Building the mammalian heart from two sources of myocardial cells

© 2005 Nature Publishing Group

*Department of Developmental Biology, CNRS URA 2578, Pasteur Institute, 25 Rue du Dr Roux, 75015 Paris, France.‡Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, United Kingdom.§These authors contributed equally to this work.Correspondence to M.B.e-mail: [email protected]:10.1038/nrg1710

MYOCARDIUMThe striated muscle of the heart, which provides the contractile force that is necessary to pump blood around the body.

In vertebrates, the heart is the first organ to form, and has a vital role in the distribution of nutrients and oxygen in the embryo. Initially it functions as a car-diac tube and is principally composed of a contractile MYOCARDIUM that is essential for its action as a central pump. Subsequently, regionalization of this structure takes place, and in adult birds and mammals this leads to the formation of the four-chambered heart.

Recent studies have fundamentally altered our view of cardiogenesis: instead of there being a single source of myocardial progenitor cells, as was previ-ously assumed, it has now been shown that there are two distinct sources of these cells. Here we provide a brief overview of the cellular processes that take place during early cardiogenesis, before discussing the experiments that led to the identification of the sec-ond source of myocardial cells. We then reassess the classical models of heart development in this context and discuss the implications for the interpretation of mutant phenotypes in myocardial regulatory genes.

Cellular aspects of early cardiogenesisMyocardial cells are derivatives of the MESODERM, which emerges from the PRIMITIVE STREAK during GASTRULATION. Cardiac progenitor cells have been mapped to the anterior region of the streak in both chick and mouse

embryos1,2; at this stage their commitment to a cardiac fate remains plastic. Later, they leave the streak and migrate in an anterior-lateral direction to positions under the head folds, forming two groups of cells on either side of the midline2,3 (FIG. 1). Myocardial mark-ers are first detected at this stage. The cells then extend across the midline to form a crescent-shaped epithe-lium, the cardiac crescent, which fuses at the midline to form the early heart tube. This tube undergoes rightward looping, with its posterior region moving anteriorly. The heart is shaped by the looping process and by expansion of the myocardium, which leads to the formation of recognizable cardiac chambers.

The classical view of how regional identity is estab-lished in the cardiac tube was based on experiments in chick embryos. These experiments indicated that cells in the primitive streak are pre-patterned, in terms of their contribution to the cardiac tube, along the arte-rial–venous axis, with outf low progenitors of the arterial pole positioned more anteriorly1,4. Such anterior–posterior pre-patterning is retained in the car-diac crescent and developing cardiac tube5–8, so that each region of the heart represents a distinct cellular unit. These observations led to a segmental model of cardio-genesis (FIG. 2). However, more recent experiments show little regionalization until the cardiac-crescent stage in

BUILDING THE MAMMALIAN HEART FROM TWO SOURCES OF MYOCARDIAL CELLSMargaret Buckingham*, Sigolène Meilhac‡§ and Stéphane Zaffran*§

Abstract | Cardiogenesis is an exquisitely sensitive process. Any perturbation in the cells that contribute to the building of the heart leads to cardiac malformations, which frequently result in the death of the embryo. Previously, the myocardium was thought to be derived from a single source of cells. However, the recent identification of a second source of myocardial cells that make an important contribution to the cardiac chambers has modified the classical view of heart formation. It also has an important influence on the interpretation of mutant phenotypes in the mouse, with consequences for the classification and prognosis of human congenital heart defects.

826 | NOVEMBER 2005 | VOLUME 6 www.nature.com/reviews/genetics

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MESODERMOne of the three layers of cells in the early embryo that, together with the endoderm and ectoderm, provides the source of all subsequent cell types that appear during embryogenesis. Mesoderm gives rise to the skeletomuscular system, connective tissues, blood, and internal organs such as the heart.

PRIMITIVE STREAKA transitory embryonic structure, which is present as a strip of cells, that pre-figures the anterior–posterior axis of the embryo. During gastrulation embryonic cells progress through the streak.

GASTRULATIONA crucial process in embryogenesis when major cellular movements lead to the involution of cells through the primitive streak, and subsequently give rise to the internal organs. As a result, the embryo contains three cell regions or germ layers: a middle layer of mesoderm surrounded by an outer layer of ectoderm and an inner layer of endoderm.

PHARYNGEAL MESODERMThe mesoderm that is situated below the head in the pharynx, which is the part of the embryo where the developing respiratory and digestive systems are present.

CHICKQUAIL GRAFTAn experimental approach to studying cell fate that is based on the ability to distinguish between chick and quail cells and on the similar embryonic development of the two species. Cells can be transplanted from one to the other in ovo and their subsequent contribution to the developing embryo monitored.

the chick embryo9–12. These different views probably partly reflect the technical limitations of earlier exper-iments, and the available evidence indicates that pre-patterning in the primitive streak is better described as a tendency of cells to contribute to specific regions of the heart1. Studies in mice also indicate the absence of early segmental regionalization13; clonally related myocardial cells are dispersed along the length of the heart tube as it begins to loop.

As the cardiac tube grows, chambers begin to form by expansion of the myocardium (FIG. 1d). This might involve differential cell growth at the outer curvature of the cardiac tube14,15, whereas orientated cell growth is probably important in shaping the chambers16.

Identification of novel heart fieldsIn addition to growth of myocardial cells within the cardiac tube (and possibly of rare undifferentiated cells17), studies in the chick6,8 and mouse18,19 indicated that further recruitment of heart progenitor cells occurs at the poles of the tube. Here we discuss more recent findings that characterize the sources of such progenitor cells. This involves the concept of a heart field, which is defined as a discrete embryonic region in which cells that have myocardial potential are located.

Heart fields in the chick embryo. The posterior addi-tion of myocardium to the venous pole, which gives rise to the inflow region (FIG. 1), was thought to arise from progenitors in the bilaterally separated extremi-ties of the cardiac crescent4,11. More recently, a distinct region of PHARYNGEAL MESODERM was identified by two groups as the source of outflow-tract myocardium in the chick embryo20,21. Ablation of the cardiac crescent did not completely abolish formation of the cardiac tube, and explant experiments, CHICKQUAIL GRAFTS and fate mapping showed the location of a second source of myocardial cells in the pharyngeal mesoderm.

Perhaps because of the different experimental approaches, conclusions about the precise location of this second source of progenitors differed between the two groups concerned (FIG. 3a). One group described it as the ‘secondary heart field’, which is situated imme-diately adjacent and posterior to the outflow-tract region behind the heart tube21, whereas the other identified an ‘anterior heart field’, which includes more cranial pharyngeal mesoderm that extends into the PHARYNGEAL ARCHES20.

Heart fields in the mouse embryo. Studies in mice also led to the identification of an anterior heart field as a source of outflow-tract myocardium22. This was revealed by using a lacZ transgene under the control of regulatory elements of the Fgf10 gene (fibroblast growth factor 10), which resulted in lacZ transcrip-tion in part of the pharyngeal mesoderm. Cells that had expressed the transgene were shown to contribute to the myocardium at the arterial pole of the heart. This was confirmed by DII LABELLING. A contribution of this anterior heart field to right-ventricular as well as outflow-tract myocardium was shown more recently by explant experiments and further Di-I labelling studies23. These studies also showed that the early cardiac tube in the mouse embryo has an essentially left-ventricular identity23.

