Human Molecular Genetics, 2001, Vol. 10, No. 9 997-1002
© 2001 Oxford University Press
Recovery from arterial growth delay reduces penetrance of cardiovascular defects in mice deleted for the DiGeorge syndrome region
1Department of Pediatrics (Cardiology) and 2Department of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA
Received 17 January 2001; Revised and Accepted 20 February 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Chromosome 22q11.2 heterozygous deletions cause the most common deletion syndrome, including the DiGeorge syndrome phenotype. Using a mouse model of this deletion (named Df1) we show that the aortic arch patterning defects that occur in heterozygously deleted mice (Df1/+) are associated with a differentiation impairment of vascular smooth muscle in the 4th pharyngeal arch arteries (PAAs) during early embryogenesis. Using molecular markers for neural crest, endothelial cells and vascular smooth muscle, we show that cardiac neural crest migration into the 4th arch and initial formation of the 4th PAAs are apparently normal in Df1/+ embryos, but affected vessels are growth-impaired and do not acquire vascular smooth muscle. As in humans, not all deleted mice present with cardiovascular defects at birth. However, we found, unexpectedly, that all Df1/+ embryos have abnormally small 4th PAAs during early embryogenesis. Many embryos later overcome this early defect, coincident with the appearance of vascular smooth muscle differentiation, and develop normally. Embryos born with aortic arch patterning defects probably represent a more severely affected group that fails to attain sufficient 4th PAA growth for normal remodelling of the PAA system. Our data indicate that Df1/+ embryos are able to overcome a localized arterial growth impairment and thereby reduce the penetrance of birth defects.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DiGeorge syndrome (DGS) (1), is most frequently caused by a heterozygous deletion of 22q11.2 (del22q11). DGS is characterized by aortic arch and conotruncal heart defects, aplasia/hypoplasia of the thymus and parathyroids and craniofacial anomalies. This spectrum of clinical features suggests that del22q11 causes abnormal development of the derivatives of the pharyngeal arches (2,3). As the mesenchyme of the 3rd and 4th pharyngeal arches is neural crest-derived, it has led to a hypothesis that del22q11 impairs neural crest function (4,5). An alternative hypothesis advocates a primary vascular defect of the pharyngeal arch arteries (PAAs), whereby insufficient blood perfusion impairs development of the pharyngeal arch derivatives (6). To date, there are no experimental data to support either hypothesis and the pathogenesis of the disorder is unknown. In order to investigate the pathogenesis of the del22q11 phenotype, we recently developed a mouse model that carries an engineered
1 Mb deletion of a segment of the homologous murine region, which contains many of the genes deleted in del22q11 (7). Heterozygous mice show reduced penetrance (30%) of del22q11-like cardiovascular abnormalities, including interrupted aortic arch type B, aberrant right subclavian artery, right aortic arch and, less frequently, overriding aorta, pulmonary stenosis and ventricular septal defects (7). The most frequently observed abnormalities are attributable to defective development of the 4th PAAs during embryogenesis (7). In order to gain insight into the nature of these defects, we have studied the development of the 4th PAAs in Df1/+ embryos using a range of molecular markers and have thereby identified morphological differences between mutant and wild-type embryos in the development of these arteries. During this analysis, we found unexpectedly that the penetrance of the 4th PAA phenotype in Df1/+ embryos at embryonic day 10.5 (E10.5) is complete and that the majority of embryos overcome the 4th PAA defects and undergo normal cardiovascular development. It is this ability to overcome the primary embryological defect, rather than the initial penetrance or severity of the defect, which appears to be a key factor in determining the final penetrance of cardiovascular defects at term. The mechanisms underlying incomplete penetrance of birth defects associated with gene mutations are unknown. Clearly, understanding the molecular basis of reduced penetrance may provide tools to identify genetic and pharmacological modifiers capable of modulating or even neutralizing the effects of gene deficiency.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The 4th PAAs form but do not grow in Df1/+ embryos
