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Human Molecular Genetics, 2004, Vol. 13, No. 15 1577-1585
DOI: 10.1093/hmg/ddh176
Human Molecular Genetics, Vol. 13, No. 15 © Oxford University Press 2004; all rights reserved

Full spectrum of malformations in velo-cardio-facial syndrome/DiGeorge syndrome mouse models by altering Tbx1 dosage

Jun Liao1, Lazaros Kochilas2, Sonja Nowotschin1, Jelena S. Arnold1, Vimla S. Aggarwal1, Jonathan A. Epstein3, M. Christian Brown4, Joe Adams4 and Bernice E. Morrow1,*

1Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, 2Department of Pediatrics, Brown University Medical School, Providence, RI 02912, USA, 3Departments of Medicine and Cell and Developmental Biology, University of Pennsylvania, 954 Biomedical Research Building (BRB) II/III, 421 Curie Boulevard, Philadelphia, PA 19104, USA and 4Department of Otology and Laryngology, Harvard Medical School, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA

Received March 22, 2004; Accepted May 26, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is associated with de novo hemizygous 22q11.2 deletions and is characterized by malformations attributed to abnormal development of the pharyngeal arches and pouches. The main physical findings include aortic arch and outflow tract heart defects, thymus gland hypoplasia or aplasia and craniofacial anomalies. The disorder varies greatly in expressivity; while some patients are mildly affected with learning disabilities and subtle craniofacial malformations, others die soon after birth with major cardiovascular defects and thymus gland aplasia. In addition to the main clinical features, many other findings are associated with the disorder such as chronic otitis media and hypocalcemia. Tbx1, a gene encoding a T-box transcription factor, which is hemizygously deleted on chromosome 22q11.2, was found to be a strong candidate for the equivalent of human VCFS/DGS in mice. Mice hemizygous for a null allele of Tbx1 had mild malformations, while homozygotes had severe malformations in the affected structures; neither precisely modeling the syndrome. Interestingly, bacterial artificial chromosome (BAC) transgenic mice overexpressing human TBX1 and three other transgenes, had similar malformations as VCFS/DGS patients. By employing genetic complementation studies, we demonstrate that altered TBX1 dosage and not overexpression of the other transgenes is responsible for most of the defects in the BAC transgenic mice. Furthermore, the full spectrum of VCFS/DGS malformations was elicited in a Tbx1 dose dependent manner, thus providing a molecular basis for the pathogenesis and varied expressivity of the syndrome.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), occurring with an incidence of 1 in 4000 live births (1), is characterized by a wide spectrum of malformations including outflow tract cardiovascular defects, thymus gland hypoplasia or aplasia and mild craniofacial anomalies including submucous cleft palate, occurring with phenotypic variability (MIM 192430/188400). Most of the structures affected derive from the pharyngeal apparatus of the developing embryo, suggesting that the defects arise during embryogenesis. Most VCFS/DGS patients have a 1.5–3 Mb (million base pair) hemizygous deletion of chromosome 22q11.2 (2), indicating that haploinsufficiency of a gene(s) within the 1.5 Mb region is responsible for its etiology.

Over 25 genes map to the 1.5 Mb interval deleted in patients with the syndrome (3). To identify candidate genes out of the 25, a series of mouse mutants with overlapping hemizygous deletions on the mouse homologous region on chromosome 16 were generated using cre/loxP strategies (4–8). Mice carrying hemizygous deletions of the proximal half of the region from Idd-Arvcf were normal (6). This eliminated genes from the Idd-Arvcf half of the region as being solely responsible for the etiology of the disorder. The distal half of the interval, Arvcf-Hira, was implicated in carrying genes relevant to the pathogenesis of this disorder. Mice with a large deletion spanning the 1.5 Mb interval from Idd-Hira, had mild cardiac outflow tract defects. These defects were rescued by crossing with bacterial artificial chromosome (BAC) transgenic mice containing 1–2 copies of human transgenes including GP1BB (9), PNUTL1 (10), TBX1 (11) and WDR14 (12) (BAC 316.27), thus reducing the number of candidates to four (8). Of the four, only one of them, TBX1, is expressed in the pharyngeal arches, the precursor to the structures affected in VCFS/DGS (13). On the basis of its pattern of expression, TBX1 was the most likely candidate gene responsible for the syndrome. This is further supported by the fact that Tbx1 +/– mice had similar cardiovascular anomalies as hemizygously deleted mice (5,7,8). Tbx1 –/– mice had a more severe phenotype than the Tbx1 heterozygotes, and they died at birth with malformations of the structures relevant to those in VCFS/DGS patients (14). They had a single cardiac outflow tract, absent thymus and parathyroid glands and a cleft palate (14). Tbx1 is expressed in the non-neural crest mesoderm (head or splanchnic mesoderm and core mesoderm of pharyngeal arches 1 and 2) and endoderm of the pharyngeal arches (13,15). In addition to the defects derived from the pharyngeal arches, Tbx1 –/– mice had a missing outer and middle ear, with a very malformed inner ear, lacking a cochlea or vestibular system (16,17). This paralleled the expression pattern of Tbx1 in both the otic vesicle and the surrounding periotic mesenchyme (13,16).

