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Wnt/Wg and Heart Development

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The heart is the first functional organ in a developing embryo, which ensures the distribution of vital nutrients within the growing organism. For decades, scientists have been intrigued by the well-orchestrated morphological and molecular events that result in the formation of this complex organ, which is composed of differently specialized cells derived from lateral plate mesoderm. Dissecting the genetic pathways involved in cardiogenesis revealed crucial roles for members of various growth factor families in this process. This chapter highlights major events in invertebrate and vertebrate cardiogenesis and focuses on the role of Wnt/Wg signaling in the induction of cardiac fate during early development. In contrast to the essential function of Wg in Drosophila cardiogenesis, the role of Wnt genes in vertebrate heart development has not yet been fully elucidated. To this date major contributions regarding Wnt function in vertebrate cardiogenesis have come from studies in avian embryos and in Xenopus and therefore will be discussed in more detail.

Introduction to Drosophila Cardiogenesis

Despite its obvious morphological and functional differences, the Drosophila heart has proven to be a rich source of knowledge about early molecular events in the determination of cardiac fate. Its tubular morphology, which is why the Drosophila heart is also referred to as the dorsal vessel, resembles the primary heart tube in vertebrates. Another feature of cardiogenesis shared by invertebrates and vertebrates is the formation of the heart from two cardiac primordia. The mesodermal progenitors that differentiate into cardiac tissue move towards the dorsal midline where they fuse (because of the reversal of the dorsal-ventral body plan between vertebrates and invertebrates the Drosophila heart forms dorsally). There are two major cell types that define the Drosophila heart: the cardial and the pericardial cells. The latter form the outer layer of the dorsal vessel and do not express muscle-specific structural genes. The contractile cardial cells form the inner layer and generate the lumen of the heart (Fig. 1 A). In contrast to the vertebrate heart, the Drosophila heart lacks an endocardium because its hemolymph is pumped through the body in an open circulatory system and blood vessels are absent. Although major progress has been made in the elucidation of cardiogenic molecular events, we are still far from understanding how various signaling pathways act together to initiate cardiac differentiation in a specific subset of mesodermal cells.

Figure 1. (A) As in vertebrates, the Drosophila heart arises from two cardiac primordia, which, in the fly, fuse at the dorsal midline to form a linear tube (dorsal vessel).

Figure 1

(A) As in vertebrates, the Drosophila heart arises from two cardiac primordia, which, in the fly, fuse at the dorsal midline to form a linear tube (dorsal vessel). The Drosophila heart consists of two cell types. The cardial cells (CC), which form the (more...)

One of the crucial molecules in cardiogenesis is the homeobox-containing transcription factor tinman. Tinman (tin) is initially expressed in the entire presumptive mesoderm and becomes restricted to the dorsal mesoderm after gastrulation.1,2 Since the dorsal mesoderm gives rise to the heart, gut muscles and dorsal skeletal muscle, flies that are mutant for tin not only lack a heart but in addition fail to form visceral mesoderm. Hence, tin alone is not sufficient to confer cardiac identity to the cells in which it is expressed and additional factors are required. One of these factors is decapentaplegic (dpp), which belongs to the TGFβ family of secreted growth factors. The protein encoded by dpp is secreted from the dorsal ectoderm and is required for the maintenance of tin expression in the underlying mesoderm (Fig. 1B).2,3 The restricted expression of dpp within the dorsal ectoderm is maintained by DJun and consequently, tin.expression in flies that are mutant for the effector of DJun kinase signaling is either reduced or absent.4,5 It is of further importance to note that activation of the JNK pathway in the dorsalmost cells is regulated by Wg.6 Flies that do not express dpp exhibit the same phenotype as tin mutants, that is, they lack cardiac as well as visceral mesoderm. Moreover, although dpp and tin are necessary for heart formation, they are not sufficient as they can not significantly expand the cardiac primordia when ectopically expressed.2,7,8 These results indicate that multiple signaling pathways are involved in the specification of cardiac fate. Since a detailed description of additional regulatory proteins involved in Drosophila heart development can not be considered here, the reader is referred to the corresponding literature.9-13

