Human Molecular Genetics, 2002, Vol. 11, No. 8 915-922
© 2002 Oxford University Press
Tbx1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways
1Department of Pediatrics (Cardiology) 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TBX1 is the major candidate gene for DiGeorge syndrome (DGS). Mouse studies have shown that the Tbx1 gene is haploinsufficient, as expected for a DGS candidate gene, and that it is required for the development of pharyngeal arches and pouches, as predicted by the DGS clinical phenotype. However, a detailed analysis of the cardiovascular phenotype associated with Tbx1 mutations has not been reported. Here we show that Tbx1 deficiency causes a number of distinct vascular and heart defects, suggesting multiple roles in cardiovascular development – specifically formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Comparison of phenotype and gene expression using a Tbx1–lacZ reporter allele supports a cell-autonomous function in the growth of the pharyngeal apparatus, and a cell non-autonomous function in the growth and early remodeling of the pharyngeal arch arteries. Our data do not support a direct role of neural crest cells in the pathogenesis of the Tbx1 mutant phenotype; however, these cells, and the cranial nerves, are misdirected. We hypothesize that this is due to the lack of a guidance role from the pouch endoderm, which is missing in these mutants.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DiGeorge syndrome (DGS) (1) is characterized by aortic arch patterning defects, conotruncal heart defects, thymus and parathyroid aplasia/hypoplasia, and craniofacial anomalies. This phenotypic complex has suggested a role for pharyngeal arch/pouch maldevelopment in the pathogenesis of DGS, because the organs affected are developmentally related to the pharyngeal apparatus (reviewed in 2). The pharyngeal apparatus is composed of derivatives of all three embryonic germ layers, patterned by the anteroposterior growth and dorsolateral outpouching of the pharyngeal endoderm. Mesodermal and ectomesenchymal (neural-crest-derived) cells colonize the region between the pouches to form the pharyngeal arches, from which connective tissue and nerves of the face and neck develop. Arteries form within these arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation.
DGS is mostly caused by a heterozygous chromosomal deletion of chromosome 22q11.2 (referred to as del22q11). del22q11 is estimated to be the most common chromosomal deletion associated with birth defects in humans (3). Unveiling the genetic basis of DGS is important for understanding the disease pathogenesis and the developmental genetics of the pharyngeal apparatus. To this end, we have generated a mouse model of DGS by deleting part of the murine region on chromosome 16 that is homologous to del22q11. Heterozygously deleted mice, referred to as Df1/+ mice, present with cardiovascular defects (4), mild thymic and parathyroid abnormalities (5), and behavioral abnormalities (6) characteristic of the human syndrome. The haploinsufficient gene, within Df1, responsible for the cardiovascular abnormalities is Tbx1 (7–9). Homozygous mutation of Tbx1 causes severe developmental defects of the pharyngeal arches and pouches (7,8). However, a detailed analysis of the effects of Tbx1 deficiency on the cardiovascular system has not been reported, and the role of neural crest cells (NCCs), postulated to be of pathogenetic importance in DGS, has not been assessed in Tbx1 mutants. Here we show that, consistent with the patients' phenotype, the cardiovascular phenotype in Tbx1 mutants is multifaceted and includes a substantial intracardiac phenotype. Potentially related to these phenotypic abnormalities is the discovery of previously unreported Tbx1 expression domains in the outflow tract. We show that Tbx1 is unlikely to have a function in NCCs, but the paths of the caudal streams of NCCs, and of the cranial nerves that are derived from these cells, are profoundly affected, probably secondary to the lack of the caudal arches and pouches.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tbx1 is required for patterning and growth of the pharyngeal endoderm
To correlate Tbx1 gene expression and the mutant phenotype, we have used a mutant Tbx1 allele carrying a knocked-in nuclear–lacZ reporter gene. The generation and establishment of mice with this allele, named Tbx1tm1Bld, has been described elsewhere (8), but its suitability as a Tbx1 expression reporter had not been tested. To establish whether the ß-galactosidase (ß-gal) activity recapitulates the expression of the wild-type (WT) Tbx1 allele, we compared ß-gal activity (detected by X-gal staining) and Tbx1 expression (detected by RNA in situ hybridization) in Tbx1wt/tm1Bld embryos, hereinafter referred to as Tbx1+/-. Results did not identify any significant differences between the two patterns (Fig. 1A and B) indicating that, in these mutants, ß-gal activity recapitulates Tbx1 gene expression.