The murine anterior heart field is located ante-riorly and also dorsally to the cardiac tube (FIG. 3b). At the cardiac-crescent stage, cells that are marked by the Fgf10–lacZ transgene lie medial to the crescent on either side of the midline. As morphogenesis pro-ceeds, the sides of the crescent move to the midline, placing these labelled cells in the dorsal mesocardium behind the fusing heart tube. They are in contact with the dorsal surface of the tube, which will become the inner curvature as the heart loops. Some labelled cells also come to lie more anteriorly. The apparent location

Figure 1 | Morphogenesis of the mouse heart. a | Myocardial progenitor cells originate in the primitive streak (PS), from where they migrate to the anterior of the embryo at about embryonic day E6.5. b | These cells come to lie under the head folds (HF) and form the cardiac crescent, where differentiated myocardial cells are now observed (E7.5). c | The early cardiac tube forms through fusion of the cardiac crescent at the midline (ML) (E8). d | It subsequently undergoes looping (E8.5). e | By E10.5 the heart has acquired well-defined chambers, but is still a tube (upper panel, ventral view; lower panel, dorsal view). f | In the fetal heart (E14.5) the chambers are now separated as a result of septation and are connected to the pulmonary trunk (PT) and aorta (Ao), which ensure the separate pulmonary and systemic circulation of the blood, respectively, after birth. Deoxygenated blood enters the heart through the right atrium (RA) and is pumped to the lungs through the pulmonary trunk by the right ventricle (RV). Oxygenated blood returns to the left atrium (LA) and is pumped by the left ventricle (LV), through the aorta, to the systemic circulation that serves the whole body. Anterior (A)–posterior (P) and right (R)–left (L) axes are indicated. AA, aortic arch; AVC, atrioventricular canal; IFT, inflow tract; IVC, inferior vena cava; IVS, interventricular septum; OFT, outflow tract; PLA primitive left atrium; PRA, primitive right atrium; PV, pulmonary vein; SVC, superior vena cava; Tr, trabeculae.

NATURE REVIEWS | GENETICS VOLUME 6 | NOVEMBER 2005 | 827

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PHARYNGEAL ARCHESThe embryonic structures that are present as a series of pouches that bud out from the pharynx. As development proceeds, the arches become incorporated into the different structures of the head or anterior trunk. The mesodermal core of the arches gives rise to cells that form anterior skeletal muscles and some myocardial progenitors.

DII LABELLINGA classical embryological approach to lineage tracing, which follows cell movement. The fluorescent dye Di-I is injected into cells of the embryo, which can subsequently be identified by stable labelling of their membranes.

RETROSPECTIVE CLONAL ANALYSISThis is a genetic approach to lineage analysis that is based on random labelling of precursor cells. In the examples cited in this review, the method used a lacZ reporter carrying a duplication (laacZ) that renders it non-functional. Rare spontaneous intragenic recombination removes the duplication so that cells that express the reporter become β-galactosidase-positive, allowing clonal analysis.

of these anterior heart-field cells in the mesodermal core of the pharyngeal arches22,24 probably results from earlier morphogenetic movements at the time of arch formation. Cells that lie dorsally and anteriorly to the early tube ultimately assume a right-ventricular iden-tity, whereas more anterior cells contribute, slightly later, to outflow-tract myocardium23.

An extension of the contribution of this population of myocardial progenitors to the heart was revealed by studies of Isl1 (insulin gene enhancer protein, a LIM homeodomain transcription factor) expression in cells that are present in the murine anterior heart field, and that also extend more posteriorly25. Lineage tracing showed that these cells contribute to the venous as well as the arterial pole of the cardiac tube. This dem-onstrates that the inflow region is also formed by cells from the novel heart field, which includes the anterior heart field that is marked by the Fgf10–lacZ transgene. This is referred to as the second heart field to distinguish it from the classical first heart field (FIG. 3b). The first heart field, which gives rise to the differentiated myocar-dial cells of the cardiac crescent and early cardiac tube, is difficult to locate precisely owing to a lack of markers (it is represented in FIG. 3b in the region of the crescent). From a spatial point of view, the cardiac crescent might be thought of as the differentiating anterior-lateral edge of a single field of progenitor cells that extends more medially. As the outer edges of this field move together to form the initial cardiac tube, which mainly comprises the future left-ventricular region in the mouse, progeni-tor cells that are situated dorsally, posteriorly and ante-riorly continue to become integrated into the heart. However, we think that it is more useful to differenti-ate between the first and second heart fields, because the progenitor cells of the latter are distinguished by the expression of specific markers.

First and second myocardial cell lineagesThe distinction between the two heart fields is re inforced by findings from a mouse cell-lineage study that is based on β-galactosidase labelling in a RETROSPECTIVE

CLONAL ANALYSIS26. The concept of myocardial lineage centres on the history of a cell and its descendents, and the characterization of their clonal contribution to the heart. The spatial distribution of clonally related β-galactosidase positive cells at embryonic day E8.5 revealed two categories of clones with distinct pat-terns of regionalization in the cardiac tube (FIG. 3c). One lineage contributes to both ventricles, the atrio-ventricular canal and both atria; the other contributes to the outflow tract and all other heart regions, except the left ventricle. This second lineage corresponds to the contribution of the second heart field, as shown with ISL1-marked cells25. Some labelled cells of the second lineage are also detected in the dorsal mesocar-dium and are more frequent on the inner curvature of the tube, as would be expected if there is recruitment from the underlying mesoderm. Both lineages are present in the right ventricle, atrioventricular canal and atria, where there is considerable overlap, although in some regions (notably in the right atrium at E8.5) clonal domains of the first and second lineages seem to be complementary.

The acquisition of regional identity can be equated with clonal restriction to a single compartment. The number of labelled cells in such a clone gives an indication of the timing of regionalization, with the assumption that proliferation is homogenous before E8.5. The atrial region becomes clonally distinct ear-lier than the right ventricle and outflow tract, which is consistent with experiments in the chick embryo that indicate that atrial regional identity is acquired at the cardiac-crescent stage27. The contribution of

Figure 2 | The segmental model of myocardial cell regionalization. The segmental model proposes that each region of the heart is composed of a separate unit and that these are derived from pre-patterned progenitor cells. a | Grafting experiments in the chick embryo identified ordered subdomains of mesoderm in the anterior primitive streak at stage 3 as the source of different segments of the developing heart tube at stage 12. These subdomains are shown here for the outflow region (the anterior region is shown in yellow and the posterior region is shown in orange) and the rest of the heart tube (shown in grey). b | At later stages in development, labelling experiments in the early heart tube of the chick embryo (stage 9) indicated the presence of two segments — the primitive right ventricle (PRV) and primitive left ventricle (PLV). Addition of further segments was thought to take place at stage 12; these are the primitive outflow region (PO), primitive ventricular inlet/atrioventricular canal (PVI), primitive right atrium (PRA) and primitive left atrium (PLA). c | Right and left views of the dissected adult heart are shown with the same colour code as in part b for the equivalent adult compartments. The findings that are illustrated in a and b led to the suggestion that the segments, which relate to the formation of a specific compartment of the heart, are independent units, with no cell mixing. This cellular model was extrapolated to the molecular level, such that each segment and cardiac compartment (chamber) was thought to have its own genetic programme86. All views are frontal and the anterior (A)–posterior (P) axis is indicated. sv/a, primitive sinus venosus and atria; v, primitive ventricles. Part a is adapted, with permission, from REF. 1 © (1993) Sinauer Associates. Part b is adapted, with permission, from REF. 5 © (1999) Birkhauser Verlag.


