Published online before print December 5, 2002, doi: 10.1161/01.RES.0000050587.76563.A5
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© 2003 American Heart Association, Inc.
Molecular Medicine |
The Homeobox Gene Lbx1 Specifies a Subpopulation of Cardiac Neural Crest Necessary for Normal Heart Development
From the Institute of Physiological Chemistry, University of Halle-Wittenberg, Halle, Germany.
Correspondence to Thomas Braun, MD, PhD, Institute of Physiological Chemistry, Hollystr. 1, Halle 06097, Germany. E-mail thomas.braun{at}medizin.uni-halle.de
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Abstract |
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Cardiac neural crest cells are known to play multiple roles during development of the inflow and outflow tract of the heart and the aortic arch. In addition, cardiac neural crest is required for normal heart tube looping and regulation of myocardial cell proliferation, as well as differentiation and function of the myocardium. We show that the homeobox gene Lbx1 is expressed in a subpopulation of the cardiac neural crest during tubular heart formation. Inactivation of the Lbx1 gene in mice resulted in defects in heart looping, changes in gene expression pattern, and increased cell proliferation ensuing in myocardial hyperplasia. We found that the activity of the Lbx1 promoter, as indicated by a LacZ reporter gene, is upregulated in the hearts of Lbx1+/-:splotch1H/splotch1H and Lbx1-/- mice, indicating that Pax3 and Lbx1 participate in a negative regulatory feedback that might be necessary for normal differentiation and function of the myocardium during early heart development. Because migration of Lbx1-expressing neural crest cells was not altered in Lbx1-/- embryos, we postulate that Lbx1 gene function is critical for specification of a subpopulation of cardiac neural crest subsequent to migration.
Key Words: Lbx1 • cardiac neural crest • mouse mutants • hyperplasia • Pax3
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Introduction |
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Neural crest contains a population of pluripotent cells that originate from almost the entire length of the neural tube. Neural crest cells migrate to various locations in the developing embryo and give rise to several different cell types.1 The cranial neural crest, which is located anterior to somite 5, has a particularly high potential to generate mesenchymal cells compared with caudal neural crest. The cardiac neural crest is a subpopulation of the cranial neural crest. It originates from posterior rhombencephalon (rhombomers 6 to 8) between the otic placode and the third somite and migrates via the circumpharyngeal region to the branchial arches 3, 4, and 6 and from there to the heart.2 Cardiac neural crest migrates between the distal end of the outflow tract of the heart and the pharyngeal endoderm and then invades the outflow tract, leading to the formation of the septal tissue that divides the aorta ascendens and truncus pulmonalis.
Ablation of premigratory cardiac neural crest in avian embryos results in malformations of the cardiac outflow tract including persistent truncus arteriosus (PTA) and double outlet right ventricle (DORV).3 Interestingly, heart defects after cardiac neural crest ablation in chicken are not exclusively linked to septation defects of the outflow tract. Well before malformation of the outflow tract would affect heart function, severe abnormalities of the myocardial developmental program occur, such as decrease of the ejection fraction of the heart, an abnormal morphology of the heart loop, and abnormally elevated heart cell proliferation.4
Mutation of several genes affects cardiac neural crest development and partially or completely mimics neural crest ablation. For example, Pax3 mutant mice, such as the naturally occurring splotch mutation, suffer from a PTA5 and thus provide a model in which the role of cardiac neural crest in heart development can be studied. The function of Pax3 for cardiac neural crest development, however, is not fully understood. Until lately, downstream genes through which Pax3 regulates cardiac neural crest development were unknown. Only recently, Msx2 was identified as an immediate downstream effector of Pax3.6 It became apparent that the Msx2 gene is upregulated in Pax3 mutant mice. This derepression of Msx2 seems to be a critical parameter for development of conotruncal heart defects because targeted inactivation of Msx2 selectively rescued development of cardiac neural crest in Splotch mutant mice.6
Despite a long list of molecules involved in cardiac neural crest development, no molecules have been identified that specify specific subsets of cardiac neural crest and thus partially mimic the neural crest ablation phenotype after gene inactivation. In the present study, we have focused on the role of the homeobox gene Lbx1 in cardiac neural crest development. Lbx1 is a homeodomain-containing transcription factor related to the Drosophila ladybird genes. In Drosophila, the ladybird genes are expressed in a specific subset of cardioblasts and have been shown to be important components of the cardiogenic pathway required for the diversification of heart precursor cells.7 In vertebrates, expression of Lbx1 has been described in the CNS and in migrating muscle precursor cells.8 Like Msx2, Lbx1 appears to be downstream of Pax3 in the genetic hierarchy because Pax3 is required to initiate expression of Lbx1 in the lateral tip of the dermomyotome that gives rise to limb muscle precursor cells.9 We found that Lbx1 is expressed in a subset of cardiac neural crest cells and that its inactivation in mice resulted in heart malformations that are similar to defects observed in chicken embryos after early neurocrest ablation including defects in heart looping, changes in gene expression pattern, and increased cell proliferation.
