a domain adjacent to that of Nkx2–5 (Fig. 2g, h and Supplementary Fig. 3d). In gastruloids, between day 5 and day 7, both Tcf21 and Ebf3 are expressed in overlapping domains with Isl1 and Tbx1 (Fig. 2g, h, k, l). In day 7 gastruloids, Ebf3 and Tcf21 were also found in a close domain of expression adjacent to the cardiomyocytes (Tnnt2 + ) (Supplementary Fig. 3f). Similarly, in day 11 gastruloids, Tbx1 were also found in a close domain of expression adjacent to Tnnt2+ cells (Fig. 2m, n). Among all the gastruloids analyzed for Tnnt2 expression, 100% showed a positive expression domain at both day 7 (n = 14 across 5 independent experiments) and day 11 (n = 23 across 5 independent experiments) (Fig. 2o). Taken together, these findings indicate that CPM and cardiomyocyte markers are expressed in a similar spatio-temporal pattern in both gastruloids and mouse embryos.

To further investigate whether myoblast differentiation occurs and is faithfully recapitulated in gastruloids, we also compared the expression of myoblast markers in both models. In stage E9.5 mouse embryos, Myf5 positive cells marked skeletal myoblasts, notably in the somites (Fig. 2p). Similarly, Myf5 were detected close to Tcf21 positive cells in gastruloids collected at day 11 (Fig. 2q). Detailed analysis revealed that 84.4% of the gastruloids analyzed showed Myf5 expression (n = 45 across 6 independent experiments) (Fig. 2r). Similarly, we analyzed with HCR, the expression of Myh3, a marker of committed skeletal myoblasts, together with Myl7, a marker of cardiomyocytes. At E10.5, Myh3 is detected in the first pharyngeal arch (Fig. 2s). On day 11, gastruloids also showed Myl7 and Myh3 expression. Myh3 expression domain was located slightly more posterior than Myl7 expression domain (Fig. 2t). Similar to Tnnt2, Myl7 expression was detected in all gastruloids analyzed at day 11 (n = 34 in >5 independent experiments) (Fig. 2u). Myh3 was not detected in all gastruloids, but 85.7% of gastruloids analyzed showed co-expression of Myl7 and Myh3 (n = 14 in 4 independent experiments) (Fig. 2u). These data support the co-existence of skeletal myoblasts and cardiomyocytes in gastruloids cultured for over 7 days. Together, this comparison shows that gastruloids and mouse embryos display similar spatio-temporal gene expression during CPM development.

Single-cell transcriptomic reveals CPM subpopulations in gastruloids

To further assess the potential of gastruloids to form CPM and investigate in detail the different cell populations, we performed single-cell RNA-sequencing (scRNA-seq) on gastruloids following a time course. Gastruloid cells were collected at day 4, day 5, day 6 and day 11. At each time point, at least 8 gastruloids were dissociated into single-cell suspensions, and approximately 7,000 cells per sample were sequenced. After quality controls, we analyzed 6,646 cells at day 4, 6,704 cells at day 5, 5,284 cells at day 6 and 8,024 cells at day 11. We performed Leiden clustering and differential gene expression analysis at each time point (Fig. 3 and Supplementary Data 1). Integration of the gastruloid single-cell data with the published atlas of mouse embryonic cells ranging from E6.5 to E8.5⁴⁰ (see Methods 4–5) showed that cells collected at day 4, day 5 and day 6 likely overlap with cells of E7.25-E7.75, E8.0-E8.25 and E8.5 embryos respectively (Supplementary Fig. 4), in agreement with our fluorescent in situ hybridization experiments (Fig. 2a–l). However, gastruloid cells collected at day 11 do not overlap in the UMAP with cells of the atlas, likely reflecting distinct transcriptional profiles (Supplementary Fig. 4j, k). Gastruloid cells at day 11 might thus reflect more differentiated states, usually found after E8.5 in mouse embryos. This observation indicates that while our gastruloid single-cell dataset from day 4, day 5 and day 6 can be confidently compared with the cells of the atlas, distinct annotation criteria had to be applied to day 11 clusters. Cell type annotation transfer, from the atlas, was first applied to the primary clusters of day 4, 5 and 6 (See Methods 4). Manual annotation was then performed based on this label transfer and on differential gene expression (Supplementary Data 1). The day 11 clusters were annotated manually based on differential gene expression analysis (Supplementary Data 1) and reference to tissue database (https://tissues.jensenlab.org/About).

