- Email: [email protected]
Early steps in vertebrate cardiogenesis
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Early steps in vertebrate cardiogenesis Tim Mohun* and Duncan Sparrowt Heart formation provides an excellent model for studying the molecular basis of cell determination in vertebrate embryos. By combining molecular assays with the experimental approaches of classic embryology, a model for the cell signalling events that initiate cardiogenesis is emerging. Studies of chick, amphibian, and fish embryos demonstrate the inductive role of dorso-anterior endoderm in specifying the cardiac fate of adjacent mesoderm. A consequence of this signalling is the onset of cardiomyogenesis and several transcription factors - Nkx2-5-related, HAND, GATA and MEF-2 families - contribute to these events.
Addresses
National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 tAA, UK "e-mail: [email protected] +e-mail: [email protected] Current Opinion in Genetics & Development 1997, 7:628-633
http://biomednet.com/elecref/O959437X00700628 © Current Biology Ltd ISSN 0959-437X Abbreviations atrial natriuretic peptide ANP basic helix-loop-helix bH LH bone morphogenetic protein BMP B-type natriuretic peptide BNP fibroblast growth factor FGF MCM1, agamous, deficiens, serum response factor MADS myosin heavy chain MHC TGF-[3 transforming growth factor-~
Introduction In vertebrate embryos, the heart originates from mesodermal tissue and initially forms as a simple tube comprising an outer myocardial epithelium separated by cardiac jelly from the inner endocardial layer. This simple contractile vessel is patterned into atrial and ventricular portions and is then transformed by elongation, looping and locally distinct patterns of differentiation into the mature multi-chambered heart that is characteristic of each vertebrate species. Here, we review recent advances in our understanding of the earliest steps in this process; the origins of cardiac progenitor cells, the inductive tissue interactions necessary for commitment to a cardiac fate, the molecular signals responsible, and the probable identity of key regulatory" genes in the cardiomyogenic program.
The origins of cardiac progenitors Despite the profound differences between different vertebrate species in embryo morphology and the mechanisms of gastrulation [1"], all show a common origin for cardiac progenitors. In each species, cardiac precursors comprise a pair of bilaterally symmetrical regions of dorso-lateral
mesoderm in the gastrula embryo. These migrate medially and fuse to form the heart tube on the anterior ventral midlinc. Historically, studies of heart formation have largely been restricted to chick and amphibian embryos because of their convenience for manipulation. In both, the relative topological pattern of mesodermal precursors revealed in blastula fate maps is maintained during gastrulation [2]. Recent studies show the same to bc true of mouse embryos. Mouse cardiac progenitors map to posterior lateral regions of the epiblast and, after ingression through the primitive streak, migrate to the proximal anterior position at which the heart tube is formed [3]. Cardiac
cell lineages
Precursors of both the endocardia] and myocardial layers are present within the cardiac mesoderm. In the mouse embryo, myocardial and cndocardial precursors have been mapped to the same regions of the mouse epiblast bflt their lineage relationship has yet to be established [4"]. Retroviral labelling studies in chick [5] and cell lineage tracing in zebrafish embryos [6], however, both indicate that myocardial and endocardial lineages are distinct prior to gastrulation. The clonal cell line QCE-6, which is derived from the cardiogenic mesoderm of quail embwos, behaves like a lineage precursor of both cell types and will differentiate into a mixed population of epithelial and mesenchymal cells [7-9]. The former express myocardial markers whereas the latter express an endothelial-specific epitope which identifies endocardial cells of the embryo prior to heart tube formation [I0]. In embryos of the zebrafish mutant cloche, the endocardial layer of the heart and anterior endothelial tissue are absent whereas myocardial differentiation is unaffected [11]. The cloche mutation appears to affect an early step in formation of the endothelial cell lineage as expression of the endothelial-specific receptor tyrosine kinase genes ./tk-1 and tie are also affected [12]. Within the chick embryo myocardial precursors, distinct populations giving rise to atrial and ventricular myocytes have been distinguished [2,13]. These correspond to posterior and anterior regions of the cardiac mesoderm in the early gastrula, the distinct fates of which are specified by this stage. No direct evidence for the early divergence of atrial and ventricular myocyte lineages is available for other species but it is noteworthy that genetic screens for early developmental mutants in zebrafish have identified mutations in which the ventricle is either selectively reduced or absent [14°,15°]. Studies of the chick have identified a further lineage division that
Early steps in
separates precursors of the Purkinje fibres from ventricular myocytes [16"] but the lineage relationships between myocytes and the central cardiac conduction system are currently unknown.