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Tbx1 homozygous mutants (Df1/Tbx1- or Tbx1-/-) present with hypoplasia of the pharynx, which has a simple tube-like appearance and lacks the characteristic segmented pattern (7,8) (Fig. 1C and D); pouches 2–4 and arches 3–6 are not recognizable. At E8.0, the earliest stage tested, Tbx1 is expressed in the pharyngeal endoderm and in the mesodermal core of the 1st pharyngeal arch. At E9.5, the strongest expression is observed in the endoderm lining the 3rd pouch and 3rd arch (Fig. 2A), which are the most posterior pharyngeal segments at this stage. At E10.5, when the pharynx has grown to include the 4th arch, 4th pouch, and 6th arch, Tbx1 in the endoderm is again expressed most strongly in the posterior segments (Fig.2B). In addition to this anteroposterior ‘gradient’ of expression, we observed higher expression towards the dorsolateral folds of the pharynx. At E11.5, endodermal expression persists almost exclusively in the 4th pouch (Fig. 2C). Hence, Tbx1 is expressed following anterior-to-posterior and medial-to-lateral gradients. These are also the directions of growth of the pharynx, suggesting that Tbx1 may be involved in the growth process. To test whether cell proliferation is affected, we calculated the mitotic index in the pharyngeal endoderm and in the mesenchyme of pharyngeal arches 1 and 2 of Tbx1+/- and Df1/Tbx1- E9.75 embryos. In the endoderm, the mitotic index was 42% (n=1084) in the heterozygous, and 39% (n=1065) in the homozygous mutant. The mitotic index of the mesenchyme was 53% (n=1082) in heterozygous mutants and 51% (n=1103) in homozygous mutants. These differences were not statistically significant.
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With the exception of the central, mesodermal core of the 1st, 2nd and 3rd arches, no Tbx1 expression was detected in the pharyngeal arch mesenchyme. To establish whether pharyngeal hypoplasia in homozygous mutants could be due to increased cell death, we performed the TUNEL assay, but no difference could be found at the stage tested (E10.0, not shown).
Tbx1 is required for normal neural crest cell distribution and normal patterning of the peripheral nervous system in the pharyngeal apparatus
Although there is no significant overlap between Tbx1 expression pattern and cardiac neural crest cells (NCC) in the pharyngeal apparatus, the anatomical defects in homozygous mutants predict abnormal NCC distribution. Crabp1, a marker of migrating NCCs, shows that NCCs in homozygous mutants are initially organized in apparently normal migratory streams after exiting the hindbrain. However, the Crabp1 expression pattern is abnormal in the pharyngeal arch region, with absence of the caudal expression domain (Fig. 3A and B) and ectopic expression (arrows in Fig. 3B). In situ hybridization on tissue sections confirmed the abnormal distribution of NCCs presumably destined to the caudal arches, which are absent in the homozygous mutant (not shown). At E10, the circumpharyngeal stream migrates caudal to the 4th arch in heterozygous animals (arrowhead in Fig. 3C), while scattered ectopic expression persists and is also observed enveloping the otocyst of the homozygous littermates (Fig. 3D). The expression pattern of Dlx2, a marker of postmigratory NCCs in the anterior arches, demonstrated that in Tbx1 homozygous mutants, NCCs are present in the first and second arches, although, in the latter, in a considerably reduced number (Fig. 3E and F) consistent with hypoplasia of the arch. Cranial nerves derive in part from NCCs and are thought to grow into the pharyngeal apparatus following similar migratory streams. Cranial nerves V, VII, IX and X supply the derivatives of arches 1, 2, 3 and 4, respectively. To observe the fate of cranial nerves in homozygous mutants, we immunostained embryos to detect neurofilament-M. Results reveal a number of abnormalities. The mandibular branch of the trigeminal (V) is abnormally directed caudally and fuses with the facial (VII) nerve fibers (compare Fig. 3G and H). The fibers of the glossopharyngeal (IX) nerve are either hypoplastic or bundled to the fibers of the vagus (X) nerve, and the axonal projections show defasciculation and disarray (bracket in Fig. 3H). Terminal projections of the vagus and the accessory (XI) nerves are misdirected rostrally (Fig. 3H). Hence, cranial nerves are formed but their migration paths are abnormal. These results indicate that NCCs are able to migrate and differentiate, but they appear to lack appropriate directional cues. Peripheral nervous system abnormalities are not limited to NCC-derived structures. Indeed, the distal ganglia of the IX and X, which derive from the ectodermal placodes, are abnormally fused (Fig. 3H, red arrow), consistent with an extensive disorganization of the pharyngeal apparatus in Tbx1 homozygous mutants.
