Most internal organs in the body are made up of several different kinds of cells. Understanding where these cells come from and how these different cells develop from a single cell in an embryo could help to guide regenerative therapies, where tissues grown in the laboratory are used to repair damage that the body cannot repair itself.

The existence of a single heart progenitor cell that can produce all of the heart's structures has long been predicted, but has so far escaped discovery. Currently, it is known that two distinct sets of heart precursor cells exist in mammals, which each produce cells for different parts of the heart. Work performed in mouse embryos has hinted that both sets of cells develop from cells that produce a protein called Mesp1. This protein controls when many genes—including those involved in heart development—are activated.

Devine et al. marked a small number of Mesp1-producing cells and followed the fate of these cells through development to see where their descendants would end up within the embryo—and specifically within the mature heart. Labeling occurred at a very early stage of development, called gastrulation, when the embryonic cells first begin to organize themselves into three tissue layers that will go on to form all the different parts of the organism. Devine et al. found that shortly after gastrulation begins, heart precursor cells are present and are already assigned to particular regions of the heart. This means that if there is a single pool of heart precursor cells, it specializes into different populations very early in the development of an embryo.

Devine et al. show that during gastrulation, heart precursor cells are already split into two distinct populations: one containing the cells that go on to form the atria and left ventricle of the heart; the other consisting of the cells that will make up the right ventricle and the ‘outflow tract’ that will eventually form the great vessels leading into and out of the heart. These two populations are separated by a boundary, which Devine et al. suggest is established very early on, and will go on to form the septum that separates the left and right ventricles in the developed heart. As defects in the septum are the source of many congenital heart defects, a better understanding of the heart cell precursor populations and how they interact could help develop treatments for these conditions.

Mammalian heart development involves the allocation of cardiac progenitors in a discrete spatial and temporal order (Evans et al., 2010; Bruneau, 2013). Understanding the identity and regulation of these progenitors is critical to understanding the origins of congenital heart defects and may lead to novel cell-based regenerative therapies for heart disease (Bruneau, 2008; Xin et al., 2013). The existence of an early and specific multipotent progenitor for all anatomic and cellular components of the heart has been predicted (Parameswaran and Tam 1995; Tam et al., 1997; Meilhac et al., 2004b; Kinder et al., 2001), but the identity of this progenitor and when it arises in embryonic development has remained undefined. At least two sets of molecularly and morphologically distinct cardiac precursors have been identified in the mammalian embryo, referred to as the first and second heart fields; these populations contribute to distinct anatomical structures within the heart (Evans et al., 2010; Buckingham et al., 2005). Separation of the left and right ventricles is dependent on a single structure, the interventricular septum (IVS) and it has been postulated that the IVS myocardium has a dual contribution from these two heart fields (Bruneau et al., 1999; Takeuchi et al., 2003). The existence of these two cardiac progenitor ‘fields’ raises the question of when cardiac precursors are allocated to these populations and their contributions to mature structures in the heart, such as the IVS.

Previous studies have suggested that a population of early mesoderm expressing the transcription factor Mesp1 precedes the establishment of the anatomically and molecularly distinct ‘heart fields’ (Saga et al., 1996, 1999; Bondue et al., 2008; Lindsley et al., 2008) that ultimately will differentially populate the great vessels, RV and atria or the LV and atria. However it is clear that Mesp1+ cells can also contribute to a broad range of mesodermal derivatives that include, but are not restricted to, the developing heart (Chan et al., 2013; Yoshida et al., 2008). A population of mesoderm labeled by Eomesodermin has also been shown to contribute to the developing heart, but again these cells have broad contributions in the embryo (Arnold and Robertson, 2009). Retrospective lineage analysis supports the distinct origins of segments of the heart from individual precursor pools (Meilhac et al., 2003; Buckingham et al., 2005; Meilhac et al., 2004b), but several questions remain regarding the timing and molecular progression of cardiac specification (Meilhac et al., 2004b). For example, do early mesodermal cells become ‘locked into’ a cardiac fate early on and when do they become ‘assigned’ to an anatomical location? Is there a multipotent, specified cardiac progenitor that anticipates the currently understood heart fields?