Commitment to a cardiac cell fate Whereas distinct endocardial, atrial, and ventricular lineages are probably established by the onset of gastrulation in the vertebrate embryo, commitment to a cardiac fate is thought to result from inductive interactions during the course of gastrulation. Many studies have suggested a role for endoderm in promoting cardiac differentiation from explants of cardiac mesoderm (see [17,18] and references therein). It is now clear that, in addition to this permissive role, endoderm adjacent to the cardiac precursors is a source of instructive signalling capable of respecifying non-cardiac mesoderm to a cardiac fate [18,19]. In the chick, this signalling capacity has been detected during mid- to late-gastrula stages in anterior endoderm from both lateral and central locations [18]. Cardiac precursors are only located in antero-lateral mesoderm and become specified during the same period ([20]; see below). In the amphibian Xenopus, deep dorso-anterior endoderm abuts the newly ingressing mesoderm and remains associated with the cardiac precursors throughout gastrulation. Cardiac mesoderm becomes specified by mid-gastrula as a result of signals from both this and from organiser tissue. The inductive capacity of dorso-anterior cndoderm can be detected using explant recombinations derived from early gastrulae [19]. In the mouse embryo, migration of the cardiac mesoderm from a distal posterior location to the proximal anterior site of heart tube formation mirrors the movement of definitive endoderm, with the result that the cardiac mesoderm and foregut (pharyngeal) endoderm remain intimately associated throughout gastrulation [3,21]. Although the timing of cardiac specification has not been established, heterochronic transplantation studies show that non-cardiac portions of the mouse epiblast will contribute to the heart when transplanted directly to the proximal anterior location of heart tube formation [4"]. This remarkable result demonstrates that participation in the morphogenetic movements of gastrulation is not essential for cells to acquire a cardiac fate; furthermore, any inductive signalling from the foregut endoderm continues until the late primitive streak stage, after cardiac precursors have reached the site of heart tube formation.
vertebratecardiogenesisMohun and Sparrow
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incomplete when experimentally induced in non-cardiac rather than cardiac mesoderm. Individual cardiac lineages may possess intrinsic differences that permit differing responses to inductive signalling from the endoderm and such differences may be essential for complete cardiogenesis. T h e results of Spemann 'organiser' grafts in amphibian embryos might appear to contradict this possibilit'y, as a heart is induced as part of the secondary axis on the ventral side of the embryo. Axis duplication, however, requires the presence of organiser tissue as well as the associated dorsal deep endoderm in the donor graft [19] and involves dorsal respecification of ventral mesoderm. An alternative possibility is suggested from the findings that the relative positions of individual tissue precurs o r s - a n d , indeed, myocardial l i n e a g e s - - m a p p e d in the blastula stage embryo remain essentially unchanged by the events of gastrulation, irrespective of its particular mechanism (see [1 °] and references above). In this proposal, individual cardiac lineages represent discrete geographical domains that subsequently experience distinct inductive signals, either as a result of positional information or by exposure to differing signalling molecules. Non-cardiac mesoderm could replace these lineages as it would be exposed to the same spatial patterning influences that form part of the endodermal inductive influence. Establishing the identity of endodermal signals will help resolve this issue.
Signalling molecules regulating cardiac differentiation In the chick, explants of non-cardiac (anterior medial) mesoderm from the mid/late gastrula embryo have been induced to undergo terminal cardiac differentiation by exposure to the TGF-[3-family growth factors, BMP-2 and BMP-4 [22"]. Inclusion of adjacent neural plate and notochord in the culture blocked terminal differentiation, although early markers of cardiac fate were still activated. Implantation of BMP-2-soaked heparin-acrylamide beads into the anterior mesoderm of cultured embryos also induced localised ectopic expression of early cardiac molecular markers and the extent of terminal differentiation depended on the proximity to the neural tube and notochord. These results suggest that BMPs act as positive inducers of cardiac fate whereas terminal differentiation is blocked by other signals from axial structures. Consistent with this view, cells from the neural tube have been found to inhibit differentiation of cardiac precursors in chick embryo cell reaggregation experiments [23].
Cell lineages and endodermal signalling If cardiac mesoderm specification is the result of instructive rather than permissive signals from localised regions of the endoderm, what is to be made of the existence of distinct cardiac lineages as early as the blastula stage? Lineage-specific markers have not actually been used in studies of the inductive capacity of antero-lateral endoderm and it may be that cardiac differentiation is
In normal development, precardiac mesoderm is indeed in contact with BMP-expressing tissue during the period when it becomes specified to a cardiac fate: ectoderm overlying the precardiac mesoderm first expresses BMP-4 transcripts and then, in addition, those of BMP-7; BMP-2 RNA is present in the endoderm which underlies the precardiac mesoderm [22"]. Furthermore. the differentiation
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of specified cardiac mesoendoderm in explant culture is blocked by exposure to the amphibian BMP antagonist noggin, giving direct evidence that BMP activity is necessary for cardiogenesis. In the mouse, homozygous mutations have been generated individually in BMP-2, BMP-4 and BMP-7 but it has proved difficult to assess their impact on heart formation because of variability in phenotype and the likelihood of some functional redundancy (see [22"°,24] for discussions of this point). BMP-2 was incapable of inducing cardiac differentiation in posterior mesodermal tissue, either by implantation of BMP-2 beads or by culture of explants in the presence of soluble protein. This is in marked contrast to the inducing properties of antero-lateral endoderm [18] and suggests that this tissue possesses a second inducing activity that specifies an anterior mesodermal domain [22"1. Treatment of posterior lateral mesoderm from early chick neurula embryos with a combination of BMP-2 and FGF-4 causes terminal differentiation whereas neither factor alone has any effect. FGF-like proteins have been detected in the anterior endoderm, raising the possibility that one of these may act as the second endodermal signal [25]. Another candidate is the secreted protein, cerberus, transcripts for which are Iocalised to the leading edge of the gastrulating mesendoderm and hence in the anterior endodermal tissue that lies adjacent to the mesodermal cardiac precursors throughout gastrulation. Ectopic expression of cerberus has been reported to induce ectopic heads and, in some cases, the duplication of heart and liver [26"]. Transcription f a c t o r s a n d c a r d i a c m y o g e n e s i s Several families of transcription factors have been implicated in the processes of cardiac commitment and differentiation. The best-studied examples are members of the Nk2 class of homeobox genes that appear to be vertebrate counterparts of the Drosophila tinman gene (reviewed in [27"]). Nkx2-5 and related proteins Nkx2-5 is expressed in the cardiac mesoderm of vertebrate embryos and provides the earliest marker of cardiac mesoderm induction. Transcripts are also expressed in other tissues during development, including foregut, pharyngeal region, and tongue. In Xenopus embryos, ectopic expression of Nkx2-5 causes a general increase in heart size but no ectopic cardiac differentiation has been detected [28"]. A similar increase in heart size has been reported in zebrafish embryos, which occasionally also show ectopic MHC-positive cells [29°]. Forced expression of nkx2.5 in a zebrafish fibroblast cell line induces cardiac differentiation markers but no comparable result has been reported with mammalian cell lines. In some circumstances, therefore, vertebrate Nkx2-5 genes may show cardiomyogenic properties but these are not comparable to the potent skeletal myogenic activities of the MyoD transcription factor family.