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NEURAL TUBEA structure that is formed from ectoderm during neurulation. It extends from the brain to the posterior part of the body, and becomes the adult spinal cord. The neural tube also gives rise to motor neurons of the PNS.

the two lineages to most parts of the heart, and the fact that most regions do not become clonally distinct until later, challenges the segmental model of heart development.

The retrospective clonal analysis showed that the first and second lineages segregate from a com-mon progenitor before the crescent stage, probably at the onset of gastrulation. This is indicated by the presence of large clones that have labelled cells in both lin eage compartments of the tube and derive

from early common progenitors. The first lineage is so called because it is the first to segregate from the common progenitors of both lineages. This temporal lineage distinction is probably accompanied by some degree of spatial sorting and progressive restriction of cell dispersion, rather than the establishment of a pre-pattern of adjacent regions of the cardiac tube (seg-mental model) that would result in their strict clonal segregation. Consistent with this, most compartments have contributions from both lineages. The tendency towards regionalization in the primitive streak that was described for the chick embryo1 showed that outflow-tract progenitor cells come from a distinct location. It is not clear whether the second lineage that contributes to the chick outflow tract also colonizes other parts of the avian cardiac tube. The mammalian second lineage may have acquired a more extensive role in cardiogenesis. Based on the clonal analysis of myocar-dial cells at E8.5 in the mouse, it has been suggested that the second lineage accounts for about a third of myocardial progenitors26, although the descendents of these cells may represent a larger part of the mature myocardium.

In conclusion, the myocardial contribution of the first and second lineages, which are distinguished by their participation in the left ventricle versus outflow tract at E8.5, relates directly to that of the first and second heart fields. The early segregation of the two lineages supports the idea that there are two heart fields with progenitor cells that make distinct regional contributions to the heart. The distinct myocardial potential of cells in the second heart field is indicated by recent molecular data. This will be discussed in the next sections, which focus on the genes that regulate cardiogenesis and the assessment of their mutant phe-notypes in light of the finding that myocardium arises from two progenitor sources.

Molecular aspects of early cardiogenesisThe earliest molecular markers for cardiac progeni-tors, the transcription factors MESP1 (mesoderm posterior 1) and MESP2 (mesoderm posterior 2) REF. 28, are expressed transiently in newly formed mesoderm at the primitive-streak stage. This is required for the movement of cells towards the ante-rior region of the embryo. Descendants of these cells colonize the whole myocardium28, so expression of MESP1 and MESP2 seems to mark both myocardial cell lineages.

Myocardial transcription factors are first detected in the cardiac crescent, where myocardial differentia-tion is initiated. Activation of key myocardial regula-tory genes, such as NKX2-5 (NK2 transcription factor related, locus 5) and GATA4 (GATA-binding protein 4), depends on positive signalling by BMPs (bone morpho-genetic proteins) and FGFs (fibroblast growth factors), whereas WNTs (wingless-related MMTV integration sites) exert a repressive effect29. The medial location of the second heart field at the cardiac-crescent stage means that it is closer to the negative influence of WNTs that emanate from the NEURAL TUBE. BMPs25 and FGFs22,25

Figure 3 | Two sources of myocardial cells in the developing heart. a | Myocardial cells in the chick embryo. The location and contribution of the ‘secondary heart field’ (orange) that was identified by Waldo et al.21 and the ‘anterior heart field’ (blue) that was identified by Mjaatvedt et al.20 are shown for a stage-16 embryo (lateral view) and at stage 22 (frontal view). Before stage 16, the cardiac crescent is present, but it is not clear where the cells of the anterior or secondary heart field are located. b | Myocardial cells in the mouse embryo. The location and contribution of the second heart field is shown in green, with the anterior heart field subdomain in dark green, and is compared with the myocardial cells that are derived from the first heart field (shown in red). Frontal views are shown for embryonic day 7.5 (E7.5) and E10.5, and lateral views for stages E8 and E8.5. c | Clones of β-galactosidase-positive myocardial cells from the cardiac actinnlaacZ/+ mouse line13 at E8.5. Frontal views are shown and clones are indicated by the numbered arrows. The central panel shows a large clone that predates segregation of the first and second lineages and colonizes all regions of the heart tube. The left and right panels show first and second lineage profiles, respectively. d | The lineage contributions to the heart at E8.5 are summarized with the same colour coding as in part b. LA, left atria; LV, left ventricle; OFT, outflow tract (D, distal; P, proximal); PA, primitive atria; PhA, pharyngeal arches; RA, right atrium; RV, right ventricle; 1, outflow region; 2, primitive right ventricle; 3, primitive left ventricle; 4/5, atrioventricular region; 6/7, primitive left and right atria. Part a is reproduced, with permission, from REF. 87 © (2004) Sinauer Associates. Part c is modified, with permission, from REF. 26 © (2004) Elsevier Science.


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CARDIAC BIFIDAThe presence of two totally or partially separated hearts, which is usually due to a failure of fusion of the cardiac crescent to form a single heart tube during embryogenesis.

are also expressed in the second heart field, where they lead to later activation of genes such as Nkx2-5 REF. 30. The two heart fields therefore seem to be regulated by similar cardiogenic signals. This is probably also the case for signals that are required for anterior–posterior and left–right patterning; these processes are mediated, respectively, by retinoic acid and its receptors27,31, and by PITX2 (paired-like homeodomain transcription fac-tor 2) REFS 32,33, which represents the transcriptional read-out of the Nodal signalling pathway.

Mutation of Gata4 REFS 34,35 and Foxp4 (fork-head box P4) REF. 36 leads to CARDIAC BIFIDA, owing to a failure of the two halves of the cardiac crescent to converge at the midline. In the Foxp4 mutant, the two unfused hearts mature further, and the second heart-field contribution seems to take place, with progenitor cells maintained bilaterally. This again indicates that the two heart fields are exposed to similar patterning signals.

When the phenotypes of null mutations in the genes that encode myocardial transcription factors were first described, it became evident that no single factor is essential for cardiomyocyte differentiation. In some cases, such as with Mef2c (myocyte enhancer factor 2c; also known as RSRF, related to serum response factor), the function might be substituted by other members of the gene family37. However, in many cases, such as with Nkx2-5 REF. 38, compensation probably operates between the different transcription factor families, which function in combination to reg-ulate downstream muscle genes39,40. The phenotypes

of these mutants show striking effects on the mor-phogenesis of the heart tube, which is often character-ized by specific loss of primitive chambers and arrest of cardiac development at the looping stage. These results were surprising because most of the genes are expressed throughout the early myocardium, which at the time these experiments were carried out was thought to derive from a single source. They can now be reassessed in terms of the myocardial con-tribution of the two heart fields. TABLE 1 presents a summary of the expression and mutant phenotypes of selected genes that regulate cardiogenesis in this context, which are discussed below.

Mutations affecting 1st heart field derivativesAs the first lineage specifically contributes the progeni-tor cells of the left ventricle, perturbations in this lineage are likely to affect the formation of this structure. In other cardiac regions, recruitment of cells from the second heart field may compensate for early defects in the first heart-field contribution.

