The deployment of cell lineages that form the mammalian heart.

The function of the mammalian heart depends on the interplay between different cardiac cell types. The deployment of these cells, with precise spatiotemporal regulation, is also important during development to establish the heart structure. In this Review, we discuss the diverse origins of cardiac cell types and the lineage relationships between cells of a given type that contribute to different parts of the heart. The emerging lineage tree shows the progression of cell fate diversification, with patterning cues preceding cell type segregation, as well as points of convergence, with overlapping lineages contributing to a given tissue. Several cell lineage markers have been identified. However, caution is required with genetic-tracing experiments in comparison with clonal analyses. Genetic studies on cell populations provided insights into the mechanisms for lineage decisions. In the past 3 years, results of single-cell transcriptomics are beginning to reveal cell heterogeneity and early developmental trajectories. Equating this information with the in vivo location of cells and their lineage history is a current challenge. Characterization of the progenitor cells that form the heart and of the gene regulatory networks that control their deployment is of major importance for understanding the origin of congenital heart malformations and for producing cardiac tissue for use in regenerative medicine.

Heart fields and cardiac morphogenesis.

In this review, we focus on two important steps in the formation of the embryonic heart: (i) the progressive addition of late differentiating progenitor cells from the second heart field that drives heart tube extension during looping morphogenesis, and (ii) the emergence of patterned proliferation within the embryonic myocardium that generates distinct cardiac chambers. During the transition between these steps, the major site of proliferation switches from progenitor cells outside the early heart to proliferation within the embryonic myocardium. The second heart field and ballooning morphogenesis concepts have major repercussions on our understanding of human heart development and disease. In particular, they provide a framework to dissect the origin of congenital heart defects and the regulation of myocardial proliferation and differentiation of relevance for cardiac repair.

Cardiac cell lineages that form the heart.

Myocardial cells ensure the contractility of the heart, which also depends on other mesodermal cell types for its function. Embryological experiments had identified the sources of cardiac precursor cells. With the advent of genetic engineering, novel tools have been used to reconstruct the lineage tree of cardiac cells that contribute to different parts of the heart, map the development of cardiac regions, and characterize their genetic signature. Such knowledge is of fundamental importance for our understanding of cardiogenesis and also for the diagnosis and treatment of heart malformations.

Itm2a is a Pax3 target gene, expressed at sites of skeletal muscle formation in vivo.

The paired-box homeodomain transcription factor Pax3 is a key regulator of the nervous system, neural crest and skeletal muscle development. Despite the important role of this transcription factor, very few direct target genes have been characterized. We show that Itm2a, which encodes a type 2 transmembrane protein, is a direct Pax3 target in vivo, by combining genetic approaches and in vivo chromatin immunoprecipitation assays. We have generated a conditional mutant allele for Itm2a, which is an imprinted gene, by flanking exons 2-4 with loxP sites and inserting an IRESnLacZ reporter in the 3' UTR of the gene. The LacZ reporter reproduces the expression profile of Itm2a, and allowed us to further characterize its expression at sites of myogenesis, in the dermomyotome and myotome of somites, and in limb buds, in the mouse embryo. We further show that Itm2a is not only expressed in adult muscle fibres but also in the satellite cells responsible for regeneration. Itm2a mutant mice are viable and fertile with no overt phenotype during skeletal muscle formation or regeneration. Potential compensatory mechanisms are discussed.

Direct molecular regulation of the myogenic determination gene Myf5 by Pax3, with modulation by Six1/4 factors, is exemplified by the -111 kb-Myf5 enhancer.

The Myf5 gene plays an important role in myogenic determination during mouse embryo development. Multiple genomic regions of the Mrf4-Myf5 locus have been characterised as enhancer sequences responsible for the complex spatiotemporal expression of the Myf5 gene at the onset of myogenesis. These include an enhancer sequence, located at -111 kb upstream of the Myf5 transcription start site, which is responsible of Myf5 activation in ventral somitic domains (Ribas et al., 2011. Dev. Biol. 355, 372-380). We show that the -111 kb-Myf5 enhancer also directs transgene expression in some limb muscles, and is active at foetal as well as embryonic stages. We have carried out further characterisation of the regulation of this enhancer and show that the paired-box Pax3 transcription factor binds to it in vitro as in vivo, and that Pax binding sites are essential for its activity. This requirement is independent of the previously reported regulation by TEAD transcription factors. Six1/4 which, like Pax3, are important upstream regulators of myogenesis, also bind in vivo to sites in the -111 kb-Myf5 enhancer and modulate its activity. The -111 kb-Myf5 enhancer therefore shares common functional characteristics with another Myf5 regulatory sequence, the hypaxial and limb 145 bp-Myf5 enhancer, both being directly regulated in vivo by Pax3 and Six1/4 proteins. However, in the case of the -111 kb-Myf5 enhancer, Six has less effect and we conclude that Pax regulation plays a major role in controlling this aspect of the Myf5 gene expression at the onset of myogenesis in the embryo.