In the mouse, targeted interruption of the Nkx2-5 gene severely disrupts heart formation but does not prevent either initial formation of the heart tube or the expression of many cardiac differentiation markers [30] . This might indicate a relatively late role for Nkx2-5 in cardiogenesis but this seems unlikely given its early expression pattern. Alternatively, it may be a result of functional redundancy amongst closely related members of the vertebrate iVk2 gene family. Two other genes that are almost identical to Nkx2-5 in homeodomain sequence are Nkx2-3 and Nkx2-7. Nkx2-3 is expressed in the gut and branchial arches in all species studied and in both chick and frog its transcripts are also prevalent in cardiac mesoderm [31-34]. nkx2.7 has been identified in zebrafish embryos where its domain of expression encompasses that of nkx2.5 but first appears several hours earlier [34]. A third relative, Nkx2-8, has recently been identified in chick (GenBank accession numbers Yl1949 and Y10655) and it is clear that the Nkx2-5-related proteins are only one group within an extensive vertebrate Nk2 gene family Downstream targets for Nkx2-5 Studies of Nkx2-5 mutant phenotype have identified several genes that lie downstream of Nbx2-5 in the cardiogenic program. In the mouse, the basic helix-loop-helix ( b H L H ) transcription factor eHAND is expressed in a strongly left dominant fashion within the myocardium as part of a complex embryonic expression pattern [35]. A similar pattern is evident in amphibian embryos (T Mohun, D Sparrow, unpublished data). In the Nkx2-5 mutant, asymmetrical expression of eHAND is disrupted [35], suggesting that eHAND expression in the myocardium is regulated, either directly or indirectl.x, by N/ex2-5.
T h e functions of eHAND in early development have been investigated, along with those of the related gene dHAND, by lack-of-function mutations. In chick embryos, antisense depletion of both HAND transcripts--but not either a l o n e - - c a u s e s arrest of cardiac development immediately prior to looping [36]. In dHAND knockout mice, heart looping is incomplete and the right ventricle fails to form [37"]. Interestingly; whereas the expression of many cardiac differentiation markers is unaffected by the mutation, transcript levels for the GATA-4 transcription factor are substantially reduced in the heart but are normal in other regions of the embryo. Most markers of cardiac muscle differentiation appear to be expressed normally in Nkx2-5 mutant mouse embryos. A notable exception is the ventricular-specific mvosin light chain gene (IIILC2v), expression of which is severely reduced. Earlier studies have identified a short (28 bp) motif from the proximal promoter that is necessary for tissue- and stage-specific expression of an ,IILC2v transgene [38]. A recently identified cardiac-specific ankyrin repeat protein, carp, may act as a negative coregulator of the MLC2v gene, binding the ubiquitous transcription factor YB-1 to the 28 bp motif [39]. Expression of C
Early steps in vertebrate cardiogenesis Mohun and Sparrow
may, in some manner, mediate the effects of Nkx2-5 on the MLC2v promoter. T h e precise nature of this regulation is unclear as the 250bp proximal promoter that contains the 28 bp motif is sufficient to confer ventricular-restricted expression on a transgene in both normal and Nkx2-5 mutant embryos [38]. Nkx2-5 and MEF-2 transcription factors
In Drosophila, DMEF-2 is a direct target for tinman [40°]. DMEF-2 encodes a MADS transcription factor necessary for cardiac and somatic muscle differentiation and similar roles are played by several proteins encoded by the MEF-2 gene family in vertebrates. In Xenopus, overexpression of XMEF-2A, like XNkx2.5, results in an enlarged heart in the tadpole [41], suggesting thatMEF-2 genes lie in a common regulatory pathway with vertebrate tinman homologues. In mice, MEF-2C is the first MEF-2 gene to be expressed in the developing heart and targeted interruption of this gene disrupts embryonic heart formation [42*]. A linear heart tube still forms in these embryos but subsequently fails to undergo looping and appears to lack the right ventricle. Molecular studies show that the levels of some cardiac differentiation markers are r e d u c e d - - t h o u g h not completely a b s e n t - - b u t many, including Nkx2-5, appear to be normal. Interestingly, expression of MEF-2B is highly upregulated in the hearts of MEF-2C mutant mice, again raising the possibility that functional redundancy between MEF-2 family members ameliorates the severity of the ,IIEF-2C mutant phenotype. GATA factors in cardiogenesis
