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Regulation of Cardiac Development by Receptor Tyrosine Kinases
Regulation of CardiacDevelopment by Receptor TyrosineKinases Lino Tessarolloand BarbaraL. Hempstead* The development of a functional heart depends on the coordinated growth, differentiation, migration, and apoptosis of cell populations of diverse embryological origins. These processes are regulated in part by soluble polypeptide growth factors that exert their efects via binding to cell su+ace receptors with intrinsic ty~osine kinase activity. In particula~ members of this class of receptors and their ligands have been shown to regulate the development of distinctive regions of the heart, such as the mesodermally den”vedcardiac myocyte, the endocardium, and outjlow tract and septa, which depend on cardiac neural crest. The hepatocyte growth factor receptov c-met the fibroblast growth factor receptors; and the neuregulin receptors have been shown to influence cardiomyocyte proliferation andlor differentiation. Receptors binding to vascularendothelialcell growth factor or angiopoietinhave been implicated in the development of the endocardium. Finally,gene-targeting experiments in the mouse have demonstrated functional roles for neurotrophins and their cognate trk receptor tyrosine kinases in the development of outflow tract, septa, and valves that are structures derivedfrom cardiac neuralcrest. (Trends Cardiovasc Med 1998;8:34-40). @ 1998, Elsevier Science Inc.
ther overexpression or targeted deletion of specific genes has contributed to our understanding of the mechanisms regulating these early events. Although the roles of transcription factors have been best studied as mediators of cardiac morphogenesis (Olson and Srivastava 1996), several classes of receptors have been implicated as well. For example, cardiovascular developmental abnormalities have been reported in mice lacking the gp130 subunit of the receptors for leukemiainhibitory factor (LIF), ciliary neuroLino Tessarollo is at the Neural Development Group, ABL-Basic Research Program, NCI- trophic facto~ interleukin(H-.)-6,oncostaFCRDC, Frederick, Maryland; Barbara L. tin-M, and cardiotrophin-1 (Yoshida et Hempstead is at the Department of Medial. 1996). Mice deficient for ligands of Gcine, Cornell University Medical College, protein+oupled receptors, such as enNew York, New York. dothelin-1 and pre–B-cell growth-stimu* Address correspondence to: Dr.BarbaraL. Hempstead,Departmentof Medicine,Cornell lating factor/stromal cell-derived factorUniversityMedical College, 1300York Ave- 1, have also shown specific cardiac defects (Kurihara et al. 1995, Nagasawa et nue, Room C-606, New York,NY 10021. The vertebrate heart develops from splanchnic mesodermal progenitor cells and the cardiac neural crest through a regulated process of commitment, migration, proliferation, and differentiation (Clark 1990). Analysis of gene expression and in vitxo studies have implicated several classes of growth factors in the regulation of these processes. More recently the use of genetically engineered mouse models obtained by ei-
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al. 1996). Tyrosine kinase receptors and their ligands are the most rapidly emerging class of genes linked to cardiac development based on their patterns of expression and the cardiovascular abnormalities observed in mice lacking these gene products through targeted deletion. The relevance of this class of receptors in cardiac organogenesis has been further emphasized by recent reports describing severe cardiac defects in mice deficient for the guanine nucleotide exchange factor S0S1 or the GTPaseactivating protein NF1, which regulate the activity of ras, a critical member of signal transduction pathways downstream of many receptor tyrosine kinases (Brannan et al. 1994, Jaks et al. 1994, Wang et al. 1997). Protein tyrosine kinase receptors consist of a single transmembrane domain separating the intracellular kinase domains from extracellular ligand-binding domain, which typically contains one or several copies of immunoglobulin-like domains, fibronectin type III–like domains, epidermal growth factor (EGF)like domains, cysteine-rich domains, or other domains (Fantl et al, 1993) (Figure 1). Many of the Iigands for proteintyrosine kinase receptors are present as homodimers, thus exhibiting two identical receptor-binding epitopes. These epitopes can bridge two receptors, resulting in receptor oligomerization (see Figure 1). Clustered receptors undergo transphosphorylation or autophosphorylation, generating novel binding sites for cytoplasmic proteins, which, in turn, become tyrosine phosphorylated and activated, initiating signal-transduction cascades (Heldin 1995). Numerous studies have helped to identify the embryological locations of these growth factor receptors by defining their spatiotemporal expression patterns, thus predicting their physiologic potential in the regulation of cardiac development. Here, we discuss some of the recent advances in understanding the functions played by specific growth factorh-eceptor systems in developmentally distinct cardiac structures, utilizing information derived from both expression analysis and the development of animal models lacking such molecules. In particular, we emphasize the newly described roles of neurotrophins and their cognate trk receptor tyrosine kinases in cardiac development in vivo.
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neu
erb B2 erb B3 erb B4
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VEGF
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Fignre 1. Schematic representation of receptor tyrosine kinases and their ligands implicated
in cardiac development.All receptorsare type I integralmembraneproteins.Extracellular loops reflectIgG-likedomains,andfiiled boxes in thecytoplasmicdomainsrepresentthecatalytic domainof the tyrosinekinase.The branchedextracellularstructuresrepresentheparin sulfateproteoglycans.Allligandsaresecretedsolubleproteins,exceptneuregulins,whichcan be membranetetheredor proteolyticallycleaved(arrow) to a solubleform. Angie-l, angiopoietin;FGF,fibroblastgrowthfactor; FGFR,fibroblastgrowthfactorreceptor;HGF,hepatocyte growth factor.
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ReceptorsRegulatingCardiomyoqte Proliferation/Differentiation
The erbB neuregulin receptors are critical in regulating the development of the cardiomyocyte. The neuregulins consist of a family of secreted and membraneattached growth factors, expressed from alternatively spliced mRNAs of a single gene (Marchionni 1995, Meyer and Birchmeier 1995). The neuregulins mediate their actions by binding to the erbB receptor family (erbB2, erbB3, and erbB4), which belongs to the larger family of the EGF receptors (Figure 1). Neuregulins can bind to either erbB3 or erbB4; howevec only erbB4 is capable of signaling, whereas erbB3 requires dimerization with erbB2 or erbB4 to transduce a signal. Conversely, erbB2 can signal, but it is required to forma heterodimeric receptor complex with erbB3 or erbB4 to bind neuregulin (Marchionni 1995, Meyer and Birchmeier 1995). The patterns of expression of neuregulin and erbB mRNA are reciprocal in the developing murine heart, with neuregulin being expressed by the endocardium (Kramer et al. 1996, Meyer and Birchmeier 1995), and myocytes expressing erbB2 and erbB4 (Gassmann et al. 1995, Lee et al. 1995). ErbB3 mRNA is expressed at highestlevelsby cells in the endocardial cushion, but expression can be detected also in myocytes at lower levels. The timing of expression has not been ex-
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tensively characterized, although both ligand and receptor expression has been documented at E9.5-10.5 (Figure 2). The phenotypes of the neuregulin, erbB2, and erbB4 null mutant animals are strikingly similar with regard to cardiac development (Gassmann et al. 1995, Kramer et al. 1996, Lee et al. 1995, Meyer and Birchmeier 1995). In these mice, cardiac development is normal until midgestation, when a pronounced defect in ventricular trabeculation occurs at E1O–10.5,resulting in the death of the embryos. These results suggest that this growth factor receptor system is critical for myocyte differentiation. It is unclear whether neuregulins exhibit effects on the developing heart through regulation of cardiac neural crest function. Mice deficient for neuregulin or erbB4, however, demonstrate a decrease in the size of the endocardial cushion. More importantly, the phenotype of mice lacking erbB3, the receptor most highly expressed in the endocardial cushion, has not yet been described. [Note added in proof: although the erbB2 null mutant animals have been generated, a cardiac phenotype has not been described (see Riethmacher et al. study on page 725 of Nattwe,Vol. 389).] Owing to the lethal cardiac phenotype at E1O.5, it is not possible to evaluate in these mouse mutants whether neuregulins contribute to the processes of valve formation and chamber septation. The
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prominent defects in cranial neural crest structures detected in the neuregulin and erbB4 mice suggest that neuregulins play critical roles in some classes of neural crest cell populations; however, their role in cardiac neural crest function will likely require the generation of conditional targeted deletions. Among the earliest growth factor receptor tyrosine kinases to be expressed in the cardiac mesoderm is the hepatocyte growth factor (HGF) receptor, cmet (Figure 1). Unlike most soluble growth factors, HGF is secreted as an inactive ligand, which can be activated by proteolytic cleavage (Rosen et al. 1994). Hepatocyte growth factor is first expressed in the precardiac mesoderm during gastrulation, whereas the expression of its receptor, c-met, is detected soon after in both cardiac mesoderm and endoderm (Rappolee et al. 1996). During the stages of cardiac looping, both receptor and ligand are expressed by cardiac myocytes (Figure 2). Expression of both receptor and Iigand is downregulated during the later stages of cardiogenesis. Owing to this spatiotemporal pattern of expression at the precontractile stage of cardiogenesis, as well as the defined actions of HGF on epithelial cells in inducing chemokinesis, HGF had been postulated to mediate cardiac myocyte migration (Koch et al. 1996). Targeted deletion of either the cmet or HGF alleles, however, results in null mutant mice that appear to undergo normal cardiac development, although they die in utero at E13.5–15, with intrauterine growth retardation and vascular defects of the placenta (Bladt et al. 1995, Schmith et al. 1995, Uehara et al. 1995). The lack of observed cardiac defects in the null mutant HGF and c-met animals has been postulated to reflect functional redundancy with related receptors (Ron and Sea) or the presence of other, unrecognized ligands. The expression pattern of HGF overlaps significantly with one member of the fibroblast growth factor family, FGF-8 (see later here). Thus it has been proposed that as cardiac myocytes coexpress HGF and FGF-8, FGF receptor signaling could potentially rescue cells with impaired HGF signaling. A second class of receptor tyrosine kinase, the FGF receptors, is also detected at early stages of cardiac myocyte development (Orr-Urtreger et al. 1991, Pat-
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precardiac mesoderm
cardiac looping
~p
acquisition of a contractile phenotype
c1
septation valvulogenesis
birth
? ●
VEGF an io ?