When taken together, neither the Tbx1 heterozygous mice nor the Tbx1 homozygous mice precisely modeled the syndrome, as they were too mild or severe, respectively. On the other hand, BAC transgenic mice containing 8–10 copies of human TBX1 and three other transgenes PNUTL, GP1BB and WDR14 (BAC 316.23), had strikingly similar malformations as in the syndrome (8). The mice had reduced viability, a variety of aortic arch defects, thymus gland anomalies and ear defects (8,12). This suggests that Tbx1 effects are sensitive to altered gene dosage. Alternatively, the abnormalities observed in the BAC transgenic mice could also be due to a dominant negative, gain of function effect of the human TBX1 transgene, or caused by overexpression of other three human transgenes, such as PNUTL1 (12).

To determine whether overexpression of TBX1 was responsible for the phenotype in BAC transgenic mice, we performed genetic complementation experiments by crossing the BAC transgenic mice into the Tbx1 null mutant background. We found that partial normalization of the Tbx1 dosage rescued most of the malformations in BAC transgenic mice, suggesting that altered Tbx1 dosage is responsible for the mutant phenotype. Moreover, the mice manifest the full phenotypic spectrum of malformations associated with VCFS/DGS in a Tbx1 dose dependent manner, implicating this single gene not only in the molecular basis of VCFS/DGS pathogenesis but also in potentially explaining the presence of variable expressivity (phenotypic variability). Recently, hemizygous mutations in TBX1 were identified in patients with the syndrome, providing proof of the importance of this gene in the etiology of VCFS/DGS (18).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic complementation
To ascertain whether overexpression of human TBX1 is responsible for the malformations in BAC transgenic mice (8–10 copies of BAC RPCI11-361L10; line 316.23) overexpressing GP1BB, PNUTL1, TBX1 and WDR14, we performed genetic complementation studies with mice carrying a null allele of Tbx1 (8). We first crossed BAC transgenic mice (TG) with Tbx1 +/– mice, both congenic in the FVB strain. The purpose of this mating was to generate compound heterozygous mice, containing one inactive copy of Tbx1 and 8–10 copies of the BAC (Tbx1 +/–; TG). The compound heterozygotes were then crossed with Tbx1 +/– mice and six different offspring with varied Tbx1 gene dosage were generated (Table 1). We examined the expression levels of endogenous mouse and human TBX1, respectively, in a pool of five 9.5 dpc embryos for each genotype by real-time reverse transcriptase polymerase chain reaction (PCR). The total Tbx1 expression level in each genotype was obtained by combining mouse and human data normalized by wild-type levels. We also tested five individual embryos for each genotype by the same analyses and obtained similar results. The fold changes in Tbx1 expression in the various genotypes versus the wild-type littermates, ranged from 0.06 in Tbx1 –/– embryos to 5.00 in TG embryos in a wild-type background (Table 1). The fold changes of gene expression matched the transgene dosage in the mice as determined by quantitative genomic Southern blot hybridization analysis (12). In addition, the pattern of expression of the human transgenes was similar to the endogenous pattern during embryogenesis (12). Partial normalization of Tbx1 expression levels was achieved in Tbx1 +/–; TG or Tbx1 –/–; TG mice (Table 1).


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  		Table 1. Comparison of Tbx1 expression level and phenotype in six different genotypes
 
We examined viability in the offspring of the genetic crosses. While all BAC transgenic mice survived embryogenesis (8), only 29% of them survived after birth (Table 1). In contrast, 79% of BAC transgenic mice superimposed on a Tbx1 –/– background, survived (Table 1). This indicates that significant rescue occurred by partially normalizing the Tbx1 dosage. If the human transgenes acted in a dominant negative manner or as a gain of function, the lethality would not have been rescued. We next performed a series of detailed morphological studies to assess the developmental malformations in the six different genotypes in the offspring.