Several members of the GATA family of zinc finger domain transcription factors have been implicated as regulators of cardiogenic differentiation. GATA-4, -5 and -6 a r e expressed in distinct but overlapping domains that include the cardiac mcsoderm prior to heart tube formation. In later development, their expression becomes restricted to heart, gut, and visceral endoderm [43-46]. Functional GATA-binding sites have been identified in the promoters of several cardiac-specific genes--including ANP, BNP, and c*-MHC. Specific depletion of GATA-4 by antisense oligonucleotides blocks dimethyl sulfoxide induced cardiac differentiation in the mouse embryonic stem cell line, P19 [47]. Overexpression of GATA-4 in the same cells increases the number of terminally differentiated cells, as well as molecular markers such as Nkx2-5. Together, these results suggest an essential role for GATA-4 in cardiac differentiation. Targeted disruption of the mouse GATA-4 gene does not support this view, however. In GATA-4 knockout mice, the heart tube fails to form but this does not appear to be caused by a failure in cardiomyogenesis. Rather, it appears to result from a defect in rostral-to-caudal and lateral-to-ventral folding of the embryo [48°,49"]. Differentiated cardiomyocytes form in the homozygous mutant embryos and markers of terminal cardiac differentiation are expressed normally. Once again, an explanation for the
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disparity between these results and those of the P19 cell line may be functional redundancy amongst the GATA factors. Thus, the level of GATA-6 transcripts is elevated in GATA-4 knockout embryos, whereas only low levels of expression are detected in the treated P19 cells [47]. Overexpression of GATA-6 in Xenopus embryos, however, has recently been found to inhibit terminal differentiation within the myocardium [44] and it is difficult to reconcile these findings with those of the mouse studies. Combinatorial regulation of cardiac differentiation
As -yet, few direct gene targets of Nkx2-5 or its relatives have been unambiguously identified and little is known about how these transcription factors function. One probable target is the cardiac atrial natHuretic factor (ANF) gene which contains a highly conserved Nkx2-5 responsive element (the NKE) within its proximal promoter [50]. Studies of this promoter in cultured cells indicate that cardiac-specific expression may require the activity of several factors, both positive and negative, including Nkx2-5 and a GATA factor. Similarly, studies of the chick cardiac actin gene have suggested that the Nkx2-5 functions in a combinatorial manner and can interact with the MADS family transcription factor, SRF (serum response factor) [51]. Conclusions
It is now possible to formulate a common model for the earliest steps in heart formation in vertebrates. Anterior regions of the newly formed mesoderm receive at least two conceptually distinct instructive signals from immediately adjacent endoderm. One establishes a propensity or capacity for heart formation that distinguishes anterolateral mesodermal cells from more posterior neighbours. Members of the FGF family and the secreted protein cerberus are candidates for this signal. A second inductive interaction results in specification of bilateral cardiac progenitors and is probably mediated by one or more BMP(s). The precise boundaries of cardiac mesodcrm arc influenced by other signals within the embryo (e.g. noggin or chordin) that regulate BMP activity. Within each region of cardiac mesoderm, atrial, ventricular, and endothelial lineages are distinct and are probably organised in a spatial pattern that prefigures their arrangement in the linear heart tube. Understanding the mechanisms responsible for this patterning is a major goal for the future. Cardiac specification is accompanied by expression of several homeodomain proteins of the Nk2 f a m i l y - - m o s t notably Nkx2-5 - - but the number of these and their individual roles are unclear. Studies of cardiac-specific transcription suggest that these proteins function in a combinatorial manner (e.g. with GATA proteins) to regulate terminal differentiation. Nkx2-5 also appears to regulate the expression of other transcription factors (such as MEF-2 and H A N D proteins) that are active in early cardiomyogenesis. Genetic approaches will prove decisive in unravelling the relative contributions of
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these proteins and their relative positions in the cardiac regulatory program.
Note added in proof An extensive review of vertebrate heart development has been published recently [52"].
Acknowledgements T M o h u n and I) Sparrow are supported by the [\ledical Research (~ouncil and the British Heart Foundation. \Ve would like to thank Richard Harvey fl~r critical c o m m e n t s on the manuscript.
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Early steps in vertebrate c a r d i o g e n e s i s Mohun and Sparrow
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Zou Y, Evans S, Chen J, Kuo H-C, Harvey RP, Chien KR: CARP, a cardiac ankyrin repeat protein is downstream in the Nkx2-5 h o m e o b o x gene pathway. Development 1997, 124:793-804.
40. •
Gajewski K, Kim Y, Lee YM, Olson EN, Schulz RA: D-mef2 is a target for tinman activation during Drosophila heart developmenL EMBO J 1997, 16:515-522.
Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek MS, Soudais C, Leiden JM: GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev 1997, 11:1048-1060. See annotation [49"].
49. •
Molkentin JD, Lin Q, Duncan SA, Olson EN: Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev 1997, 11:1061-1072. This paper and [48 °] both describe the effects of GATA-4 knockout in the mouse. Although cardiogenesis appears to occur, as measured by the presence of markers of terminal cardiac differentiation, a gross defect in embryonic folding prevents heart tube fusion and causes a generalized disruption of the ventral body pattern. 50.