Quantitative analysis of polarity in 3D reveals local cell coordination in the embryonic mouse heart.

Anisotropies that underlie organ morphogenesis have been quantified in 2D, taking advantage of a reference axis. However, morphogenesis is a 3D process and it remains a challenge to analyze cell polarities in 3D. Here, we have designed a novel procedure that integrates multidisciplinary tools, including image segmentation, statistical analyses, axial clustering and correlation analysis. The result is a sensitive and unbiased assessment of the significant alignment of cell orientations in 3D, compared with a random axial distribution. Taking the mouse heart as a model, we validate the procedure at the fetal stage, when cardiomyocytes are known to be aligned. At the embryonic stage, our study reveals that ventricular cells are already coordinated locally. The centrosome-nucleus axes and the cell division axes are biased in a plane parallel to the outer surface of the heart, with a minor transmural component. We show further alignment of these axes locally in the plane of the heart surface. Our method is generally applicable to other sets of vectors or axes in 3D tissues to map the regions where they show significant alignment.

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity.

How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles.

Asymmetric fate of the posterior part of the second heart field results in unexpected left/right contributions to both poles of the heart.

RATIONALE: The second heart field (SHF) contains progenitors of all heart chambers, excluding the left ventricle. The SHF is patterned, and the anterior region is known to be destined to form the outflow tract and right ventricle. OBJECTIVE: The aim of this study was to map the fate of the posterior SHF (pSHF). METHODS AND RESULTS: We examined the contribution of pSHF cells, labeled by lipophilic dye at the 4- to 6-somite stage, to regions of the heart at 20 to 25 somites, using mouse embryo culture. Cells more cranial in the pSHF contribute to the atrioventricular canal (AVC) and atria, whereas those more caudal generate the sinus venosus, but there is intermixing of fate throughout the pSHF. Caudal pSHF contributes symmetrically to the sinus venosus, but the fate of cranial pSHF is left/right asymmetrical. Left pSHF moves to dorsal left atrium and superior AVC, whereas right pSHF contributes to right atrium, ventral left atrium, and inferior AVC. Retrospective clonal analysis shows the relationships between AVC and atria to be clonal and that right and left progenitors diverge before first and second heart lineage separation. Cranial pSHF cells also contribute to the outflow tract: proximal and distal at 4 somites, and distal only at 6 somites. All outflow tract-destined cells are intermingled with those that will contribute to inflow and AVC. CONCLUSIONS: These observations show asymmetric fate of the pSHF, resulting in unexpected left/right contributions to both poles of the heart and can be integrated into a model of the morphogenetic movement of cells during cardiac looping.

Tracing cells for tracking cell lineage and clonal behavior.

Reconstructing the lineage of cells is central to understanding development and is now also an important issue in stem cell research. Technological advances in genetically engineered permanent cell labeling, together with a multiplicity of fluorescent markers and sophisticated imaging, open new possibilities for prospective and retrospective clonal analysis.

How to make a heart: the origin and regulation of cardiac progenitor cells.

The formation of the heart is a complex morphogenetic process that depends on the spatiotemporally regulated contribution of cardiac progenitor cells. These mainly derive from the splanchnic mesoderm of the first and second heart field (SHF), with an additional contribution of neurectodermally derived neural crest cells that are critical for the maturation of the arterial pole of the heart. The origin and distinguishing characteristics of the two heart fields, as well as the relation of the SHF to the proepicardial organ and to a proposed third heart field are still subjects of debate. In the last ten years many genes that function in the SHF have been identified, leading to the establishment of a gene regulatory network in the mouse embryo. It is becoming increasingly evident that distinct gene networks control subdomains of the SHF that contribute to different parts of the heart. Although there is now extensive information about mutant phenotypes that reflect problems in the integration of progenitor cells into the developing heart, relatively little is known about the mechanisms that regulate SHF cell behavior. This important source of cardiac progenitor cells must be maintained as a proliferative, undifferentiated cell population. Selected subpopulations, at different development stages, are directed to myocardial, and also to smooth muscle and endothelial cell fates, as they integrate into the heart. Analysis of signaling pathways that impact the SHF, as well as regulatory factors, is beginning to reveal mechanisms that control cardiac progenitor cell behavior.

Biphasic development of the mammalian ventricular conduction system.