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

To assess the cellular heterogeneity of the gastruloids, we analyzed the different clusters obtained at each time point. At day 4, we observed a significant number of clusters of mesodermal cells (Fig. 3a, b; Supplementary Figs. 5 and 6) with four clusters showing high Mesp1 expression (clusters 1, 3, 5 and 14) (Table 1). Analysis of the single-cell data also showed high expression of the Gata4 and Gata6 transcription factors in most clusters (all clusters except clusters 8, 9 and 13) (Supplementary Fig. 7). Gata transcription factors are known to have a broad expression pattern in the anterior/cardiogenic mesoderm as well as in the endoderm⁴¹. They have a critical function during heart development⁴² and recent work in the fish has shown that they are involved in CPM specification, by promoting cardiac fate and inhibiting pharyngeal identity^43,44. Hand1, a marker of the extraembryonic mesoderm and cardiac progenitor cells in the recently identified juxtacardiac field^45,46 was also highly expressed at day 4 (except in clusters 8, 9 and 10) (Supplementary Fig. 7). Interestingly, with scRNA-seq, we detected Myl7 as early as day 4 (Fig. 3b). Cluster 9 showed expression of the pluripotency marker Pou5f1 indicative of epiblast-like cells (Supplementary Fig. 7). Mesp2, the closest homolog of Mesp1, was only expressed at very low levels in gastruloids (Supplementary Fig. 7).

Table 1 Percentage of positive cells for specific genes in scRNAseq

Full size table

At day 5, the cell type annotation transfer from the atlas was enriched in mesenchymal and pharyngeal mesodermal cells while nascent mesoderm was barely observed (Supplementary Fig. 8). We identified 5 clusters annotated as pharyngeal mesoderm (clusters 1, 3, 6, 7, and 13) (Fig. 3c). The first fully differentiated cells were found in cluster 8. This cluster corresponds to endothelial cells and is marked by the expression of Sox7 (Supplementary Figs. S8 and S9). At this stage, Mesp1 started to be downregulated while Gata6 was still highly expressed (Fig. 3d and Supplementary Fig. 9). Isl1 expression was also detected, while Tbx1 was present at a very low level and in a small number of cells (Table 1 and Supplementary Fig. 9).

At day 6, we uncovered 16 clusters (Fig. 3e, Supplementary Fig. 10). We detected two clusters of differentiating or differentiated cardiomyocytes (clusters 6 and 2) expressing high level of Myl7 and other cardiac myosin genes (Fig. 3f). All these data are consistent with the previous report on gastruloids³⁵. We also found 5 clusters of pharyngeal mesodermal cells (clusters 3, 5, 8, 10 and 11). Expression of the transcription factors involved in CPM development, including Tbx1, Tcf21, Lhx2 and Ebf3, was also detected (Fig. 3f and Supplementary Fig. 11). These data reveal the emergence of CPM in differentiating gastruloids.

Finally, to investigate the potential differentiation of CPM derivatives in gastruloids, we analyzed scRNA-seq data at day 11. We identified 20 clusters (Fig. 3g). One cluster (cluster 20) likely corresponds to visceral endoderm tissue, with expression of Epcam (Supplementary Fig. 12). We also uncovered 4 different clusters (clusters 10, 12, 13 and 17) of ectodermal cells with enrichment of genes linked with neural derivatives, including a small number of Sox10+ neural crest-like cells in cluster 13 (Supplementary Data 1 and Supplementary Fig. 12). We identified a cluster of endothelial cells (cluster 7) and a cluster of blood/hematopoietic lineages (cluster 19). Clusters 1, 2, 3, 4, 5, 11 and 14 were annotated as mesoderm and included mesenchymal cells and undifferentiated progenitors (Fig. 3g, Supplementary Data 1). The presence of high expression levels of Myl7 in clusters 6, 8 and 9 indicates that these clusters contain cardiomyocytes. Strikingly, we found 2 clusters (clusters 16 and 18) with expression of the myogenic regulatory genes Myf5 (Fig. 3g, h and Supplementary Fig. 5). Together, our data reveal different CPM subpopulations in the gastruloid model and show that markers of cardiomyocytes and skeletal myoblasts are found in this model.