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Materials and Methods |
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Mice
The generation and genotyping of Lbx1 mutant mice has been described.10 Splotch1H mice were originally obtained from Thomas Franz (University of Bonn, Germany) and kept on a C57/BL6 background for several years in our laboratory.5 In the care and use of laboratory animals, the guidelines given by the German government (animal care laws) have been followed, and the animals were routinely seen and evaluated by a veterinarian. The mice were kept in a specific pathogen-free facility.
Histology, Immunohistochemistry, and ß-Galactosidase Staining
Histological examination of tissues was performed by routine hematoxylin-eosin and trichrome staining on paraffin sections (8 µm) according to the instructions of the manufacturer (Sigma Inc). Staining for ß-galactosidase (ß-gal) activity was accomplished as described.11 ß-Galactosidase activity was quantitated in heart tissue extracts normalized for their protein content using chlorophenol red-ß-D-galactopyranoside (CPRG) as substrate as described previously.12 Extinction was measured at 575 nm after a 60-minute incubation period at 37°C. Immunohistochemical staining was achieved by reacting cryosections that have been stained for ß-gal activity with antibodies against connexin43 (connexin43 monoclonal antibody, 1:500; DAKO). To label proliferating cells, mice were injected with 120 mg BrdU/kg body weight 1 to 3 hours before dissection. Labeled cells were detected using a BrdU detection system (Roche Biochemicals) following exactly the instructions of the manufacturer.
In Situ Hybridization
Whole mount in situ hybridizations were done according to the protocol of Wilkinson.13 The Lbx1 antisense probe was synthesized from plasmid pCRII-Lbx1 that contains a 726-bp Lbx1 cDNA fragment (nucleotide positions 947 to 1673).9 The Pax3 probe was kindly provided by A. Mansouri (MPI Göttingen, Germany); the connexin 40 probe was provided by K. Willecke (University of Bonn, Germany). The FHL2 probe was a kind gift of Ju Chen (University of California, San Diego, Calif).
Quantitative Real-Time RT-PCR
Total RNA was isolated from hearts of E10.5 embryos using the TRIzol reagent (Life technologies Inc) according to the manufacturers instructions for small samples. First strand cDNA was synthesized with reverse transcriptase from Gibco BRL according to standard protocols. Real time quantitative RT-PCR was performed using the iCycler from BioRad and the Brilliant SYBR green QPCR kit from Stratagene. Plasmids containing the amplicon sequences for G3PDH and FHL2 were used to quantitate the cDNA concentration (G3PDH) and to determine the absolute copy number of FHL2 mRNA in different samples. Copy numbers describe the calculated FHL2 mRNA concentration within the RNA isolated from a complete E10.5 heart.