We have shown previously that the 4th PAAs in Df1/+ embryos may be reduced in size or non-patent to injected ink (7). To initiate a molecular definition of the artery defect, we bred Df1/+ mice with Tie2-LacZ transgenic mice that express a LacZ reporter gene specifically in the endothelial cells (8). We analyzed embryos carrying both the deletion and the transgene (Df1/+; Tie2-LacZ) at time points between E9.5 and E10.5. Within this 24 h period the 4th PAAs of wild-type embryos form and reach their mature size. The 4th pharyngeal arch and respective arch artery were not recognizable earlier than E9.75 (25–29 somites) in either wild-type or Df1/+ embryos. At E9.75, we found that ß-gal-positive endothelial cells were present in the 4th arch and were organized into recognizable vessels in both Df1/+ and wild-type embryos. However, at E10 (30–34 somites) and E10.5 (35–39 somites), one or both of the 4th PAAs of Df1/+ embryos were small compared with wild-type vessels (Fig. 1A and B). The 3rd and 6th PAAs appeared to be normal in all embryos. These results indicate that endothelial cell differentiation and capillary tube formation occur normally in Df1/+ embryos, but that after formation, growth of the 4th PAAs is impaired. The process of vessel development requires that vascular smooth muscle (VSM) progenitors are recruited from the surrounding mesenchyme to the vessel wall, where they differentiate into VSM. We evaluated VSM differentiation by immunohistochemistry using an antibody to smooth muscle
-actin (SMA) in Df1/+ and wild-type embryos. Some experiments were also carried out in Df1/+;Tie2-LacZ and +/+; Tie2-LacZ embryos, in order to facilitate analysis of reduced-size vessels. We found that at E10.5 the 4th PAA walls of wild-type embryos were strongly positive (Fig. 1C), whereas Df1/+ litter mates had no SMA-positive cells in the walls of the reduced size 4th PAAs (Fig. 1D). The 3rd PAA and dorsal aorta were strongly positive in mutant embryos and served as internal controls for SMA staining. SMA staining was normal in the rest of the Df1/+ embryo (data not shown).
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Normal neural crest migration in Df1/+ embryos
The most frequently cited hypothesis for the pathogenesis of DGS is a neural crest impairment. In the 4th pharyngeal arches, the non-endothelial cellular components of the arch arteries differentiate from the surrounding neural-crest derived mesenchyme (9–11). The observed deficiency of VSM-specific staining in mutant embryos could therefore be due to a failure of neural crest migration into the 4th pharyngeal arches or to an inability of post-migratory neural crest to differentiate into VSM, because they do not receive or cannot respond to developmental signals. To distinguish between a migratory and post-migratory problem, we bred Df1/+ mice with Wnt1-LacZ transgenic mice that specifically express ß-gal in neural crest cells (12). Results showed that at E10.5, both Df1/+ and wild-type embryos had ß-gal-positive cells in the mesenchyme of the 4th pharyngeal arches, and there was no discernable difference in number or distribution (Fig. 1E and F). Whole-mount in situ hybridization using the cardiac neural crest-specific marker CrabpI (13) showed similar results (data not shown). These results suggest that the Df1 deletion does not affect neural crest migration. To exclude the possibility that lack of VSM is due to abnormal cell death or due to reduced cell proliferation, we used the TUNEL procedure to assess apoptosis (14) and bromodeoxyuridine incorporation to assess cell proliferation. Neither study revealed differences in cell turnover in the 4th PAAs of Df1/+ and wild-type embryos (data not shown).
Genetic rescue of the 4th PAA abnormalities
The cardiovascular abnormalities observed in Df1/+ adult mice and in term embryos (E18.5) are due to reduced dosage of one or more genes located within the deleted segment (7). To confirm that the 4th PAA phenotype observed at E10.5 and described here is also due to gene haploinsufficiency, we bred Df1/+ mice with Dp1/+ mice that carry a duplication of the Df1 segment. Results showed that E10.5 Df1/Dp1 embryos are normal (n = 12), indicating that the duplication rescues the E10.5 phenotype caused by the deletion.