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Tbx1 is required for formation and early remodeling of the pharyngeal arch arteries
It has been noted that in homozygous mutant embryos (Tbx1-/- or Df1/Tbx1-) pharyngeal arch arteries (PAAs) 3, 4, and 6 do not form (7,8), while in heterozygous mutants (Df1/+ or Tbx1+/-), the 4th PAA is hypoplastic in its early stages of development (8,10). To gain insight as to how Tbx1 expression relates to early 4th PAA remodeling, we examined Tbx1+/- embryos stained with ß-gal at E9.75 and at E10.5. At E9.75, the nascent 4th PAA is nestled between the surface ectoderm and the pharyngeal endoderm; Tbx1 is expressed in the pharyngeal endoderm of the developing 4th pouch and, to a lesser extent, on the surface ectoderm and in the 4th arch mesoderm (Fig. 4A and B). At this stage, no phenotypic difference could be detected between WT and mutant embryos, consistent with previously reported data obtained with the Df1/+ mutant (10). At E10.5, when the 4th PAAs are hypoplastic in heterozygous mutants, Tbx1 expression in the pharyngeal region is restricted to the endoderm (Fig. 4C). The same expression pattern is observed in Dp1/Tbx1- embryos (which carry two functional copies of the Tbx1 gene), but in these animals, the size of the 4th PAA is normal (Fig. 4D). These data suggest that the role of Tbx1 in early remodeling of this artery is likely to operate through expression in endodermal cells. However, we also considered an alternative hypothesis, since we detected a few Tbx1-positive cells in the vessel wall near the confluence of the 4th artery into the dorsal aorta (Fig. 4E and F). Because Tbx1 is expressed in a subpopulation of vascular smooth muscle (vsm) cells in the dorsal aorta (DAo) and inner carotid, it is possible that Tbx1-expressing cells may contribute directly to building the vessel vsm wall. It has been noted that the hypoplastic 4th PAAs in Df1/+ embryos lack vsm cells (10). First, we tested whether Tbx1 gene dosage may affect the number of X-gal positive cells in the 4th PAAs. To do this, we compared Dp1/Tbx1- and Tbx1+/- embryos, but we found no difference (n=6). Next, we examined whether Tbx1 is required for vsm cell differentiation. Results showed that X-gal-positive cells around the DA and inner carotids (pharyngeal arch arteries do not form in homozygous Tbx1 mutants) stain normally with anti-
-smooth muscle actin (sma) antibody (Fig. 4G–J). Hence, it is unlikely that Tbx1 has a cell-autonomous role in building the structure of the vessel.