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Lbx1 Is Expressed in a Subpopulation of Neural Crest Cells
Previous studies have described expression of Lbx1 in the CNS and in migrating muscle precursor cells.8,9 So far, no expression of Lbx1 has been reported in the heart, although the Drosophila homolog ladybird is expressed in a specific subset of cardioblasts in Drosophila and is required for diversification of heart precursor cells.7 Examination of the heart region of wild-type mouse embryos by whole mount in situ hybridization between E9.0 and E10.5 revealed a stream of Lbx1-expressing cells that emerged from the dorsal region of the embryo and migrated ventrally to the developing heart (Figure 1D). In splotch mutant mice but not in Lbx1 homozygous mutants, this stream of Lbx1-expressing cells was missing (Figure 1N). This cell population does not represent cardiac neural crest but a population of myogenic precursor cells that will populate the hypoglossal cords and form the muscles of the neck.14 Although we sometimes found a diffuse Lbx1 staining after whole mount in situ hybridization in the heart (Figure 1E), we were unable to validate the presence of noteworthy amounts of Lbx1 transcripts in the hearts of mouse embryos.
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Despite the failure to detect Lbx1 mRNA in the heart, we observed a small population of Lbx1-LacZ–positive cells that migrated from the neural tube (Figures 1F and 1K) to the caudal branchial arch and the truncus arteriosus (Figures 1A and 1B) between E9.0 and E9.5. This cell population does represent only a small fraction of the large amounts of cardiac neural crest that has been shown to invade the branchial arches and the truncus arteriosus.15 Because only single ß-gal–expressing cells were found, it is hard to distinguish whether the detection of these cells is due to a higher sensitivity supplied by the Lbx1-LacZ allele or whether the stable ß-gal enzyme fate-mapped cells that have abated Lbx1 expression before they reached the heart. At E9.0, Lbx1-LacZ–positive cells were still relatively rare (Figures 1H and 1M). At E11.0, Lbx1-LacZ–positive cells were also detected in small numbers in the left ventricle (Figures 1J and 1O; see also Figure 8). Because cells of neural crest origin have never been shown to contribute to the myocardium,15 Lbx1-LacZ expression in the myocardium might formally reflect a de novo Lbx1 expression in the myocardium or a separate cell population that is distinct from neural crests cells as previously defined.
Lbx1 Mutant Mice Show Alterations in Heart Looping but Form Normal Inflow and Outflow Tracts
The expression of Lbx1 in cells that give rise to heart neural crest prompted us to screen for heart defects that are caused by deficiencies in neural crest development. We found that approximately 15% of homozygous Lbx1 mutant embryos showed irregularities in heart looping, including straight heart tubes (Figure 2B), incomplete looping (Figure 2C), variable dilations along the heart tube (Figure 2D), and a disorganized appearance (Figures 2D through 2F). The observed variability of the Lbx1 heart phenotype resembled the variability seen after cardiac neural crest ablation in chicken embryos4 and in Pax3 mutant mice. The alteration in heart looping in Lbx1 mutant mice did not correlate with a reduced presence of Lbx1-LacZ cells in the heart because these cells were readily detectable in the heart tube of homozygous Lbx1 mutant embryos (Figures 2E and 2F).
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We next analyzed the hearts of homozygous mutant Lbx1 embryos between E14.5 and P0 for defects in the outflow tract. Because those Lbx1 mutant embryos that displayed defects in heart looping were arrested in development and died between E9.5 and E11.5, such an analysis was restricted to embryos without an initial heart looping phenotype. As shown in Figure 3, the remaining Lbx1 mutant mice showed a normal morphology of the outflow tract with no signs of DORV, malformations of heart valves or the great vessels such as the aorta ascendens and the truncus pulmonalis. In addition, no malformation of the inflow tract was found. The septation between aortic and pulmonary vessels and between the ventricles was normal, and no PTA was observed.