Here we show that early cardiac progenitors are assigned to a specific developmental path prior to or shortly after the initiation of gastrulation. We identify a population of specified cardiac precursors arising from these mesodermal progenitors that express the chromatin remodeling factor Smarcd3 prior to the onset of expression of known cardiac progenitor markers (Nkx2-5, Isl1, and Tbx5). Clonal labeling of early cardiac precursors highlights the heterogeneity among Mesp1+ progenitors, including the existence of precursors that are restricted in their anatomical contribution, especially to distinct ventricular chambers. Finally, inducible genetic marking of early Tbx5+ and Mef2c-AHF + populations highlights this early segregation of cardiac progenitors and suggests that the compartment boundary that exists between the right and left ventricles arises from an early clonal boundary, prior to the onset of septum morphogenesis. Overall our findings delineate the progression and molecular identity of cardiac precursors in the early mouse embryo.

In reassessing the in vivo differentiation potential of Mesp1+ cells, we find that this population contributes broadly to several mesodermal derivatives, (Figure 1A), consistent with other reports (Yoshida et al., 2008). We reasoned that among this diverse mesodermal population, a more specific population destined for the cardiac lineage exists. To test this model, we performed in vivo clonal analysis by generating mosaic mice in which very few Mesp1+ cells were labeled at isolated clonal density via the mosaic analysis with double markers (MADM) system (Zong et al., 2005; Hippenmeyer et al., 2010) (Figure 1B–C). This approach is particularly advantageous because labeling events are rare, labeling is permanent, and one can identify labeled daughter cells (twin spots) based on color (Figure 1—figure supplement 1A). We analyzed in fetuses (E12.5-E14.5) the anatomic distribution and cellular constituents of clones induced by Mesp1^Cre (which is active in mesoderm from ∼E6.0 to E7.5) (Saga et al., 1999). While we did not use a conditional Cre allele to control the timing of Cre activity, we confirmed the timing of Cre expression by in situ hybridization (Figure 1—figure supplement 1B). By the late head fold stage (LHF), we see a downregulation of Cre mRNA and localization to the base of the allantois. We see no expression in the area of forming cardiogenic mesoderm. In addition, we counted the number of labeling events in embryos at E8.5 and E14.5 (Figure 1—figure supplement 1D–E and Statistical Analysis) and saw no change in the distribution of labeled clusters, suggesting that no additional recombination events have occurred over this time interval. Finally, a complementary lineage labeling approach using a Mesp1-rtTA transgenic allele (Lescroart et al., 2014) defines a functional window of Mesp1 activity based on the timing of doxycycline administration between E6.25-E7.5, again supporting the narrow timing of Mesp1 activity.

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In order to ensure an accurate description of clone locations throughout the embryo, a thorough external examination of embryos was performed followed by removal and, in many instances, immunostaining of dissected hearts for labeled twin spots. Coherent clusters of uniquely colored cells separated by > 100 µm were classified as a twin spot derived from a single labeling event. Because the majority of hearts (32 of 38 embryos) contained three or fewer uniquely colored clusters, determining lineage relationships between and among clusters was straightforward. A subset of clones uniquely label the heart (Figure 1D–E and Figure 2), demonstrating the existence of an early, cardiac-specific progenitor. We also found specimens with clones of cells in other mesodermal derivatives but with no apparent clones within the heart (Figure 1—figure supplement 1F–G and Figure 2), conclusively demonstrating that within the population of Mesp1+ mesoderm a dedicated population of cardiac progenitors exists.