Cardiovascular defects
Cardiovascular defects, deriving from the outflow tract and pharyngeal arch arteries (PAA) occur in ~60–75% of VCFS/DGS patients (19,20). The most common defects are tetralogy of fallot (TOF), ventricular septal defect (VSD), interrupted aortic arch type B (IAA-B), pulmonary atresia (PA), right-sided aortic arch (RAA), retroesophageal subclavian artery (RSA), double outlet right ventricle (DORV) and persistent truncus arteriosus (PTA) (19–21). Tbx1 –/– embryos displayed the most severe defect and all had a PTA (14) (Table 1). Tbx1 +/– mice survived in normal Mendelian ratios; however, 10% of them had outflow tract and PAA defects including TOF, RSA and DORV (Table 1). The BAC 316.23 transgenic embryos at 17.5 dpc had predominantly two forms of PAA defects, IAA-B and RAA (8) (Table 1). Although these are rare anomalies, they are common in VCFS/DGS patients (19,21). The malformations in the TG mice were significantly rescued by introducing the Tbx1 null allele, thus supporting the role of TBX1 in the pathogenesis of aortic arch anomalies. In examining the range of malformations detected at the varying expression levels of Tbx1, we noted a Tbx1 dose dependency. When the dosage was moderately elevated above normal levels, the mice were predominantly affected with TOF, PS and RSA, which are all common defects in VCFS/DGS. By altering Tbx1 dosage, we were thus able to elicit the full range of outflow tract defects occurring in VCFS/DGS patients, suggesting that alteration of Tbx1 levels might contribute to the significant clinical variability in VCFS/DGS patients.

Pharyngeal arches 3–6 do not develop properly in the Tbx1 homozygotes, which is a more severe developmental abnormality than those occurring in any other genotype (Fig. 1D). Defects in growth and development of the left 4th PAA have been implicated in the etiology of the aortic arch malformations in VCFS/DGS patients and found in mice carrying deletions of the homologous region of the human 22q11.2 interval on the mouse chromosome 16 (5,7) and in Tbx1 +/– heterozygotes (7,8,14) (Fig. 1B). We evaluated the PAAs in the BAC transgenic embryos to ascertain whether they had similar defects. We found that the PAAs 1–3 were normal in all BAC transgenic embryos, but both sides of 4th PAAs were severely hypoplastic (Fig. 1C). This suggests that elevated Tbx1 expression may be responsible for these cardiovascular defects through a similar pathogenic mechanism as in the mutants with decreased or absent Tbx1 expression.



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  		Figure 1. Examination of the 4th PAA in Tbx1 mutant embryos. Sagittal histological sections through the pharyngeal arch region in (A) Tbx1+/+, (B) Tbx1+/–, (C) BAC transgenic and (D) Tbx1 –/– embryos at 10.5 dpc. Note the hypoplasia of the 4th PAA in the Tbx1 +/– and BAC transgenic embryos, and the absence of the 3rd and 4th PAAs in the Tbx1 –/– embryo. 3, 3rd PAA; 4, 4th PAA.

 
Thymus, parathyroid and thyroid anomalies
A subset of VCFS/DGS patients have immune deficiencies associated with hypoplasia or occasionally aplasia of the thymus gland (20). Tbx1 –/– mutants had thymus gland aplasia (14), while Tbx1 +/– mice had no significant thymus gland abnormalities (Table 1). In contrast to these two extremes, TG mice had thymus gland defects that ranged from very subtle such as asymmetry and migration defects to more severe such as hypoplasia or aplasia of the gland (Table 1) (8). Most thymus gland anomalies were significantly rescued in Tbx1 –/–; TG embryos, indicating again that TBX1 overexpression was responsible for these defects (Table 1). The presence and severity of thymus gland anomalies increased with increasing Tbx1 levels, suggesting a significant Tbx1 dosage dependency (Table 1).

A subset of human patients with VCFS/DGS has severe neonatal hypocalcemia, perhaps secondary to parathyroid anomalies (19,20). Just as with the thymus gland, Tbx1 –/– embryos lacked parathyroid glands (Fig. 2D) (14), while in Tbx1 +/– (8/10) and BAC transgenic (6/8) embryos, the parathyroid glands were either missing or ectopically located (Fig. 2B and C).



