Retinoic acid signalling regulates branchiomeric neck muscle development at the head/trunk interface.

Skeletal muscles of the head and trunk originate in distinct lineages with divergent regulatory programmes converging on activation of myogenic determination factors. Branchiomeric head and neck muscles share a common origin with cardiac progenitor cells in cardiopharyngeal mesoderm (CPM). The retinoic acid (RA) signalling pathway is required during a defined early time window for normal deployment of cells from posterior CPM to the heart. Here, we show that blocking RA signalling in the early mouse embryo also results in selective loss of the trapezius neck muscle, without affecting other skeletal muscles. RA signalling is required for robust expression of myogenic determination factors in posterior CPM and subsequent expansion of the trapezius primordium. Lineage-specific activation of a dominant-negative RA receptor reveals that trapezius development is not regulated by direct RA signalling to myogenic progenitor cells in CPM, or through neural crest cells, but indirectly through the somitic lineage, closely apposed with posterior CPM in the early embryo. These findings suggest that trapezius development is dependent on precise spatiotemporal interactions between cranial and somitic mesoderm at the head/trunk interface.

Emergence of heart and branchiomeric muscles in cardiopharyngeal mesoderm.

Branchiomeric muscles of the head and neck originate in a population of cranial mesoderm termed cardiopharyngeal mesoderm that also contains progenitor cells contributing to growth of the embryonic heart. Retrospective lineage analysis has shown that branchiomeric muscles share a clonal origin with parts of the heart, indicating the presence of common heart and head muscle progenitor cells in the early embryo. Genetic lineage tracing and functional studies in the mouse, as well as in Ciona and zebrafish, together with recent experiments using single cell transcriptomics and multipotent stem cells, have provided further support for the existence of bipotent head and heart muscle progenitor cells. Current challenges concern defining where and when such common progenitor cells exist in mammalian embryos and how alternative myogenic derivatives emerge in cardiopharyngeal mesoderm. Addressing these questions will provide insights into mechanisms of cell fate acquisition and the evolution of vertebrate musculature, as well as clinical insights into the origins of muscle restricted myopathies and congenital defects affecting craniofacial and cardiac development.

Single Cell Approaches to Understand the Earliest Steps in Heart Development.

PURPOSE OF REVIEW: Cardiac progenitors are the building blocks of the heart. Our knowledge, on how these progenitors build the heart, has considerably increased over the last two decades with the development of single cell approaches. We discuss the lessons learnt from clonal analyses and from single cell sequencing technologies on the understanding of the earliest steps of cardiac specification and lineage segregation. RECENT FINDINGS: While experiments were initially performed at the population level, the development of approaches to investigate heart development at the single cell resolution has clearly demonstrated that cardiac progenitors are highly heterogeneous, with different progenitors contributing to different cardiac regions and different cardiac cell types. Some critical transcriptional determinants have also been identified for cardiac progenitor specification. Single cell approaches have finally provided insights into the spatio-temporal specification of unipotent and multipotent cardiac progenitors and provided a framework for investigating congenital heart defects.

Ectopic expression of Hoxb1 induces cardiac and craniofacial malformations.

Members of the large family of Hox transcription factors are encoded by genes whose tightly regulated expression in development and in space within different embryonic tissues confer positional identity from the neck to the tips of the limbs. Many structures of the face, head, and heart develop from cell populations expressing few or no Hox genes. Hoxb1 is the member of its chromosomal cluster expressed in the most rostral domain during vertebrate development, but never by the multipotent neural crest cell population anterior to the cerebellum. We have developed a novel floxed transgenic mouse line, CAG-Hoxb1,-EGFP (CAG-Hoxb1), which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP overexpression. When induced within the neural crest lineage, pups die at birth. A variable phenotype develops from E11.5 on, associating frontonasal hypoplasia/aplasia, micrognathia/agnathia, major ocular and forebrain anomalies, and cardiovascular malformations. Neural crest derivatives in the body appear unaffected. Transcription of effectors of developmental signaling pathways (Bmp, Shh, Vegfa) and transcription factors (Pax3, Sox9) is altered in mutants. These outcomes emphasize that repression of Hoxb1, along with other paralog group 1 and 2 Hox genes, is strictly necessary in anterior cephalic NC for craniofacial, visual, auditory, and cardiovascular development.

Clonal analysis reveals a common origin between nonsomite-derived neck muscles and heart myocardium.