E6
E7.5
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geted deletions of FGF (Feldman et al. 1995)and the fact that the strategyused in the chick study inhibited the function of severalFGF receptor isoforms.
E12
E14
E20
Receptor TyrosineKinases Regulatingthe Formation of the Endocardium
Two families of receptor tyrosine kinases have been implicated in the regulation theircognateligandsduringcardiacdevelopment.Therelativeorderingof initiationof signal- of the endocardium and in vasculogeneing reflectsthe publishedspatiotemporalpatternsof expressionduringrodentdevelopment. sis, the vascular endothelial growth facThedurationof receptoractivityhasnot beendeterminedfor severalligand:receptorpairs,as tor (VEGF) receptors fit-l and flk-1, and targeteddeletionsresultin embryoniclethality,precludingevaluationof laterstagesof cardiac the angiopoetin receptors tie-1 and tie-2. development(indicatedby a question mark). FGF, fibroblast growth factor; HGF, hepatocyte VEGF was initially identified as a factor growth factor; VEGF, vascular endothelial growth factor; neu, neuregulin; angio, angiopoietin; that promoted vascular permeability and NT, neurotrophins. proliferation of endothelial cells (Majesky 1996, Yang et al. 1996). The biological stone et al. 1993, Sugi et al. 1995). The FGF function in cardiogenesis. First, the actions of VEGF are mediated by two related receptor tyrosine kinases, fit-l and FGF family of growth factors represents bFGF-receptor interaction is promoted a large family of at least nine related by coexpression of heparin sulfate (Fig- flk-1, which differ in their affinity of ligands and four related receptor ty- ure 1). Thus, the effectiveness of ligand binding [reviewed by Majesky (1996)]. Although flk-1 receptor expression is first rosine kinases, which can generate addi- signaling may require the coexpression tional isoforms by alternative mRNA of ligand with an appropriate extracellu- detected in precardiac mesoderrn at E7.O splicing [reviewed by Mason (1994)]. lar matrix, functioning to promote ligand of the mouse, the reciprocal patterns of Given this complexity, a complete de- stability, presentation, or a high-affinity expression of VEGF by the developing scription of ligand and receptor localiza- ligand–receptor interaction (Ruoslahti myocardium and of flk-1 in the develoption during cardiovascular development and Yamaguchy 1991). In addition, the ing endothelium have been detected at is lacking. Early studies using immuno- relative insensitivity of in situ hybridiza- E8.5 and are maintained through E15 histochemical or in situ hybridization tion analysis to detect FGF expression, (Dumont et al. 1995, Yamaguchi et al. 1993). Fit-l mRNA is not detected in the analysis, however, support the colocal- as compared with immunohistochemisization of the basic FGF ligand and re- try, has complicated the studies of ligand developing heart, and its expression apceptor by cardiac myocytes, suggesting localization. Alternatively the relative pears limited to the extraembryonic tisan autocrine or local paracrine mecha- ease in detecting the FGF proteins may sues through E15 (Figure 2). The critical roles of fit-l and flk-1 in nism of action in midgestation. Although reflect a longer half-life of ligand bound vasculogenesis have been defined by the an extensiveanalysisof the relativebind- to the heparin-rich extracellular matrix. early embryonic lethality of targeted ing affinitiesof the FGF receptors for the Despite these potentially confounding different FGF ligands has been under- issues, bFGF has been documented as gene deletion in mice (Carmeliet et al. taken, the distinctive spatial and tempo- providing a proliferative signal in cardiac 1996, Ferrara et al. 1996, Fong et al. ral patterns of expression may be more myocytes in in vitro studies using anti- 1995, Shalaby et al. 1995). Indeed, the relevantthan modest differences in the af- sense oligonucleotides to inhibit FGR sig- profound impairment in vasculogenesis finity of receptor–ligand interactions in naling (Choy et al. 1996, Sugi et al. 1993). in midsomite embryos limits analysis to regulating biological function (Ruo- These results have been confirmed in all but the earliest processes of ventricuslahti and Yamaguchy 1991). For exam- vivo by overexpression of dominant neg- lar fusion and looping. Nonetheless, the ple, the expression of the FGF receptor ative FGF receptors that inhibited myo- flk-1 null mutant animals lack endocarcek 1 is widespread in the chick develop- cyte proliferation (Mima et al. 1995). In- dium before their death at E9.5 (Shalaby ing heart, whereas the cek 2 receptor terestingly,inhibition of FGF receptor 1 et al. 1995). The fit-l null mutant anidisplays a more constrained temporal signaling through retrovirus-mediated mals display a distinctive phenotype and spatial expression in developing overexpression of a dominant negative with endocardial cells filling up the venvalves (Uehara et al. 1995). In the FGFreceptor 1 induces defects in myocyte tricular chamber, perhaps resulting from mouse, the most relevant members of mitogenesis only at relativelyearly devel- a loss of contact inhibition of the enthe FGF/FGF receptor families in car- opmental stages (before E7 in the chick), docardium at E9-E1l (Fong et al. 1995). diogenesis may be FGF-8, which is ex- suggestingthatFGFsmay exerttheirfunc- The VEGF-deficient animals are more pressed transiently at E7.5–9, and the tions in early cardiogenesis (Mima et al. severely affected than animals lacking bec receptor, which is expressed by car- 1995). Unfortunately,these observations either VEGF receptor (Carmeliet et al. diac myocytes at E8-13 (Figure 2) (Orr- have not been extended to the examina- 1996, Ferrara et al. 1996). These mice Urtreger et al. 1991). tion of several mouse FGF null mutant exhibit profound defects in vasculogeneSeveral additional considerations models. In part, these differences may re- sis because they lack dorsal aortae and have complicated the interpretation of flect the early embryonic lethality of tar- yolk sac vessels. Thus, these deficiencies Figure 2. Schematicrepresentation of thetimingof activationof receptortyrosinekinasesby
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full
full trunc
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Figure 3. Schematic representation of the neurotrophins andtrk receptor tyrosinekinases. The major isoform of each trk receptor is indicated with full, the full-length kinase-active isoform; trunc, the truncated, kinase-inactive isoform; and insert, the foil-length kinase-active isoform, bearing an insert in the kinase domain. The dominant ligand–receptor interactions are indicated by heavy arrows. Interactions that reflect reduced efficiency or affinity are indicated with broken arrows. BDNF, brain-derived neurotrophic factor; NGF,nerve growth factor; NT-3, neurotrophin-3
preclude the analysis of cardiogenic processes. Even the reduction in expression of VEGF in animals heterozygous for the targeted allele results in lethality in midembryonic development; however, these mice showed a delay in the development of the common atria and primitiveventricles, no evidence of endocardial formation, and a marked reduction in ventricular wall thickness. The defects seen in both endocardium and myocardium in the VEGF null mutant animals suggest that the absence of endocardium in these animals could limit the supply of normal trophic factors provided by the endocardium to the adjacent developing myocytes; this could result in hypoplasia and developmental delay in myocardiogenesis. The second receptor tyrosine kinase family that is involved in the regulation of vasculogenesis and the endocardium consists of the tie-1 and tie-2 receptor tyrosine kinases. Initially classified as orphan receptors, the tie-2 ligand has been recently identified as angiopoietin-1, a unique growth factor with coiled-coil and fibrinogen-like domains (Davis et al. 1996). A related ligand, angiopoietin-2, acts as a competitive inhibitor of angiopoetin with the tie-2 receptor (Maisonpierre et al. 1997). The ligand(s) for tie-1 have not been identified. The tie-1 receptor is first expressed in the endocardium (E8), followed by expression in the aorta and allantois, whereas the tie-2 receptor