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Lbx1 Mutant Mice Develop Myocardial Hyperplasia and Show Increased Cell Proliferation in the Heart
Another consequence of the ablation of neural crest in chicken embryos is an increase of proliferating myocardial cells that gives rise to a disorganization of myofibrils in the heart.4 To determine whether the lack of Lbx1 is able to induce cardiac hyperplasia and/or hypertrophy, ventricular wall thickness and ventricular myocyte width was assessed in Lbx1-deficient mice and in heterozygous and wild-type control littermates at P0. As shown in Figure 4, the ventricular walls of newborn Lbx1 mutant mice (n=6) were significantly wider than in control animals (n=10), while no differences in body weight among the groups were observed. Mutant animals showed a 80±15% increase of the diameter of the left ventricular wall, a 50±25% increase of the diameter of the right ventricular wall, and a 70±20% increase of the interventricular septum compared with control animals. Concomitant with the increase of the thickness of the ventricular walls an enlargement of the coronary vessels of mutant mice became apparent (Figures 4I and 4J). In contrast, the width of ventricular myocytes was not increased in Lbx1-deficient mice indicating that the enlargement of heart walls was due to hyperplasia and not to hypertrophy (Figures 4E through 4H).
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To obtain a more comprehensive profile of morphological changes during heart development, we assessed the number of proliferating cells in the myocardium by BrdU incorporation. BrdU-positive cells were counted at different sites in the heart of E14.5, E15.5, E17.5, and P0 wild-type and Lbx1 mutant animals. As shown in Figure 5I, a significant increase of proliferating cells was detected at E14.5 in the wall at the outer curvature and the apex of the heart. At E15.5, a strong increase of BrdU-incorporating cells was noted at the apex but not at the outer curvature of the heart of Lbx1-deficient mice. At E17.5, the situation was reversed with an increase of proliferating cells at the outer curvature but normal numbers of BrdU-positive cells at the apex. At P0 a strong increase of proliferating cells was found at both locations in Lbx1-deficient compared with wild-type mice. Such differences might reflect the varying ability of different areas of the myocardium to respond to growth stimuli, which might be necessary to create the normal shape of the heart.
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Loss of Lbx1 Results in Changes of Gene Expression in the Myocardium Including a Downregulation of FHL2
The restricted expression of Lbx1 in developing hearts suggested that increased myocardial cell proliferation in Lbx1-deficient mice might be caused by secondary effects evoked by an erroneous programming of neural crest cells. We therefore analyzed the expression of selected regulatory factors, such as BMP-10, Msx1, and LIM proteins,16,17,18 in the myocardium of wild-type and Lbx1 embryos by whole mount in situ hybridization (WISH). Although no significant differences in the expression levels of BMP-10, Msx1, and the LIM protein FHL1 were detected, we found a strong decrease in the expression of the LIM protein FHL2 in Lbx1-deficient embryos at E10.5. As shown in Figure 6, expression of FHL2 mRNA in hearts of Lbx1-deficient mice was below the detection limit of WISH. In contrast, a normal FHL2 expression was found in a second unrelated expression domain that represented the prospective urogenital tract. To validate the loss of FHL2 expression, we determined the absolute copy number of FHL2 mRNA molecules by quantitative real-time RT-PCR in hearts of Lbx1-/- embryos at E10.5 and in wild-type control animals. As shown in Figure 6G, virtually no FHL2 mRNAs were present in hearts of Lbx1 mutant embryos, whereas the concentration of FHL2 mRNAs ranged between 3x107 and 8x107 molecules per E10.5 heart in wild-type embryos.
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E/H); lane 2: WT1; lane 3: WT2; lane 4: mut1; lane 5: mut2; lane 6: complete wild-type E10.5 embryo; lane 7: 105 copies of plasmid DNA of the analyzed gene (FHL2 or G3PDH); lane 8: no template control; and lane 9: size standard (pUC18 Sau3AI).In addition, alterations in the expression of structural proteins of cardiomyocytes were noted. In the rodent heart, expression Cx40, a gap junction protein, is upregulated during cardiac development, reaching a maximum at E14.5.19 In Lbx1 mutant embryos, however, the accumulation of Cx40 mRNA was significantly delayed. We detected a significant reduction of Cx40 expression at E13.0 in the atria and left ventricle of Lbx1 mutant mice compared with wild-type mice, whereas at later developmental stages no significant differences were observed, indicating a distorted temporal development of myocardial cells and the cardiac atrioventricular conductions system but not a complete developmental arrest (data not shown).