Df1/+ embryos can overcome the 4th PAA growth impairment, thereby reducing penetrance of cardiovascular defects at term
In examining Df1/+ embryos at E10.5, it became apparent that the penetrance of the 4th PAA defects was much higher than would be predicted by the low penetrance of cardiovascular abnormalities at E18.5 (7). We therefore analyzed the vascular anatomy of E10.5 and E11.5 embryos and related our findings to the anatomical heart defects seen at term. E18.5 embryos were examined by direct inspection, and E10.5 and E11.5 embryos by intracardiac India ink injection. The normal morphology of the 4th PAAs at E10.5 is shown in Figure 2A. Abnormalities scored were ‘absence’, i.e. non-patency to injected ink (Fig. 2B) or size reduction (Fig. 2C and D) of the 4th PAAs. In E18.5 embryos, we scored the predicted outcomes of absence of the right or left 4th PAAs, which are aberrant origin of the right subclavian artery and interrupted aortic arch type-B or right aortic arch, respectively. No arch artery abnormalities were seen in wild-type litter mates (n = 164 for all stages). Surprisingly, at E10.5, 100% of Df1/+ embryos had abnormalities of one or both 4th PAAs (Fig. 3; n = 48), with the right side more commonly affected than the left (P < 0.01). At E11.5, 34.5% of Df1/+ embryos were normal (Fig. 3; n = 29), revealing a significantly lower penetrance of the phenotype compared to E10.5 (P < 0.001). At this time point too, the right side was more commonly affected than the left side (P < 0.05). Non-patent vessels were identified in 77% of E10.5 embryos and in 48.3% of E11.5 embryos, suggesting that although most arteries are growth delayed, some vessels fail to grow. The trend towards ‘normalization’ of arch artery development was confirmed by the analysis of embryos at term, which showed that 68% of Df1/+ embryos (Fig. 3; n = 76) had normal aortic arch anatomy. This is a significant decrease in penetrance compared to E11.5 (P < 0.01), and the right side continued to be more frequently affected than the left (P < 0.02). The reduced penetrance of the phenotype is not a survival/selection effect caused by loss of mutant embryos, because the ratio of mutant:wild-type embryos remained constant at approximately 1:1 at all stages tested. To understand whether the growth that occurs in Df1/+ embryos between E10.5 and E11.5, and that restores 30% of affected vessels to normality, correlates with normalization of VSM differentiation, we analyzed SMA expression at E11.5. We found that out of seven Df1/+ embryos analyzed, four had normal size 4th PAAs with normal SMA expression (not shown), similar to that shown on Figure 4A (wild-type embryo). The other three embryos had one normal and one reduced-size 4th PAA. Of the three reduced-size arteries, the larger had scanty SMA positive cells that did not form continuous or multiple layers (Fig. 4B), whereas the two smaller vessels were devoid of SMA positive cells, except at the junctions with the dorsal aorta and aortic sac (Fig. 4C). These data indicate that partial normalization of the 4th PAA phenotype in E11.5 embryos correlates with onset of VSM differentitation. Whether the onset of VSM differentiation results from, or causes, the growth recovery is to be determined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Del22q11 causes the most frequent chromosomal deletion syndrome in humans [1 in 4000 live births (15,16)]. Congenital heart disease affects
70–80% of patients ascertained and is responsible for virtually all the deaths caused by DGS (17). Most abnormalities are aortic arch patterning defects or conotruncal heart defects (reviewed in 3,18). Based on data collected using our del22q11 mouse model and presented here, we propose the following pathogenesis for the aortic arch patterning defects. The development of the 4th PAAs is the key to the genesis of aortic arch patterning defects in patients with del22q11 and in mice with the Df1 deletion. We have shown that initially, arteries of mutant and wild-type embryos are indistinguishable, indicating that capillary tube formation is not affected by the mutation. However, mutant arteries do not grow normally. We refer to these abnormal arteries as growth-impaired because our data suggest that whereas some arteries are simply growth delayed, others (those non-patent to ink at E11.5) fail to grow. The arterial growth impairment does not correlate with abnormalities of cell proliferation or apoptosis, but does correlate with a delay (and