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is expressed by the endothelium of major vessels at the same developmental stages (Korhonen et al. 1994, Sato et al. 1995) (Figure 2). Angiopoietin-1 exhibits a reciprocal pattern of expression to tie2, with angiopoietin-1 mRNA detectable in the myocardium (E9–11) and in the mesenchyme surrounding developing vessels (at El 3) in mouse embryos (Davis et al. 1996). No angiopoetin-2 mRNA is detected in embryonic mouse hearts (Maisonpierre et al. 1997). This pattern of gene expression suggests that the angiopoietin-l/tie-2 receptor functions at a developmentally later stage than the VEGF/flk-l receptor system. Recombinant angiopoetin-1 fails to induce the proliferation of cells expressing tie-2 receptor or to induce tubule formation in vitro. The critical roles of angiopoietin-1 and the tie-1 and tie-2 receptors in angiogenesis, however, have been confirmed by generating null mutant mice lacking expression of any of these three gene products (Puri et al. 1995, Sato et al. 1995, Suri et al. 1996). All three null mutant mice die in utero at E9.5–13 and display marked vascular defects such as microvascular hemorrhage (tie-1) or abnormal vascular networks (tie-2 and angiopoietin-1). In addition, all animals demonstrate cardiac defects with retardation in the growth of the hearts, although specific defects vary.The tie-l–deficient animals have no gross myocardial defects, but ultrastruc-
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turally,the endocardium exhibits a more immature phenotype. Tie-2 mutant animals exhibit defects in the development of cardiac vessels and die at E9.5–E1O.5, precluding analysis at later stages. The angiopoietin- 1–deficient mice exhibit slightly less severe defects, with retardation in the growth of the heart first notable at E 10.5. The endocardium is immature and less intricately folded in animals lacking angiopoietin-1 as compared with wild-type littermates, and before their death at E12.5, a marked decrease in ventricular trabeculations is noted. Although the tie receptors appear to act later than the flk-1/flt-l receptors, the lack of endocardium and reduction in myocardial growth are strikingly similar.The reciprocal patterns of expression of angiopoietin-1 and VEGF in the myocardium, potentially regulating the development of the endocardium, and neuregulins in the endocardium, potentially regulating myocyte development, suggests that the trophic support of the developing heart is achieved by local paracrine actions of these classes of growth factors.
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Receptor Tyosine Kinases Regulatingthe Cardiac Neural Crest
A third embryologically distinct class of cells, the cardiac neural crest, is required for normal cardiac development. The cardiac neural crest cells, originating from the neural tube at the level of the mitotic placode to the third somite, migrate along the pharyngeal arches to reside in the cardiac outflow tracts and the endocardial cushions. Cardiac crest ablation studies in the avian model system have amply demonstrated the fundamental role of this cell population in the septation of the truncus arteriosus, the formation of the truncal cushion, the membranous portion of the interventricular septum, and the pulmonic valves (Kirby 1993). Although the neural crest has been studied in detail, little is known about the specific growth factors that regulate the cardiac neural crest. Leukemia inhibitory factor promotes the survival of chicken cardiac neural crest cells in vitro (Kirby et al. 1993), and extracellular matrix proteins such as fibronectin and laminin induce directed cell migration of neural crest precursors (Bronner-Fraser 1994, Noden 1991). Car-
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diac abnormalities, however, have not yet been reported in mice lacking LIF or its low-affinity receptor. Recently we and others have reported (Hiltunen et al. 1996, Donovan et al. 1996) the expression of another family of growth factors, the neurotrophins, during cardiac development. Neurotrophins are members of a highly homologous family of growth factors that contribute to the regulation of proliferation, survival, and differentiation of cell populations in the mammalian nervous system. This family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin3 (NT-3), neurotrophin-4/5 (NT-4/5) and neurotrophin-6 (NT-6) [reviewed by Lewin and Barde (1996)] (Figure3). Neurotrophins bind and activate trk receptor tyrosine kinases, with NGF binding only to trkA, BDNF and NT4/5 binding to trkB, and NT-3 binding predominantly to trkC, but also activating trkA and trkB with lower efficiency in biochemical assays (Figure 2). AU neurotrophins also interact with the p75 neurotrophin receptor, which lacks intrinsic enzymatic activity but influences high-affinity NGF receptor formation, ligand internalization, and has been shown to play a role in the induction of apoptosis (Chao and Hempstead 1995). Trk receptors, the mediators of neurotrophins’ biological activities, are highly expressed in the central and peripheral nervous system. Thus studies on neurotrophin actions have focused on the developing nervous system, with little attention to neurotrophin function in nonneuronal tissues. Expression of neurotrophins and their receptors has been demonstrated in the developing cardiovascular system of vertebrates including Xenopus, chick, rat, and mouse (Donovan et al. 1995, Hiltunen et al. 1996, Scarisbrick et al. 1993, Tessarollo et al. 1993, Williams et al. 1993). Mouse and rat have been most extensively examined for the spatiotemporal patterns of gene expression. In the rat, all neurotrophins have been detected in the developing cardiovascular system during ontogenesis, and their presence has been correlated with the sensory and sympathetic innervation pattern of great vessels and the heart. Indeed, neurotrophin expression has been detected in the tunica media of the aorta, pulmonary and other major elastic arteries of the thorax and abdomen at