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  		Figure 2. Thyroid and parathyroid gland anomalies in Tbx1 mutant mice. (A–D) Transverse sections at the level of thyroid and parathyroid glands of (A) Tbx1 +/+, (B) Tbx1 +/–, (C) BAC transgenic and (D) Tbx1 –/– embryos at 17.5 dpc. Note the bilaterally absent parathyroid glands in Tbx1 +/–, Tbx1 –/–, and BAC transgenic embryos, hypoplastic thyroid gland in Tbx1 –/– embryo. In Tbx1 +/+ embryo, only one side parathyroid gland can be seen at this level. tr, thyroid; pt, parathyroid. (E and F) Transverse sections showing the thyroid primordia in (E) Tbx1 +/+ and (F) Tbx1 –/– at 10.5 dpc. Note that the thyroid primordium was evident in Tbx1 –/– embryo. ph, pharynx; tp, thyroid primordium. (G and H) Transverse sections at the level of thymus and ultimobranchial bodies in (G) Tbx1 +/+ and (H) Tbx1 –/– embryos at 12.5 dpc. Note the absence of thymus and ultimobranchial body in Tbx1 –/– embryo. tm, thymus; ub, ultimobranchial body.

 
Hypothyroidism is also occasionally detected as part of the phenotype of VCFS/DGS patients (20). We found that Tbx1 –/– embryos had hypoplasia of the thyroid gland. Instead of a bi-lobed structure in wild-type animals, a single lobe thyroid gland was present in Tbx1 –/– embryos (Fig. 2D). As the thyroid gland is formed by the fusion of the thyroid diverticulum from the floor of the pharynx and the ultimobranchial body from the 4th pharyngeal pouch, we examined these two structures during development. We found that the thyroid primordium was formed appropriately (Fig. 2F), but the ultimobranchial body never developed (Fig. 2H). The failure of the ultimobranchial body to form may have contributed to the thyroid malformations detected in Tbx1 –/– embryos (Fig. 2D). Compared with Tbx1 –/– mice, no thyroid defect was found in mice belonging to the other genotypes.

Craniofacial malformations
VCFS/DGS patients have craniofacial malformations that include a receding or abnormal jaw, widely spaced eyes, broad nasal root, midface hypoplasia, as well as occult submucous cleft palate and vertical maxillary excess (22). Some of these defects would be difficult to score in the mouse because of their mild nature. Overt cleft palate occurs in ~10% of patients (19,20), which can be easily detected in the mouse. All of the Tbx1 –/– embryos had an overt cleft palate, while only 21% of TG mice and <5% of the embryos with intermediary Tbx1 dosage had one (Table 1; Fig. 3B and C). Thus, embryos having either extreme of Tbx1 dosage had an overt cleft palate. The palatal abnormalities were rescued by partially normalizing Tbx1 gene dosage, again suggesting that these defects in the BAC transgenic mice are caused by TBX1 overexpression and not by the other three transgenes. We examined the 17.5 dpc embryos of all six genotypes to assess the types of craniofacial defects that can result from altered Tbx1 dosage. Only Tbx1 –/– embryos had additional detectable bone malformations of the skull and craniofacial region, all in derivatives of the 1st through 3rd pharyngeal arches, as previously reported (14).



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  		Figure 3. Craniofacial malformations in Tbx1 mutant mice with varied Tbx1 dosage. (A–C) Coronal histological sections through the heads of (A) Tbx1 +/+, (B) Tbx1 –/– and (C) BAC transgenic embryos at 14.5 dpc. Note the cleft palate in Tbx1 –/– and BAC transgenic embryos. ns, nasal septum; ps, palatal shelves; t, tongue. (D–G) Transverse sections through the heads of (D and F) wild-type and (E and G) Tbx1 –/– embryos at 17.5 dpc. Note the aplasia of masseter and pterygoid muscles in Tbx1 –/– embryos. m, masseter muscle; mc, mandibular condyle; mt, molar tooth; p, pterygoid muscle.

 
Assessment of the craniofacial musculature to determine the presence of structural abnormalities suspicious of causing vertical maxillary excess, revealed that the Tbx1 –/– embryos were missing the masseter and pterygoid muscles (Fig. 3E and G). In contrast, the mylohyoid and genioglossus muscles were intact. This implicates Tbx1 directly in the development of a subset of muscles derived from the 1st pharyngeal arch, whose abnormal development can represent the basis of the human equivalent of vertical maxillary excess in mouse. However, all Tbx1 +/– and BAC transgenic mice had normal masseter and pterygoid muscles, suggesting that the development of these muscles is affected only by the complete loss of Tbx1.