Wg Signaling in Drosophila Cardiogenesis

In addition to Dpp, the dorsal ectoderm is the source of another secreted signaling molecule, the segment polarity gene wingless (wg), which is the homolog of vertebrate Wnt-1. Wg is expressed in 15 transversal stripes, perpendicular to the dpp expression domain, in the trunk region of the embryo.14 Wg, like Dpp, is involved in a plethora of patterning events during Drosophila development. However, with respect to mesoderm specification, wg mutants fail to form cardiac progenitor cells whereas formation of visceral mesoderm is unaffected. Some somatic mesoderm also forms, albeit abnormally patterned. By using a temperature-sensitive wg allele Wu et al.15showed that the cardiogenic function of zygotic wg is distinct from its role in segmentation and neurogenesis and that it is required for heart precursor formation at the stage when tin is restricted to dorsal mesoderm. Hence, wg provides additional spatial cues leading to a further subdivision of the dpp and tin expressing dorsal mesoderm (Fig. 1B). Although wg is also transiently expressed in the dorsal mesoderm during gastrulation, the crucial positional information as to where the heart progenitors are formed is provided by the wg signal emitted from the dorsal ectoderm.16 A more detailed investigation of the spatiotemporal relationship between Wg, dpp, and tin expression demonstrates very nicely that their concerted dynamic expression pattern determines which cells become specified to enter the cardiac lineage. Misexpression studies showed that only tin expressing cells that have been exposed to simultaneous Wg and Dpp signaling at defined stages during development undergo cardiac specification.5 As one would predict, examination of components of the Wg signal transduction cascade identified dishevelled (dsh), armadillo (arm), and dTCF to also positively regulate heart development.17,18 Embryos that are mutant for both maternal and zygotic dsh or arm lack any heart precursors.17 A similar dramatic effect was observed in flies overexpressing a dominant-negative form of dTCF.18 Additionally, overexpression of Dsh results in an increase of heart precursor cells.17 The fact that Dsh becomes hyperphosphorylated either in response to Wg signaling or when it is overexpressed has been opportune to show that dsh is epistatic to wg and is required autonomously within the mesoderm for heart development.17,19

The situation is somewhat more complex for zeste-white3/shaggy (zw3), which is regulated by Dsh. Zw3 acts antagonistically to Dsh and arm and therefore, one would expect that overexpression of zw3 suppresses heart precursor formation and loss of zw3 function results in an increase thereof. Neither is the case though. A more detailed investigation of the role of zw3 in cardiogenesis showed that zw3 has two distinct functions in mesoderm specification and thus the observed phenotypes require careful interpretation.18 The initial requirement of zw3 function to specify dorsal mesoderm is obvious in zw3 mutants, which are characterized by severely reduced tin and fasciclin III expression domains, the latter being a marker for visceral mesoderm.18 Hence, the loss of cardiac progenitor cells is primarily due to a failure of dorsal mesoderm formation. To further elucidate the role of zw3, Park et al.18 performed overexpression experiments using a zw3 construct, which is under the control of a heat-shock promoter. Excess zw3 function elicited both cardiac hypertrophy and failure of heart formation. The first observation is consistent with the function of zw3 as specifier of dorsal mesoderm, the latter indicates an inhibitory role for zw3 in cardiogenic wg signaling. These important findings suggest two temporally distinct functions for zw3, which would be consistent with studies on the temporal requirement for wg itself.15 In vertebrates, a similar scenario regarding the role of GSK3 and β-catenin (zw3 and arm in Drosophila) in patterning the dorsal-ventral axis and in heart formation has to be considered and will be discussed in more detail later.