sometimes failure) of differentiation of VSM from neural crest-derived mesenchyme. The differentiation defect is restricted to the 4th PAAs, suggesting that the development of these arteries may rely on a specific set of signals, which is genetically separable from that responsible for the development of the other PAAs. Alternatively, the 4th PAAs may be more sensitive to reduced gene dosage than the other PAAs. The restricted nature of the defects does not support the possibility of a general problem of angiogenesis, and it is not attributable to failed neural crest migration. We propose that Df1 affects a local signaling pathway between endothelial cells and neural crest-derived mesenchyme, or between pharyngeal endoderm and mesenchyme in the 4th arches of mutant embryos, and that as a consequence, mesenchymal cells fail to receive or fail to respond to signals that mediate their differentiation into VSM. Interestingly, the PAA defects in Df1/+ embryos are significantly more frequent on the right than on the left at all stages tested. While this may not be surprising, because the two arteries have a different (asymmetric) developmental program, the finding is significant because it is apparent at a stage of development when the paired aortic arch arteries are still symmetrical. This suggests that the gene(s) haploinsufficient in Df1/+ may be involved in the process of asymmetric remodeling of the PAAs, a process that is poorly understood. Our data show that most of the 4th PAAs that are growth-impaired eventually develop normally. However, 32% of Df1/+ embryos fail to overcome this growth impairment, resulting in the permanent vascular defects observed at E18.5. Presumably, the more severely growth-impaired arteries cannot respond to developmental cues that initiate the major remodeling of the PAA system that occurs between E11.5 and E13.5. This scenario indicates that developmental growth delay, though simple in concept, is not inconsequential, as failure to reach key developmental milestones within the tight time frames of mammalian development may, as in the case of Df1/+ embryos, have severe and even lethal consequences.
The ability of Df1/+ embryos to overcome the 4th PAA growth delay is, at least in part, reminiscent of MyoD–/– mice (19), in which initiation of epaxial muscle development is delayed by 2 days (from E11.5 to E13.5), due to delayed gene compensation by Myf5. A similar delay in intervention of a compensatory gene could be acting in the case of Df1/+ embryos. However, in contrast to the MyoD–/– phenotype, the Df1/+ phenotype is due to gene haploinsufficiency. Therefore, we favor the hypothesis that the product of the haploinsufficient gene(s) needs to accumulate over time in order to reach threshold levels sufficient for biological activity. Another possibility is that the activation of the haploinsufficient gene(s) itself is delayed. Indeed, a theoretical model of stochastic gene expression predicts that the loss of one allele may cause a delay in gene activation, and the heterozygous cell will temporarily behave as a homozygous mutant (20).
Reduced penetrance and variable expressivity are important features of the del22q11-associated cardiovascular phenotype (17,21,22). Using an animal model of this phenotype, we believe that we have shown for the first time that embryos can overcome specific localized developmental delays and thereby reduce the penetrance of the disease phenotype. This kind of self repair is a novel concept in our understanding of reduced penetrance of birth defects, and it carries important implications, as clarification of its molecular basis may lead to novel preventive strategies so far unexplored. Df1/+ mice will be an excellent tool to measure the efficiency of such self repair under different genetic, environmental or pharmacological interventions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse breeding
Df1/+ embryos were obtained from crosses between +/+ and Df1/+ animals in a mixed genetic background (129SvEvBrd x C57BL/6) and genotyped using DNA extracted from yolk sacs. The Mendelian ratio was as expected for all crosses and embryonic stages. Df1/+;Tie2-LacZ embryos were obtained from crosses between Tie2-LacZ/+ (Jackson Laboratory) and Df1/+ animals. Tie2-LacZ/+ transgenic mice carry a Tie2 promoter-LacZ reporter transgene which is specifically expressed in endothelial cells (8,23). Df1/+;Wnt1-LacZ embryos were obtained from crosses between Wnt1-LacZ/+ (12) and Df1/+ animals.