Ear disorders
The majority of VCFS/DGS patients have chronic otitis media and associated conductive hearing loss, while a small subset has sensorineural hearing loss as well (23–25). We previously found that two BAC 316 transgenic mouse lines (BAC 316.27, 1–2 copies and BAC 316.23, 8–10 copies) (8) had chronic otitis media. The high copy line, the focus of this report, also had an abnormal stapes footplate and shortened cochlea (12). The most severe ear defects occurred in Tbx1 –/– mice, which consisted of a missing outer and middle ear structures and lack of defined inner ear structures (16). We found that the Tbx1 +/– mice had frequent middle ear abnormalities including chronic otitis media, infiltration of inflammatory cells, thickening of the middle ear submucosa, thickening of the bony wall of the bulla, middle ear fluid accumulation, hyperplasia of ciliated cells and associated hearing loss (Table 1; Fig. 4). These results implicate altered Tbx1 dosage in the etiology of chronic otitis media. Although other mouse models exist for chronic otitis media (26,27), to our knowledge, this is the first mouse model for chronic middle ear inflammation in direct association with a human genetic syndrome.



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  		Figure 4. Middle ear defects in Tbx1 mouse mutants. Middle ear and cochlea of two Tbx1 –/–; TG mice (TG or Tbx1 +/– show the same anomalies). In (A) there is no chronic otitis media but in (B) there is. The tympanic membrane (TM in A and B) separates the middle ear space from the external auditory canal. MAL is the malleus. E. Tube indicates the entrance to the Eustachian tube. In (A) the middle ear space is air-filled, but in (B) the space is largely occupied by inflammatory cells and remnants of fluid that filled the cavity, as indicated by the uniform pink staining of proteins that were part of that fluid. The boxes labeled C–H in (A) and (B) indicate the sites of higher magnification images that appear below indicated by their respective letters. (C) The tympanic membrane at the left with elongated inflammatory cells adjacent to it. Congregations of large and smaller infiltrating inflammatory cells are present within the field of eosinophilic proteinaceous exudate. mal indicates the malleus with its surrounding mucosa showing clear signs of hyperplasia. (D) A thick aggregation of monocytes (MON) adjacent to the tensor tympani muscle (TT) that are covered by a greatly thickened mucosal layer (MUC). (E) Ciliated cells (arrowhead) from the non-inflamed ear in a site where they are not usually found in healthy wild-type animals. (F) Similar cells at that site in the inflamed ear. (G) The normal squamous mucosal lining that overlies the bone in non-inflamed ears. The arrowhead indicates the nucleus of a squamous mucosal cell. This state is contrasted with the grossly thickened mucosa at the same site in the inflamed ear shown in (H).

 
We then ascertained whether the ear abnormalities in the BAC transgenic mice could be rescued by introducing the Tbx1 null allele, but there was no obvious rescue. Middle ear abnormalities were frequent in all the transgenic animals (Table 1) although we found a modest reduction in the presence of chronic otitis media in BAC transgenic mice homozygous for the Tbx1 null allele (Tbx1 –/–; TG, Fig. 4). A frequent abnormality included an abnormal stapes footplate (six out of six ears for Tbx1 +/+; TG and six out of six ears for Tbx1 –/–; TG). One out of 6 ears from Tbx1 –/–; TG mice had a shortened cochlea, compared with a greater number of inner ear dysmorphisms in the transgenic mice (12), suggesting only partial rescue. However, all of the mice had auditory brain stem response (ABR) hearing loss (Table 1). The amount of the loss as quantified by the ABR thresholds was least for the Tbx –/–; TG mice and greatest for the TG mice. Most transgenic mice also exhibited circling behavior indicating an abnormal vestibular system. The reason for the failure of the rescue experiment in the ear may be the partial normalization of Tbx1 expression levels in Tbx1 +/– or Tbx1 –/– mice that carried the BAC and/or the higher sensitivity of ear to increased Tbx1 dosage compared with other affected organs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Differential sensitivity to altered Tbx1 dosage
In this report, we compared the phenotypes of mice with different levels of Tbx1 mRNA expression to gain more insight into the role of this gene during embryonic development. We previously generated and characterized low and high copy BAC transgenic lines (1–2 copies, 316.27; 8–10 copies, 316.23) harboring four human genes from the 22q11.2 region hemizygously deleted in VCFS/DGS patients (8). Mice of the low copy BAC transgenic line had ear disorders but no other malformations, while mice of the high copy BAC transgenic line had reduced viability and multiple anomalies that were similar to humans with VCFS/DGS (8,12). To determine whether the phenotype in the high copy TG mice was due to overexpression of TBX1, and not overexpression of the other transgenes on the BAC, we crossed them into the Tbx1 null mutant background.