Deregulation of Lbx1 Gene Expression in the Heart of Lbx1 and Splotch Mutant Mice
The replacement of the Lbx1 coding region by the bacterial LacZ gene enabled us to analyze the distribution of Lbx1-LacZ–positive cells as well as Lbx1 gene activity in Lbx1 mutant hearts. Surprisingly, the number of LacZ-positive cells was greatly enhanced in Lbx1 homozygous mutants compared with heterozygous mice at E13.0 (Figure 7). Lbx1-LacZ activity increased sharply between E12.5 to E13.0 in the hearts of Lbx1 homozygous (Figures 7C, 7F, and 7I and Table) versus heterozygous embryos (Figures 7A, 7D, and 7G and Table). We next generated Lbx1+/-:splotch1H/splotch1H mutant embryos. Again, we observed a marked increase of Lbx1-LacZ activity at E13.0 in the hearts of Lbx1+/-:splotch1H/splotch1H mutant embryos (Figure 7H and Table). To analyze whether the presence of additional Lbx1-LacZ–expressing cells is due to enhanced proliferation of Lbx1-LacZ cells that were fate-mapped to the heart but declined Lbx1 expression before they reached their destination, we performed mRNA in situ hybridization. Lbx1 mRNA expression was strongly upregulated in homozygous splotch1H mutant embryos (Figure 7L), suggesting a de novo Lbx1 expression in the myocardium and/or an amplification of an otherwise rare Lbx1 mRNA-expressing cell population in the absence of either Pax3 or Lbx1.
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Defects in a Subpopulation of Cardiac Neural Crest in Lbx1-Deficient Embryos
Deficiencies of cardiac neural crest might affect heart development in several ways. Relatively easy to understand are defects in structures that are directly derived from cardiac neural crest cells such as the outflow tract. Other defects, which have been observed after neural crest ablation in chicken in tissues not directly built by neural crest are more difficult to explain. These changes include defects in heart looping and cardiomyocyte proliferation.4 Because such changes of the myocardia can be monitored well before neural crest cells had the opportunity to contact the myocardium, it has been speculated that neural crest cells affect myocardial development from a distance during migration. Cardiac neural crest has been hypothesized to serve inductive roles in heart development including (1) control of myocardial growth by modulation of endodermal signaling and (2) induction of maturation of the heart conduction systems, which is linked to the arrival of neural crest cells through the dorsal myocardium.4,20,21 Different mechanisms have been put forward to explain these findings: (1) release of a factor produced by cardiac neural crest cells or by another cell population that interacts with neural crest cells; (2) activation of deposited growth factors by neural crest cells as transient apoptosis-prone cells; and (3) suppression of a factor that is produced by a third population of cells. Namely, an unnaturally prolonged period of release of a FGF-like factor by the pharyngeal endoderm has been proposed,4 because the pharyngeal endoderm is a well-known source of secreted factors that affect heart development during the time when neural crest cells populate the caudal pharyngeal arches. The normal development of the outflow tract in Lbx1-deficient mice along with the restricted expression of Lbx1 in the normal heart also argues for the absence of an activating factor or the failure of a properly timed suppression of a factor released from another cell population as the cause for the heart phenotype in Lbx1-deficient mice. In contrast, a cell autonomous defect of cardiac neural crest in Lbx1-deficient mice appears less likely.
Lbx1 is expressed in a small subpopulation of neural crest cells. The failure to detect a gross migration defect of cardiac neural crest in Lbx1-deficient mice was therefore not unexpected. Apparently, the lack of Lbx1 affects only a small subpopulation of neural crest cells, which is a quite different situation compared with neural crest ablated chicken embryos where all cardiac neural crest cells are absent. It appears feasible that the affected neural crest subpopulation has a special role to turn down pharyngeal signaling or to supply a positive factor for cardiac development although this is purely speculative at the moment.