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Tbx1 is required for growth and septation of the conotruncus
Prior to E11.5, there is a common outflow tract, the truncus arteriosus, which connects the heart to the systemic circulation. Subsequently, the outflow undergoes septation to separate aortico (systemic) and pulmonary flows. Formally, the septum is divided in three components (from distal to proximal, relative to the heart): (a) the aortico-pulmonary (AP) septum, (b) the truncal septum and (c) the conal septum (intracardiac), which is also important for the closure of the interventricular foramen. Tbx1 homozygous mutation causes disruption of all three components, ultimately leading to truncus arteriosus communis (TAC) in all homozygous mutant embryos examined at term (n=9) (Fig. 5A and B), while the semilunar valve leaflets (which divide the truncal from the conal region) form normally (Fig. 5C). The AP septum agenesis is caused by the severe abnormalities of the pharyngeal region and the aortic sac, from which the AP septum originates (Fig. 5D and E). In homozygous mutants, the conotruncal conduit is considerably reduced in diameter as early as E9.5 (Fig. 5H and I). X-gal staining revealed for the first time a Tbx1 expression domain in the muscular wall of the outflow of both heterozygous and homozygous mutants (Fig. 5J and K), these X-gal-positive cells become more abundant later in development (E11.5, Fig. 5L, N, P, R). Another expression domain is also found in the outflow myocardium near the inner curvature of the heart from E10.5 through at least E12.5 (arrowheads in Fig. 5F and U). Immunostaining confirmed that these X-gal-positive cells are muscle cells, since they are
-sma-positive (Fig. 5G, M, Q). Anti--sma staining was maintained in homozygous mutants (Fig. 5O and S), indicating that Tbx1 function is not required for differentiation of muscle cells of the outflow tract. No X-gal staining was detected in the truncal swellings, which are populated by neural-crest-derived mesenchymal cells from about E10.0 (11,12). From E11.5 onwards, we have identified another expression domain in the endothelial and sub-endothelial layers of the septating outflow (Fig. 5T). This cell population becomes more abundant at E12.5 in the walls of the ascending aorta and pulmonary trunk (Fig. 5U). Interestingly, these cells were absent in homozygous mutants, indicating that induction, localization or survival of these cells is dependent upon Tbx1 function. Alternatively, it is possible that these cells originate from the pharyngeal arch artery-derived vessels, and consequently these Tbx1-positive cells are absent since the arch arteries do not form.
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The most proximal component of the outflow septum, the conal septum, is also severely affected in homozygous Tbx1 mutants. In these mutants at term, the single arterial orifice is connected exclusively to the right ventricle, while the left ventricle communicates to the right ventricles through a large ventricular septal defect (VSD) that encompasses the perimembranous and infundibular areas (Fig. 5V). We also found that, in contrast to the normal arrangement, homozygous mutants lack continuity between the truncal leaflets and the mitral valve, indicating a failure of alignment between the atrioventricular canal and the outflow tract. Tbx1 expression is consistent with these phenotypic findings. In heterozygous embryos, after septation (E12.0), Tbx1-expressing cells are localized below the outflow valves, between the left and right outlets (Fig. 5W). In the homozygous mutant, this region of the heart has a simplified structure, and Tbx1-expressing cells are localized in the wall of the non-septated outflow (Fig. 5X). We interpret this abnormal morphology as persistence of an earlier (approximately E10.5) embryonic morphology.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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While the evidence that mutation of TBX1 alone can cause DGS is not yet available, mouse studies provide compelling evidence that Tbx1 mutations can cause severe and gene-dosage-dependent pharyngeal arch and pouch abnormalities that are strikingly similar to the DGS phenotype (7–9). These studies, however, have left a number of questions open. Expression studies of Tbx1 using conventional in situ hybridization (7–9,13,14) have not reported expression in the conotruncus, begging the question of whether TBX1 is a good candidate for the non-aortic arch cardiovascular defects commonly observed in patients, and whether the truncus arteriosus observed in Tbx1-/- mutants (7) may in fact be secondary to the severe disruption of the pharyngeal arch system. A potentially related question is what is the role of neural crest cells in the pathogenesis of the Tbx1 mutant phenotype. To begin addressing these questions, we initiated the study presented here. Our results show for the first time that Tbx1 gene expression is consistent not only with a role in arch artery formation and early remodeling, but also with a role in the development of the cardiac outflow tract. Tbx1 has two expression domains in the conotruncus: an early expression domain in the muscular wall of the outflow tract and a later