For a better understanding of the role of wg in heart formation it is necessary to put wg signaling in the context of other segment patterning genes. Initial molecular events during Drosophila development subdivide the ectoderm into segmentally repeated units, each of which is composed of an anterior (A) and a posterior (P) compartment. Within each segment, wg expression is confined to the A compartment, whereas the P compartment is characterized by the expression of hedgehog (hh), another secreted segmentation gene product that positively regulates Wg transcription (Fig. 1 B).20,21 Like wg, hh is required for heart formation during early gastrula.14 In contrast to wg null mutants, however, flies lacking hh expression possess some cardiac precursor cells. Furthermore, overexpression of wg in a hh mutant background demonstrated that wg signaling determines cardiac fate directly and does not exert its function by regulating other segmentation genes.17 It is also noteworthy that the mesodermal heart precursors form right beneath the ectodermal wg expression domains, which is consistent with the notion that the secreted wg gene product acts on neighboring cells. For example, expression of the homeobox transcription factor ladybird (lb) requires high Wg concentrations and as a consequence is restricted to heart cells that are located underneath dorsal ectodermal cells expressing wg (Fig. 1B).22 Within the P compartment, hh signaling suppresses lb expression.22 The gene products of lb (lb early and lb late) also play a role in the diversification of cardiac lineages where during the segregation of heart precursors lb genes act to specify a subset of cardioblasts and pericardial cells.22 In addition to wg, the forkhead transcription factor sloppy paired (slp) is expressed in the ectodermal and mesodermal portions of each A compartment. The identification of putative binding sites for the Wg effector Pangolin (the Drosophila homolog of vertebrate LEF-1/TCF) within the 5'-flanking region of the slp gene, implicates that slp is a direct target of Wg signaling.20 Sloppy paired plays an important role in the specification of cardiac and visceral mesoderm. It is required for the formation of heart progenitor cells, as fliesm mutant for slp do not form a heart.17 A likely explanation for this effect is the fact that ectodermal wg expression requires slp and hence, the loss of cardiac precursors results from the lack of wg signaling.20,23 It is known that in the ectoderm wg and slp act in an autoregulatory feed back loop.20 slp function is also required to suppress visceral mesoderm formation by inhibiting bagpipe (bap) expression, which is consistent with the observation that loss of wg signaling is accompanied by an expansion of visceral mesoderm.24 Furthermore, wg and slp cooperate in cardiogenesis as neither wg or slp alone can rescue the loss of heart progenitors in embryos mutant for slp and wg, respectively.20 Consistent with this finding, only coexpression of wg and slp in the mesoderm resulted in a significant increase of heart progenitor cells.20 The experiments also revealed the complexity of the positive regulatory role of Wg in heart development. Although slp is required for mediating the Wg signal it is not sufficient. This suggests that cells expressing slp are rendered competent to respond to subsequent wg signals.20

The Drosophila mutant heartless (htl) demonstrates the requirement of FGF signaling in heart formation. heartless codes for an FGF receptor, which is coexpressed with tin throughout the mesoderm. Unlike the factors discussed so far, htl is not required for the processes that specify cardiac mesoderm but rather for the migration of mesodermal cells towards the dorsal side of the embryo.25,26 Previous findings indicate that domains of the dorsal mesoderm with overlapping activities of Dpp, tin, and Wg could create an environment that makes those cells competent to respond to FGF receptor signaling.

In summary, these findings demonstrate the crucial role of Wg signaling in Drosophila cardiogenesis and they outline the position of Wg within this complex regulatory network of molecules involved in cardiac differentiation.

Introduction to Vertebrate Cardiogenesis

As in Drosophila, the vertebrate heart derives from distinct patches of mesodermal cells that, under the influence of multiple signaling events, have been specified to form the cardiac primordia. These primordia are initially located on either side of the embryonic organizer (Fig. 2A,B). As development progresses they eventually reach their final destination, the ventral midline where they fuse to form the primary heart tube. The heart tube is composed of an outer, contractile layer, the myocardium, which encloses the inner endocardium. During development, the latter becomes continuous with the endothelium of the blood vessels. The heart tube is composed of the outflow tract (bulbus cordis), ventricle, atrium, and sinus venosus, which are arranged in a linear order along the anteroposterior axis of the embryo. Soon after its formation, the primary heart tube undergoes major morphological rearrangements, which place the distinct functional units at their proper positions to give rise to the multichambered heart. Explant studies and transplantation experiments have provided initial clues as to the sources of the molecules involved in cardiogenesis. These areas comprise the organizer region including more lateral domains as well as the underlying endoderm.2729

Figure 2. (A) Schematic drawing of a gastrula stage Xenopus embryo, depicting the dorsal blastoporus and the bilaterally established cardiac primordia adjacent to the Organizer region.

Figure 2

(A) Schematic drawing of a gastrula stage Xenopus embryo, depicting the dorsal blastoporus and the bilaterally established cardiac primordia adjacent to the Organizer region. (B) Schematic drawing of a gastrula stage chicken embryo. The cardiac primordia (more...)