Figure 2

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To determine if these early cardiac progenitors represent a common precursor for the two heart fields, we analyzed the anatomic distribution of Mesp1^Cre-MADM twin spot clones within the heart. In contrast to previous retrospective clonal analysis (Meilhac et al., 2004b), we did not observe twin spots that populated anatomic structures classically thought to derive from first and second heart field progenitors, for example spanning the left and right ventricles, or contributing to the left ventricle and outflow tract. Rather, we saw twin spots that populated discreet anatomic locations including the left ventricle (Figure 1F–G and Figure 1—figure supplement 3A–B), right ventricle (Figure 1D and Figure 1—figure supplement 3C–D), outflow tract (Figure 1N–O and Figure 1—figure supplement 3E–F), atria-left ventricle (Figure 1L–M), and interventricular septum (Figure 1H). Notably absent from our clonal analysis were twin spots that spanned the right and left ventricles. Based on the total number of clonal observations made (n = 96), the probability that a common progenitor for the left and right ventricles does not exist, assuming a binomial distribution (success, or 1, = the progenitor is observed; failure, or 0 = the progenitor is not observed), can be calculated using Jeffreys interval. The upper and lower limits of a 95% confidence interval was calculated such that the upper limit is 0.019 (See Statistical Methods). Thus, we can be quite confident given the number of observations made that such a common progenitor does not exist within the Mesp1+ population. An explanation for the discrepancy between our Mesp1^Cre-MADM clonal analysis and prior retrospective clonal analyses lies with the timing of the labeling events. Mesp1 is transiently expressed ((Saga et al., 1999) and Figure 1—figure supplement 1B), thus all of our clones are induced over a narrow window of time during gastrulation. The retrospective clonal analysis employed a cardiomyocyte-specific promoter, so an early labeling event (in the epiblast for example) that would contribute broadly throughout the embryo would only be visualized in the heart, giving the impression of a specific common progenitor cell. Indeed, when we perform MADM clonal analysis using the epiblast-specific Cre line Sox2::Cre, we see many clones in the embryos, including large, dispersed clones throughout the looping heart tube (Figure 1—figure supplement 2) that could be interpreted as a labeling event in a single, common cardiac progenitor. We cannot definitively conclude, however, that these labeled cells within the heart are all clonally related because of the large number of labeling events outside of the heart. In summary, our results suggest that soon after a heart field progenitor is specified, shortly after the initiation of gastrulation, the anatomic destiny of daughter cells quickly diverge, especially those destined to occupy /contribute to the left or right ventricle, and these fates remain fixed.

In analyzing the cellular composition of Mesp1^Cre-MADM clones, we noticed that while most twin spots give rise to homogenous cellular progeny (cardiomyocytes or endothelial cells), individual cardiac progenitors (<5%, see Figure 2) can contribute to multiple cell types, for example myocardium and endocardium (Figure 1J–K and Figure 1—figure supplement 3J–L), or endothelium and smooth muscle (Figure 1O). While a multipotent progenitor had been predicted from in vitro ES cell-based differentiation models (Wu et al., 2006; Moretti et al., 2006) this is the first evidence of the existence of such a multipotent progenitor in vivo.