We found that most of the malformations in the BAC transgenic embryos were rescued significantly in the Tbx1 –/–; TG mice. This suggests that TBX1 and not the other transgenes had the greatest role in the etiology of abnormalities. We uncovered differential sensitivities of developing systems to Tbx1 mRNA levels in a dosage dependent manner. The most obvious was a change in viability, which increased from 29% to 79%, as dosage was partially normalized. The greatest change in viability, from 29% to 69%, occurred as the Tbx1 mRNA dosage decreased from 5- to 4.5-fold overexpression (Tbx1 +/+; TG to Tbx1 +/–; TG). There are two possible explanations for this disparity between rescue of viability and mRNA levels. One explanation is that there is a threshold above normal levels of Tbx1, which needs to be achieved to detect alterations in viability as caused by the presence or frequency of a life-threatening malformation. From 4.5- to 5-fold overexpression, the presence of cleft palate and selected outflow tract defects became more commonplace, implicating these defects in the change in viability of the mice. Alternatively, although Tbx1 mRNA levels change modestly, protein levels might change more significantly, resulting in the differences in phenotype.

Most of the animals described in this report have various levels of overexpression of TBX1. This is despite the fact that they share striking phenotypic overlap with the deletion disorder, VCFS/DGS, especially when all the genotypes are considered. These results suggest that similar pathogenic pathways may be affected by both decreased and increased gene dosage. For example, the 4th PAAs are hypoplastic both in Tbx1 heterozygous (7) and in BAC transgenic mice, suggesting that the aortic arch defects are mediated by similar pathogenic mechanisms. Analyzing candidate downstream target genes in mice with varying levels of Tbx1 will make it possible to uncover whether the observed malformations share common genetic alterations downstream of Tbx1 as well. We do realize however, that in some developmental systems in the mouse, the situation might be more complex, such as overexpression of Tbx1 may affect the same structures but in different or opposite ways, as we found for inner ear development (17). In the case of the inner ear, in Tbx1 –/– mice, the sensory organs fail to form, but an enlarged VIII cranial ganglion is generated, while the opposite situation occurs in the BAC transgenic mice. Overexpression results in the presence of supernumerary or ectopic inner ear sensory organs (12) and a small VIII ganglion (17). Another possible example is the palate where a different mechanism in cleft palate closure might occur (compare Fig. 3B with Fig. 3C). More careful examination of the affected structures with molecular and protein markers at timed developmental stages as was done in the ear (17) will be necessary to clarify the precise role of Tbx1 dosage in the development of the structures described in this report.

T-genes and haploinsufficiency
Tbx1 is a member of the phylogenetically conserved family of dosage sensitive transcription factors, that share a common palindromic DNA-binding domain, named the T-box and is required for proper embryonic development (28). Haploinsufficiency of other T-box family members, Brachyury or T (29), TBX3 (30) and TBX5 (31,32) are associated with congenital anomalies that occur with variable expressivity.

The exact molecular mechanism that underlies the developmental abnormalities and their variable expressivity associated with haploinsufficiency of T-box genes is not known. Specific hypotheses to explain the defects associated with altered dosage of transcription factors have been proposed, based on the fact that most act as part of protein complexes (33,34). The cooperative behavior of the protein components in such functional complexes may have a synergistic effect on a given promoter (transcriptional synergy) (34). Thus, a narrow window exists, outside of which slight changes in the level of expression of the transcription factors cause an imbalance that lead to a significant impact on target gene transcription levels. As T-box genes have been shown to act as homodimers (35,36) and heterodimers (37,38) to regulate transcription, we believe that transcriptional synergy may be responsible for TBX1 haploinsufficiency in VCFS/DGS patients. Stochastic effects or genetic modifiers, altering the levels of the transcription factor, would enable the imbalance to have phenotypic consequences with variable levels of expressivity (39). The highly variable expressivity in VCFS/DGS patients can be explained by slightly altered levels of TBX1 expression among different individuals carrying same size of deletions that may be caused by random environmental events or sequence variations of genetic modifiers such as PAX3 (40), CRKL (41), VEGF (42) and RALDH2 (43). We also suggest that similar mechanisms could occur in mice overexpressing TBX1.