embryonic day 13, when innervation has been established (Scarisbrick et al. 1993). In the rat heart, NT-3 is first detected in the developing conduction system in the interventricular septum as early as El 1 before cardiac innervation is established. This pattern of NT-3 expression has led to the suggestion that NT-3 may function as a chemoattractant, as well as a trophic factor, for developing cardiac ganglia neurons expressing trkC and innervating conduction system cells (Hiltunen et al. 1996). In mouse, we have been able to detect trkC immunoreactivity in myocytes within the ventricle, atria, and cardiac outflow tract as early as E9.5. Furthermore, we have detected trkC mRNA and protein in the migrating cardiac neural crest at the same developmental stage (Donovan et al. 1996, Tessarollo et al. 1993). Detection of trkC in the mouse at earlier stages than reported in the rat maybe due to different sensitivity in methodologies employed or by intrinsic differences of rodent systems (Hiltunen et al. 1996). In fact, although mouse and rat have similar gestational periods of about 20–21 days, some of the critical developmental stages of cardiac development occur at different gestational days. The detection of trkC in mouse cardiac myocytes and migrating cardiac neural crest cells long before innervation occurs suggests that diverse roles are played by neurotrophins and their receptors in the development of the cardiovascular system. Recent reports have demonstrated chemotactic roles for neurotrophins on vascular smooth muscle cells (Donovan et al. 1995) and melanocytes (Hermann et al. 1993). Furthermore, although no studies to date have been performed with cardiac crest, trunk neural crest cells express the trkB and trkC neurotrophin receptors. These ceils respond mitogenically to BDNF and NT-3 and undergo NT-3–induced differentiation in vitro (Henion et al. 1995). These data support the hypothesis that neurotrophins can modulate neural crest development. Data from analyses of cardiac development in mice bearing targeted deletions of NGF, BDNF, and NT4/5, and trkA and trkB receptors are not available to date. We have recently reported (Donovan et al. 1996), however, that mice lacking NT-3 show developmental anomalies of the great vessels, including develop-
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mental delay in the primitive myofibril organization of the truncus arteriosus evident as early as E9.5. These defects are associated with reduced levels of expression of trkC in myocytes within the ventricles, atria, and cardiac outflow tracts. These early developmental defects before the onset of cardiac innervation are consistent with a direct role of NT-3 and trkC in cardiac development. More data are needed to elucidatewhether NT-3 and trkC function directly in cardiac myocyte differentiation. More striking cardiac defects were detected in the atria and ventricular septation, valves, and outflow tracts of both trkC and NT-3 mutant mice (Donovan et al. 1996, Tessarollo et al. in press). In some cases, these mutant mice developed defects resembling some of the most common congenital malformations in humans, including ventricular septal defects and tetralogy of Fallot. The observed defects were consistent with abnormalities in the survival and/ or migration of cardiac neural crest early in embryogenesis. Although the majority of studies defining the contributions of cardiac neural crest cells to normal cardiac development have been performed in the avian model because of ease of manipulation of early embryos, the abnormalities of the homozygous trkC or NT-3 null mutant mice follow these avian predictions. . Future Directions Although the studies reviewed here underscore the importance of several distinct classes of receptor tyrosine kinases in regulating specific cell types in the developing mammalian heart, further work is needed to address three important questions: (a) What are the upstream and downstream gene products that regulate, or are regulated by the activation of receptor tyrosine kinases? (b) Do receptor tyrosine kinases continue to support the survival and function of the myocardium and endocardium postnatally, under normal physiological, or in pathological, conditions? (c) Can the information gained from the analysis of mouse mutants further the understanding of human congenital cardiac defects? The rapidly emerging diversity of transcription factors that regulate distinctive aspects of early cardiogenesis should provide the means to assess
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which factors directly activate the genes encoding specific receptor tyrosine kinases and their ligands. Information regarding the timing and patterns of expression of these transcription factors, as well as the identification of their target consensus sequences, should aid in these studies, as the promoter regions of most of the ligands and receptors described here are under study. Similarly, an analysis of genes specifically induced by an activated receptor tyrosine kinase is likely to provide insight into their biological functions as well as the molecular basis for the cardiac dysfunction noted in null mutant animals. In addition, these types of studies could yield information on the potential use of specific growth factors to ameliorate degenerative cardiac conditions. The vast majority of studies of receptor tyrosine kinases in the heart have focused on their roles in development, rather than in regulating cardiac physiology in postnatal animals. Given the embryonic lethality of targeted deletions in many of these genes (Figure 2), alternative strategies, using chimeric animal models, or conditional targeted gene deletions will be necessary to explore their potential roles in later embryonic stages. The prominent roles for receptor tyrosine kinases in maintaining the survival and appropriate function of cells in other organs suggest that these signaling molecules could play critical roles in maintaining cardiac myocyte function in the adult. Finally, studies are needed correlating the specific phenotypes observed in animals with targeted gene deletions with that of children with similar congenital cardiac abnormalities. The early embryonic lethality of some mutations in mice may preclude analysis of comparable human tissue. Mutations that yield a cardiac phenotype in the mouse due to haploinsufficiency or that induce phenotypes that are compatible with postnatal survival, such as cardiac crest abnormalities, should be amenable to comparative analysis.
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Acknowledgments
Weill-Caulier Trust (to B.L.H.), and by the National Cancer Institute, Department of Health and Human Services, under contract with Advanced Bioscience Laboratories (to L.T.).
References Bladt F, Riethmacher D, Isenmann S, Aguzzi A, Birchmeier C: 1995. Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature 376:768-771. Brannan CI, Perkins AS, Vogel KS, et al.: 1994. Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev 8:1019-1029. Bronner-Fraser M: 1994. Neural crest cell migration in the developing embryo. FASEB J 8:699-706. Carmeliet P, Ferreira V, Breler G, et al.: 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380:435439. Chao MV, Hempstead BL: 1995. p75 and Trk: a two-receptor system. Trends Neurosci 18:321-326. Choy M, Oltjen SL, Otani YS, Armstrong MT, Armstrong PB: 1996. Fibroblast growth factor-2 stimulates embryonic cardiac mesenchymal cell proliferation. Dev Dyn 206: 193-200. Clark EB: 1990. Growth, morphogenesis and function: the dynamic of cardiac development. Zn Moller JH, Neal WA, eds. Fetal, Neonatal and Infant Cardiac Disease. Norwalk, CT,Appleton and Lange, pp 3–33. Davis S, Aldrich TH, Jones PF, et al.: 1996. Isolation of angiopoietin-l, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87:1 161–1169. Donovan MJ, Miranda RC, Kraemer R, et al.: 1995. Neurotrophins and neurotrophin receptors in vascular smooth muscle cells: regulation of expression in response to injury. Am J Pathol 147:309–324. Donovan MJ, Hahn R, Tessarollo L, Hempstead BL: 1996. Identification of an essential nonneuronal function of neurotrophin3 in mammalian cardiac development. Nat Genet 14:210-213. Dumont D, Fong G, Puri M, Gradwohl G, Alitalo K, Breitman M: 1995. Vascularization of the mouse embryo: a study of the flk-1, tek, tie and vascular epithelial growth factor expression during development. Dev Dyn 203:80-92.
We thank Esta Sterneck for critical reading of the manuscript. This work was supported in part by grants from the American Heart Association (National and New York Affiliate), by the Hirschl/
Fantl WJ, Johnson DE, Williams LT: 1993 Signaling by receptor tyrosinekinases.Annu Rev Biochem 62:453481.