Durocher D, Chen CY, Ardati A, Schwartz RJ, Nemer M: The atrial natriuretic factor promoter is a downstream target for Nkx2-5 in the myocardium. Mo/Cell Bio/1996, 16:4648-4655.
51.
Chen CY, Croissant J, Majesky M, Topouzis S, McQuinn 1", Frankovsky M J, Schwartz RJ: Activation of the cardiac alphaactin promoter depends upon serum response factor, tinman homologue, Nkx-2.5, and intact serum response elements. Dev Gen 1996, 19:119-130.
52. •o
Fishman MC, Chien KR: Fashioning the vertebrate heart: earliest embryonic decisions. Development 1997, 124:20992117. In addition to discussing the early events of cardiac specification, this review also covers later steps in morphogenesis - such as chamber formation - that are beyond the scope of our own review.
Early steps in vertebrate cardiogenesis Tim Mohun* and Duncan Sparrowt Heart formation provides an excellent model for studying the molecular basis of cell determination in vertebrate embryos. By combining molecular assays with the experimental approaches of classic embryology, a model for the cell signalling events that initiate cardiogenesis is emerging. Studies of chick, amphibian, and fish embryos demonstrate the inductive role of dorso-anterior endoderm in specifying the cardiac fate of adjacent mesoderm. A consequence of this signalling is the onset of cardiomyogenesis and several transcription factors - Nkx2-5-related, HAND, GATA and MEF-2 families - contribute to these events.
Addresses
National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 tAA, UK "e-mail: [email protected] +e-mail: [email protected] Current Opinion in Genetics & Development 1997, 7:628-633
http://biomednet.com/elecref/O959437X00700628 © Current Biology Ltd ISSN 0959-437X Abbreviations atrial natriuretic peptide ANP basic helix-loop-helix bH LH bone morphogenetic protein BMP B-type natriuretic peptide BNP fibroblast growth factor FGF MCM1, agamous, deficiens, serum response factor MADS myosin heavy chain MHC TGF-[3 transforming growth factor-~
Introduction In vertebrate embryos, the heart originates from mesodermal tissue and initially forms as a simple tube comprising an outer myocardial epithelium separated by cardiac jelly from the inner endocardial layer. This simple contractile vessel is patterned into atrial and ventricular portions and is then transformed by elongation, looping and locally distinct patterns of differentiation into the mature multi-chambered heart that is characteristic of each vertebrate species. Here, we review recent advances in our understanding of the earliest steps in this process; the origins of cardiac progenitor cells, the inductive tissue interactions necessary for commitment to a cardiac fate, the molecular signals responsible, and the probable identity of key regulatory" genes in the cardiomyogenic program.
The origins of cardiac progenitors Despite the profound differences between different vertebrate species in embryo morphology and the mechanisms of gastrulation [1"], all show a common origin for cardiac progenitors. In each species, cardiac precursors comprise a pair of bilaterally symmetrical regions of dorso-lateral
mesoderm in the gastrula embryo. These migrate medially and fuse to form the heart tube on the anterior ventral midlinc. Historically, studies of heart formation have largely been restricted to chick and amphibian embryos because of their convenience for manipulation. In both, the relative topological pattern of mesodermal precursors revealed in blastula fate maps is maintained during gastrulation [2]. Recent studies show the same to bc true of mouse embryos. Mouse cardiac progenitors map to posterior lateral regions of the epiblast and, after ingression through the primitive streak, migrate to the proximal anterior position at which the heart tube is formed [3]. Cardiac
cell lineages
Precursors of both the endocardia] and myocardial layers are present within the cardiac mesoderm. In the mouse embryo, myocardial and cndocardial precursors have been mapped to the same regions of the mouse epiblast bflt their lineage relationship has yet to be established [4"]. Retroviral labelling studies in chick [5] and cell lineage tracing in zebrafish embryos [6], however, both indicate that myocardial and endocardial lineages are distinct prior to gastrulation. The clonal cell line QCE-6, which is derived from the cardiogenic mesoderm of quail embwos, behaves like a lineage precursor of both cell types and will differentiate into a mixed population of epithelial and mesenchymal cells [7-9]. The former express myocardial markers whereas the latter express an endothelial-specific epitope which identifies endocardial cells of the embryo prior to heart tube formation [I0]. In embryos of the zebrafish mutant cloche, the endocardial layer of the heart and anterior endothelial tissue are absent whereas myocardial differentiation is unaffected [11]. The cloche mutation appears to affect an early step in formation of the endothelial cell lineage as expression of the endothelial-specific receptor tyrosine kinase genes ./tk-1 and tie are also affected [12]. Within the chick embryo myocardial precursors, distinct populations giving rise to atrial and ventricular myocytes have been distinguished [2,13]. These correspond to posterior and anterior regions of the cardiac mesoderm in the early gastrula, the distinct fates of which are specified by this stage. No direct evidence for the early divergence of atrial and ventricular myocyte lineages is available for other species but it is noteworthy that genetic screens for early developmental mutants in zebrafish have identified mutations in which the ventricle is either selectively reduced or absent [14°,15°]. Studies of the chick have identified a further lineage division that
Early steps in
separates precursors of the Purkinje fibres from ventricular myocytes [16"] but the lineage relationships between myocytes and the central cardiac conduction system are currently unknown.