Nkx2-5 and Hand1. The Nkx2-5 mutant pheno-type41,42, although open to other interpretations, seems to reflect a first lineage defect. This is indicated by the loss of ventricular tissue and the absence of Hand1 (heart and neural crest derivatives expressed tran-script 1) expression at the cardiac-crescent stage and later in the cardiac tube43, where it would normally label the left ventricle (FIG. 4b). Hand2, which later marks the right ventricle44, is still expressed45.

Table 1 | Mutant phenotypes of genes that are involved in myocardial development

Gene Crescent expression*

Second heart-field expression*

Cardiac tube expression*

Mutant phenotype in the cardiac tube References

Nkx2-5 Yes Yes Yes Single atrial and ventricular compartments; loss of ventricular tissue; no Hand1 expression

41–43

Hand1 Yes No LV (high in outer curvature)

LV disrupted 44–47

Tbx5 Yes No LV, A, INF Sinoatrial defects; hypoplastic LV 50–51

Fgf10 No Yes No No early phenotype detected 22

Fgf8 No Yes No OFT defects 21,22,54,55,62

Tbx1 Yes Yes No OFT defects 57–61,63,64

Isl1 No Yes OFT, RV (LV), A, INF

Single atrial and ventricular compartments; Hand1 and Tbx5 expression, which indicates that LV identity is intact; no OFT; atria at the venous pole are abnormal

25

Foxh1 No Yes No OFT reduced or absent; RV does not develop 69

Mef2c (Yes?) Yes Yes OFT reduced; RV does not develop (Hand2 is downregulated); INF abnormalities

37,74

Hand2 Yes Yes RV RV abnormalities 77,78

Hand1/Hand2 Yes Yes RV/LV No ventricle; only atrial chamber forms 43,49

Tbx20 Yes Yes OFT, RV, LV Chambers do not develop; no Hand1 expression; hypoplastic RV; OFT disrupted

68,80–82

*Very low levels of expression or expression in only a few cells have not been included. In the cardiac tube, initial expression patterns can be dynamic, showing a tendency towards regionalization before this becomes well-defined. Only well-defined expression patterns are indicated in the table. A, atria; Fgf8/10, fibroblast growth factor 8 and 10; Foxh1, forkhead box H1; Hand1/2, heart and neural crest derivatives expressed transcript 1 and 2; INF, inflow region; Isl1, insulin gene enhancer protein, a LIM homeodomain transcription factor; LV, left ventricle; Mef2c, myocyte enhancer factor 2C (also known as RSRF, related to serum response factor); Nkx2-5, NK2 transcription factor related, locus 5; OFT, outflow tract; RV, right ventricle; Tbx1/5/20, T-box 1, 5 and 20.


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Nkx2-5Nkx2-5–/––/–

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Isl1Isl1+/–+/–


Isl1Isl1+/–+/–


Nkx2-5Nkx2-5+/–+/– Nkx2-5–/– Nkx2-5+/–

h hh

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HYPOPLASIAThis term is used to describe the reduced size of a tissue or organ that is due to a deficit in its cell population. During embryogenesis this may be due to a failure of a progenitor cell population to contribute to the tissue, or to a defect in proliferation or apoptosis.

Hand1 mutants die from extra-embryonic defects46,47, but examination of chimaeras of mutant and wild-type cells shows that cells that lack HAND1 fail to contrib-ute to the left ventricle46,48. Furthermore, a conditional Hand1 mutation, which is probably activated at the heart tube stage, results in left-ventricular HYPOPLASIA49. Cells that express Hand1 at early stages are not lost, as other markers are expressed normally in the cardiac crescent and no cell death is detectable in the Nkx2-5 mutant43. Instead, there may be a first lineage proliferation defect. Effects on the second lineage contribution may also occur, as indicated by the shortened outflow tract in these mutants. However, this may be a secondary con-sequence of abnormal cardiogenesis, which affects later recruitment of cells to the arterial pole.

Tbx5. Tbx5 (T-box 5) mutant mice50 show severe defects in the atria-inflow region of the heart and left-ventricle hypoplasia, although left-ventricular markers such as HAND1 are still detectable. Despite their abnormal morphology, the outflow tract and right ventricle con-tinue to grow. This indicates an effect on first lineage proliferation. Ectopic expression of Tbx5 in the myo-cardium in transgenic mice perturbs right-ventricular development and leads to a reduction in the overall size of the heart. Hand1 is upregulated and Hand2 expres-sion is absent51. This indicates a role for TBX5 in left-ventricular specification, potentially through negative effects on expansion of other regions of the heart.

There is a lack of molecular markers for the first heart field that define the progenitor-cell population and distinguish it from the differentiating cardiomyo-cytes of the crescent. This makes it difficult to separate later effects of mutants on the regionalized myocar-dium of the chambers in the heart tube from specific effects on cells of the first myocardial lineage. By con-trast, increasing numbers of markers for the second heart field are emerging.

Mutations affecting 2nd heart field derivativesAs the second myocardial cell lineage alone contrib-utes the progenitor cells of the outflow tract, pertur-bations in this lineage are likely to specifically affect the formation of this compartment. Elsewhere in the heart, the first lineage might compensate for early defects in the second heart-field contribution.

Fgf8 and Fgf10. As described previously, the first indi-cation of a second heart field in the mouse came from the expression of an Fgf10–lacZ transgene. Fgf8 (fibro-blast growth factor 8) has a similar expression pattern to Fgf10 in the second heart field22, although unlike Fgf10 it is expressed in the ectoderm and endoderm of the pharyngeal arches, but not in the mesoderm21.

Fgf10 mutant mice have no evident early cardiac phenotype52,53 and the outflow tract seems to be normal (F. Bajolle, S.Z., R. Kelly and M.B., unpublished obser-vations). Fgf8-null mutants die at gastrulation, but an Fgf8 hypomorph54,55 dies later of cardiac failure that is due to malformation of the outflow region. FGF signal-ling may be necessary for the proliferation of cells in the second heart field, as shown in the chick embryo21. Malformations of the aorta and pulmonary trunk (FIG. 1f) that also occur in this mutant54 probably reflect an effect on NEURAL CREST CELLS, which are required for the septation and normal development of these struc-tures. Fgf8 is not expressed in the neural crest, but neural crest cells migrate from the mesenchyme of the pharyngeal arches to the outflow region, where they are probably compromised by abnormal FGF signalling in the mutant. The anterior part of the second heart field is in close contact with the neural crest, and interference with the migration of neural crest cells might affect this field. This was indicated by neural crest ablation experiments in the chick embryo, which resulted in a reduced number of myocardial cells being recruited to the arterial pole of the heart56.