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Feldman B, Poueymirou W, Papaioannou VE, SeChaira TM, Goldfarb M: 1995. Requirement for FGF-4 for postimplantation mouse development. Science 267:246-249. Ferrara N, Carver-Moore K, Chen H, et al.: 1996. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439442. Fong GH, Rossant J, GertensteinM, Breitman ML: 1995. Role of the Fit-l receptor tyrosine kinase in regulating the assembly of vascuIar endothelium. Nature 376:66–70. Gassmann M, Casagranda F, Orioli D, et al.: 1995. Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature 378:390–394. Heldin C-H: 1995. Dimerization of cell surface receptors in signal transduction. Cell 80:213-223. Henion PD, Gamer AS, Large TH, Weston JA: 1995. Trk-mediated NT-3 signaling is required for the early development of a subpopulation of neurogenic neural crest cells. Dev Biol 172:602–613. Hermann JL, Menter DG, Hamada J, Marchetti D, Nakajima M, Nicolson GL: 1993. Mediation of NGF-stimulated extracellular matrix ““invasion by the human melanoma low-affinity p75 neurotrophin receptor: melanoma p75 functions independently of trkA. Mol Biol Cell 4:12051216. Hiltunen JO, Arumae U, Moshnyakov M, Saarma M: 1996. Expression of rnRNAsfor neurotrophins and their receptors in developing rat heart. Circ Res 79:930–939. Jaks T, Shih TS, Schmitt EM, Bronson RT, Bernards A, Weimberg RA: 1994. Tumor predisposition in mice heterozygous-for a ta~geted mutation in NF1. Nat Genet 7:353-361. Kirby ML: 1993. Cellular and molecular contributions of the cardiac neural crest to cardiovascular development. Trends Cardiovasc Med 3:18-23. Kirby ML, Kumiski DH, Myers T, Cerjan C, Mishima N: 1993. Backtransplantation of chick cardiac neural crest cells cultured in LIF rescues heart development. Dev Dyn 198:296-31 1. Koch AE, Halloran MM, Hosaka S, et al.: 1996. Hepatocyte growth factor: a cytokine mediating endothelial migration in inflammatory arthritis. Arthritis Rheum 39: 1566– 1575. Korhonen L, Polvi A, Partanen J, Alitalo K: 1994. The mouse tie receptor tyrosine kinase gene: expression during embryonic angiogenesis. Oncogene 9:395403. Kramer R, Bucay N, Kane DJ, Martin LE, Tarpley JE, TheiIl LE: 1996. Neuregulins with an IG-like domain are essential for mouse myocardial and neuronal develop-
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ment. Proc Natl Acad Sci USA 93:4833– 4838. Kurihara Y, Kurihara H, Oda H, et al.: 1995. Aortic arch malformations and ventricular septal defect in mice deficient in endotheIin-1. J Clin Invest 96:293-300. Lee K-F, Simon H, Chen H, Bates B, Hung M-C, Hauser C: 1995. Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378:394–398. Lewin GR, Barde Y-A: 1996. Physiology of neurotrophins. Annu Rev Neurosci 19:289– 317. Maisonpierre PC, Suri C, Jones PF, et al.: 1997. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277:55-60. Majesky MA: 1996. A little VEGF goes a long way. Circulation 94:3062–3064. Marchionni MA: 1995. CellLcellsignaling, new tack on neuregulin.Nature378:334-335. Mason I: 1994. The ins and outs of fibroblast growth factors. Cell 78:547–552. Meyer D, Birchmeier C: 1995. Multiple essential functions of neuregulin in development. Nature 378:334–335. Mima T, Ueno H, Fischman DA, Williams LT, Mikawa T: 1995. Fibroblast growth factor receptor is required for in vivo cardiac myocyte proliferation at early embryonic stages of heart development. Proc Natl Acad Sci USA 92:467-471. Nagasawa T, Hirota S, Tachibana K, et al.: 1996. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-l. Nature 382:635-638. Noden DM: 1991. Origins and patterning of avian outflow tract endocardium. Development 111:867–876.
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Sato TN, Tozawa Y, Deutsch U, et al.: 1995. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376:70–74. Scarisbrick IA, Jones EG, Isackson PJ: 1993. Coexpression of mRNAs for NGF, BDNF, and NT-3 in the cardiovascular system of the pre- and postnatal rat. J Neurosci 13:875-893. Schmith C, Bladt F, Goedecke S, et al.: 1995. Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373:699-702.
Uehara Y, Minowa O, Mori C, et al.: 1995. Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/ scatter factor. Nature 373:702–705. Wang DZM, Hammond VE, Abud HE, et al.: 1997. Mutation in Sosl dominantly enhances a weak allele of the EGFR, demonstrating a requirement for Sosl in EGFR signaling and development. Genes Dev 11:309-320.
Shafaby R, Rossant J, Yamaguchi TP, et al.: 1995. Failure of blood island formation and vasculogenesis in Flk-1 deficient mice. Nature 376;62–66.
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Sugi Y, Sasse J, Lough J: 1993. Inhibition of precardiac mesoderm cell proliferation by antisense oligonucleotide complementary to fibroblast growth factor-2. Dev Biol 157: 28-37.
Yamaguchi TP, Dumont DJ, Conlon RA, Breitman ML: 1993. flk-1, An fZ-related receptor tyrosine kinase is an early marker for endothelial cell precursors. Development 118:489-498.
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Suri C, Jones PF, Patan S, et al.: 1996. Requisite role of Angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87:1 171-1180.
Yoshida K, Taga T, Saito M, et al.: 1996. Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardiaf and hematological disorders. Proc Natl Acad Sci USA 93:407-41 1.
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ther overexpression or targeted deletion of specific genes has contributed to our understanding of the mechanisms regulating these early events. Although the roles of transcription factors have been best studied as mediators of cardiac morphogenesis (Olson and Srivastava 1996), several classes of receptors have been implicated as well. For example, cardiovascular developmental abnormalities have been reported in mice lacking the gp130 subunit of the receptors for leukemiainhibitory factor (LIF), ciliary neuroLino Tessarollo is at the Neural Development Group, ABL-Basic Research Program, NCI- trophic facto~ interleukin(H-.)-6,oncostaFCRDC, Frederick, Maryland; Barbara L. tin-M, and cardiotrophin-1 (Yoshida et Hempstead is at the Department of Medial. 1996). Mice deficient for ligands of Gcine, Cornell University Medical College, protein+oupled receptors, such as enNew York, New York. dothelin-1 and pre–B-cell growth-stimu* Address correspondence to: Dr.BarbaraL. Hempstead,Departmentof Medicine,Cornell lating factor/stromal cell-derived factorUniversityMedical College, 1300York Ave- 1, have also shown specific cardiac defects (Kurihara et al. 1995, Nagasawa et nue, Room C-606, New York,NY 10021. The vertebrate heart develops from splanchnic mesodermal progenitor cells and the cardiac neural crest through a regulated process of commitment, migration, proliferation, and differentiation (Clark 1990). Analysis of gene expression and in vitxo studies have implicated several classes of growth factors in the regulation of these processes. More recently the use of genetically engineered mouse models obtained by ei-
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al. 1996). Tyrosine kinase receptors and their ligands are the most rapidly emerging class of genes linked to cardiac development based on their patterns of expression and the cardiovascular abnormalities observed in mice lacking these gene products through targeted deletion. The relevance of this class of receptors in cardiac organogenesis has been further emphasized by recent reports describing severe cardiac defects in mice deficient for the guanine nucleotide exchange factor S0S1 or the GTPaseactivating protein NF1, which regulate the activity of ras, a critical member of signal transduction pathways downstream of many receptor tyrosine kinases (Brannan et al. 1994, Jaks et al. 1994, Wang et al. 1997). Protein tyrosine kinase receptors consist of a single transmembrane domain separating the intracellular kinase domains from extracellular ligand-binding domain, which typically contains one or several copies of immunoglobulin-like domains, fibronectin type III–like domains, epidermal growth factor (EGF)like domains, cysteine-rich domains, or other domains (Fantl et al, 1993) (Figure 1). Many of the Iigands for proteintyrosine kinase receptors are present as homodimers, thus exhibiting two identical receptor-binding epitopes. These epitopes can bridge two receptors, resulting in receptor oligomerization (see Figure 1). Clustered receptors undergo transphosphorylation or autophosphorylation, generating novel binding sites for cytoplasmic proteins, which, in turn, become tyrosine phosphorylated and activated, initiating signal-transduction cascades (Heldin 1995). Numerous studies have helped to identify the embryological locations of these growth factor receptors by defining their spatiotemporal expression patterns, thus predicting their physiologic potential in the regulation of cardiac development. Here, we discuss some of the recent advances in understanding the functions played by specific growth factorh-eceptor systems in developmentally distinct cardiac structures, utilizing information derived from both expression analysis and the development of animal models lacking such molecules. In particular, we emphasize the newly described roles of neurotrophins and their cognate trk receptor tyrosine kinases in cardiac development in vivo.