expression domain in endothelial and subendothelial cells. The conotruncal muscle cells derive from a region of the splanchnic mesoderm underlying the caudal pharynx (15–17). We have shown here that Tbx1 is not required for muscle cell differentiation. However, it is tempting to speculate that the early conotruncal hypoplasia in Tbx1-deficient embryos, at a stage preceding NCC migration, may be due to a reduction in the number of the abovementioned mesodermal precursor cells. The expression of Tbx1 in endothelial and subendothelial cells of the conotruncus could be related to the septation defects in homozygous mutants, although it is also possible that the reduction of NCCs in the truncal swellings, which are hypoplastic, could contribute to the pathogenesis of these defects. As a consequence of the loss of septation throughout the outflow tract, Tbx1 deficiency causes truncus arteriosus and a large perimembranous/infundibular VSD. The truncus communicates with the right ventricle only, implying a defective alignment of the conal septum with the truncal septum, potentially due to failed rotation of the outflow tract. Interestingly, perimembranous VSD was also detected in the haploinsufficient mouse model Df1/+ (4) and is commonly seen in DGS patients. The conal alignment defect observed in homozygous mutants is developmentally related to the overriding of the aorta seen in some Df1/+ mutants (4) and in DGS patients. The fact that related defects are observed in the haploinsufficiency model indicates that these defects are not secondary to the severe anatomical disruption of the pharyngeal apparatus in homozygous mutants. The expression of Tbx1 in a subpopulation of vsm cells of the inner carotids is intriguing because del22q11 patients often present with abnormalities (mainly tortuosity and malpositioning) of inner carotid arteries (18). However, we have not observed such abnormalities in Tbx1 mouse mutants (heterozygous or homozygous), suggesting that in humans TBX1 plays a more critical role in carotid morphology, or that this particular phenotype is caused by deletion of another gene.
DiGeorge syndrome is a classical pharyngeal arch/pouch disorder (2). Whether or not NCCs play a pathogenetic role has been the subject of speculation, but there are no experimental data to answer this question conclusively. Data from genetically modified mice do not support a NCC migration defect, in either the heterozygous multigene deletion mutant (10) or in the homozygous Tbx1 mutant, as shown here. However, we show that the distribution of neural-crest-derived cells, as detected by migratory, postmigratory, and differentiation markers, is profoundly disrupted in homozygous mutants. This disruption is also reflected in abnormalities of the peripheral nervous system, particularly of cranial nerves IX and X, which may be consistent with the common finding of velopharyngeal incompetence (VPI) in del22q11 patients (19). The aberrant distribution of neural-crest-derived cells, however, should be seen in the context of a general disruption of the pharyngeal architecture that involves essentially all the arches and pouches, although most dramatically the caudal segments, which fail to form. Several observations support a primary role of Tbx1 in the endoderm, rather than in the neural-crest-derived tissues: (a) the characteristic expression of Tbx1 in the pharyngeal endoderm, (b) the lack of expression of Tbx1 in the neural-crest-derived cells of the pharyngeal arches and (c) the fact that Tbx1 deficiency causes severe hypoplasia and loss of segmentation of the embryonic pharynx. The segmentation of the pharyngeal apparatus is thought to be dependent upon intrinsic properties of the pharyngeal endoderm, and does not require neural-crest-derived mesenchymal cells (20,21). The endoderm provides signaling molecules with inductive abilities on the underlying mesenchyme (22), which is consistent with the apparently cell non-autonomous role of Tbx1 in early remodeling of the 4th PAA.
In summary, Tbx1 mutations cause one of the most dramatic and yet relatively specific pharyngeal phenotypes so far reported. To our knowledge, there is only one other example of a similar phenotype, namely the zebrafish mutant van gogh (23). While the gene mutated in van gogh has still to be identified, the phenotype of this mutant also supports a primary role of the pharyngeal endoderm in the development of the pharyngeal apparatus. We speculate that Tbx1 plays at least two roles in pharyngeal development: an early, cell-autonomous role in pharyngeal endoderm growth and patterning, and a later, cell non-autonomous role in supporting the development of pharyngeal arch and pouch derivatives. The latter role may be mediated through signaling to neural crest-derived cells. In vivo conditional expression of Tbx1 should allow us to dissect further the role of this gene in pharyngeal development. Understanding of the mechanisms of action of Tbx1 and the definition of the genetic pathways involved are important because abnormal development of the pharyngeal apparatus is the basis for a wide range of birth defects. These include craniofacial abnormalities, palatal clefts, thymic and parathyroid defects, and some of the most common cardiovascular defects.