Figure 4 | Examples of mutant phenotypes that show first and second lineage defects. a | A mutant that shows a putative first lineage defect. Nkx2-5 (NK2 transcription factor related, locus 5) mutant mouse embryos (Nkx2-5–/–) are compared with control heterozygote embryos (Nkx2-5+/–) at embryonic day E8.5. The left and right panels show frontal and lateral views, respectively. Nkx2-5–/– embryos show morphological defects in the heart (h) with loss of ventricular myocardium. In the right panel, Hand1 (heart and neural crest derivatives expressed transcript 1) expression, which marks the left ventricle, is seen as purple colouration. This is lost in the Nkx2-5–/– embryo45. The strong labelling of this marker in the Nkx2-5+/– embryo occurs in the endoderm (en). b | A mutant that reflects a putative second lineage defect. Scanning electron micrographs of mouse embryos at E9.5 are shown in the left-hand panels. The central panels show whole-mount in situ hybridization with an MLC2a (myosin light chain 2α) probe, which marks the whole myocardium. The right-hand panels show sections that are labelled with this marker. Isl1 (insulin gene enhancer protein, a LIM homeodomain transcription factor) mutant embryos (Isl1–/–) show loss of the outflow tract and right ventricle compared with control heterozygote (Isl1+/–) embryos, and have a single ventricular (V) and atrial (A) chamber. LA, left atrium; LV, left ventricle; OFT, outflow tract; RA, right atrium; RV, right ventricle. Left panel of part a is courtesy of Richard Harvey, University of New South Wales. Right panel of part a is reproduced, with permission, from REF. 45 © (1997) Cold Spring Harbor Laboratory Press. Part b is reproduced, with permission, from REF. 25 © (2003) Elsevier Science.


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NEURAL CREST CELLSProgenitor cells present in vertebrates that arise from the dorsal neural tube and migrate to other sites in the embryo. These include the mesenchymal cells that contribute to septation in the heart.

CRELOXP SYSTEMA genetic approach for producing conditional mutants or examining cell fate. This uses a specifically expressed Cre recombinase that recognizes loxP sites that are introduced into the gene to be targeted, which results in the recombination and removal of the intervening sequence between sites.

A conditional mutation targeted to the second heart field will be necessary to determine the specific role of FGF8 in myocardial progenitors.

Tbx1. The cardiac phenotype of Tbx1 (T-box 1) mutant mice57–60 is similar to that of the Fgf8 hypo-morph54,55. However, the neural crest defect in Tbx1 mutants is more severe, reflecting its expression in the endoderm and core mesoderm of the pharyngeal arches. Analysis of this mutant, together with gain-of-function experiments, indicates that Fgf8 REFS 6163 and Fgf10 REFS 64,65 may be targets of Tbx1. This has been confirmed for Fgf8 by the identification of a TBX1-dependent regulatory sequence61.

Lineage-tracing experiments using the CRELOXP SYSTEM confirmed the role of TBX1 in the second heart field and showed extensive labelling of the myocardial wall of the outflow tract, with some additional positive cells in the right ventricle62,63. This indicates that the early myocardial contribution of cells that express Fgf8, Fgf10 or Tbx1 is similar, although Fgf10-expressing cells may have a more extensive right-ventricular contribution23. Interestingly, the Tbx1 tracing experiment showed that labelled left-ventricular cells become apparent at later stages of development, and in the adult the whole heart is labelled62. This suggests that cells of the second lineage expand throughout the heart at later stages, as clonal analysis indicates that the second lineage is not initially represented in the left ventricle26. Alternatively there may be as yet undetected later reactivation of Tbx1.

The function of TBX1 in the second heart field is revealed by lineage-tracing experiments on a Tbx1-null background. These show a diminished contribution of cells of the second heart field, which normally express this gene, to the outflow-tract myocardium63. Targeted deletion of Tbx1 in the second heart field causes a pro-liferation defect63, which would explain this reduced contribution. Overexpression of Tbx1 in myocardial cells results in upregulation of Fgf8 and Fgf10, with an increase in outflow-tract myocardium61. TBX1 may therefore regulate proliferation in the second heart field by activating these genes. However, whether substitu-tion of the TBX1-coding sequence by FGF8 and FGF10 sequences rescues the Tbx1 mutant phenotype remains to be determined.

The transcription factors FOXC1 and FOXC2 (fork-head box C1 and C2) are potential upstream activa-tors of Tbx1, and a Foxc1 Foxc2 double mutant shows defects in the anterior heart field (REF. 66; T. Kume, per-sonal communication). Tbx1 is also a target of FOXA2 (forkhead box A2), which is itself regulated by TBX1 in pharyngeal mesoderm61,67.

Isl1. In Isl1 mutant embryos (FIG. 4b), the heart tube seems to have only two chambers. Genetic markers demonstrate the presence of atrial and ventricular myocardium, and labelling with Hand1 and Tbx5 probes shows the left-ventricular identity of the ante-rior compartment. The outflow tract is absent, and right-ventricular markers are not expressed, whereas posterior atrial myocardium seems to be reduced.

This is consistent with Isl1 expression in cells of the second heart field, which contribute to the venous pole of the heart as well as the arterial pole (FIG. 3c).

ISL1 may have several roles in the second heart field. In its absence, migration into the cardiac tube is not observed, proliferation is affected and cells undergo apoptosis25. In addition to loss of expression that is due to cell death, BMP4, BMP7 and FGF10 are specifically downregulated. A potential effect on myo-cardial cell specification is indicated by ISL1-mediated regulation of an Nkx2-5 enhancer that is active in the second heart field68.

Lineage tracing of cells that express Isl1 using the Cre–loxP strategy showed that in addition to coloniz-ing the outflow tract, the right ventricle and part of the atria25, labelled cells are also present in the inner curvature of the left ventricle. The contribution of these second heart-field cells to all parts of the heart was confirmed by further lineage-tracing experiments using a more efficient Isl1–Cre; here unlabelled cells were seen only on the outer curvature of the left ven-tricle and in part of the right atrium in the looped heart tube (S. Evans, personal communication). This is in contrast to the retrospective clonal analysis, which had indicated that the second lineage does not contribute to the primitive left ventricle26.

A minor contribution of second lineage cells to the left ventricle, possibly through the inner curvature, might have been missed in the retrospective clonal analysis. However, as the end-point of this analysis was at an early stage of heart development, the second lineage may subsequently expand into other regions of the heart, as discussed in the context of the Tbx1 lineage-tracing study62,63. An important caveat of this approach is that cells that express Isl1 (or Tbx1) might be present within the myocardium, which would inter-fere with interpretation. In the case of Isl1, such cells have recently been shown to be present in the adult heart17. They are rare and have the properties of cardiac progenitor cells. To some extent, their distribution cor-responds to that of the second heart-field contribution that was indicated by the lineage study25, as numbers of these ISL1-positive cells are lowest in the left ventricle and highest in the outflow tract. This raises the pos-sibility that they are derived from the second heart field, and it will be interesting to determine whether they also express Tbx1. Although such cells are rare, if they self-renew, they might make a substantial con-tribution to myocardial growth during heart develop-ment, as indicated by the extensive labelling of adult hearts in the Tbx1 lineage-tracing experiment62.

Foxh1. In Foxh1 (forkhead box H1) mutants69, right-ventricular and outflow-tract markers are not detected, although a truncated outflow region is present. This indicates that FOXH1 is essential for right-ventricle formation, but is only partially required for that of the outflow tract. The venous pole seems normal in Foxh1 mutants and the left ventricle is present, as indicated by the expression of genes such as Hand1 and Tbx5. Markers that are associated with left-ventricular


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Gata4

Mef2c

Bop

Hand2

Tbx20

Nkx2-5

Isl1

Foxh1

Foxc1/c2 (Foxa2)

Tbx1

Fgf10 Fgf8

HISTONE METHYL TRANSFERASEAn enzyme that adds methyl groups to histones — DNA-binding proteins — that are involved in the regulation of gene accessibility to transcription. Their modification by methylation affects this function.

maturation, such as Nppa (natriuretic peptide precur-sor type A), are not expressed, which is consistent with arrested development of this region. Foxh1 is expressed in the anterior part of the second heart field70,71. In its absence there is downregulation of Fgf8 expression and a later effect on Fgf10, whereas Isl1 expression is unaffected. FOXH1 transduces TGFB (transforming growth factor-β) signals69, and acts in conjunction with SMADs70; it might therefore affect the second heart field through these signalling pathways.