Commitment to a cardiac cell fate Whereas distinct endocardial, atrial, and ventricular lineages are probably established by the onset of gastrulation in the vertebrate embryo, commitment to a cardiac fate is thought to result from inductive interactions during the course of gastrulation. Many studies have suggested a role for endoderm in promoting cardiac differentiation from explants of cardiac mesoderm (see [17,18] and references therein). It is now clear that, in addition to this permissive role, endoderm adjacent to the cardiac precursors is a source of instructive signalling capable of respecifying non-cardiac mesoderm to a cardiac fate [18,19]. In the chick, this signalling capacity has been detected during mid- to late-gastrula stages in anterior endoderm from both lateral and central locations [18]. Cardiac precursors are only located in antero-lateral mesoderm and become specified during the same period ([20]; see below). In the amphibian Xenopus, deep dorso-anterior endoderm abuts the newly ingressing mesoderm and remains associated with the cardiac precursors throughout gastrulation. Cardiac mesoderm becomes specified by mid-gastrula as a result of signals from both this and from organiser tissue. The inductive capacity of dorso-anterior cndoderm can be detected using explant recombinations derived from early gastrulae [19]. In the mouse embryo, migration of the cardiac mesoderm from a distal posterior location to the proximal anterior site of heart tube formation mirrors the movement of definitive endoderm, with the result that the cardiac mesoderm and foregut (pharyngeal) endoderm remain intimately associated throughout gastrulation [3,21]. Although the timing of cardiac specification has not been established, heterochronic transplantation studies show that non-cardiac portions of the mouse epiblast will contribute to the heart when transplanted directly to the proximal anterior location of heart tube formation [4"]. This remarkable result demonstrates that participation in the morphogenetic movements of gastrulation is not essential for cells to acquire a cardiac fate; furthermore, any inductive signalling from the foregut endoderm continues until the late primitive streak stage, after cardiac precursors have reached the site of heart tube formation.
vertebratecardiogenesisMohun and Sparrow
629
incomplete when experimentally induced in non-cardiac rather than cardiac mesoderm. Individual cardiac lineages may possess intrinsic differences that permit differing responses to inductive signalling from the endoderm and such differences may be essential for complete cardiogenesis. T h e results of Spemann 'organiser' grafts in amphibian embryos might appear to contradict this possibilit'y, as a heart is induced as part of the secondary axis on the ventral side of the embryo. Axis duplication, however, requires the presence of organiser tissue as well as the associated dorsal deep endoderm in the donor graft [19] and involves dorsal respecification of ventral mesoderm. An alternative possibility is suggested from the findings that the relative positions of individual tissue precurs o r s - a n d , indeed, myocardial l i n e a g e s - - m a p p e d in the blastula stage embryo remain essentially unchanged by the events of gastrulation, irrespective of its particular mechanism (see [1 °] and references above). In this proposal, individual cardiac lineages represent discrete geographical domains that subsequently experience distinct inductive signals, either as a result of positional information or by exposure to differing signalling molecules. Non-cardiac mesoderm could replace these lineages as it would be exposed to the same spatial patterning influences that form part of the endodermal inductive influence. Establishing the identity of endodermal signals will help resolve this issue.
Signalling molecules regulating cardiac differentiation In the chick, explants of non-cardiac (anterior medial) mesoderm from the mid/late gastrula embryo have been induced to undergo terminal cardiac differentiation by exposure to the TGF-[3-family growth factors, BMP-2 and BMP-4 [22"]. Inclusion of adjacent neural plate and notochord in the culture blocked terminal differentiation, although early markers of cardiac fate were still activated. Implantation of BMP-2-soaked heparin-acrylamide beads into the anterior mesoderm of cultured embryos also induced localised ectopic expression of early cardiac molecular markers and the extent of terminal differentiation depended on the proximity to the neural tube and notochord. These results suggest that BMPs act as positive inducers of cardiac fate whereas terminal differentiation is blocked by other signals from axial structures. Consistent with this view, cells from the neural tube have been found to inhibit differentiation of cardiac precursors in chick embryo cell reaggregation experiments [23].