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HGF
c-met
FGF
FGFR
neu
erb B2 erb B3 erb B4
angio-1
tie 2 tie 1
VEGF
Fit-l Flk-1
NT
trk A trk B trk C
Fignre 1. Schematic representation of receptor tyrosine kinases and their ligands implicated
in cardiac development.All receptorsare type I integralmembraneproteins.Extracellular loops reflectIgG-likedomains,andfiiled boxes in thecytoplasmicdomainsrepresentthecatalytic domainof the tyrosinekinase.The branchedextracellularstructuresrepresentheparin sulfateproteoglycans.Allligandsaresecretedsolubleproteins,exceptneuregulins,whichcan be membranetetheredor proteolyticallycleaved(arrow) to a solubleform. Angie-l, angiopoietin;FGF,fibroblastgrowthfactor; FGFR,fibroblastgrowthfactorreceptor;HGF,hepatocyte growth factor.
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ReceptorsRegulatingCardiomyoqte Proliferation/Differentiation
The erbB neuregulin receptors are critical in regulating the development of the cardiomyocyte. The neuregulins consist of a family of secreted and membraneattached growth factors, expressed from alternatively spliced mRNAs of a single gene (Marchionni 1995, Meyer and Birchmeier 1995). The neuregulins mediate their actions by binding to the erbB receptor family (erbB2, erbB3, and erbB4), which belongs to the larger family of the EGF receptors (Figure 1). Neuregulins can bind to either erbB3 or erbB4; howevec only erbB4 is capable of signaling, whereas erbB3 requires dimerization with erbB2 or erbB4 to transduce a signal. Conversely, erbB2 can signal, but it is required to forma heterodimeric receptor complex with erbB3 or erbB4 to bind neuregulin (Marchionni 1995, Meyer and Birchmeier 1995). The patterns of expression of neuregulin and erbB mRNA are reciprocal in the developing murine heart, with neuregulin being expressed by the endocardium (Kramer et al. 1996, Meyer and Birchmeier 1995), and myocytes expressing erbB2 and erbB4 (Gassmann et al. 1995, Lee et al. 1995). ErbB3 mRNA is expressed at highestlevelsby cells in the endocardial cushion, but expression can be detected also in myocytes at lower levels. The timing of expression has not been ex-
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tensively characterized, although both ligand and receptor expression has been documented at E9.5-10.5 (Figure 2). The phenotypes of the neuregulin, erbB2, and erbB4 null mutant animals are strikingly similar with regard to cardiac development (Gassmann et al. 1995, Kramer et al. 1996, Lee et al. 1995, Meyer and Birchmeier 1995). In these mice, cardiac development is normal until midgestation, when a pronounced defect in ventricular trabeculation occurs at E1O–10.5,resulting in the death of the embryos. These results suggest that this growth factor receptor system is critical for myocyte differentiation. It is unclear whether neuregulins exhibit effects on the developing heart through regulation of cardiac neural crest function. Mice deficient for neuregulin or erbB4, however, demonstrate a decrease in the size of the endocardial cushion. More importantly, the phenotype of mice lacking erbB3, the receptor most highly expressed in the endocardial cushion, has not yet been described. [Note added in proof: although the erbB2 null mutant animals have been generated, a cardiac phenotype has not been described (see Riethmacher et al. study on page 725 of Nattwe,Vol. 389).] Owing to the lethal cardiac phenotype at E1O.5, it is not possible to evaluate in these mouse mutants whether neuregulins contribute to the processes of valve formation and chamber septation. The
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prominent defects in cranial neural crest structures detected in the neuregulin and erbB4 mice suggest that neuregulins play critical roles in some classes of neural crest cell populations; however, their role in cardiac neural crest function will likely require the generation of conditional targeted deletions. Among the earliest growth factor receptor tyrosine kinases to be expressed in the cardiac mesoderm is the hepatocyte growth factor (HGF) receptor, cmet (Figure 1). Unlike most soluble growth factors, HGF is secreted as an inactive ligand, which can be activated by proteolytic cleavage (Rosen et al. 1994). Hepatocyte growth factor is first expressed in the precardiac mesoderm during gastrulation, whereas the expression of its receptor, c-met, is detected soon after in both cardiac mesoderm and endoderm (Rappolee et al. 1996). During the stages of cardiac looping, both receptor and ligand are expressed by cardiac myocytes (Figure 2). Expression of both receptor and Iigand is downregulated during the later stages of cardiogenesis. Owing to this spatiotemporal pattern of expression at the precontractile stage of cardiogenesis, as well as the defined actions of HGF on epithelial cells in inducing chemokinesis, HGF had been postulated to mediate cardiac myocyte migration (Koch et al. 1996). Targeted deletion of either the cmet or HGF alleles, however, results in null mutant mice that appear to undergo normal cardiac development, although they die in utero at E13.5–15, with intrauterine growth retardation and vascular defects of the placenta (Bladt et al. 1995, Schmith et al. 1995, Uehara et al. 1995). The lack of observed cardiac defects in the null mutant HGF and c-met animals has been postulated to reflect functional redundancy with related receptors (Ron and Sea) or the presence of other, unrecognized ligands. The expression pattern of HGF overlaps significantly with one member of the fibroblast growth factor family, FGF-8 (see later here). Thus it has been proposed that as cardiac myocytes coexpress HGF and FGF-8, FGF receptor signaling could potentially rescue cells with impaired HGF signaling. A second class of receptor tyrosine kinase, the FGF receptors, is also detected at early stages of cardiac myocyte development (Orr-Urtreger et al. 1991, Pat-
35
precardiac mesoderm
cardiac looping
~p
acquisition of a contractile phenotype
c1
septation valvulogenesis
birth
? ●
VEGF an io ?
E6
E7.5
E1O
geted deletions of FGF (Feldman et al. 1995)and the fact that the strategyused in the chick study inhibited the function of severalFGF receptor isoforms.