Intracardiac ink injection
Ink injection was performed as described previously (7). The 4th PAA anatomy was analyzed by visual inspection and the vessels classified into one of three categories: normal (indistinguishable from wild-type), reduced (visibly smaller than wild-type vessels; Fig. 4C and D) and absent (non-patent to ink; Fig. 4B).
Immunohistochemistry and in situ hybridization
We fixed embryos in PBS containing 4% paraformaldehyde. For immunohistochemical staining, embryos were embedded in paraffin and cut into 10 µm thick sections. Immunohistochemical staining with alkaline phosphatase conjugated anti-SMA (Sigma) was performed using a standard protocol (24). Staining was evaluated in serial sections over the entire length of the PAAs in all embryos. For E10.5 embryos, we considered PAAs to be normal if there was a complete or near complete monolayer of positively stained cells around the vessels, and abnormal if there was scant or absence of staining. PAAs of E11.5 embryos were designated as normal if there was a multilayer of positively stained cells around the vessels, and abnormal if there was scant or absence of staining. Whole-mount in situ hybridization was performed according to published protocols (25).
ß-galactosidase staining
We fixed embryos in PBS containing 4% paraformaldehyde for 30 min. at 4°C. Fixed embryos were washed several times in PBS and equilibrated for 10 min in PBS containing 0.02% NP-40 and 0.01% Na-deoxycholate. We stained embryos overnight at room temperature in X-gal (1 mg/ml; 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), K4Fe(CN)6 (5 mM), K3Fe(CN)6 (5 mM), 0.02% NP-40 and 0.01% Na-deoxycholate. After staining, we rinsed embryos in PBS, refixed in PBS containing 4% paraformaldehyde for 10 min at 4°C, rinsed again in PBS and stored in 70% ethanol. Embryos were embedded in paraffin and cut into 10 µm thick sections.
Statistical analyses
Data were analyzed using the 
2 test for two independent samples.
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ACKNOWLEDGEMENTS |
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Wnt1-LacZ/+ mice were kindly provided by Dr A.P. McMahon. We thank Tuong Huynh for technical assistance. This work was funded in part by grants from the American Heart Association, Texas Affiliate (0060099Y to E.A.L.) and the National Institutes of Health (HL51524 and HL64832 to A.B.).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Pediatrics (Cardiology), Baylor College of Medicine, 1 Baylor Plaza, 833E, Mail Stop BCM320, Houston, TX 77030, USA. Tel: +1 713 798 8286; Fax: +1 713 798 1483; Email: elindsay@bcm.tmc.edu
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F. Vitelli, I. Taddei, M. Morishima, E. N. Meyers, E. A. Lindsay, and A. Baldini A genetic link between Tbx1 and fibroblast growth factor signaling Development, January 10, 2002; 129(19): 4605 - 4611. [Abstract] [Full Text] [PDF]
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R. Abu-Issa, G. Smyth, I. Smoak, K.-i. Yamamura, and E. N. Meyers Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse Development, January 10, 2002; 129(19): 4613 - 4625. [Abstract] [Full Text] [PDF]
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 F. VITELLI, E.A. LINDSAY, and A. BALDINI Genetic Dissection of the DiGeorge Syndrome Phenotype Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 327 - 332. [Abstract] [PDF]
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I. Taddei, M. Morishima, T. Huynh, and E. A. Lindsay Genetic factors are major determinants of phenotypic variability in a mouse model of the DiGeorge/del22q11 syndromes PNAS, September 13, 2001; (2001) 201127298. [Abstract] [Full Text] [PDF]
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I. Taddei, M. Morishima, T. Huynh, and E. A. Lindsay Genetic factors are major determinants of phenotypic variability in a mouse model of the DiGeorge/del22q11 syndromes PNAS, September 25, 2001; 98(20): 11428 - 11431. [Abstract] [Full Text] [PDF]
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