Mef2c. Expression of MEF2C REF. 73 was originally described in the cardiac crescent72. However, analysis of a lacZ transgene74 that is under the control of an extensive Mef2c regulatory region indicates that early expression is medial to the crescent, and subsequently continues in the second heart field. Mef2c mutants37 have a second heart-field phenotype: as with the Foxh1 mutant, the outflow tract is detectable but reduced, whereas the right ventricle does not develop. The sinus venosus is absent, which indicates that MEF2C is required for the second heart-field contribution to the venous pole as well as the arterial pole.

Mef2c seems to be a target of ISL1, as indicated by an enhancer element within the Mef2c gene that is regu-lated by ISL1 and GATA4 REF. 74. This enhancer directs transgene expression to the second heart field, where it partially overlaps with Isl1 expression, and to the out-flow tract and right ventricle. This myocardial expres-sion might be due to perduration of β-galactosidase, as in the case of the Fgf10–lacZ transgene22, marking the contribution of second heart-field cells in which the transgene was transcribed to these parts of the heart. MEF2C has been implicated in cell-cycle regulation in other tissues, so deregulation of Mef2c may contribute to the proliferative defect in Isl1 mutants25.

Mef2c may also be a target of FOXH1 which, in conjunction with NKX2-5, functions on another Mef2c regulatory element69. MEF2C itself activates the Bop (also known as Smyd1, SET and MYND domain containing 1) gene, which encodes a putative HISTONE

METHYL TRANSFERASE and is expressed in the second heart field and myocardium75, with a mutant phenotype that is similar to that of Mef2c REF. 76.

Hand2. Hand2 is a potential target of BOP76. Hand2 mutants show right-ventricular hypoplasia77,78, whereas Hand1 Hand2 REFS 43,49 double mutants show a more dramatic phenotype. Here only one cardiac chamber is formed, which expresses atrial markers; there is vir-tually no ventricular myocardium. This is consistent with the loss of first lineage cells in the left ventricle in the absence of HAND1. The severe right-ventricular phenotype in the double mutant probably reflects the loss of both first and second heart-field contributions to this chamber.

In the Hand1 Hand2 double mutant, small numbers of ventricular cells are detectable in the outer curva-ture of the heart tube, where apoptosis is seen. These may correspond to cells that are derived from the sec-ond heart field, which die in the absence of HAND2. The anti-apoptotic action of HAND2 is evident in the mesenchyme of the pharyngeal arches, where it is also expressed and where extensive cell death is seen in its absence79. This results in neural crest defects that affect the formation of the aorta and pulmonary trunk78. As discussed previously, such perturbations probably also affect the second heart field. Again, a conditional mutation that specifically affects this field should distinguish a direct effect on these cells.

Tbx20. Four recent papers describe the complex pheno-type of Tbx20 (T-box 20) mutants68,80–82. Contributions from both heart fields seem to be affected, and the expansion of myocardial cells within the heart tube is also abnormal. This latter defect is probably due to the continued expression of Tbx2, which is normally repressed by TBX20 as the myocardial chambers grow15,83,84. The absence of Hand1 expression in Tbx20 mutants indicates that the first lineage contribution to the left ventricle is affected, which probably reflects a direct effect of TBX20 on the maintenance of Nkx2-5 expression. Tbx20 mutants also show hypoplasia of the future right ventricle and lack a morphologically distinct outflow tract, which indicates effects on the myocardial contribution from the second heart field, where Tbx20 expression is detected68.

TBX20 synergizes with ISL1 and GATA4 to activate the Mef2c enhancer74, and a similar synergy is seen in vitro for an Nkx2-5 enhancer. Furthermore, TBX20 can function as an Isl1 repressor. In the Tbx20 mutant, Isl1 expresson is unaltered in the second heart field, but is observed throughout the heart tube, indicating its maintenance in cells that are derived from this field and activation in cells of the first lineage in the absence of TBX20.

Figure 5 | Regulatory networks in the second heart field. The solid lines indicate that direct in vivo activation of regulatory sequences has been demonstrated. Dotted lines indicate genetic data or in vitro activation. Foxa2 (forkhead box A2) is expressed in the mesenchyme of the pharyngeal arches, but is shown here as it has a putative role in the regulatory networks that are shown. Bop, also known as Smyd1, SET and MYND domain containing 1; Fgf8/10, fibroblast growth factor 8 and 10; Foxc1/c2/h1, forkhead box C1, C2 and H1; Gata4, GATA-binding protein 4; Hand2, heart and neural crest derivatives expressed transcript 2; Isl1, insulin gene enhancer protein, a LIM homeodomain transcription factor; Mef2c, myocyte enhancer factor 2C (also known as RSRF, related to serum response factor); Nkx2-5, NK2 transcription factor related, locus 5; Tbx1/20, T-box 1 and 20.


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ConclusionsThe classical view of cardiogenesis has been modified by the demonstration that there are two myocardial cell lineages and by the identification of a second heart field, which is characterized by specific regulatory networks (FIG. 5). We have concentrated here on genes for which mutant phenotypes have been described. However, an increasing number of second heart-field markers are emerging. Re-examination of the mutant phenotypes of other genes that are expressed in the developing heart may reveal their roles in the second lineage.

The identification of the second heart field is likely to have an important effect on the classification and interpretation of human congenital heart defects. Until now these malformations have been considered

mainly in the context of the segmental model of heart development85,86. Malformations of the outflow region, which are observed in 0.3% of live births, can now be examined by a candidate-gene approach that is based on what is known about the regulation of the second heart field. Null mutations in the main cardiac-regu-latory genes are likely to be lethal, as in the mouse. However, some congenital heart defects have already been linked to haploinsufficiency of these genes, as in the case of Holt–Oram syndrome for Tbx5 REF. 50 and DiGeorge syndrome for Tbx1 REFS 5860. In the future, other genes that are expressed in the second heart field may prove to be linked to malformations of the human heart, highlighting the importance of understanding its role in cardiac development.

1. Garcia-Martinez, V. & Schoenwolf, G. C. Primitive-streak origin of the cardiovascular system in avian embryos. Dev. Biol. 159, 706–719 (1993).

2. Tam, P. P., Parameswaran, M., Kinder, S. J. & Weinberger, R. P. The allocation of epiblast cells to the embryonic heart and other mesodermal lineages: the role of ingression and tissue movement during gastrulation. Development 124, 1631–1642 (1997).

3. Rawles, M. E. The heart-forming areas of the early chick blastoderm. Physiol. Zool. 22–42 (1943).

4. Rosenquist, G. C. Location and movements of cardiogenic cells in the chick embryo: the heart-forming portion of the primitive streak. Dev. Biol. 22, 461–475 (1970).

5. de la Cruz, M. & Markwald, R. (eds) Living Morphogenesis of the Heart (Birkhauser, Boston, 1999).

6. de la Cruz, M. V., Sanchez Gomez, C., Arteaga, M. M. & Arguello, C. Experimental study of the development of the truncus and the conus in the chick embryo. J. Anat. 123, 661–686 (1977).