The existence of a dedicated population of cardiac progenitors within gastrulating mesoderm suggests that these cells may have a unique molecular signature. To define the order and timing of early cardiac gene expression in vivo, we examined by in situ hybridization and by ß-galactosidase reporter activity early markers of cardiac differentiation (Mef2c, Tbx5, Isl1, Mesp1, and Smarcd3) at the late streak (LS) and early head fold (EHF) stages. We find that Smarcd3 expression precedes Isl1 and Tbx5 in a domain that lies at the anterior-proximal region of the embryo and extends into the extraembryonic tissues (Figure 3B,D–E). This expression domain appears coincident temporally but non-overlapping with Mesp1 expression (Figure 3A–B and Figure 3—figure supplement 1), but is within the Mesp1^Cre-derived lineage (Figure 4O–P). Activity of the Mef2cAHF enhancer (Verzi et al., 2005) is also detected at the LS stage (Figure 3C) prior to the expression of Tbx5 and Isl1. Previous studies have shown that activity of this enhancer is dependent on Isl1 (Dodou et al., 2004). In order to confirm an absence of Isl1 expression at the LS stage, we used a reporter allele where a nuclear lacZ (Isl1^nLacZ) has replaced a short segment of the coding sequence, including the endogenous start codon (Sun et al., 2007). While there was robust ß-galactosidase reporter activity at the cardiac crescent stage, no detectable staining was seen in LS stage embryos (Figure 3—figure supplement 2A–B), suggesting that initiation of Mef2cAHF enhancer expression precedes Isl1 expression and its initiation may be independent of Isl1. Several hours later (EHF stage), weak Isl1 and Tbx5 expression is detectable, Mef2cAHF activity remains, and Smarcd3 expression restricts to the embryo proper (Figure 3G–J). The VEGF-A receptor Flk1 labels multipotent progenitors that can differentiate into hematopoietic, endothelial, smooth muscle and cardiac lineages (Ishitobi et al., 2011; Kattman et al., 2006), thus we looked at co-localization of Mesp1, Flk1, and Smarcd3 in late streak embryos. While we find minimal overlap between Mesp1 and Flk1 and Mesp1 and a reporter of early Smarcd3 expression (Figure 3—figure supplement 1A,C–D), there is significant overlap of Flk1 with this same reporter of early Smarcd3 expression (see below) in late streak stage embryos. Taken together, we see an orderly progression of gene expression that follows the progressive commitment of nascent mesoderm, from early expression of Mesp1 to the intermediate expression of Smarcd3, Flk1, and the Mef2cAHF enhancer, and finally the later cardiac lineage-specific expression of Tbx5 and Isl1 (Figure 3K).

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In comparing human and mouse sequences at the Smarcd3 locus, we identified several regions of conservation in noncoding sequences upstream of the Smarcd3 transcriptional start site. We tested an ∼9 kb genomic element we called Smarcd3-F1 in a transgenic mouse reporter assay and found that it was sufficient to recapitulate early endogenous Smarcd3 expression (Figure 4A–C). Expression was maintained in the cardiac crescent and looping heart at later stages of development (Figure 4E–I), however expression in extraembryonic tissues was noted. Given the broad expression domain of Smarcd3 mRNA and our Smarcd3-F1::lacZ reporter lines, we sought to define an enhancer fragment that might uniquely label early cardiac progenitors. Through deletion analysis of the 9 kb enhancer/promoter region (Smarcd3-F1), we defined an ∼2.5 kb genomic element (Smarcd3-F6) that is expressed only in the embryo proper, in a more restricted pattern than Smarcd3-F1 (Figure 4D and J–N and Figure 4—figure supplement 1).