E12
E14
E20
Receptor TyrosineKinases Regulatingthe Formation of the Endocardium
Two families of receptor tyrosine kinases have been implicated in the regulation theircognateligandsduringcardiacdevelopment.Therelativeorderingof initiationof signal- of the endocardium and in vasculogeneing reflectsthe publishedspatiotemporalpatternsof expressionduringrodentdevelopment. sis, the vascular endothelial growth facThedurationof receptoractivityhasnot beendeterminedfor severalligand:receptorpairs,as tor (VEGF) receptors fit-l and flk-1, and targeteddeletionsresultin embryoniclethality,precludingevaluationof laterstagesof cardiac the angiopoetin receptors tie-1 and tie-2. development(indicatedby a question mark). FGF, fibroblast growth factor; HGF, hepatocyte VEGF was initially identified as a factor growth factor; VEGF, vascular endothelial growth factor; neu, neuregulin; angio, angiopoietin; that promoted vascular permeability and NT, neurotrophins. proliferation of endothelial cells (Majesky 1996, Yang et al. 1996). The biological stone et al. 1993, Sugi et al. 1995). The FGF function in cardiogenesis. First, the actions of VEGF are mediated by two related receptor tyrosine kinases, fit-l and FGF family of growth factors represents bFGF-receptor interaction is promoted a large family of at least nine related by coexpression of heparin sulfate (Fig- flk-1, which differ in their affinity of ligands and four related receptor ty- ure 1). Thus, the effectiveness of ligand binding [reviewed by Majesky (1996)]. Although flk-1 receptor expression is first rosine kinases, which can generate addi- signaling may require the coexpression tional isoforms by alternative mRNA of ligand with an appropriate extracellu- detected in precardiac mesoderrn at E7.O splicing [reviewed by Mason (1994)]. lar matrix, functioning to promote ligand of the mouse, the reciprocal patterns of Given this complexity, a complete de- stability, presentation, or a high-affinity expression of VEGF by the developing scription of ligand and receptor localiza- ligand–receptor interaction (Ruoslahti myocardium and of flk-1 in the develoption during cardiovascular development and Yamaguchy 1991). In addition, the ing endothelium have been detected at is lacking. Early studies using immuno- relative insensitivity of in situ hybridiza- E8.5 and are maintained through E15 histochemical or in situ hybridization tion analysis to detect FGF expression, (Dumont et al. 1995, Yamaguchi et al. 1993). Fit-l mRNA is not detected in the analysis, however, support the colocal- as compared with immunohistochemisization of the basic FGF ligand and re- try, has complicated the studies of ligand developing heart, and its expression apceptor by cardiac myocytes, suggesting localization. Alternatively the relative pears limited to the extraembryonic tisan autocrine or local paracrine mecha- ease in detecting the FGF proteins may sues through E15 (Figure 2). The critical roles of fit-l and flk-1 in nism of action in midgestation. Although reflect a longer half-life of ligand bound vasculogenesis have been defined by the an extensiveanalysisof the relativebind- to the heparin-rich extracellular matrix. early embryonic lethality of targeted ing affinitiesof the FGF receptors for the Despite these potentially confounding different FGF ligands has been under- issues, bFGF has been documented as gene deletion in mice (Carmeliet et al. taken, the distinctive spatial and tempo- providing a proliferative signal in cardiac 1996, Ferrara et al. 1996, Fong et al. ral patterns of expression may be more myocytes in in vitro studies using anti- 1995, Shalaby et al. 1995). Indeed, the relevantthan modest differences in the af- sense oligonucleotides to inhibit FGR sig- profound impairment in vasculogenesis finity of receptor–ligand interactions in naling (Choy et al. 1996, Sugi et al. 1993). in midsomite embryos limits analysis to regulating biological function (Ruo- These results have been confirmed in all but the earliest processes of ventricuslahti and Yamaguchy 1991). For exam- vivo by overexpression of dominant neg- lar fusion and looping. Nonetheless, the ple, the expression of the FGF receptor ative FGF receptors that inhibited myo- flk-1 null mutant animals lack endocarcek 1 is widespread in the chick develop- cyte proliferation (Mima et al. 1995). In- dium before their death at E9.5 (Shalaby ing heart, whereas the cek 2 receptor terestingly,inhibition of FGF receptor 1 et al. 1995). The fit-l null mutant anidisplays a more constrained temporal signaling through retrovirus-mediated mals display a distinctive phenotype and spatial expression in developing overexpression of a dominant negative with endocardial cells filling up the venvalves (Uehara et al. 1995). In the FGFreceptor 1 induces defects in myocyte tricular chamber, perhaps resulting from mouse, the most relevant members of mitogenesis only at relativelyearly devel- a loss of contact inhibition of the enthe FGF/FGF receptor families in car- opmental stages (before E7 in the chick), docardium at E9-E1l (Fong et al. 1995). diogenesis may be FGF-8, which is ex- suggestingthatFGFsmay exerttheirfunc- The VEGF-deficient animals are more pressed transiently at E7.5–9, and the tions in early cardiogenesis (Mima et al. severely affected than animals lacking bec receptor, which is expressed by car- 1995). Unfortunately,these observations either VEGF receptor (Carmeliet et al. diac myocytes at E8-13 (Figure 2) (Orr- have not been extended to the examina- 1996, Ferrara et al. 1996). These mice Urtreger et al. 1991). tion of several mouse FGF null mutant exhibit profound defects in vasculogeneSeveral additional considerations models. In part, these differences may re- sis because they lack dorsal aortae and have complicated the interpretation of flect the early embryonic lethality of tar- yolk sac vessels. Thus, these deficiencies Figure 2. Schematicrepresentation of thetimingof activationof receptortyrosinekinasesby
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full
full trunc
full trunc insert
Figure 3. Schematic representation of the neurotrophins andtrk receptor tyrosinekinases. The major isoform of each trk receptor is indicated with full, the full-length kinase-active isoform; trunc, the truncated, kinase-inactive isoform; and insert, the foil-length kinase-active isoform, bearing an insert in the kinase domain. The dominant ligand–receptor interactions are indicated by heavy arrows. Interactions that reflect reduced efficiency or affinity are indicated with broken arrows. BDNF, brain-derived neurotrophic factor; NGF,nerve growth factor; NT-3, neurotrophin-3
preclude the analysis of cardiogenic processes. Even the reduction in expression of VEGF in animals heterozygous for the targeted allele results in lethality in midembryonic development; however, these mice showed a delay in the development of the common atria and primitiveventricles, no evidence of endocardial formation, and a marked reduction in ventricular wall thickness. The defects seen in both endocardium and myocardium in the VEGF null mutant animals suggest that the absence of endocardium in these animals could limit the supply of normal trophic factors provided by the endocardium to the adjacent developing myocytes; this could result in hypoplasia and developmental delay in myocardiogenesis. The second receptor tyrosine kinase family that is involved in the regulation of vasculogenesis and the endocardium consists of the tie-1 and tie-2 receptor tyrosine kinases. Initially classified as orphan receptors, the tie-2 ligand has been recently identified as angiopoietin-1, a unique growth factor with coiled-coil and fibrinogen-like domains (Davis et al. 1996). A related ligand, angiopoietin-2, acts as a competitive inhibitor of angiopoetin with the tie-2 receptor (Maisonpierre et al. 1997). The ligand(s) for tie-1 have not been identified. The tie-1 receptor is first expressed in the endocardium (E8), followed by expression in the aorta and allantois, whereas the tie-2 receptor
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is expressed by the endothelium of major vessels at the same developmental stages (Korhonen et al. 1994, Sato et al. 1995) (Figure 2). Angiopoietin-1 exhibits a reciprocal pattern of expression to tie2, with angiopoietin-1 mRNA detectable in the myocardium (E9–11) and in the mesenchyme surrounding developing vessels (at El 3) in mouse embryos (Davis et al. 1996). No angiopoetin-2 mRNA is detected in embryonic mouse hearts (Maisonpierre et al. 1997). This pattern of gene expression suggests that the angiopoietin-l/tie-2 receptor functions at a developmentally later stage than the VEGF/flk-l receptor system. Recombinant angiopoetin-1 fails to induce the proliferation of cells expressing tie-2 receptor or to induce tubule formation in vitro. The critical roles of angiopoietin-1 and the tie-1 and tie-2 receptors in angiogenesis, however, have been confirmed by generating null mutant mice lacking expression of any of these three gene products (Puri et al. 1995, Sato et al. 1995, Suri et al. 1996). All three null mutant mice die in utero at E9.5–13 and display marked vascular defects such as microvascular hemorrhage (tie-1) or abnormal vascular networks (tie-2 and angiopoietin-1). In addition, all animals demonstrate cardiac defects with retardation in the growth of the hearts, although specific defects vary.The tie-l–deficient animals have no gross myocardial defects, but ultrastruc-
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turally,the endocardium exhibits a more immature phenotype. Tie-2 mutant animals exhibit defects in the development of cardiac vessels and die at E9.5–E1O.5, precluding analysis at later stages. The angiopoietin- 1–deficient mice exhibit slightly less severe defects, with retardation in the growth of the heart first notable at E 10.5. The endocardium is immature and less intricately folded in animals lacking angiopoietin-1 as compared with wild-type littermates, and before their death at E12.5, a marked decrease in ventricular trabeculations is noted. Although the tie receptors appear to act later than the flk-1/flt-l receptors, the lack of endocardium and reduction in myocardial growth are strikingly similar.The reciprocal patterns of expression of angiopoietin-1 and VEGF in the myocardium, potentially regulating the development of the endocardium, and neuregulins in the endocardium, potentially regulating myocyte development, suggests that the trophic support of the developing heart is achieved by local paracrine actions of these classes of growth factors.
