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.

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.

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.

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.

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.

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.

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 clonal origin of myocardial cells in different regions of the embryonic mouse heart.

When and how cells form and pattern the myocardium is a central issue for heart morphogenesis. Many genes are differentially expressed and function in subsets of myocardial cells. However, the lineage relationships between these cells remain poorly understood. To examine this, we have adopted a retrospective approach in the mouse embryo, based on the use of the laacZ reporter gene, targeted to the alpha-cardiac actin locus. This clonal analysis demonstrates the existence of two lineages that segregate early from a common precursor. The primitive left ventricle and the presumptive outflow tract are derived exclusively from a single lineage. Unexpectedly, all other regions of the heart, including the primitive atria, are colonized by both lineages. These results are not consistent with the prespecification of the cardiac tube as a segmented structure. They are discussed in the context of different heart fields and of the evolution of the heart.

Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis.

During heart morphogenesis, cardiac chambers arise by differential expansion of regions of the primitive cardiac tube. This process is under the control of specific transcription factors such as Tbx5 and dHAND. To gain insight into the cellular mechanisms that underlie cardiogenesis, we have used a retrospective clonal approach based on the spontaneous recombination of an nlaacZ reporter gene targeted to the murine alpha-cardiac actin locus. We show that clonal growth of myocardial cells is oriented. At embryonic day (E) 10.5, the shape of clones is characteristic of a given cardiac region and reflects its morphology. This is already detectable in the primitive cardiac tube at E8.5, and is maintained after septation at E14.5 with additional modulations. The clonal analysis reveals new subdivisions of the myocardium, including an interventricular boundary region. Our results show that the myocardium, from the time of its formation, is a polarized and regionalized tissue and point to the role of oriented clonal cell growth in cardiac chamber morphogenesis.

Rotation of the myocardial wall of the outflow tract is implicated in the normal positioning of the great arteries.

Congenital heart defects frequently involve a failure of outflow tract (OFT) formation during development. We analyzed the remodeling of the OFT, using the y96-Myf5-nlacZ-16 transgene, which marks a subpopulation of myocardial cells of the pulmonary trunk. Expression analyses of reporter transcript and protein suggest that the myocardial wall of the OFT rotates before and during the formation of the great arteries. Rotational movement was confirmed by Di-I injection experiments with cultured embryos. We subsequently examined the expression of the transgene in mouse models for OFT defects. In hearts with persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), or transposition of the great arteries, rotation of the myocardial wall of the OFT is arrested or fails to initiate. This is observed in Splotch (Pax3) mutants with PTA or DORV and may be a result of defects in neural crest migration, known to affect OFT septation. However, in Pitx2deltac mutant embryos, where cardiac neural crest cells are present in the heart, PTA and DORV are again associated with a rotation defect. This is also seen in Pitx2deltac mutants, which have transposition of the great arteries. Because Pitx2c is involved in left-right signaling, these results suggest that embryonic laterality affects rotation of the myocardial wall during OFT maturation. We propose that failure of normal rotation of OFT myocardium may underlie major forms of congenital heart disease.

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.

The anterior heart-forming field: voyage to the arterial pole of the heart.

Studies of vertebrate heart development have identified key genes and signalling molecules involved in the formation of a myocardial tube from paired heart-forming fields in splanchnic mesoderm. The posterior region of the paired heart-forming fields subsequently contributes myocardial precursor cells to the inflow region or venous pole of the heart. Recently, a population of myocardial precursor cells in chick and mouse embryos has been identified in pharyngeal mesoderm anterior to the early heart tube. This anterior heart-forming field gives rise to myocardium of the outflow region or arterial pole of the heart. The amniote heart is therefore derived from two myocardial precursor cell populations, which appear to be regulated by distinct genetic programmes. Discovery of the anterior heart-forming field has important implications for the interpretation of cardiac defects in mouse mutants and for the study of human congenital heart disease.

Congenital heart defects in Fgfr2-IIIb and Fgf10 mutant mice.

OBJECTIVE: Myocardial progenitor cells expressing Fgf10 give rise to the outflow tract and right ventricle of the mammalian heart. In order to define the role of fibroblast growth factor (FGF) signaling in this process we investigated whether Fgf10 or the major Fgf10 receptor Fgfr2-IIIb are required for normal heart development. METHODS: The cardiac phenotype of Fgf10 and Fgfr2-IIIb mutant mice was analysed by histology, scanning electron microscopy and gene and transgene expression studies. RESULTS: Outflow tract formation from Fgf10 expressing progenitor cells occurs normally in Fgf10 mutant embryos and in the majority of Fgfr2-IIIb mutant embryos; a proportion of Fgfr2-IIIb mutant embryos, however, display outflow tract and right ventricular hypoplasia. The predominant cardiac defects in Fgfr2-IIIb mutant embryos are ventricular septal defects associated with overriding aorta or double outlet right ventricle. In addition, loss of Fgfr2-IIIb is associated with ventricular anomalies including a thin myocardial wall, abnormal trabeculation and muscular ventricular septal defects. In contrast, Fgf10 is required to correctly position the heart in the thoracic cavity but not for outflow tract septation. Both Fgf10 and Fgfr2-IIIb mutant embryos lack pulmonary arteries and veins. CONCLUSIONS: Fgfr2-IIIb and Fgf10 mutant mice have distinct roles during cardiac morphogenesis, although neither gene is essential for outflow tract elongation from Fgf10 expressing progenitor cells. Fgfr2-IIIb and Fgf10 mutant mice provide new models for common components of congenital heart disease.

Right ventricular myocardium derives from the anterior heart field.

The mammalian heart develops from a primary heart tube, which is formed by fusion of bilateral cardiac territories in which myocardial and endothelial cells have already begun to differentiate from splanchnic mesoderm. A population of myocardial precursors has been identified in pharyngeal mesoderm, anterior to the early heart tube. Cell labeling studies have indicated that this novel territory, called the anterior heart field (AHF), gives rise to the myocardial wall of the outflow tract. We now report that not only the myocardium of the outflow tract but also myocardial cells of the embryonic right ventricle are derived from this source. Explants of pharyngeal mesoderm or of the early heart tube were cultured from transgenic mice in which transgene expression marks different regions of the heart. Pharyngeal mesoderm from 5 to 7 somite embryos gives rise to cardiomyocytes with right ventricular and outflow tract identities, whereas the heart tube as this stage has an essentially left ventricular identity. DiI labeling confirms that the early heart tube is destined to contribute to the embryonic left ventricle and indicates that right ventricular myocardium is added from extracardiac mesoderm. Retrospective clonal analysis of the heart at embryonic day (E) 10.5 reveals the existence of a clonal boundary in the interventricular region, which appears during ventricular septation, underlining different origins of the two ventricular compartments. This study demonstrates the differences in the embryological origin of right and left ventricular myocardium, which has important implications for congenital heart disease.

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.

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.