Different subtypes of cardiomyocytes differentiate in gastruloids

To investigate whether the cardiomyocyte clusters at day 11 represent different cardiac subpopulations, we performed a detailed analysis of the clusters 6, 8, and 9, i.e., the three clusters containing cardiomyocytes. Among the genes differentially expressed in cluster 6, we found Actg1, Cald1, Actb, Acta2, Vsnl1, Gucy1a1, Shox2 and Ptn (Fig. 4a, b and Supplementary Table 1). This cluster thus showed the expression of Cald1, Acta2, expressed in smooth-muscle cells and throughout the myocardium of the immature heart tube^47,48 as well as genes involved in sinus venosus/sinus atrial node development (Vsn1, Shox2)^49,50,51 (Fig. 4a, b). In cluster 9, Nppa, Itga6, Myl7, Myh6 were among the genes differentially expressed (Fig. 4c, d and Supplementary Data 1). These genes are usually enriched in atrial cardiomyocytes. Cluster 8, on the other hand, was enriched in genes such as Myl2, Myl3, Myh7, Mpped2, Pln (Fig. 4e, f and Supplementary Data 1). These genes were previously identified as signatures of ventricular cardiomyocytes in different independent studies^52,53,54. Our scRNA-seq analysis thus identified 3 cardiomyocyte clusters with distinct transcriptional signatures. We can further speculate that gastruloids contain ventricular, atrial and conductive-like cardiomyocytes.

To validate the presence of atrial and ventricular cardiomyocytes in gastruloids we performed in situ hybridization using the HCR method with specific ventricular and atrial probes. We first showed that at E10.5, Myl2 is indeed restricted to ventricular cardiomyocytes, while Myl7 is expressed in all cardiomyocytes but enriched in atrial cardiomyocytes (Fig. 4g). We then performed HCR experiments in wholemount gastruloids or followed by flow cytometry (see Methods). We found clearly distinct domains of Myl2 and Myl7 expression, each exhibiting a unique but seemingly random organization (n = 13/15 – Fig. 4h) or in two well organized domains (n = 2/15 – Fig. 4h’). We also found a significant shift of the cloud of cells, in the flow cytometry chart, indicative that a significant proportion of cells are Myl7-positive alone (corresponding to clusters 6, 8, and 9 of the sc-RNAseq data) or Myl7-positive in combination with Myl2 (differentially expressed in cluster 8) in gastruloids at day 11 (Fig. 4i). This result suggests that Myl7 + , Myl2+ ventricular-like cardiomyocytes and Myl7+,Myl2- non-ventricular cardiomyocytes are present in gastruloids. This result further validates the scRNAseq analysis and confirms the existence of different subpopulations of cardiomyocytes in gastruloids.

In order to support these results, we performed lineage tracing with a cell line, that labels specific subpopulations of cardiomyocytes in gastruloids. For that purpose, we rederived mESCs from Mef2c-AHF-enhancer-Cre; Rosa^tdTomato/+ mouse blastocysts. Mef2c-AHF-enhancer-Cre mouse line is known to specifically label progenitors that contribute to outflow tract, right ventricle and a subpopulation of venous pole myocardium (dorsal mesenchymal protrusion) as well as non-cardiac CPM derivatives^55,56,57 (Fig. 4j). Mef2c-AHF-enhancer-Cre; Rosa^tdTomato/+ (Mef2c-Cre; Rosa-tdTomato) derived mESCs were able to form beating gastruloids (Supplementary Video 2) with expression of tdTomato in their anterior domain as expected. Flow cytometry at day 11 revealed that Mef2c-Cre; Rosa-tdTomato-derived gastruloids contain a significant proportion of cardiomyocytes, as shown by the expression of cardiac troponin T (cTnT) (Fig. 4k). Thus, tdTomato+ cardiomyocytes represented an average of 58.2% (ranging from 30 to 74.4%) of all cardiomyocytes in gastruloids (Fig. 4l). Flow cytometry in E10.5/E11.5 mouse embryos showed less variability with between 0.9 and 1.6% of tdTomato+ cells. In addition, staining of mouse embryos with cTnT antibody showed that Mef2c-AHF-enhancer-Cre derived (tdTomato + ) cardiomyocytes accounted for about 22.9% of all cardiomyocytes in embryos (Fig. 4m). These data indicate that gastruloids can undergo differentiation into at least two distinct cardiomyocyte fates: either a tdTomato positive SHF fate (outflow tract, right ventricle for example) or a first heart field (FHF) enriched tdTomato negative fate.

We additionally performed in situ hybridization, with the ventricular specific Myl2 probe, to discriminate left ventricular versus right ventricular fates in Mef2c-AHF-enhancer-Cre; Rosa^tdTomato/+ gastruloids collected at day 11 (Fig. 4n). We observed the presence of both Myl2 + /tdTomato+ and Myl2 + /tdTomato- domains (Fig. 4o), supporting the specification of right and left ventricular cardiomyocytes respectively. Myl2-/Myl7 + /tdTomato+ cells also present in gastruloids might correspond to outflow tract-like cardiomyocytes. Interestingly, we also observed the expression of Foxc2 and Hoxb1, markers of the anterior and posterior SHF respectively⁵⁸, in gastruloids collected at day 4 (Supplementary Fig. 13). In day 6 and day 11 gastruloids, we identified Tbx5 + /Tnnt2+ double positive cells, as potential FHF-derived cardiomyocytes and Tbx5 + /Tnnt2- cells as potential posterior SHF progenitors⁵⁷ (Supplementary Fig. 13). Together these data validate the existence of different subpopulations of cardiomyocytes (left ventricular, right ventricular and atrial) in gastruloids.