The combinatorial expression of cardiac transcription factors characterizes cells that are specified to form cardiac tissue. Signaling molecules that act upstream of these transcription factors have been identified by their ability to induce cardiac differentiation in non-cardiogenic mesoderm. For example, the combination of bone morphogenetic proteins 2/4 (BMP-2/4), which belong to the TGFβ-family, and FGF-4 is able to convert avian posterior mesoderm, which normally does not contribute to the heart, into cardiac tissue in chicken.30,31 In Xenopus, BMP signaling is necessary for the maintenance of XNkx2.5 expression, terminal differentiation, and normal cardiac morphogenesis.32-34 The most dramatic effect of a secreted molecule on early cardiogenesis has been observed with cripto-1, an EGF-like signaling protein. Mice that are mutant for cripto-1 lack cardiomyocytes.35

Although the cardiac-promoting role of some growth factors has been thoroughly investigated, our current knowledge about regulatory events leading to heart formation is mostly based on previously characterized transcription factors that belong to very different gene families. Exploiting the advantages of different model organisms, studies of these transcription factors have provided major insight into various steps of cardiogenesis. These range from the specification of cardiac fate within a particular population of mesodermal cells to the establishment of diverse structures, which constitute the adult heart. One of the biggest challenges up to this date is to identify molecules that regulate these cardiac transcription factors and to understand their intertwined regulatory relationships in this context.

The crucial function of tinman during Drosophila heart development initiated the search for its vertebrate homolog and was found in Nkx2.5 This homeobox transcription factor of the NK2 family is one of the earliest cardiac marker genes expressed in vertebrate embryos. RNA injections of a dominant-negative Nkx2.5 construct inhibit formation of the linear heart tube and terminal differentiation of cardiomyocytes in Xenopus embryos.36 Nkx2.5 null mutant mice produce a lethal phenotype due to aberrant heart development.37 The phenotype is characterized by defective looping morphogenesis and reduced or eliminated cardiac marker gene expression.37-40 It needs to be noted though, that none of the phenotypes elicited by negative interference with Nkx2.5 expression in any vertebrate embryo is as dramatic as the complete failure of heart formation observed in Drosophila. This is likely due to the presence of additional Nkx2 genes in vertebrates whose functions appear to be redundant. It also implicates that the action of various genes is required for cardiac specification. In fact, a regulatory circuit has been demonstrated for Nkx2.5 and GATA factors in the heart.41-44 The GATA family of zinc finger transcription factors comprises six members, three of which, GATA-4, -5, and -6, have been shown to function in cardiogenesis.43,45 Like Nkx2.5, expression of GATA-4/5/6 commences during gastrulation when cardiac progenitors are specified. GATA proteins can regulate the transcription of multiple cardiac genes, for example, overexpression of GATA-4 in Xenopus embryos results in a premature activation of genes encoding α-myosin heavy chain and cardiac α-actin.45 Introduction of antisense GATA-4 transcripts into the mouse embryonic carcinoma stem cell line P19 blocks the DMSO-inducible expression of cardiac muscle markers.46 Targeted mutations in the GATA-4 gene in mice disturb normal migration of cardiac cells, which results in cardia bifida.47,48

A thorough description of all known factors involved in vertebrate heart development would extend beyond the topic of this chapter and therefore we refer the reader to refs. 4954.

Wnt Signaling in Vertebrate Cardiogenesis

In contrast to the well-investigated roles of the signaling molecules described above, the function of members of the Wnt gene family in vertebrate cardiogenesis has only started to be under thorough examination. But already, the previously published data on the role of Wnt proteins in heart development has revealed the complexity of this subject, which arises from the existence of different Wnt signal transduction cascades.

Among the known Wnt proteins, several are expressed in a spatiotemporal pattern that implicates their involvement in heart development. The conserved expression pattern of Wnt-11 in precardiac mesoderm in birds, mouse, and Xenopus makes this gene the best candidate molecule for playing a role in signaling events leading to cardiac differentiation.55-58 In mouse and chick, Wnt-11 expression is also conserved in the primary heart tube and transcripts of human Wnt-11 are detected in the adult heart.59-61 In the early mouse gastrula, Wnt-2 expression overlaps with that of Wnt-11.62 Later in development mWnt-2 expression becomes restricted to the pericard.63 Mouse Wnt-8 is only weakly expressed during early embryogenesis, followed by a peak of expression at stage 10.5 when its transcripts are detected in the myocardium at high levels.63 Additional avian and mouse Wnt genes expressed within or lateral to the primitive streak during early gastrula are Wnt-3a, Wnt-5a, Wnt-5b, and Wnt-8c.64-66 Four other Wnt genes are expressed in the tubular heart of the chick: Wnt-2b, Wnt-14, Wnt-5a, and Wnt-7a.60,67 To this date, only Wnt-11 has been ascribed a promoting role in cardiac differentiation. This does not exclude positive regulatory roles for other Wnts expressed in cardiac tissue unfortunately there is no well-documented experimental evidence for this hypothesis yet available. Targeted gene disruptions of different Wnt genes in mice suggest that the functions of these proteins are interchangeable.