Interestingly, in humans there have been reports about hypertrophic cardiomyopathy in association with lentiginosis as manifested in the leopard syndrome and other disorders of the neural crest tissue further emphasizing the potential role of neural crest in the development of a hyperplastic or hypertrophic heart disease.22 Because Lbx1-deficient mice decease shortly after birth when skin pigmentation is not yet completed, we were unable to access the distribution of skin pigments in Lbx1 mutant mice. However, in some cases hypopigmented spots were observed on the back or the belly of heterozygous Lbx1 mutant mice, which might indicate a haploinsufficiency of the Lbx1 gene in regard to migration of melanocytes. It should be pointed out, however, that the frequency of such observations was low (<1:500).
Lbx1 and Pax3 in Cardiac Development
The increase of Lbx1 expression in hearts of Pax3-deficient mice was an unexpected finding, in particular because Pax3 is necessary for Lbx1 activation in migrating limb muscle precursor cells.9 The same upregulation of Lbx1 promoter activity as in Pax3 mutants was observed in Lbx1-deficient mice, suggesting a common regulatory mechanism. Apparently, the expression of Lbx1 and Pax3 in the heart is linked by a regulatory feedback loop that is more complex than a simple epistatic relation. We propose that Pax3 and Lbx1 participate in the activation of a repressor that restricts the expression of Pax3 and Lbx1 depending on the tissue and the developmental stage. Such a negative regulatory feedback loop would explain why Lbx1 is upregulated in the heart of Lbx1 and splotch mutants at E13.5. Interestingly, recent experiments in splotch-deficient mice have disclosed a negative regulatory role of Pax3 on the homeobox gene Msx2, leading to an upregulation of Msx2 expression in Pax3-deficient mice.6 In turn, targeted mutation of Msx2 leads to suppression of embryonic lethality of the homozygous splotch mutation, suggesting a critical role of deregulated Msx2 expression for the development of cardiac neural crest defects. The upregulation of Msx2 resembles the upregulation of Lbx1 in splotch mutant mice but with some noteworthy differences. Whereas Kwang et al6 showed that Pax3 probably represses Msx2 by a direct effect on a conserved Pax3 binding site in the Msx2 promoter, the situation for Lbx1 seems to be more complex. We have shown previously that Pax3 is sufficient to induce expression of Lbx123 excluding a simple repressing function of Pax3 on the Lbx1 promoter. Either Pax3 functions both as a repressor and activator of Lbx1 transcription depending on the cellular context or the upregulation of Lbx1 in splotch mutant mice occurs indirectly via erroneous programming of the cardiac neural crest. The upregulation of Lbx1 in the myocardium at a time when Pax3 expression has long ceased as well as the upregulation of Lbx1-promoter activity in Lbx1 homozygous mutant argues for the latter possibility.
What Are the Components of the Molecular Machinery Leading to Hyperplasia in Lbx1-Deficient Hearts?
During pre- and postnatal cardiac development as well as during ventricular remodeling the size, shape, and composition of the heart tissue changes, a process that involves hypertrophy and hyperplasia of cardiomyocytes (see review24). Stimuli might result in either hyperplasia or hypertrophy depending on the developmental stage at which the stimulus acts.25 Hyperplastic and hypertrophic responses are relatively common reactions of the myocardia to adverse conditions and might be mediated by numerous signal transduction cascades. The LIM protein FHL2 belongs to the LIM-only (LMO) subgroup of this superfamily and functions as a transcriptional modulator that is involved in cardiac hypertrophy and stress response. We have demonstrated that FHL2 is downregulated in Lbx1-deficient hearts that are prone to hyperplasia, suggesting that it might be part of an Lbx1-induced machinery, which restricts cardiac growth during development.26 It appears unlikely that Lbx1 regulates FHL2 and Cx40 directly because Lbx1 is expressed only in a small subset of cardiac neural crest cells.
It will be a future challenge to decipher the cellular and molecular interactions that mediate the erroneous programming of a subset of cardiac neural crest cells in Lbx1-deficient mice to the myocardia and therefore results in defects in heart looping, changes in gene expression, and myocardial hyperplasia.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft, the "Wilhelm-Roux-Program" for research of the Martin-Luther-University Halle-Wittenberg.
Received June 21, 2002; revision received October 14, 2002; accepted November 12, 2002.
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