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Based on the temporal and spatial expression of Smarcd3, prior to expression of the canonical heart field markers Tbx5 and Isl1, and within a small subset of Mesp1^Cre-derived cells, we hypothesized that Smarcd3+ cells may represent cardiac precursors. To define the lineage potential of Smarcd3+ cells and to compare the lineage potential of the cell populations marked by our two enhancer elements, we performed temporally regulated genetic fate-mapping using mice expressing a tamoxifen inducible CreERT2 under control of the Smarcd3-F1 and Smarcd3-F6 sequences (Smarcd3-F1CreERT2 and Smarcd3-F6CreERT2 mice; Figure 5—figure supplement 1A–H). Lineage labeling of Smarcd3-F1CreERT2; Rosa^R26R at E5.5 and observation at E10.5 marked cells that contribute predominantly to the heart and anterior forelimb (Figure 5A–B), with scattered cells in the trunk and cranial mesoderm (Figure 5A and data not shown); observation at earlier time points revealed significant labeling in extraembryonic tissues (Figure 5—figure supplement 2A–C). Within the heart, labeled cells contribute to cardiomyocyte, endocardial, and pericardial layers (Figure 5C). Labeling one day earlier (E4.5) marks a similar distribution of cells but with reduced labeling (Figure 5—figure supplement 2D–F). Earlier induction (E3.5) and non-injected animals have minimal to no detectable labeling (Figure 5—figure supplement 2G–K and data not shown). Given the restricted expression pattern of the Smarcd-F6 enhancer, we hypothesized that marking these cells early on would label a more restricted population of cells later in the embryo. Indeed, lineage tracing with Smarcd3-F6CreERT2 labeled a much smaller number of more spatially defined cells, including the heart and a small group of cells in the anterior forelimb (Figure 5D–E). There was minimal to no labeling in head/trunk mesoderm or in extraembryonic tissues, and within the heart Smarcd3-F6CreERT2 labeled primarily the myocardial layer (Figure 5F and Figure 5—figure supplement 3A–C), consistent with this enhancer labeling a more restricted population of cells. We addressed the clonal potential of early Smarcd3+ cells with limiting doses of tamoxifen to induce small numbers of well-isolated labeled cells in Smarcd3-F1CreERT2;Rosa^R26R embryos (Figure 5G–H). The shape and distribution of the labeled patches is reminiscent of previous work describing the oriented clonal cell growth throughout the myocardium (Meilhac et al., 2004a). In order to confirm the clonal nature of these patches induced with our Smarcd3-F1CreERT2 allele, we attempted to perform MADM analysis with this particular allele. Unfortunately, the recombination frequency was too low to identify any clones in several litters of embryos and we proceeded with using the Rosa^Confetti multicolor reporter (Snippert et al., 2010). We identified 6 Smarcd3-F1CreERT2;Rosa^Confetti embryos from several litters where tamoxifen was administered at E5.5, which contained rare or infrequent labeling events; none of these examples showed a labeling pattern that would support a common progenitor contributing to both right and left ventricles. Instead, we saw isolated, single-color clones (Figure 5I and Figure 5—figure supplement 3D) that contributed to a single chamber. While the number of embryos we examined is not sufficient to reach statistical significance, the results are highly supportive of our conclusions from the Mesp1Cre-MADM analysis. We conclude that Smarcd3 expression in the late gastrulating embryo labels a defined population of specified cardiac precursors that are fated to occupy unique anatomic structures within the mature heart.

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Given the restricted expression and lineage potential of cells marked by the Smarcd3-F6 enhancer, we sought to characterize the global molecular signature that uniquely identifies this population. We generated a mouse ES cell line with a targeted insertion of the Smarcd3-F6 enhancer driving expression of a fluorescent reporter and we differentiated these cells into cardiomyocytes using an established directed differentiation protocol (Figure 6A and Figure 6—figure supplement 1) (Wamstad et al., 2012). In this system, we find expression of Mesp1 is rapidly induced at early mesoderm stage (Day 3-Day 4) while expression of the cardiac precursor markers Nkx2-5 and Tbx5 initiate about a day later (Figure 6C). Expression of Smarcd3 as well as the Smarcd3-F6 reporter are detectable in ES cells and gradually decline until both are sharply induced at Day 4. At the same time, Mesp1 expression is rapidly downregulated (Figure 6B). As a result, Mesp1 and Smarcd3 mRNA are largely non-overlapping, temporally. We isolated total RNA from three biological replicates of sorted cells shortly after the initiation of reporter activity at the mesoderm stage (D4.75) and compared the gene expression profiles of GFP + to GFP- cells by RNA-seq (Figure 6D). GFP + cells expressed genes associated with early cardiovascular progenitors (e.g. Hand2, Gata4, and Meis1), while GFP- cells expressed genes associated with hematopoietic and other lineages. Among the GFP + population, we attempted to identify markers that were unique to either cardiomyocyte or endocardial progenitors. While cell type-specific markers for differentiated cells are well documented (eg. contractile proteins for cardiomyocytes vs Nfatc1 for endocardial cells) such markers are less well defined at the early stages when we are isolating mRNA. In addition, our differentiation protocol pushes cells towards a cardiomyocyte fate, thus we may not expect to see any endocardial cell markers. One gene of interest that appears to be depleted in our GFP + population/enriched in our GFP- population is Tal1/Scl. This gene had been implicated as a critical component of endocardial morphogenesis (Bussmann et al., 2007) and more recently has been shown to repress cardiomyogenesis in yolk sac vasculature and endocardium (Van Handel et al., 2012). A complete list of differentially expressed genes meeting a strict false discovery rate (FDR) of 0.02 (2%) is presented in Figure 6—Source data 1. Overall, these results indicate that cells labeled by the Smarcd3-F6 enhancer, both in vivo and in vitro, represent an early cardiac progenitor population.