Neck muscles constitute a transition zone between somite-derived skeletal muscles of the trunk and limbs, and muscles of the head, which derive from cranial mesoderm. The trapezius and sternocleidomastoid neck muscles are formed from progenitor cells that have expressed markers of cranial pharyngeal mesoderm, whereas other muscles in the neck arise from Pax3-expressing cells in the somites. Mef2c-AHF-Cre genetic tracing experiments and Tbx1 mutant analysis show that nonsomitic neck muscles share a gene regulatory network with cardiac progenitor cells in pharyngeal mesoderm of the second heart field (SHF) and branchial arch-derived head muscles. Retrospective clonal analysis shows that this group of neck muscles includes laryngeal muscles and a component of the splenius muscle, of mixed somitic and nonsomitic origin. We demonstrate that the trapezius muscle group is clonally related to myocardium at the venous pole of the heart, which derives from the posterior SHF. The left clonal sublineage includes myocardium of the pulmonary trunk at the arterial pole of the heart. Although muscles derived from the first and second branchial arches also share a clonal relationship with different SHF-derived parts of the heart, neck muscles are clonally distinct from these muscles and define a third clonal population of common skeletal and cardiac muscle progenitor cells within cardiopharyngeal mesoderm. By linking neck muscle and heart development, our findings highlight the importance of cardiopharyngeal mesoderm in the evolution of the vertebrate heart and neck and in the pathophysiology of human congenital disease.

The novel transmembrane protein Tmem2 is essential for coordination of myocardial and endocardial morphogenesis.

Coordination between adjacent tissues plays a crucial role during the morphogenesis of developing organs. In the embryonic heart, two tissues - the myocardium and the endocardium - are closely juxtaposed throughout their development. Myocardial and endocardial cells originate in neighboring regions of the lateral mesoderm, migrate medially in a synchronized fashion, collaborate to create concentric layers of the heart tube, and communicate during formation of the atrioventricular canal. Here, we identify a novel transmembrane protein, Tmem2, that has important functions during both myocardial and endocardial morphogenesis. We find that the zebrafish mutation frozen ventricle (frv) causes ectopic atrioventricular canal characteristics in the ventricular myocardium and endocardium, indicating a role of frv in the regional restriction of atrioventricular canal differentiation. Furthermore, in maternal-zygotic frv mutants, both myocardial and endocardial cells fail to move to the midline normally, indicating that frv facilitates cardiac fusion. Positional cloning reveals that the frv locus encodes Tmem2, a predicted type II single-pass transmembrane protein. Homologs of Tmem2 are present in all examined vertebrate genomes, but nothing is known about its molecular or cellular function in any context. By employing transgenes to drive tissue-specific expression of tmem2, we find that Tmem2 can function in the endocardium to repress atrioventricular differentiation within the ventricle. Additionally, Tmem2 can function in the myocardium to promote the medial movement of both myocardial and endocardial cells. Together, our data reveal that Tmem2 is an essential mediator of myocardium-endocardium coordination during cardiac morphogenesis.

Lineage tree for the venous pole of the heart: clonal analysis clarifies controversial genealogy based on genetic tracing.

RATIONALE: Genetic tracing experiments and cell lineage analyses are complementary approaches that give information about the progenitor cells of a tissue. Approaches based on gene expression have led to conflicting views about the origin of the venous pole of the heart. Whereas the heart forms from 2 sources of progenitor cells, the first and second heart fields, genetic tracing has suggested a distinct origin for caval vein myocardium, from a proposed third heart field. OBJECTIVE: To determine the cell lineage history of the myocardium at the venous pole of the heart. METHODS AND RESULTS: We used retrospective clonal analyses to investigate lineage segregation for myocardium at the venous pole of the mouse heart, independent of gene expression. CONCLUSIONS: Our lineage analysis unequivocally shows that caval vein and atrial myocardium share a common origin and demonstrates a clonal relationship between the pulmonary vein and progenitors of the left venous pole. Clonal characteristics give insight into the development of the veins. Unexpectedly, we found a lineage relationship between the venous pole and part of the arterial pole, which is derived exclusively from the second heart field. Integration of results from genetic tracing into the lineage tree adds a further temporal dimension to this reconstruction of the history of venous myocardium and the arterial pole.

Clonal analysis reveals common lineage relationships between head muscles and second heart field derivatives in the mouse embryo.