RATIONALE: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. OBJECTIVE: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. METHODS AND RESULTS: A retrospective clonal analysis using the alpha-cardiac actin(nlaacZ/+) mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using connexin40-driven enhanced green fluorescent protein expression. Two classes of clusters containing conductive cells were obtained. Mixed clusters, composed of conductive and working myocytes, reveal that both cell types develop from common progenitor cells, whereas smaller unmixed clusters, composed exclusively of conductive cells, show that proliferation continues after lineage restriction to the conduction system lineage. Differences in the working component of mixed clusters between the right and left ventricles reveal distinct progenitor cell histories in these cardiac compartments. These results are supported by genetic fate mapping using Cre recombinase revealing progressive restriction of connexin40-positive myocytes to a conductive fate. CONCLUSIONS: A biphasic mode of development, lineage restriction followed by limited outgrowth, underlies establishment of the mammalian ventricular conduction system.

Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries.

RATIONALE: The genes encoding fibroblast growth factor (FGF) 8 and 10 are expressed in the anterior part of the second heart field that constitutes a population of cardiac progenitor cells contributing to the arterial pole of the heart. Previous studies of hypomorphic and conditional Fgf8 mutants show disrupted outflow tract (OFT) and right ventricle (RV) development, whereas Fgf10 mutants do not have detectable OFT defects. OBJECTIVES: Our aim was to investigate functional overlap between Fgf8 and Fgf10 during formation of the arterial pole. METHODS AND RESULTS: We generated mesodermal Fgf8; Fgf10 compound mutants with MesP1Cre. The OFT/RV morphology in these mutants was affected with variable penetrance; however, the incidence of embryos with severely affected OFT/RV morphology was significantly increased in response to decreasing Fgf8 and Fgf10 gene dosage. Fgf8 expression in the pharyngeal arch ectoderm is important for development of the pharyngeal arch arteries and their derivatives. We now show that Fgf8 deletion in the mesoderm alone leads to pharyngeal arch artery phenotypes and that these vascular phenotypes are exacerbated by loss of Fgf10 function in the mesodermal core of the arches. CONCLUSIONS: These results show functional overlap of FGF8 and FGF10 signaling from second heart field mesoderm during development of the OFT/RV, and from pharyngeal arch mesoderm during pharyngeal arch artery formation, highlighting the sensitivity of these key aspects of cardiovascular development to FGF dosage.

Pax3:Foxc2 reciprocal repression in the somite modulates muscular versus vascular cell fate choice in multipotent progenitors.

Maintenance of multipotency and how cells exit this state to adopt a specific fate are central questions in stem cell biology. During vertebrate development, multipotent cells of the dorsal somite, the dermomyotome, give rise to different lineages such as vascular smooth and skeletal muscle, regulated by the transcription factors Foxc2 and Pax3, respectively. Here we show reciprocal inhibition between Pax3 and Foxc2 in the mouse embryo. Using both genetic approaches and manipulation of external signals in somite explants, we demonstrate that the Pax3:Foxc2 ratio modulates myogenic versus vascular cell fates. This provides insight into how cell fate choices are orchestrated by these lineage genes in the dermomyotome.

Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program.

Pax3/7-dependent stem cells play an essential role in skeletal muscle development. We now show that Fgfr4 lies genetically downstream from Pax3 and is a direct target. In chromatin immunoprecipitation (ChIP)-on-chip experiments, Pax3 binds to a sequence 3' of the Fgfr4 gene that directs Pax3-dependent expression at sites of myogenesis in transgenic mouse embryos. The activity of this regulatory element is also partially dependent on E-boxes, targets of the myogenic regulatory factors, which are expressed as progenitor cells enter the myogenic program. Other FGF signaling components, notably Sprouty1, are also regulated by Pax3. In vivo manipulation of Sprouty expression reveals that FGF signaling affects the balance between Pax-positive progenitor cells and committed myoblasts. These results provide new insight into the Pax-initiated regulatory network that modulates stem cell maintenance versus tissue differentiation.

Atrial myocardium derives from the posterior region of the second heart field, which acquires left-right identity as Pitx2c is expressed.

Splanchnic mesoderm in the region described as the second heart field (SHF) is marked by Islet1 expression in the mouse embryo. The anterior part of this region expresses a number of markers, including Fgf10, and the contribution of these cells to outflow tract and right ventricular myocardium has been established. We now show that the posterior region also has myocardial potential, giving rise specifically to differentiated cells of the atria. This conclusion is based on explant experiments using endogenous and transgenic markers and on DiI labelling, followed by embryo culture. Progenitor cells in the right or left posterior SHF contribute to the right or left common atrium, respectively. Explant experiments with transgenic embryos, in which the transgene marks the right atrium, show that atrial progenitor cells acquire right-left identity between the 4- and 6-somite stages, at the time when Pitx2c is first expressed. Manipulation of Pitx2c, by gain- and loss-of-function, shows that it represses the transgenic marker of right atrial identity. A repressive effect is also seen on the proliferation of cells in the left sinus venosus and in cultured explants from the left side of the posterior SHF. This report provides new insights into the contribution of the SHF to atrial myocardium and the effect of Pitx2c on the formation of the left atrium.