7. Rosenquist, G. C. & DeHaan, R. L. Migration of precardiac cells in the chick embryo: a radioautographic study. Contrib. Embryol. 38, 111–121 (1966).

8. Stalsberg, H. & DeHaan, R. L. The precardiac areas and formation of the tubular heart in the chick embryo. Dev. Biol. 19, 128–159 (1969).

9. Inagaki, T., Garcia-Martinez, V. & Schoenwolf, G. C. Regulative ability of the prospective cardiogenic and vasculogenic areas of the primitive streak during avian gastrulation. Dev. Dyn. 197, 57–68 (1993).

10. Patwardhan, V., Fernandez, S., Montgomery, M. & Litvin, J. The rostro-caudal position of cardiac myocytes affect their fate. Dev. Dyn. 218, 123–135 (2000).

11. Redkar, A., Montgomery, M. & Litvin, J. Fate map of early avian cardiac progenitor cells. Development 128, 2269–2279 (2001).

12. Satin, J., Fujii, S. & DeHaan, R. L. Development of cardiac beat rate in early chick embryos is regulated by regional cues. Dev. Biol. 129, 103–113 (1988).

13. Meilhac, S. M. et al. A retrospective clonal analysis of the myocardium reveals two phases of clonal growth in the developing mouse heart. Development 130, 3877–3889 (2003).

14. Christoffels, V. M. et al. Chamber formation and morphogenesis in the developing mammalian heart. Dev. Biol. 223, 266–278 (2000).

15. Christoffels, V. M. et al. T-box transcription factor Tbx2 represses differentiation and formation of the cardiac chambers. Dev. Dyn. 229, 763–770 (2004).

16. Meilhac, S. M., Esner, M., Kerszberg, M., Moss, J. E. & Buckingham, M. E. Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis. J. Cell Biol. 164, 97–109 (2004).

17. Laugwitz, K. L. et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433, 647–653 (2005).

18. Viragh, S. & Challice, C. E. Origin and differentiation of cardiac muscle cells in the mouse. J. Ultrastructure Res. 42, 1–24 (1973).

19. De Vries, P. A. Evolution of Precardiac and Splanchnic Mesoderm in Relationship to the Infundibulum and Truncus 31–48 (Raven, New York, 1981).

20. Mjaatvedt, C. H. et al. The outflow tract of the heart is recruited from a novel heart-forming field. Dev. Biol. 238, 97–109 (2001).

This paper demonstrates the existence of a second source of myocardial cells in pharyngeal mesoderm in the avian embryo; these cells constitute a novel heart field that contributes to the outflow-tract region of the heart.

21. Waldo, K. L. et al. Conotruncal myocardium arises from a secondary heart field. Development 128, 3179–3188 (2001).Similar to reference 20, this study describes manipulations in the chick embryo that showed the presence of a novel heart field that contributes to outflow-tract myocardium. These cells are reported to be located anteriorly to the heart tube and immediately adjacent to it.

22. Kelly, R. G., Brown, N. A. & Buckingham, M. E. The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev. Cell 1, 435–440 (2001).This study in mice demonstrates the existence of a second source of myocardial cells in pharyngeal mesoderm that contribute to the outflow-tract myocardium at the arterial pole of the heart. These cells initially lie medially to the cardiac crescent before assuming a position that is dorsal and anterior to the heart tube.

23. Zaffran, S., Kelly, R. G., Meilhac, S. M., Buckingham, M. E. & Brown, N. A. Right ventricular myocardium derives from the anterior heart field. Circ. Res. 95, 261–268 (2004).This paper extends the observations reported in reference 22 to show that pharyngeal mesoderm also contributes to right-ventricular, as well as outflow-tract, myocardium. In addition, the authors show that the primitive heart tube in the mouse has an essentially left-ventricular identity.

24. Kelly, R. G. & Buckingham, M. E. The anterior heart-forming field: voyage to the arterial pole of the heart. Trends Genet. 18, 210–216 (2002).

25. Cai, C. L. et al. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877–889 (2003).This paper provides evidence for a more extensive second heart field. The authors show that Isl1 is expressed and required in myocardial progenitor cells that contribute to the venous as well as the arterial pole of the mouse heart.

26. Meilhac, S. M., Esner, M., Kelly, R. G., Nicolas, J. F. & Buckingham, M. E. The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell 6, 685–698 (2004).This paper presents a retrospective clonal analysis of myocardial cells in the early mouse heart and demonstrates the existence of two lineages that segregate early. The second lineage contribution can be compared to that of the second heart field, marked by Isl1, whereas the first lineage colonizes all myocardium except that of the outflow tract.

27. Yutzey, K. E., Rhee, J. T. & Bader, D. Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart. Development 120, 871–883 (1994).

28. Kitajima, S., Takagi, A., Inoue, T. & Saga, Y. MesP1 and MesP2 are essential for the development of cardiac mesoderm. Development 127, 3215–3226 (2003).

29. Brand, T. Heart development: molecular insights into cardiac specification and early morphogenesis. Dev. Biol. 258, 1–19 (2003).

30. Brown, C. O. 3rd et al. The cardiac determination factor, Nkx2-5, is activated by mutual cofactors GATA-4 and Smad1/4 via a novel upstream enhancer. J. Biol. Chem. 279, 10659–10669 (2004).

31. Hochgreb, T. et al. A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field. Development 130, 5363–5374 (2003).

32. Franco, D. & Campione, M. The role of Pitx2 during cardiac development. Linking left-right signaling and congenital heart diseases. Trends Cardiovasc. Med. 13, 157–163 (2003).

33. Liu, C. et al. Pitx2c patterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions. Development 129, 5081–5091 (2002).

34. Kuo, C. T. et al. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 11, 1048–1060 (1997).

35. Molkentin, J. D., Lin, Q., Duncan, S. A. & Olson, E. N. Requirement for the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 11, 1061–1072 (1997).

36. Li, S., Zhou, D., Lu, M. M. & Morrisey, E. E. Advanced cardiac morphogenesis does not require heart tube fusion. Science 305, 1619–1622 (2004).

37. Lin, Q., Schwarz, J., Bucana, C. & Olson, E. N. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science 276, 1404–1407 (1997).

38. Tanaka, M., Schinke, M., Liao, H. S., Yamasaki, N. & Izumo, S. Nkx2.5 and Nkx2.6, homologs of Drosophila tinman, are required for development of the pharynx. Mol. Cell. Biol. 21, 4391–4398 (2001).

39. Small, E. M. & Krieg, P. A. Molecular regulation of cardiac chamber-specific gene expression. Trends Cardiovasc. Med. 14, 13–18 (2004).

40. Habets, P. E., Moorman, A. F. & Christoffels, V. M. Regulatory modules in the developing heart. Cardiovasc. Res. 58, 246–263 (2003).

41. Lyons, I. et al. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev. 9, 1654–1666 (1995).

42. Tanaka, M. et al. Complex modular cis-acting elements regulate expression of the cardiac specifying homeobox gene Csx/Nkx2.5. Development 126, 1439–1450 (1999).

43. Yamagishi, H. et al. The combinatorial activities of Nkx2.5 and dHAND are essential for cardiac ventricle formation. Dev. Biol. 239, 190–203 (2001).

44. Srivastava, D. HAND proteins: molecular mediators of cardiac development and congenital heart disease. Trends Cardiovasc. Med. 9, 11–18 (1999).