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Figure 6—Source data 1

Complete list of differentially expressed genes meeting strict FDR of 0.02.

A mouse ESC line with the Smarcd3-F6 ^nlsEGFP reporter was differentiated toward the cardiac lineage (see methods for complete details). 18 hours after replating, GFP + cells were sorted from GFP- cells. Total RNA was extracted and analyzed by RNA-sequencing. Following bioinformatics analysis (see methods) a list of differentially expressed transcripts meeting a strict FDR of 0.02 was selected. This list of transcripts was used to generate the heatmap in Figure 3K.

https://doi.org/10.7554/eLife.03848.018

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One class of clones identified in our Mesp1^Cre-MADM clonal analysis included twin spots within the interventricular septum (IVS) between the left and right ventricles (Figure 7A–D and Figure 2). These twin spots formed a sharp boundary within the IVS, reminiscent of compartment boundaries classically defined in the Drosophila wing imaginal disc and mammalian midbrain-hindbrain boundary (Lawrence and Struhl, 1996; Dahmann and Basler, 1999). To determine if and when this boundary between the right ventricle and left ventricle is established, we performed temporally regulated genetic fate-mapping to mark early cardiac progenitors and followed their contribution later in the mature IVS. In order to mark cells that would contribute to the right ventricle, we used the Mef2c-AHF enhancer (Verzi et al., 2005) to drive expression of a fusion of the Dre recombinase (Anastassiadis et al., 2009) to the tamoxifen inducible ERT2 protein (Mef2cAHF^DreERT2). We found that this enhancer element is expressed very early in the embryo, in a domain that appeared to partially overlap with that of Smarcd3 (Figure 3C,H). Previous work using a constitutive Cre recombinase expressed under the control of the Mef2cAHF enhancer suggested that endothelial and myocardial components of the outflow tract, right ventricle, and IVS are derived from this population (Verzi et al., 2005). To label the complementary population of progenitors that contribute predominantly to the left ventricle, we targeted a tamoxifen inducible Cre recombinase to the Tbx5 locus (Tbx5^CreERT2) (Figure 7—figure supplement 1A–B). In the looped heart, expression of Tbx5 is restricted to the left ventricle and atria with very little expression in the right ventricle or outflow tract (Bruneau et al., 1999). Early labeling of Tbx5+ cells marked cells that were predominantly restricted to the left ventricle and atria (Figure 7E–F and 7I–L); scattered surface labeling on the right ventricle (Figure 7—figure supplement 1I–J) was also seen with early tamoxifen injection, but myocardial labeling was limited to the LV up to the junction with the RV at the IVS. Earlier observation at E8.5 following a pulse of tamoxifen at E6.5 revealed labeling in a restricted population of cells within the presumptive left ventricle (Figure 7—figure supplement 2), suggesting that the Tbx5 lineage restriction arises early and is not a consequence of later sorting out of cells. Labeling Mef2cAHF + cells at E6.5 marked a complementary population of cells that were largely restricted to the right ventricle and outflow tract (Figure 7G–H and Figure 7—figure supplement 3A). Using an intersectional reporter that responds to the combined activity of Cre and Dre we confirmed that these early Mef2cAHF + cells are also within the Smarcd3-F1 lineage (Figure 7M and Figure 7—figure supplement 3B–C). The expression of Tbx5 and the Mef2AHF enhancer appears to label complementary populations that are established prior to morphogenesis, which correspond, in part, to left and right ventricular precursors, respectively.