Head muscle progenitors in pharyngeal mesoderm are present in close proximity to cells of the second heart field and show overlapping patterns of gene expression. However, it is not clear whether a single progenitor cell gives rise to both heart and head muscles. We now show that this is the case, using a retrospective clonal analysis in which an nlaacZ sequence, converted to functional nlacZ after a rare intragenic recombination event, is targeted to the alpha(c)-actin gene, expressed in all developing skeletal and cardiac muscle. We distinguish two branchiomeric head muscle lineages, which segregate early, both of which also contribute to myocardium. The first gives rise to the temporalis and masseter muscles, which derive from the first branchial arch, and also to the extraocular muscles, thus demonstrating a contribution from paraxial as well as prechordal mesoderm to this anterior muscle group. Unexpectedly, this first lineage also contributes to myocardium of the right ventricle. The second lineage gives rise to muscles of facial expression, which derive from mesoderm of the second branchial arch. It also contributes to outflow tract myocardium at the base of the arteries. Further sublineages distinguish myocardium at the base of the aorta or pulmonary trunk, with a clonal relationship to right or left head muscles, respectively. We thus establish a lineage tree, which we correlate with genetic regulation, and demonstrate a clonal relationship linking groups of head muscles to different parts of the heart, reflecting the posterior movement of the arterial pole during pharyngeal morphogenesis.

Mesp1 controls the chromatin and enhancer landscapes essential for spatiotemporal patterning of early cardiovascular progenitors.

The mammalian heart arises from various populations of Mesp1-expressing cardiovascular progenitors (CPs) that are specified during the early stages of gastrulation. Mesp1 is a transcription factor that acts as a master regulator of CP specification and differentiation. However, how Mesp1 regulates the chromatin landscape of nascent mesodermal cells to define the temporal and spatial patterning of the distinct populations of CPs remains unknown. Here, by combining ChIP-seq, RNA-seq and ATAC-seq during mouse pluripotent stem cell differentiation, we defined the dynamic remodelling of the chromatin landscape mediated by Mesp1. We identified different enhancers that are temporally regulated to erase the pluripotent state and specify the pools of CPs that mediate heart development. We identified Zic2 and Zic3 as essential cofactors that act with Mesp1 to regulate its transcription-factor activity at key mesodermal enhancers, thereby regulating the chromatin remodelling and gene expression associated with the specification of the different populations of CPs in vivo. Our study identifies the dynamics of the chromatin landscape and enhancer remodelling associated with temporal patterning of early mesodermal cells into the distinct populations of CPs that mediate heart development.

Cardiopharyngeal Progenitor Specification: Multiple Roads to the Heart and Head Muscles.

During embryonic development, the heart arises from various sources of undifferentiated mesodermal progenitors, with an additional contribution from ectodermal neural crest cells. Mesodermal cardiac progenitors are plastic and multipotent, but are nevertheless specified to a precise heart region and cell type very early during development. Recent findings have defined both this lineage plasticity and early commitment of cardiac progenitors, using a combination of single-cell and population analyses. In this review, we discuss several aspects of cardiac progenitor specification. We discuss their markers, fate potential in vitro and in vivo, early segregation and commitment, and also intrinsic and extrinsic cues regulating lineage restriction from multipotency to a specific cell type of the heart. Finally, we also discuss the subdivisions of the cardiopharyngeal field, and the shared origins of the heart with other mesodermal derivatives, including head and neck muscles.

Hox-dependent coordination of mouse cardiac progenitor cell patterning and differentiation.

Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression of Hoxb1 in the anterior SHF results in hypoplastic right ventricle. Activation of Hoxb1 in embryonic stem cells arrests cardiac differentiation, whereas Hoxb1-deficient mouse embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking both Hoxb1 and its paralog Hoxa1 results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.

Hox and Tale transcription factors in heart development and disease.

Hox genes are highly conserved transcription factors with critical functions during development, in particular for patterning the antero-posterior axis of the embryo. Their action is very often associated with cofactors including the TALE family transcription factors. From Drosophila to vertebrates, Hox genes have been shown to have a major role in heart development. In this review, we focus on the increasing evidence implicating the anterior Hox genes and the Tale family members during heart development both in the cardiac mesoderm and in neural crest cells. Congenital heart defects are the leading cause of death in the first year of life and a better understanding of the role of Hox and Tale factors is highly relevant to human pathologies and will provide novel mechanistic insights into the underlying defects.

Universality of clone dynamics during tissue development.

The emergence of complex organs is driven by the coordinated proliferation, migration and differentiation of precursor cells. The fate behaviour of these cells is reflected in the time evolution their progeny, termed clones, which serve as a key experimental observable. In adult tissues, where cell dynamics is constrained by the condition of homeostasis, clonal tracing studies based on transgenic animal models have advanced our understanding of cell fate behaviour and its dysregulation in disease (1, 2). But what can be learned from clonal dynamics in development, where the spatial cohesiveness of clones is impaired by tissue deformations during tissue growth? Drawing on the results of clonal tracing studies, we show that, despite the complexity of organ development, clonal dynamics may converge to a critical state characterized by universal scaling behaviour of clone sizes. By mapping clonal dynamics onto a generalization of the classical theory of aerosols, we elucidate the origin and range of scaling behaviours and show how the identification of universal scaling dependences may allow lineage-specific information to be distilled from experiments. Our study shows the emergence of core concepts of statistical physics in an unexpected context, identifying cellular systems as a laboratory to study non-equilibrium statistical physics.

Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq.

Mouse heart development arises from Mesp1-expressing cardiovascular progenitors (CPs) that are specified during gastrulation. The molecular processes that control early regional and lineage segregation of CPs have been unclear. We performed single-cell RNA sequencing of wild-type and Mesp1-null CPs in mice. We showed that populations of Mesp1 CPs are molecularly distinct and span the continuum between epiblast and later mesodermal cells, including hematopoietic progenitors. Single-cell transcriptome analysis of Mesp1-deficient CPs showed that Mesp1 is required for the exit from the pluripotent state and the induction of the cardiovascular gene expression program. We identified distinct populations of Mesp1 CPs that correspond to progenitors committed to different cell lineages and regions of the heart, identifying the molecular features associated with early lineage restriction and regional segregation of the heart at the early stage of mouse gastrulation.

Mesp1 controls the speed, polarity, and directionality of cardiovascular progenitor migration.

During embryonic development, Mesp1 marks the earliest cardiovascular progenitors (CPs) and promotes their specification, epithelial-mesenchymal transition (EMT), and cardiovascular differentiation. However, Mesp1 deletion in mice does not impair initial CP specification and early cardiac differentiation but induces cardiac malformations thought to arise from a defect of CP migration. Using inducible gain-of-function experiments during embryonic stem cell differentiation, we found that Mesp2, its closest homolog, was as efficient as Mesp1 at promoting CP specification, EMT, and cardiovascular differentiation. However, only Mesp1 stimulated polarity and directional cell migration through a cell-autonomous mechanism. Transcriptional analysis and chromatin immunoprecipitation experiments revealed that Mesp1 and Mesp2 activate common target genes that promote CP specification and differentiation. We identified two direct Mesp1 target genes, Prickle1 and RasGRP3, that are strongly induced by Mesp1 and not by Mesp2 and that control the polarity and the speed of cell migration. Altogether, our results identify the molecular interface controlled by Mesp1 that links CP specification and cell migration.

Uncovering the Number and Clonal Dynamics of Mesp1 Progenitors during Heart Morphogenesis.

The heart arises from distinct sources of cardiac progenitors that independently express Mesp1 during gastrulation. The precise number of Mesp1 progenitors that are specified during the early stage of gastrulation, and their clonal behavior during heart morphogenesis, is currently unknown. Here, we used clonal and mosaic tracing of Mesp1-expressing cells combined with quantitative biophysical analysis of the clonal data to define the number of cardiac progenitors and their mode of growth during heart development. Our data indicate that the myocardial layer of the heart derive from ∼250 Mesp1-expressing cardiac progenitors born during gastrulation. Despite arising at different time points and contributing to different heart regions, the temporally distinct cardiac progenitors present very similar clonal dynamics. These results provide insights into the number of cardiac progenitors and their mode of growth and open up avenues to decipher the clonal dynamics of progenitors in other organs and tissues.

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.

Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.

Cardiac development arises from two sources of mesoderm progenitors, the first heart field (FHF) and the second (SHF). Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors common for both heart fields. Here, using clonal analysis of the earliest prospective cardiovascular progenitors in a temporally controlled manner during early gastrulation, we found that Mesp1 progenitors consist of two temporally distinct pools of progenitors restricted to either the FHF or the SHF. FHF progenitors were unipotent, whereas SHF progenitors were either unipotent or bipotent. Microarray and single-cell PCR with reverse transcription analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Together, these results provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors that independently express Mesp1 at different time points during their specification, revealing that the regional segregation and lineage restriction of cardiac progenitors occur very early during gastrulation.

Cell lineages, growth and repair of the mouse heart.

The formation of the heart involves diversification of lineages which differentiate into distinct cardiac cell types or contribute to different regions such as the four cardiac chambers. The heart is the first organ to form in the embryo. However, in parallel with the growth of the organism, before or after birth, the heart has to adapt its size to maintain pumping efficiency. The adult heart has only a mild regeneration potential; thus, strategies to repair the heart after injury are based on the mobilisation of resident cardiac stem cells or the transplantation of external sources of stem cells. We discuss current knowledge on these aspects and raise questions for future research.