45. Biben, C. & Harvey, R. P. Homeodomain factor Nkx2–5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development. Genes Dev. 11, 1357–1369 (1997).

46. Riley, P., Anson-Cartwright, L. & Cross, J. C. The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis. Nature Genet. 18, 271–275 (1998).

47. Firulli, A. B., McFadden, D. G., Lin, Q., Srivastava, D. & Olson, E. N. Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1. Nature Genet. 18, 266–270 (1998).


R E V I E W S


48. McFadden, D. G. et al. A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart. Development 127, 5331–5341 (2000).

49. McFadden, D. G. et al. The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner. Development 132, 189–201 (2005).

50. Bruneau, B. G. et al. A murine model of Holt–Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106, 709–721 (2001).

51. Takeuchi, J. K. et al. Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis. Development 130, 5953–5964 (2003).

52. Min, H. et al. Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev. 12, 3156–3161 (1998).

53. Sekine, K. et al. Fgf10 is essential for limb and lung formation. Nature Genet. 21, 138–141 (1999).

54. Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K. & Meyers, E. N. Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse. Development 129, 4613–4625 (2002).

55. Frank, D. U. et al. An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome. Development 129, 4591–4603 (2002).

56. Yelbuz, T. M. et al. Myocardial volume and organization are changed by failure of addition of secondary heart field myocardium to the cardiac outflow tract. Dev. Dyn. 228, 152–160 (2003).

57. Garg, V. et al. Tbx1, a DiGeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development. Dev. Biol. 235, 62–73 (2001).

58. Jerome, L. A. & Papaioannou, V. E. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nature Genet. 27, 286–291 (2001).

59. Lindsay, E. A. et al. Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410, 97–101 (2001).

60. Merscher, S. et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104, 619–629 (2001).

61. Hu, T. et al. Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors. Development 131, 5491–5502 (2004).This paper uses a genetic approach to show a role of TBX1 in the second heart field, distinguishing effects on outflow-tract myocardium from those on pharyngeal-arch development. The authors also identify a TBX1-dependent enhancer that functions on the Fgf8 gene, so providing evidence for a direct link between these markers of the second heart field.

62. Brown, C. B. et al. Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse. Dev. Biol. 267, 190–202 (2004).With the caveat that Tbx1 is not re-expressed in the myocardium, these Cre–loxP experiments indicate that the second heart field makes an extensive contribution to the adult heart.

63. Xu, H. et al. Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract. Development 131, 3217–3227 (2004).This paper, as with reference 64, points to a role for Tbx1 in the anterior part of the second heart field and shows that Tbx1-expressing progenitor cells contribute to the arterial pole of the heart. Tbx1 affects the proliferation of these cells, possibly through an effect on Fgf10.

64. Vitelli, F. et al. A genetic link between Tbx1 and fibroblast growth factor signaling. Development 129, 4605–4611 (2002).

65. Kochilas, L. et al. The role of neural crest during cardiac development in a mouse model of DiGeorge syndrome. Dev. Biol. 251, 157–166 (2002).

66. Kume, T., Jiang, H., Topczewska, J. M. & Hogan, B. L. The murine winged helix transcription factors, Foxc1 and Foxc2, are both required for cardiovascular development and somitogenesis. Genes Dev. 15, 2470–2482 (2001).

67. Yamagishi, H. et al. Tbx1 is regulated by tissue-specific forkhead proteins through a common Sonic hedgehog-responsive enhancer. Genes Dev. 17, 269–281 (2003).

68. Takeuchi, J. K. et al. Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development. Development 132, 2463–2474 (2005).

69. von Both, I. et al. Foxh1 is essential for development of the anterior heart field. Dev. Cell 7, 331–345 (2004).These authors show that FOXH1, expressed in the anterior part of the second heart field, has a role in the formation of outflow-tract and right-ventricular myocardium. They also propose that Mef2c may be regulated by FOXH1, in conjunction with NKX2-5.

70. Chen, X. et al. Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389, 85–89 (1997).

71. Weisberg, E. et al. A mouse homologue of FAST-1 transduces TGF-β superfamily signals and is expressed during early embryogenesis. Mech. Dev. 79, 17–27 (1998).

72. Edmondson, D. G., Lyons, G. E., Martin, J. F. & Olson, E. N. Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development 120, 1251–1263 (1994).

73. Pollock, R. & Treisman, R. Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes Dev. 5, 2327–2341 (1991).

74. Dodou, E., Verzi, M. P., Anderson, J. P., Xu, S. M. & Black, B. L. Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development. Development 131, 3931–3942 (2004).This paper describes an enhancer that controls expression of the Mef2c gene. The enhancer is active in the second heart field and is activated by ISL1 and GATA4, so providing an example of a regulatory network in this field.

75. Phan, D. et al. BOP, a regulator of right ventricular heart development, is a direct transcriptional target of MEF2C in the developing heart. Development 132, 2669–2678 (2005).

76. Gottlieb, P. D. et al. Bop encodes a muscle-restricted protein containing MYND and SET domains and is essential for cardiac differentiation and morphogenesis. Nature Genet. 31, 25–32 (2002).

77. Srivastava, D., Cserjesi, P. & Olson, E. N. A subclass of bHLH proteins required for cardiac morphogenesis. Science 270, 1995–1999 (1995).

78. Srivastava, D. et al. Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nature Genet. 16, 154–160 (1997).

79. Thomas, T. et al. A signaling cascade involving endothelin-1, dHAND and msx1 regulates development of neural-crest-derived branchial arch mesenchyme. Development 125, 3005–3014 (1998).

80. Singh, M. K. et al. Tbx20 is essential for cardiac chamber differentiation and repression of Tbx2. Development 132, 2697–2707 (2005).

81. Stennard, F. A. et al. Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation. Development 132, 2451–2462 (2005).

82. Cai, C. L. et al. T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis. Development 132, 2475–2487 (2005).

83. Harrelson, Z. et al. Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development 131, 5041–5052 (2004).

84. Christoffels, V. M., Burch, J. B. & Moorman, A. F. Architectural plan for the heart: early patterning and delineation of the chambers and the nodes. Trends Cardiovasc. Med. 14, 301–307 (2004).

85. Clark, E. B. in The Genetics of Cardiovascular Diseases (eds Pierpont, M. E. & Moller, J. M.) 3–11 (Martinus-Nijoff, Boston, 1986).

86. Fishman, M. & Olson, E. Parsing the heart: genetic modules for organ assembly. Cell 17, 153–156 (1997).

87. Abu-Issa, R., Waldo, K. & Kirby, M. L. Heart fields: one, two or more? Dev. Biol. 272, 281–285 (2004).

AcknowledgementsWe are grateful to members of the Buckingham laboratory for fruitful discussion. Work on cardiogenesis in the laboratory of M.B. is supported by grants from the Pasteur Institute and the Centre National de la Recherche Scientifique. S.M.M. is sup-ported by a Marie Curie Intra-European Fellowship within the Sixth European Framework Programme.

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneFgf8 | Fgf10 | Foxh1 | Foxp4 | GATA4 | Hand1 | Hand2 | Isl1 | Mef2c | MESP1 | MESP2 | NKX2-5 | PITX2 | Tbx1 | Tbx5 | Tbx20OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM DiGeorge syndrome | Holt–Oram syndromeAccess to this interactive links box is free online.


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Building the mammalian heart from two sources of myocardial cells

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