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MATERIALS AND METHODS |
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Mouse mutants and breeding
Heterozygous Tbx1 mice carrying the lacZ knock-in allele Tbx1tm1Bld (here referred to as Tbx1+/-) were obtained as described previously (8). Mutants were maintained and analyzed on a C57BL/6x129SvEvBrd(129S5) mixed genetic background and were crossed to each other and to mice carrying a chromosomal deletion or duplication that includes the Tbx1 locus [Df1/+ and Dp1/+, respectively (4)] or wild-type mice of the same genetic background. Embryos were collected at various time points, considering the day of observation of a vaginal plug to be embryonic day (E) 0.5. Embryo stage was also confirmed by somite count. Mice and embryos were genotyped by PCR of DNA extracted from tail biopsies or yolk sacs, respectively, using previously published PCR primer pairs (8). Because to date we could not identify phenotypic differences between Tbx1-/- and Df1/Tbx1- animals, we selected the latter genotype for this work because it carries a single copy of the lacZ reporter gene and is hence more directly comparable with Tbx1+/- animals. The term ‘homozygous Tbx1 mutant’ throughout this paper refers to the genotype Df1/Tbx1-, unless otherwise specified. At least three embryos were analyzed per time point per genotype and per phenotyping procedure.
ß-Galactosidase detection, histology and immunohistochemistry
ß-Gal activity was detected in paraformaldehyde-fixed embryos using the X-gal substrate, according to standard procedures. Stained embryos were photographed as whole mounts and then embedded in paraffin and cut into 10 µm histological sections. Sections were counterstained with Nuclear Fast Red. Immunohistochemistry using an anti--smooth muscle actin (sma) monoclonal antibody (Clone 1A4, Sigma) was used to identify muscle cells. We used the monoclonal antibody 2H3 (Developmental Studies Hybridoma Bank) to detect neurofilament-M (165 kDa). To assay cell proliferation, pregnant females were injected with 5 mg/100 g body weight of bromodeoxyuridine (BrdU) and sacrificed 1 h after injection to harvest embryos. Embryos were fixed in ethanol, embedded in paraffin and cut into 7 µm sections. BrdU incorporation was detected on histological sections using an anti-BrdU monoclonal antibody (Clone # 85-2C8, Novacastra). Apoptosis was assayed by TUNEL analysis using a commercial kit (Roche).
In situ hybridization
Radioactive or non-radioactive in situ hybridization experiments were performed on sectioned or whole-mount embryos, respectively, using a published protocol (24). Sense and antisense riboprobes were prepared by reverse transcription of DNA probes and labeled by incorporation of digoxigenin-conjugated UTP (Roche) or 35S-UTP (ICN). A Tbx1 probe (13) was obtained from Dr V. Papaioannou. Crabp1 transcripts were detected using a probe described previously (25). Dlx2 transcripts were detected using the Expressed Sequence Tag clone BG228249.
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ACKNOWLEDGEMENTS |
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We acknowledge the valuable technical support of Tuong Huynh and Hedda Sobotka. This research has been supported by Grants RO1-HL51524, RO1-HL64832 and PO1-HL67155 from the US National Heart Lung and Blood Institute, NIH (to A.B.) and by Grant 0060099Y from the American Heart Association Texas Affiliate (to E.A.L.).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Pediatrics (Cardiology), Baylor College of Medicine, One Baylor Plaza, Room 830E, Mail stop BCM320, Houston, TX 77030, USA. Tel: +1 713 798 6519; Fax: +1 713 798 1483; Email: baldini{at}bcm.tmc.edu
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