The Wnt signal is transduced after the ligand has bound to its putative Frizzled (Fz) receptor. Transcripts of different Fz have been detected in cardiac tissue including cardiac mesoderm and adult heart as well as in cardiac neural crest cells. These are human Fz-1, -2, -7, -8, -9, mouse Fz-2, -4, -9, and Xenopus Fz-7, -8, -10a, -10b.68-75

Vertebrate Cardiogenesis Involves Different Wnt Signal Transduction Cascades

Secreted signals from the embryonic organizer are essential for the formation of the vertebrate heart. For example, Xenopus embryos lacking the organizer region fail to form cardiac tissue despite the presence of presumptive cardiac mesoderm.27 Recent studies in chick and Xenopus demonstrated that cardiac tissue formation can be initiated in non-cardiogenic posterior ventral mesoderm by overexpression of the Wnt inhibitors dickkopf-1 (Dkk-1) and crescent, which are secreted by the embryonic organizer.76,77 Ectopic expression of Dkk-1 and crescent not only induce cardiac marker gene expression but also stimulate the formation of beating tissue. In contrast to Dkk-1, crescent induces a contractile phenotype to a much lesser extent. Interestingly, ectopic cardiac differentiation is not elicited by other Wnt inhibitors, such as WIF, FrzA and Sizzled or dominant-negative Wnt-8.77 A possible explanation could be that these inhibitors differ in their abilities to block signaling of different Wnts.78-81 Dkk-1, for example, is a very potent inhibitor of Wnts that activate the β-catenin signaling pathway.82 Overexpressi on of GSK3β RNA, which inhibits maternal β-catenin-mediated Wnt signaling in Xenopus posterior ventral mesoderm, phenocopied the effect of Dkk-1, further suggesting that cardiac induction is initiated simply by blocking β-catenin signaling. This assumption is supported by the finding that activation of zygotic β-catenin signaling in the heart-forming regions of Xenopus embryos by Wnt-3a and Wnt-8 inhibits endogenous cardiac gene expression. 77 The conclusion, however, is challenged by two observations. First, it needs to be noted that GSK3β is part of a multi-protein complex, whose regulation is influenced by multiple signals in a temporally regulated manner and in dependence on the cellular context.83,84 If the mere inhibition of β-catenin signaling was indeed sufficient to induce cardiogenesis, the introduction of a dominant-negative mutant of LEF-1/TCF should phenocopy the effect of Dkk-1 and GSK3β. This specific block of β-catenin signaling on the level of transcription, however, did not induce cardiogenesis in posterior ventral mesoderm suggesting that additional signaling events must be involved in this process.85 These events could be triggered by Wnts that do not act through β-catenin. This idea is supported by data demonstrating that overexpression of Wnt-5a and Wnt-11, which activate non-canonical signaling pathways, in the presumptive heart region had no inhibitory effect on heart formation in vivo.77

Before we discuss the cardiac-promoting role of Wnt-11, another feature of β-catenin signaling during Xenopus development needs to be stressed. It is important to distinguish between the role of maternal and zygotic β-catenin-mediated signaling. Maternally provided β-catenin is essential for the formation of dorso-anterior tissue. UV irradiation of Xenopus eggs or LiCl treatment of early cleavage stage embryos lead to ventralization or hyperdorsalization of the tissue. Both effects are elicited by interference with β-catenin signaling, which concomitantly leads to changes in the expression of genes characteristic for either tissue type.86 Hence,with regard to cardiogenesis, the expression domains of GATA-4,-5,-6 are expanded in LiCl-treated embryos, whereas UV-irradiated embryos lack expression of these cardiac genes.45 Likewise, embryos that were hyperdorsalized by dorsal injections of XWnt-8 RNA had expanded expression domains of XNkx2.5 (own observation). Indeed, evidence for a dual role for β-catenin signaling in Xenopus development has already been provided with regard to head formation. Here, maternal β-catenin signaling is initially required to determine dorso-anterior cell fate, subsequently however, Wnt/β-catenin signaling has to be inhibited for head formation to occur.87 In fact, ectopic activation of zygotic β-catenin signaling by XWnt-8 interferes with head development.88 Last but not least, the dual role of vertebrate β-catenin resembles the dual role of Drosophila zw3, which subsequently regulates arm function in the formation of the invertebrate heart.