Skeletal myogenesis in gastruloids

To ascertain whether gastruloids could generate skeletal muscle cells, we analyzed the clusters 16 and 18, which are characterized by the expression of genes associated with myoblast differentiation (Fig. 5a). Differential gene expression between clusters 16 and 18 was not conclusive (Supplementary Data 2). We first analyzed the cell cycle and uncover that the myoblast cluster 16 was more enriched in cells in G2/M and S phases than cluster 18 (Fig. 5b). This suggests that cluster 16 might correspond to a more proliferative state than cluster 18. We thus decided to investigate in detail the genes involved in cardiopharyngeal and/or somitic mesoderm muscle differentiation and their expression in the two myoblast clusters (Fig. 5c). We noticed an increased expression of genes associated with muscle precursors such as Pax7 and Myf5 in cluster 16 compared to cluster 18, as shown by the violin plots (Fig. 5c, d). Although Met has been shown to be downregulated during differentiation in some particular CPM- or somites-derived myoblasts^59,60, it was expressed in more cells and at higher levels in cluster 16 compared to cluster 18 (Fig. 5d). In contrast, markers of more committed myoblasts such as MyoD, Myog and Myh3 were enriched in cluster 18 (Fig. 5d). These data, together with our findings on the cell cycle, indicate that myogenesis occurs in the gastruloid model and that clusters 16 and 18 express markers of muscle precursors and committed myoblasts, respectively.

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

To dissect the CPM versus somitic origin of these myoblasts, we investigated the expression of transcription factors that were specific to either the CPM or somitic muscle progenitors (Fig. 5c). Expression of the CPM genes, Isl1, Pitx2, Tbx1 and MyoR, was found in both cluster 16 and 18 but only in a small number of cells (Fig. 5e). Tcf21 was expressed only in the myoblast cluster 16 in a limited number of cells. Pax3 expression was also recorded in about 40 out of 243 cells of cluster 16 and in 2 out of 67 cells of cluster 18 (Fig. 5e). Deeper analysis of cluster 16 showed a mutually exclusive expression of Tbx1 and Pax3 in the population of muscle precursors, however a significant number of Tbx1+ or Pax3+ cells co-express Myf5 and/or Myod (Fig. 5f, g). This observation suggests the activation of distinct myogenic programs. Our scRNA-seq analysis at day 11 thus indicates that both CPM and somitic progenitors undergo myogenesis in the gastruloid model.

To validate this data and further support the existence of myogenesis in gastruloids, we performed in situ hybridization with skeletal myocyte specific probes either in wholemount or followed by flow cytometry. Flow cytometry interestingly showed a shift in the cloud of cells with two cell populations expressing Myog (Fig. 5h). As found in the single-cell data, we identified a subpopulation of cells expressing Myog alone (cluster 16) and a subpopulation of cells expressing Myog and Myh3 (cluster 18). Using immunofluorescence, we showed the expression of MyoG at the protein level, further supporting the specification and differentiation of skeletal myoblasts (Fig. 5i). Wholemount RNAscope experiments revealed Myf5 expression near the Pax3 and Tcf21 expression domains (Fig. 5j). Furthermore, wholemount RNAscope experiments with Pax3, Tbx1 and Myf5 probes showed the expression of Tbx1 and Pax3 in distinct domains (Fig. 5k) and co-expression of Myf5 with either Pax3 (Fig. 5k’) or Tbx1 (Fig. 5k”), further supporting the scRNAseq dataset. Overall, these observations demonstrate the existence of convergent CPM and somitic myogenic programs in the gastruloid model.