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Given the seemingly complementary lineage contributions of early Tbx5 and Mef2cAHF expressing progenitors to the mature heart, we sought to map their contributions and overlap within the interventricular septum. We performed simultaneously lineage tracing of Tbx5+ and Mef2cAHF + cells using separate reporters for Cre (Rosa^mTmG) and Dre (Rosa^nKmB) activity and found that the two populations of labeled cells appeared largely mutually exclusive, except perhaps a narrow stripe of cells at the border between the forming left and right ventricles (Figure 7N). We confirmed the existence of a small population of double positive cells early in development using the intersectional Cre/Dre reporter described previously. Labeling at E6.5 marked a small population of cells that would eventually go on to form a narrow ring of cells at the border between the left and right ventricles in the forming IVS (Figure 7O–P and Figure 7—figure supplement 3D). In summary, genetic lineage labeling of the earlyTbx5+ and Mef2cAHF + precursors along with our clonal analysis of early Mesp1+ progenitors that contribute to the IVS suggest that a compartment boundary between the future left and right ventricles is established early, prior to cardiac morphogenesis, and that early progenitors positive for both Tbx5 and Mef2cAHF will go on to contribute specifically to the forming IVS.

The existence of a multipotent cardiac progenitor that can contribute to all anatomic and cellular components of the mature heart has been predicted, but the identity and origins of such a progenitor has remained undefined. Our findings, summarized in Figure 8, pinpoint the existence of specified cardiac precursors in gastrulating mesoderm, and also highlight an unanticipated early segregation of first and second heart field progenitors in their contribution to distinct chambers of the developing heart. Further, we define the orderly progression of gene expression that parallels the commitment of nascent mesoderm to a cardiovascular fate. In particular, the expression of Smarcd3 labels a population primarily comprised of the earliest specified precursors, which can be identified in vivo and in ES cell-derived differentiating cells.

Figure 8

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Our results show that mesoderm is rapidly specified at or before E6.0 to E7.5 into discrete fates that anticipate anatomical localization. Retrospective lineage tracing using a cardiac marker suggested the existence of a precursor that could contribute to all chambers of the heart (Meilhac et al., 2004b). Our results, and those of Lescroart et al. (2014), suggest that such a precursor could only exist prior to gastrulation, likely in the epiblast or shortly thereafter, and that early mesoderm is rapidly patterned such that specific cells are shunted towards a unique path that assigns them to a specific cardiac compartment. Whether these mesodermal cells retain plasticity and could adopt different fates if transplanted elsewhere in the embryo is not clear. We also cannot at this point distinguish between a scenario where Mesp1+ cells are pre-patterned or the gradual restriction of Mesp1+ cells to different lineage fates during gastrulation in response to signaling cues. Understanding which molecular cues induce the emergence and patterning of this population of early cardiac precursors will be of considerable interest, particularly in the context of regenerative cell therapy, and especially given the apparent heterogeneity of early cardiac progenitors.

The Smarcd3-expressing cardiac precursor population is already specified into separate populations that will contribute to the left ventricle and atria, as marked by Tbx5, and the right ventricle and outflow tract, as labeled by the Mef2cAHF enhancer. These results are consistent with genetic tracing of cardiac precursors expressing Hcn4, which contribute to the left ventricle and conduction system (Später et al., 2013), and we additionally show, at both a population and single cell level, that both left and right ventricular progenitors are independently established early in development and are separated by a compartment boundary. This compartment boundary is derived from a unique population of early progenitors that express Tbx5 as well as a subpopulation of cells co-expressing the Mef2cAHF enhancer, suggesting that a relatively small number of cells may be important in establishing and/or maintaining this border between the left and right ventricles. Understanding the molecular and cellular basis of this compartment boundary, and its importance in ventricular septation, will be of great relevance to congenital heart defects. This knowledge will also be relevant for disease modeling from stem cells, or for regenerative strategies, since knowing whether a progenitor cell is committed to become a right ventricular cardiomyocyte vs an atrial cardiomyocyte, for example, could be critical depending upon the anatomic or cellular defect one is attempting to correct.