With respect to cardiogenesis the data described above suggests that zygotic β-catenin signaling activated by Wnt-8 and/or Wnt-3a may be required to restrict the heart forming domains within the dorsolateral mesoderm. It is known that the initial heart field, which comprises cells that have the potential to become cardiac tissue is broader than the actual heart forming area.89-91 Additionally, it has been demonstrated that signals from the neural tube and notochord repress cardiogenesis.90,92-94 Results obtained from ablation and explant studies in chicken are consistent with such a role for Wnt-8c and Wnt-3a, which are expressed in neural tissue (Fig. 2B). In detail, removal of the anterior neural tube initiated cardiogenesis in head mesenchyme, whereas the cardiac inducing property of anterior endoderm in the overlying mesoderm was inhibited in cocultures of anterior mesoderm with neural tube and notochord.95

Studying the role of Wnt-11 in cardiac induction was pioneered by Eisenberg et al.57 who used the quail mesoderm cell line QCE-6 as a culture model. The QCE-6 cell line is representative of early nondifferentiated mesoderm cells and has the potential to differentiate into cardiomyocytes, endothelial or red blood cells.96 Differentiation of QCE-6 cells is initiated by treating the cells with a combination of retinoic acid, bFGF, TGFβ2, and TGFβ3.96 Most importantly though, the endogenous expression of Wnt-11 in this cell line is a requisite for myocardial differentiation, as Wnt-11-minus cells did not immunoreact with an antibody against muscle myosin heavy chain.57 This cardiogenic function of Wnt-11 was further elucidated in quail embryos.97 Explants of anterior precardiac mesendoderm dissected at stage 5 or 6 differentiate into cardiac tissue, i.e. they become contractile in culture, whereas explants of posterior mesendoderm do not differentiate into beating tissue. Interestingly, explants of the posterior mesendoderm do become contractile and express sarcomeric proteins when dissected at stage 4.97 To test the ability of Wnt-11 to ectopically activate a cardiac developmental program, posterior non-cardiogenic mesendoderm was exposed to Wnt-11. Indeed, addition of Wnt-11 promoted cardiogenesis in posterior tissue dissected at stage 5 or 6.97 These results and the fact that Wnt-11 is the only Wnt gene known to this date to be expressed in avian precardiac mesoderm demonstrate the importance of Wnt-11 activated signaling pathways in heart development. As in the avian embryo, Xenopus Wnt-11 (XWnt-11) is present at the right time and in the appropriate areas to play a role in heart development. In Xenopus, cardiac specification of mesodermal cells is completed by the end of gastrulation. XWnt-11 RNA is present maternally and is post-transcriptionally regulated, which leads to an accumulation of XWnt-11 protein on the dorsal side of the embryo as early as the 64-cell stage.58 After the onset of zygotic transcription, Xwnt-11 mRNA first becomes detectable in the dorsal marginal zone where high levels persist during gastrulation.55 Loss-of-function studies of XWnt-11 finally demonstrate a crucial function for this gene in cardiogenesis, since inhibition of XWnt-11 signaling, for example by morpholino antisense oligonucleotides, dramatically decreases cardiac marker gene expression. 85 Overexpression of XWnt-11 in ectodermal tissue explants demonstrated that this gene is sufficient to induce early cardiac genes, such as GATA-4, GATA-6 and XNkx2.5, as well as genes coding for structural proteins of the myocardium.85 Most importantly, ectopic expressionof XWnt-11 in ventral marginal zone (VMZ) explants is sufficient to convert this non-cardiogenic tissue to contractile tissue.