Muscular trajectories are found in gastruloids over time

Time series scRNA-seq data allows the reconstruction of transcriptional trajectories from a progenitor cell state toward differentiated cell states⁶¹. We hypothesize that we could similarly infer the transcriptional trajectories from CPM progenitors toward cardiomyocyte and myoblast cell states. We merged the single-cell data from the 4 different time points to create a transcriptional time series of the growing gastruloids (Fig. 6a). We first used CellRank (see Methods)⁶². We focused on a differentiated cell state (called macrostate), that includes cells from the cardiomyocyte cluster 8 from the day 11 dataset, and computed the fate probabilities towards this macrostate. Interestingly, we found high fate probability with cells from clusters 6 and 9 from day 11, as well with clusters annotated as cardiomyocytes at day 6 (Fig. 6b). We then focused on a second macrostate that includes cells from the myoblast cluster 18 from the day 11 dataset. We found high fate probabilities with cells from the myoblast cluster 16 also from day 11 (Fig. 6c). These data indicate the existence of muscular trajectories toward the cardiac and skeletal muscle states.

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

To reconstruct fate decision trees for the various populations, we used URD⁶³ (see Methods) as a computational reconstruction method, with the merge of all time points (days 4, 5, 6 and 11). URD analysis computed transcriptional trajectories from cells at day 4 (named root) towards the different clusters at day 11 (named tips) (Fig. 6d). We observed 5 branches emerging from day 4. The first branch (branch A) heads towards the ectodermal lineages (clusters 10, 12, 13 and 17) while the last branch (E) heads towards the endoderm lineages (cluster 20). The 3 other branches (branches B, C and D) are mesodermal, and include the endothelial (cluster 7) and hematopoietic lineages (cluster 19). Notably, one of these mesodermal branches, branch D, heads towards the cardiomyocytes (clusters 6, 8 and 9) and myoblast clusters (clusters 16 and 18). Thus, the URD analysis shows that transcriptional trajectories can be found toward both the cardiomyocytes and skeletal muscles.

To further dissect these trajectories, we specifically focused on branch D. This branch also includes clusters 4 and 14. These two clusters were previously defined as mesodermal derivatives (Fig. 3g). We investigated gene expression in branches M and H to characterize further these clusters (Fig. 6d). We found that branches M and H showed Gata6 expression as well as Hand1 and Mab21l2, which mark the juxta-cardiac field^45,46 (Supplementary Fig. 14). We also showed expression of Tbx18 and Wt1 (Supplementary Fig. 14), which mark the proepicardial organ and their derivatives⁶⁴. It is tempting to speculates that these clusters contain epicardial cells but none of these markers are completely restricted to this cell lineage.

We then investigated how key markers of the CPM and their derivatives are expressed in the tree. We found that Isl1, Tcf21, but also Lhx2 and Ebf3 are expressed in branch D, in the root as well as along the trajectories towards the cardiomyocyte and myoblast clusters (Fig. 6e, f and Supplementary Fig. 14). Myl7 is specifically expressed in the three cardiomyocyte clusters (6, 8 and 9) and the cardiomyocyte branching point I (Fig. 6g), while Myl2 is expressed after the branching bifurcation only in cluster 8 (Fig. 6h). As expected Myog is expressed in branch L that heads towards the myoblast cluster 18 (Fig. 6i). Tbx1, MyoR, markers of the CPM muscle progenitors, Pax3, markers of the somitic muscle progenitors, and the early myogenic transcription factor Myf5 are all expressed in branch F that heads towards the two myoblast clusters 16 and 18 (Supplementary Fig. 14). We also explored where cells of clusters from days 5 and 6 would be found in this tree (Fig. 6j–n). Strikingly, cells of the pharyngeal mesoderm 1 cluster, identified at day 5, were found almost at the root of the tree (branch D), before the branching bifurcation between the cardiomyocytes and myoblast lineages (Fig. 6j).

Similarly, cells of the cardiopharyngeal mesoderm 2 cluster, identified at day 6, were found before the branching point (branch D) (Fig. 6k). However, we found that cells of the cardiopharyngeal mesoderm 3 cluster, identified at day 6, were restricted to the cardiomyocyte lineages (branch I). These cells are located after the branching bifurcation between the cardiomyocytes and myoblast lineages (branch D) (Fig. 6l). We then focused on the two myoblast clusters identified at day 11. Cells of cluster 16 were also found in branch F, before the branching point that heads toward the myoblast clusters 16 and 18 (Fig. 6m). Cells of cluster 18, on the other hands, were found only after branch bifurcation, in branch L (Fig. 6n). These data further confirm that myoblast cells from cluster 18 are in a more differentiated state that cells from cluster 16. Together these trajectory inferences show that muscular transcriptional trajectories are found in gastruloids. They also show that a specific branch of the tree includes the CPM and heads towards both cardiomyocytes and myoblasts clusters.

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Source: https://www.nature.com/articles/s41467-024-54466-w

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Gastruloids are competent to specify both cardiac and skeletal muscle lineages

CPM markers are expressed in gastruloids

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