●
Receptor Tyosine Kinases Regulatingthe Cardiac Neural Crest
A third embryologically distinct class of cells, the cardiac neural crest, is required for normal cardiac development. The cardiac neural crest cells, originating from the neural tube at the level of the mitotic placode to the third somite, migrate along the pharyngeal arches to reside in the cardiac outflow tracts and the endocardial cushions. Cardiac crest ablation studies in the avian model system have amply demonstrated the fundamental role of this cell population in the septation of the truncus arteriosus, the formation of the truncal cushion, the membranous portion of the interventricular septum, and the pulmonic valves (Kirby 1993). Although the neural crest has been studied in detail, little is known about the specific growth factors that regulate the cardiac neural crest. Leukemia inhibitory factor promotes the survival of chicken cardiac neural crest cells in vitro (Kirby et al. 1993), and extracellular matrix proteins such as fibronectin and laminin induce directed cell migration of neural crest precursors (Bronner-Fraser 1994, Noden 1991). Car-
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diac abnormalities, however, have not yet been reported in mice lacking LIF or its low-affinity receptor. Recently we and others have reported (Hiltunen et al. 1996, Donovan et al. 1996) the expression of another family of growth factors, the neurotrophins, during cardiac development. Neurotrophins are members of a highly homologous family of growth factors that contribute to the regulation of proliferation, survival, and differentiation of cell populations in the mammalian nervous system. This family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin3 (NT-3), neurotrophin-4/5 (NT-4/5) and neurotrophin-6 (NT-6) [reviewed by Lewin and Barde (1996)] (Figure3). Neurotrophins bind and activate trk receptor tyrosine kinases, with NGF binding only to trkA, BDNF and NT4/5 binding to trkB, and NT-3 binding predominantly to trkC, but also activating trkA and trkB with lower efficiency in biochemical assays (Figure 2). AU neurotrophins also interact with the p75 neurotrophin receptor, which lacks intrinsic enzymatic activity but influences high-affinity NGF receptor formation, ligand internalization, and has been shown to play a role in the induction of apoptosis (Chao and Hempstead 1995). Trk receptors, the mediators of neurotrophins’ biological activities, are highly expressed in the central and peripheral nervous system. Thus studies on neurotrophin actions have focused on the developing nervous system, with little attention to neurotrophin function in nonneuronal tissues. Expression of neurotrophins and their receptors has been demonstrated in the developing cardiovascular system of vertebrates including Xenopus, chick, rat, and mouse (Donovan et al. 1995, Hiltunen et al. 1996, Scarisbrick et al. 1993, Tessarollo et al. 1993, Williams et al. 1993). Mouse and rat have been most extensively examined for the spatiotemporal patterns of gene expression. In the rat, all neurotrophins have been detected in the developing cardiovascular system during ontogenesis, and their presence has been correlated with the sensory and sympathetic innervation pattern of great vessels and the heart. Indeed, neurotrophin expression has been detected in the tunica media of the aorta, pulmonary and other major elastic arteries of the thorax and abdomen at
embryonic day 13, when innervation has been established (Scarisbrick et al. 1993). In the rat heart, NT-3 is first detected in the developing conduction system in the interventricular septum as early as El 1 before cardiac innervation is established. This pattern of NT-3 expression has led to the suggestion that NT-3 may function as a chemoattractant, as well as a trophic factor, for developing cardiac ganglia neurons expressing trkC and innervating conduction system cells (Hiltunen et al. 1996). In mouse, we have been able to detect trkC immunoreactivity in myocytes within the ventricle, atria, and cardiac outflow tract as early as E9.5. Furthermore, we have detected trkC mRNA and protein in the migrating cardiac neural crest at the same developmental stage (Donovan et al. 1996, Tessarollo et al. 1993). Detection of trkC in the mouse at earlier stages than reported in the rat maybe due to different sensitivity in methodologies employed or by intrinsic differences of rodent systems (Hiltunen et al. 1996). In fact, although mouse and rat have similar gestational periods of about 20–21 days, some of the critical developmental stages of cardiac development occur at different gestational days. The detection of trkC in mouse cardiac myocytes and migrating cardiac neural crest cells long before innervation occurs suggests that diverse roles are played by neurotrophins and their receptors in the development of the cardiovascular system. Recent reports have demonstrated chemotactic roles for neurotrophins on vascular smooth muscle cells (Donovan et al. 1995) and melanocytes (Hermann et al. 1993). Furthermore, although no studies to date have been performed with cardiac crest, trunk neural crest cells express the trkB and trkC neurotrophin receptors. These ceils respond mitogenically to BDNF and NT-3 and undergo NT-3–induced differentiation in vitro (Henion et al. 1995). These data support the hypothesis that neurotrophins can modulate neural crest development. Data from analyses of cardiac development in mice bearing targeted deletions of NGF, BDNF, and NT4/5, and trkA and trkB receptors are not available to date. We have recently reported (Donovan et al. 1996), however, that mice lacking NT-3 show developmental anomalies of the great vessels, including develop-
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mental delay in the primitive myofibril organization of the truncus arteriosus evident as early as E9.5. These defects are associated with reduced levels of expression of trkC in myocytes within the ventricles, atria, and cardiac outflow tracts. These early developmental defects before the onset of cardiac innervation are consistent with a direct role of NT-3 and trkC in cardiac development. More data are needed to elucidatewhether NT-3 and trkC function directly in cardiac myocyte differentiation. More striking cardiac defects were detected in the atria and ventricular septation, valves, and outflow tracts of both trkC and NT-3 mutant mice (Donovan et al. 1996, Tessarollo et al. in press). In some cases, these mutant mice developed defects resembling some of the most common congenital malformations in humans, including ventricular septal defects and tetralogy of Fallot. The observed defects were consistent with abnormalities in the survival and/ or migration of cardiac neural crest early in embryogenesis. Although the majority of studies defining the contributions of cardiac neural crest cells to normal cardiac development have been performed in the avian model because of ease of manipulation of early embryos, the abnormalities of the homozygous trkC or NT-3 null mutant mice follow these avian predictions. . Future Directions Although the studies reviewed here underscore the importance of several distinct classes of receptor tyrosine kinases in regulating specific cell types in the developing mammalian heart, further work is needed to address three important questions: (a) What are the upstream and downstream gene products that regulate, or are regulated by the activation of receptor tyrosine kinases? (b) Do receptor tyrosine kinases continue to support the survival and function of the myocardium and endocardium postnatally, under normal physiological, or in pathological, conditions? (c) Can the information gained from the analysis of mouse mutants further the understanding of human congenital cardiac defects? The rapidly emerging diversity of transcription factors that regulate distinctive aspects of early cardiogenesis should provide the means to assess
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which factors directly activate the genes encoding specific receptor tyrosine kinases and their ligands. Information regarding the timing and patterns of expression of these transcription factors, as well as the identification of their target consensus sequences, should aid in these studies, as the promoter regions of most of the ligands and receptors described here are under study. Similarly, an analysis of genes specifically induced by an activated receptor tyrosine kinase is likely to provide insight into their biological functions as well as the molecular basis for the cardiac dysfunction noted in null mutant animals. In addition, these types of studies could yield information on the potential use of specific growth factors to ameliorate degenerative cardiac conditions. The vast majority of studies of receptor tyrosine kinases in the heart have focused on their roles in development, rather than in regulating cardiac physiology in postnatal animals. Given the embryonic lethality of targeted deletions in many of these genes (Figure 2), alternative strategies, using chimeric animal models, or conditional targeted gene deletions will be necessary to explore their potential roles in later embryonic stages. The prominent roles for receptor tyrosine kinases in maintaining the survival and appropriate function of cells in other organs suggest that these signaling molecules could play critical roles in maintaining cardiac myocyte function in the adult. Finally, studies are needed correlating the specific phenotypes observed in animals with targeted gene deletions with that of children with similar congenital cardiac abnormalities. The early embryonic lethality of some mutations in mice may preclude analysis of comparable human tissue. Mutations that yield a cardiac phenotype in the mouse due to haploinsufficiency or that induce phenotypes that are compatible with postnatal survival, such as cardiac crest abnormalities, should be amenable to comparative analysis.
●
Acknowledgments
Weill-Caulier Trust (to B.L.H.), and by the National Cancer Institute, Department of Health and Human Services, under contract with Advanced Bioscience Laboratories (to L.T.).
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