Although the molecular mechanism of Wnt-11 triggered cardiogenesis in avian embryos remained obscure at the time, several studies regarding gastrulation movements in Xenopus have demonstrated in the meantime that Wnt-11 activates non-canonical Wnt signaling pathways. 98,99 Known mediators of non-canonical signaling are PKC, Ca2+/calmodulin- dependent kinase II (CamKII) in vertebrates and Jun-N-terminal kinase (JNK) in Drosophila. In two independent experimental assays it could be demonstrated that the regulatory function of cardiac genes by XWnt-11 involves PKC and JNK but not CamKII.85 Decreased cardiac gene expression in dorsal marginal zone (DMZ) explants that include the heart forming region as a result of inhibiting XWnt-11 signaling could be rescued by activation of PKC.85 Injections of a kinase-dead mutant form of JNK phenocopied the effect of dnXWnt-11 in DMZ explants. Consistent with these findings, JNK-1 activation was shown to be regulated by XWnt-11, as XWnt-11 overexpression leads to increased levels of phosphorylated JNK-1 and injections of XWnt-11 morpholino antisense oligonucleotides resulted in a decrease thereof.85 The activation of JNK-1 was shown to be specific for XWnt-11 and could not be mimicked by XWnt-8.

The picture that emerges from the data summarized above is that cardiac induction is initiated in a region of the embryo that is characterized by low levels of β-catenin signaling activity and high levels of JNK signaling activity (Fig. 2C). This assumption is substantiated by the findings that inhibitors of the Wnt/β-catenin pathway, Dkk-1 and crescent, as well as GSK3, can also activate JNK signaling.85

The conservation of the cardiogenic function of Wnt-11 could be demonstrated using the mouse embryonic carcinoma stem cell line P19. The P19 cell line is a widely used model for investigating molecular events involved in cardiac differentiation. Addition of DMSO to the culture medium activates a cardiac developmental program in these cells and likewise, treatment with Wnt-11 conditioned medium mimics the effect of DMSO.85

In summary, the available data on Wnt function in cardiogenesis may be interpreted as follows, currently being most appropriate for Xenopus but also likely to apply for chick:

During early cleavage stages maternal β-catenin specifies dorsoanterior tissue and maternally provided XWnt-11, together with proteins of other growth factor families initiate events that are required for the establishment of the bilateral heart fields in presumptive dorsolateral mesoderm. The cardiac promoting processes regulated by these signaling molecules continue through gastrula stages when heart precursor cells become specified. The emergence of zygotically expressed Wnts in the ventrolateral mesoderm and neural tissue, Wnt-8 and Wnt-3a, that counteract cardiac development, would then be necessary to restrict the initial heart field, thereby refining the area of cells that are specified to become cardiac tissue. Obviously, β-catenin-mediated signaling by these Wnts needs to be blocked within the cardiac tissue, which is achieved by the appropriate inhibitors secreted from the organizer. At the same time, components of the non-canonical signaling pathways, PKC and JNK, that are activated, for example, by XWnt-11 and Dkk-1, promote cardiogenesis in the dorsolateral mesoderm.

Conclusion and Future Perspectives

In conclusion, multiple signaling pathways converge to specify cardiac fate in cells that have been exposed to diverse growth factors at defined stages during development. To this date, the role of Wg in the formation of the heart in Drosophila has been studied in more detail and its requirement has been demonstrated in multiple experiments. Several lines of evidence indicate an important function for Wnt genes in vertebrate heart development. Studies conducted so far have primarily demonstrated the roles of various members of this gene family in early steps of cardiogenesis. They also make clear that Wnts belonging to different classes (Wnt-1 or Wnt-5a class) may differ in their function of regulating the expression of cardiac genes. Therefore, detailed analyses of Wnt gene expression in combination with gain-and loss-of-function studies of Wnts are required to deepen our understanding of the roles of these secreted factors during early cardiogenesis as well as during later events in heart development. In the same way as findings obtained from embryological studies in various model organisms will be helpful with respect to repair processes after heart injury, the determination of molecules involved in various cardiac diseases will help to further elucidate the molecular pathways in the formation of this complex organ.


We would like to thank Daniel Maurus, Marc-Phillip Hitz and Matthias Läsche for discussion and comments on the manuscript. Work in our lab has been supported by the Deutsche Forschungsgemeinschaft (DFG, Junior Group SFB 271).


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