Integration of embryonic and fetal skeletal myogenic programs at the myosin light chain 1f/3f locus.

The genetic control of skeletal muscle differentiation at the onset of myogenesis in the embryo is relatively well understood compared to the formation of muscle during the fetal period giving rise to the bulk of skeletal muscle fibers at birth. The Mlc1f/3f (Myl1) locus encodes two alkali myosin light chains, Mlc1f and Mlc3f, from two promoters that are differentially regulated during development. The Mlc1f promoter is active in embryonic, fetal and adult fast skeletal muscle whereas the Mlc3f promoter is upregulated during fetal development and remains on in adult fast skeletal muscle. Two enhancer elements have been identified at the mammalian Mlc1f/3f locus, a 3' element active at all developmental stages and an intronic enhancer activated during fetal development. Here, using transgenesis, we demonstrate that these enhancers act combinatorially to confer the spatial, temporal and quantitative expression profile of the endogenous Mlc3f promoter. Using double reporter transgenes we demonstrate that each enhancer can activate both Mlc1f and Mlc3f promoters in vivo, revealing enhancer sharing rather than exclusive enhancer-promoter interactions. Finally, we demonstrate that the fetal activated enhancer contains critical E-box myogenic regulatory factor binding sites and that enhancer activation is impaired in vivo in the absence of myogenin but not in the absence of innervation. Together our observations provide insights into the regulation of fetal myogenesis and the mechanisms by which temporally distinct genetic programs are integrated at a single locus.

Myocardium at the base of the aorta and pulmonary trunk is prefigured in the outflow tract of the heart and in subdomains of the second heart field.

Outflow tract myocardium in the mouse heart is derived from the anterior heart field, a subdomain of the second heart field. We have recently characterized a transgene (y96-Myf5-nlacZ-16), which is expressed in the inferior wall of the outflow tract and then predominantly in myocardium at the base of the pulmonary trunk. Transgene A17-Myf5-nlacZ-T55 is expressed in the developing heart in a complementary pattern to y96-Myf5-nlacZ-16, in the superior wall of the outflow tract at E10.5 and in myocardium at the base of the aorta at E14.5. At E9.5, the two transgenes are transcribed in different subdomains of the anterior heart field. A clonal analysis of cardiomyocytes in the outflow tract, at E10.5 and E14.5, provides insight into the behaviour of myocardial cells and their progenitors. At E14.5, most clones are located at the base of either the pulmonary trunk or the aorta, indicating that these derive from distinct myocardial domains. At E10.5, clones are observed in subdomains of the outflow tract. The distribution of small clones indicates proliferative differences, whereas regionalization of large clones, that derive from an early myocardial progenitor cell, reflect coherent cell growth in the heart field as well as in the myocardium. Our results suggest that myocardial differences at the base of the great arteries are prefigured in distinct progenitor cell populations in the anterior heart field, with important implications for understanding the etiology of congenital heart defects affecting the arterial pole of the heart.

The A17 enhancer directs expression of Myf5 to muscle satellite cells but Mrf4 to myonuclei.

The myogenic regulatory factors, Myf5 and Mrf4, play key roles in the specification and differentiation of skeletal muscle, respectively. Many cis-acting regulatory elements at the Mrf4/Myf5 locus have been identified, including the A17 enhancer. During development, A17 in conjunction with the Mrf4 or Myf5 promoter, directs transgene expression either to embryonic or fetal muscles. We now show that this enhancer also regulates Mrf4/Myf5 transcription in the adult. A17 linked to the Myf5 promoter drives expression in muscle satellite cells, whereas with the Mrf4 promoter, A17 directs transgene expression to myonuclei. Interestingly, expression of A17-Mrf4-nlacZ transgenes in myonuclei varies between muscles, revealing muscle autonomous transcriptional regulation. During muscle repair, satellite cells are induced to proliferate and differentiate to provide new myonuclei. A17 directs Myf5 expression in satellite cell progeny while it only drives the Mrf4 promoter after differentiation. Importantly, therefore, this promoter-specific activity directed by A17 reflects aspects of the expression profiles of the endogenous Myf5 and Mrf4 genes.