Model for Tbx1 dosage effects
We propose a simple model for the downstream molecular effects of altered TBX1 dosage in human embryonic development. In the model (Fig. 5), we propose that TBX1 protein regulates transcription in the form of functional heterodimers. When sufficient TBX1 protein is present, functional heterodimers can bind to the promoters of direct downstream target genes. When the dosage is reduced, there is insufficient TBX1 protein and transcription of downstream targets is not activated or repressed. On the other hand, when the dosage is increased above a threshold, TBX1 forms non-functional dimers with other molecules or perhaps forms TBX1 homodimers. Such non-functional dimers can compete with the functional heterodimer for the binding-site at the promoter, thus squelching the overall activity of TBX1 protein.



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  		Figure 5. Model for TBX1 dosage effect. We hypothesize that at normal dosage, TBX1 protein (opened circles) binds with its partner (closed circle) functioning as a heterodimer in regulating the transcription of target genes (closed rectangle). Low TBX1 dosage reduces the level of heterodimers and reduces the levels of transcription of downstream targets. When TBX1 dosage is above normal levels, TBX1 forms non-functional dimers, such as TBX1 homodimers. These can compete with the functional heterodimer for the binding-site at the promoter, thus sequestering the heterodimer. It will cause the same effect as low dosage of TBX1 protein.

 
The deleterious effects of decreased or increased gene dosage, both associated with variable expressivity, is not unique to Tbx1. For example Cx43 mutations were found in patients exhibiting heart malformations resulting from visceroatrial heterotaxia. Both Cx43 null mutants and Cx43 overexpressing mice exhibit conotruncal defects with partial complementation of the Cx43 deletion in the presence of the transgene (44), as in the case with Tbx1 that we describe in this study. Another well-described example relates to Pax6 (45). Humans with hemizygous PAX6 mutations are associated with aniridia (MIM 106210). Mice carrying heterozygous inactivating alleles of Pax6, termed Sey, have iris hypoplasia and other eye defects, while homozygotes have no eyes (46). Yeast artificial chromosome (YAC) transgenic mice harboring the human PAX6 locus, were generated and they had related developmental abnormalities of the eye (45). As in this study, crossing the YAC transgenic mice, carrying human PAX6, with the Sey/+ or Sey/Sey mice provided significant genetic rescue. It was hypothesized that dosage imbalance or sequestration of protein cofactors is responsible for the adverse effects of increased gene dosage. Finally, a more complex situation of overexpression might exist where a dose dependent inhibition of transcription of the gene's downstream targets occurs as dosage increases, as for example could be the case with Fgf8 (47). Increased Fgf8 dosage was found to have the same increase in apoptosis effect as its complete loss of function, whereas the opposite effect occurred in Fgf8 +/– mice, where apoptosis was decreased. Recently, Fgf8 has been shown to act as a downstream target of Tbx1 (48), and it is conceivable that Tbx1 acts via a similarly regulated Fgf8 pathway. However, we did not detect dramatic alterations in Fgf8 expression levels in the mice generated in this study except Tbx1 –/– either by whole mount in situ hybridization or by quantitative reverse transcription–PCR (RT–PCR) analysis (data not shown), suggesting that the effects of Tbx1 dosage might be independent of the Fgf8 pathway, and that Fgf8 is only one of many downstream targets.