Cell lineages and endodermal signalling If cardiac mesoderm specification is the result of instructive rather than permissive signals from localised regions of the endoderm, what is to be made of the existence of distinct cardiac lineages as early as the blastula stage? Lineage-specific markers have not actually been used in studies of the inductive capacity of antero-lateral endoderm and it may be that cardiac differentiation is
In normal development, precardiac mesoderm is indeed in contact with BMP-expressing tissue during the period when it becomes specified to a cardiac fate: ectoderm overlying the precardiac mesoderm first expresses BMP-4 transcripts and then, in addition, those of BMP-7; BMP-2 RNA is present in the endoderm which underlies the precardiac mesoderm [22"]. Furthermore. the differentiation
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of specified cardiac mesoendoderm in explant culture is blocked by exposure to the amphibian BMP antagonist noggin, giving direct evidence that BMP activity is necessary for cardiogenesis. In the mouse, homozygous mutations have been generated individually in BMP-2, BMP-4 and BMP-7 but it has proved difficult to assess their impact on heart formation because of variability in phenotype and the likelihood of some functional redundancy (see [22"°,24] for discussions of this point). BMP-2 was incapable of inducing cardiac differentiation in posterior mesodermal tissue, either by implantation of BMP-2 beads or by culture of explants in the presence of soluble protein. This is in marked contrast to the inducing properties of antero-lateral endoderm [18] and suggests that this tissue possesses a second inducing activity that specifies an anterior mesodermal domain [22"1. Treatment of posterior lateral mesoderm from early chick neurula embryos with a combination of BMP-2 and FGF-4 causes terminal differentiation whereas neither factor alone has any effect. FGF-like proteins have been detected in the anterior endoderm, raising the possibility that one of these may act as the second endodermal signal [25]. Another candidate is the secreted protein, cerberus, transcripts for which are Iocalised to the leading edge of the gastrulating mesendoderm and hence in the anterior endodermal tissue that lies adjacent to the mesodermal cardiac precursors throughout gastrulation. Ectopic expression of cerberus has been reported to induce ectopic heads and, in some cases, the duplication of heart and liver [26"]. Transcription f a c t o r s a n d c a r d i a c m y o g e n e s i s Several families of transcription factors have been implicated in the processes of cardiac commitment and differentiation. The best-studied examples are members of the Nk2 class of homeobox genes that appear to be vertebrate counterparts of the Drosophila tinman gene (reviewed in [27"]). Nkx2-5 and related proteins Nkx2-5 is expressed in the cardiac mesoderm of vertebrate embryos and provides the earliest marker of cardiac mesoderm induction. Transcripts are also expressed in other tissues during development, including foregut, pharyngeal region, and tongue. In Xenopus embryos, ectopic expression of Nkx2-5 causes a general increase in heart size but no ectopic cardiac differentiation has been detected [28"]. A similar increase in heart size has been reported in zebrafish embryos, which occasionally also show ectopic MHC-positive cells [29°]. Forced expression of nkx2.5 in a zebrafish fibroblast cell line induces cardiac differentiation markers but no comparable result has been reported with mammalian cell lines. In some circumstances, therefore, vertebrate Nkx2-5 genes may show cardiomyogenic properties but these are not comparable to the potent skeletal myogenic activities of the MyoD transcription factor family.
In the mouse, targeted interruption of the Nkx2-5 gene severely disrupts heart formation but does not prevent either initial formation of the heart tube or the expression of many cardiac differentiation markers [30] . This might indicate a relatively late role for Nkx2-5 in cardiogenesis but this seems unlikely given its early expression pattern. Alternatively, it may be a result of functional redundancy amongst closely related members of the vertebrate iVk2 gene family. Two other genes that are almost identical to Nkx2-5 in homeodomain sequence are Nkx2-3 and Nkx2-7. Nkx2-3 is expressed in the gut and branchial arches in all species studied and in both chick and frog its transcripts are also prevalent in cardiac mesoderm [31-34]. nkx2.7 has been identified in zebrafish embryos where its domain of expression encompasses that of nkx2.5 but first appears several hours earlier [34]. A third relative, Nkx2-8, has recently been identified in chick (GenBank accession numbers Yl1949 and Y10655) and it is clear that the Nkx2-5-related proteins are only one group within an extensive vertebrate Nk2 gene family Downstream targets for Nkx2-5 Studies of Nkx2-5 mutant phenotype have identified several genes that lie downstream of Nbx2-5 in the cardiogenic program. In the mouse, the basic helix-loop-helix ( b H L H ) transcription factor eHAND is expressed in a strongly left dominant fashion within the myocardium as part of a complex embryonic expression pattern [35]. A similar pattern is evident in amphibian embryos (T Mohun, D Sparrow, unpublished data). In the Nkx2-5 mutant, asymmetrical expression of eHAND is disrupted [35], suggesting that eHAND expression in the myocardium is regulated, either directly or indirectl.x, by N/ex2-5.