In organisms with a simpler heart, such as zebrafish or Ciona (Stolfi et al., 2010; Tolkin and Christiaen, 2012; Stainier et al., 1993; Keegan et al., 2004), there is also a very early specification of cardiac precursors. Indeed, fate mapping in the chick embryo suggests distinct origins of the outflow tract vs the remainder of the heart, but does not indicate a compartmentalization of left and right ventricular progenitors, nor what molecular identity early cardiac precursors adopt (Camp et al., 2012). Our data suggest that the mouse embryo, despite the complexity of its developing heart, has a very early patterning and allocation of mesodermal cells to specific cardiac fates and anatomical derivatives. In addition, it has been hypothesized that simpler organisms have integrated cardiac and pharyngeal mesoderm into a single lineage, the so-called cardiopharyngeal mesoderm (Stolfi et al., 2010). Retrospective clonal analysis in the mouse (Lescroart et al., 2010) suggests a conservation of shared cardiac and pharyngeal lineages; caveats applicable to chamber-specific lineage allocation, including the timing of clone induction and the promoter used to visualize clones, remain. Lescroart and colleagues more recently reported rare instances of Mesp1+ clones co-labeling head muscles and the heart (Lescroart et al., 2014). In our lineage labeling experiments, using the MADM system, we do not see convincing evidence for a common heart and head muscle progenitor. Additionally, Smarcd3+ precursors contribute to heart but not facial muscle. While our collective data do not support a common precursor for heart and facial muscle, we cannot definitively rule out a rare population of common progenitors.

Having a well-defined understanding of the developmental origins of patterned organs is critical for the development of regenerative strategies, and for our understanding of the basis of congenital malformations. For the future development of cellular therapies, the knowledge that molecularly distinct populations of progenitors contribute to distinct anatomic regions of the heart will guide the selection of the most appropriate cellular and molecular signature. More broadly, our results suggest a rapid and precise patterning of progenitor populations during gastrulation.

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Author details

1. W Patrick Devine

   1. Gladstone Institute of Cardiovascular Disease, San Francisco, United States
   2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, United States
   3. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
   4. Department of Pathology, University of California, San Francisco, San Francisco, United States

   Contribution

   WPD, Developed and analyzed the Mef2cAHFDreERT2 allele, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   patrick.devine@gladstone.ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

2. Joshua D Wythe

   1. Gladstone Institute of Cardiovascular Disease, San Francisco, United States
   2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, United States

   Contribution

   JDW, Developed and analyzed the Mef2cAHFDreERT2 allele, Analyzed the Tbx5 lineage, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Matthew George

   1. Gladstone Institute of Cardiovascular Disease, San Francisco, United States
   2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, United States
   3. Developmental and Stem Cell Biology Program, University of San Francisco, San Francisco, United States

   Contribution

   MG, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Kazuko Koshiba-Takeuchi

   1. Gladstone Institute of Cardiovascular Disease, San Francisco, United States
   2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, United States

   Contribution

   KK-T, Made the Tbx5CreERT2 mouse line, Conception and design, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Benoit G Bruneau

   1. Gladstone Institute of Cardiovascular Disease, San Francisco, United States
   2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, United States
   3. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
   4. Developmental and Stem Cell Biology Program, University of San Francisco, San Francisco, United States

   Contribution

   BGB, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   bbruneau@gladstone.ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

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