Increased TBX1 dosage and dup(22)(q11.2)
In addition to VCFS/DGS, a newly recognized genetic disorder termed dup(22)(q11.2) has been described in which affected individuals have a reciprocal duplication to the region deleted in VCFS/DGS (49,50). The dup(22)(q11.2) patients were originally ascertained, because they had clinical features reminiscent of VCFS/DGS (49,50). On the basis of the results presented here, we suggest that overexpression of TBX1 might be responsible for this disorder. Further support of this hypothesis is based on previous mouse genetics work. Mice carrying a duplication of the region of homology in the proximal half of the 22q11.2 interval are physically normal, thus eliminating genes from this interval as being primarily responsible (6,8). We generated a library of BAC transgenic mice that span the distal half of the 22q11.2 interval (8). Of the lines generated, only BAC 316 transgenic mice, the focus of this report, had a phenotype on its own, and some of the malformations are similar to those in dup(22)(q11.2) syndrome patients. Thus, we propose that TBX1 overexpression might be responsible for the pathogenesis of dup(22)(q11.2) disorder. On the basis of the similarities between the phenotypic abnormalities in mice and humans, abnormal Tbx1 dosage outside the normal range seems to disrupt the same developmental pathways in mammalian species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mouse strains and genotyping
Both the Tbx1 +/– and the BAC transgenic mice are congenic in the FVB genetic background. PCR for genotyping DNA were performed by using the following PCR primer pairs: Tbx1 wild-type allele, Tbx1KO1-F (5'-TTGGTGACGATCATCTCGGT-3') and Tbx1KO2-R (5'-ATGATCTCCGCCGTGTCTAG-3'); Tbx1 mutant allele, Tbx1KO1-F and mut2-R (5'-AGGTCCCTCGAAGAGGTTCA-3'); BAC transgenes, TBX1a1-F (5'-AGGATGATTCCCTCAAACTGG-3') and TBX1a1-R (5'-ACTGGACAGCAGCACTTGG-3'). They were performed using conventional PCR conditions.

Histology
Mouse embryos were dissected in phosphate-buffered saline (PBS), and fixed in 10% neutral buffered formalin solution (Sigma) overnight. Then, they were dehydrated through graded ethanol, embedded in paraffin and sectioned (5–7 µm). For adult mouse ears, following the hearing tests the mice were deeply anesthetized with urethane (1.5 gm/kg, i.p.) and exsanguinated by cardiac perfusion with saline. This was followed by perfusion with 10% formalin for the first stage of fixation and then by perfusion with 10% formalin and 1% glacial acetic acid for the second stage of fixation. The top of the cranium was removed with a razor blade so that the cut surface was in the horizontal plane. This surface served to orient the block in the embedding mold so the specimens were cut in the horizontal plane, which is also the plane of the cochlear modiolus. Blocks of tissue containing the temporal bones were placed in the formalin/acetic acid fixative for 1 week and then were embedded in paraffin for sectioning (10 µm). All sections were stained with hemotoxylin and eosin under standard conditions.

Quantitative RT-PCR
To minimize the variability of gene expression in individual embryos, a pool of five 9.5 dpc embryos for each genotype was used to test Tbx1 expression level. Only embryos with 19–21 pairs of somites were used to obtain accurate developmental stages. Total RNA was isolated using the RNeasy Protect Mini Kit (Qiagen), and used for the first-strand cDNA synthesis with SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). The PCR reactions were performed in the LightCycler apparatus (Roche) using LightCycler-FastStart DNA Master SYBR Green I (Roche). The housekeeping gene Gapdh, was chosen for internal normalization. Specific primers used for PCR amplification were as follows: for mouse Tbx1, MTbx1RT-F (5'-ATGCACTTCAG CACAGTCAC-3') and MTbx1RT-R (5'-TTGGAACGTGGG GAACATTC-3'); for human TBX1, TBX1RT1-F (5'-TGGTCTATGTGGACCCACGC-3') and TBX1RT1-R (5'-AGGCGCTCATGAGCGGCAGT-3'); for Gapdh, Gapdh-F (5'-TTCACCACCATGGAGAAGGC-3') and Gapdh-R (5'-GGCATGGACTGTGGTCATGA-3').

ABR testing
The hearing status of adult mice (6 months of age) was assessed by obtaining ABR thresholds. ABR signals were recorded from needle electrodes inserted through the skin (vertex to ipsilateral tragus). Computer-assisted evoked potential systems were used to obtain responses to tone pips at 5, 8, 11, 16, 22, 32 and 45 kHz. Averaged responses were obtained to 512 pips of alternating polarity (tone pip duration 5 ms; repetition rate 30/s).


   ACKNOWLEDGEMENTS
 
We thank Dr Radma Mahmood and Dawn Lee for technical assistance, Dr Birgit Funke for generating BAC transgenic mice, Drs Robert Shprintzen, James Lupski, Thomas Van De Water, Steven Raft, Arthur Skoultchi and Shari Lipner for intellectual advice and helpful discussions. This work is supported by the American Heart Association, March of Dimes (1-FY02-193) and the National Institutes of Health (HD34980-08 and DC05186-01) to B.E.M.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Molecular Genetics, Albert Einstein College of Medicine, Ullmann Building, Room 1217, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Tel: +1 7184304274; Fax: +1 7184308778; Email: morrow{at}aecom.yu.edu


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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