T h e functions of eHAND in early development have been investigated, along with those of the related gene dHAND, by lack-of-function mutations. In chick embryos, antisense depletion of both HAND transcripts--but not either a l o n e - - c a u s e s arrest of cardiac development immediately prior to looping [36]. In dHAND knockout mice, heart looping is incomplete and the right ventricle fails to form [37"]. Interestingly; whereas the expression of many cardiac differentiation markers is unaffected by the mutation, transcript levels for the GATA-4 transcription factor are substantially reduced in the heart but are normal in other regions of the embryo. Most markers of cardiac muscle differentiation appear to be expressed normally in Nkx2-5 mutant mouse embryos. A notable exception is the ventricular-specific mvosin light chain gene (IIILC2v), expression of which is severely reduced. Earlier studies have identified a short (28 bp) motif from the proximal promoter that is necessary for tissue- and stage-specific expression of an ,IILC2v transgene [38]. A recently identified cardiac-specific ankyrin repeat protein, carp, may act as a negative coregulator of the MLC2v gene, binding the ubiquitous transcription factor YB-1 to the 28 bp motif [39]. Expression of C
Early steps in vertebrate cardiogenesis Mohun and Sparrow
may, in some manner, mediate the effects of Nkx2-5 on the MLC2v promoter. T h e precise nature of this regulation is unclear as the 250bp proximal promoter that contains the 28 bp motif is sufficient to confer ventricular-restricted expression on a transgene in both normal and Nkx2-5 mutant embryos [38]. Nkx2-5 and MEF-2 transcription factors
In Drosophila, DMEF-2 is a direct target for tinman [40°]. DMEF-2 encodes a MADS transcription factor necessary for cardiac and somatic muscle differentiation and similar roles are played by several proteins encoded by the MEF-2 gene family in vertebrates. In Xenopus, overexpression of XMEF-2A, like XNkx2.5, results in an enlarged heart in the tadpole [41], suggesting thatMEF-2 genes lie in a common regulatory pathway with vertebrate tinman homologues. In mice, MEF-2C is the first MEF-2 gene to be expressed in the developing heart and targeted interruption of this gene disrupts embryonic heart formation [42*]. A linear heart tube still forms in these embryos but subsequently fails to undergo looping and appears to lack the right ventricle. Molecular studies show that the levels of some cardiac differentiation markers are r e d u c e d - - t h o u g h not completely a b s e n t - - b u t many, including Nkx2-5, appear to be normal. Interestingly, expression of MEF-2B is highly upregulated in the hearts of MEF-2C mutant mice, again raising the possibility that functional redundancy between MEF-2 family members ameliorates the severity of the ,IIEF-2C mutant phenotype. GATA factors in cardiogenesis
Several members of the GATA family of zinc finger domain transcription factors have been implicated as regulators of cardiogenic differentiation. GATA-4, -5 and -6 a r e expressed in distinct but overlapping domains that include the cardiac mcsoderm prior to heart tube formation. In later development, their expression becomes restricted to heart, gut, and visceral endoderm [43-46]. Functional GATA-binding sites have been identified in the promoters of several cardiac-specific genes--including ANP, BNP, and c*-MHC. Specific depletion of GATA-4 by antisense oligonucleotides blocks dimethyl sulfoxide induced cardiac differentiation in the mouse embryonic stem cell line, P19 [47]. Overexpression of GATA-4 in the same cells increases the number of terminally differentiated cells, as well as molecular markers such as Nkx2-5. Together, these results suggest an essential role for GATA-4 in cardiac differentiation. Targeted disruption of the mouse GATA-4 gene does not support this view, however. In GATA-4 knockout mice, the heart tube fails to form but this does not appear to be caused by a failure in cardiomyogenesis. Rather, it appears to result from a defect in rostral-to-caudal and lateral-to-ventral folding of the embryo [48°,49"]. Differentiated cardiomyocytes form in the homozygous mutant embryos and markers of terminal cardiac differentiation are expressed normally. Once again, an explanation for the
631
disparity between these results and those of the P19 cell line may be functional redundancy amongst the GATA factors. Thus, the level of GATA-6 transcripts is elevated in GATA-4 knockout embryos, whereas only low levels of expression are detected in the treated P19 cells [47]. Overexpression of GATA-6 in Xenopus embryos, however, has recently been found to inhibit terminal differentiation within the myocardium [44] and it is difficult to reconcile these findings with those of the mouse studies. Combinatorial regulation of cardiac differentiation
As -yet, few direct gene targets of Nkx2-5 or its relatives have been unambiguously identified and little is known about how these transcription factors function. One probable target is the cardiac atrial natHuretic factor (ANF) gene which contains a highly conserved Nkx2-5 responsive element (the NKE) within its proximal promoter [50]. Studies of this promoter in cultured cells indicate that cardiac-specific expression may require the activity of several factors, both positive and negative, including Nkx2-5 and a GATA factor. Similarly, studies of the chick cardiac actin gene have suggested that the Nkx2-5 functions in a combinatorial manner and can interact with the MADS family transcription factor, SRF (serum response factor) [51]. Conclusions
It is now possible to formulate a common model for the earliest steps in heart formation in vertebrates. Anterior regions of the newly formed mesoderm receive at least two conceptually distinct instructive signals from immediately adjacent endoderm. One establishes a propensity or capacity for heart formation that distinguishes anterolateral mesodermal cells from more posterior neighbours. Members of the FGF family and the secreted protein cerberus are candidates for this signal. A second inductive interaction results in specification of bilateral cardiac progenitors and is probably mediated by one or more BMP(s). The precise boundaries of cardiac mesodcrm arc influenced by other signals within the embryo (e.g. noggin or chordin) that regulate BMP activity. Within each region of cardiac mesoderm, atrial, ventricular, and endothelial lineages are distinct and are probably organised in a spatial pattern that prefigures their arrangement in the linear heart tube. Understanding the mechanisms responsible for this patterning is a major goal for the future. Cardiac specification is accompanied by expression of several homeodomain proteins of the Nk2 f a m i l y - - m o s t notably Nkx2-5 - - but the number of these and their individual roles are unclear. Studies of cardiac-specific transcription suggest that these proteins function in a combinatorial manner (e.g. with GATA proteins) to regulate terminal differentiation. Nkx2-5 also appears to regulate the expression of other transcription factors (such as MEF-2 and H A N D proteins) that are active in early cardiomyogenesis. Genetic approaches will prove decisive in unravelling the relative contributions of
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these proteins and their relative positions in the cardiac regulatory program.
Note added in proof An extensive review of vertebrate heart development has been published recently [52"].
Acknowledgements T M o h u n and I) Sparrow are supported by the [\ledical Research (~ouncil and the British Heart Foundation. \Ve would like to thank Richard Harvey fl~r critical c o m m e n t s on the manuscript.
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