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Human Molecular Genetics Advance Access originally published online on October 4, 2007
Human Molecular Genetics 2008 17(1):150-157; doi:10.1093/hmg/ddm291
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

In vivo response to high-resolution variation of Tbx1 mRNA dosage

Zhen Zhang1 and Antonio Baldini1,2,*

1 Institute of Biosciences and Technology, Texas A&M University Health Sciences Center, Houston, TX 77030, USA 2 Telethon Institute of Genetics and Medicine, University of Naples Federico II, Naples, Italy

* To whom correspondence should be addressed at: Telethon Institute of Genetics and Medicine (Tigem), Via Pietro Castellino 111, Napoli I-80131, Italy. Email: baldini{at}tigem.it; abaldini{at}ibt.tamhsc.edu

Received August 12, 2007; Accepted September 28, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Mouse modeling of haploinsufficiency syndromes and, in general, of syndromes caused by gene dosage imbalance, is often unsatisfactory because loss (or gain) of one copy of the gene of interest is insufficient to recapitulate the disease phenotype. In this study, we use Tbx1 mutants, which model one of the most common haploinsufficiency disorders, the 22q11.2DS/DiGeorge/Velocardiofacial syndrome, to test the feasibility of high resolution dosage manipulation to generate mouse models that more closely resemble the human syndrome. We used nine different genotypes at the Tbx1 locus that are associated with progressively lower mRNA levels in vivo. We show that penetrance and expressivity of different phenotypic features became more severe as the dosage diminished, as expected, but the response was strikingly non-linear, with extreme examples such as neonatal lethality, which changed from 2 to 100% after a dosage reduction of just ~16%. Furthermore, heart phenotype variability, extreme in the human syndrome but very limited, or absent, in the standard knockout model, was seen when mRNA level was ~20% of normal level, suggesting that there is a threshold level associated with unstable balance, which can be perturbed by chance events. Overall, our data suggest that there are developmental process-specific gene dosage thresholds beyond which the phenotype worsens very rapidly with very small mRNA level reductions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Syndromes associated with segmental aneuploidies are caused by gene dosage imbalance. There may be one or more critical genes within the aneuploid segment, that encode different classes of proteins, but the common characteristic is that the processes in which they are involved (developmental or otherwise) are sensitive to their concentration. In most if not all cases, the mechanisms underlying dosage sensitivity are unknown. A complicating issue is that mouse modeling of these syndromes, while technically feasible in many cases, is hindered by different sensitivity to dosage of a particular gene product across species. This problem translates into the finding that similar gene dosage reduction in mice and humans (e.g. heterozygous deletion) leads to different phenotypes in the two species. Among the extensively studied (in humans and mice) segmental aneuploidies is the del22q11 deletion syndrome (22q11.2DS), which includes DiGeorge and Velocardiofacial clinical phenotypes (1). Mouse models of this deletion syndrome were generated by engineering chromosomal deletions (2) and the gene encoding the transcription factor Tbx1 was identified as the gene haploinsufficient in mice and humans (3–7). Heterozygous multigene deletion in the mouse as well as Tbx1+/– mice, presented with a less severe phenotype than the majority of 22q11.2DS patients or TBX1 mutant patients, with the caveat that mild patients may escape clinical diagnosis. Most intriguingly, the great phenotypic variability observed in human patients could not be modeled in mice, although different phenotypic penetrance, but not expressivity, has been reported as a function of strain (5,8) or genotype at interacting loci (9–11).

If phenotypic differences between human and mouse were solely due to different dosage sensitivity, then, lowering Tbx1 dosage in mice should lead to a phenotype more similar to the human disease.

The effect of increased dosages of TBX1 using multigene BAC transgenic mice has been reported (12,13). However, the effect of reduced dosage in the critical interval between 50 and 0% has not been studied. Tbx1 dosage manipulations using hypomorphic alleles have met with some success (14–16) and have revealed that different tissues have different sensitivity to Tbx1 dosage. However, the phenotypic modifications obtained were relatively modest, most likely because the mRNA dosage levels tested in vivo were close to those obtained with null alleles.

In this study, we have generated a series of genotypes associated with a nearly continuous variation of Tbx1 mRNA dosage between 0 and 100% of the wild-type level by combining two different hypomorphic alleles and a null allele. Phenotypic survey revealed a non-linear correlation between mRNA dosage and phenotypic severity. Intriguingly, the cardiovascular phenotype of embryos with ~20% of the wild-type dosage was close to that of human patients, in terms of severity and variability.

The striking differences in the dynamic response to mRNA dosage of the phenotypes tested suggest that Tbx1 may have different partners or may work differently in the various developmental processes in which it is involved.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Generation of a genotype series with near-continuous variation of Tbx1 mRNA dosage
We generated embryos with different combinations of wild-type, Tbx1Neo2 (16), Tbx1Neo (15) and Tbx1– (3) alleles (Fig. 1A). We have previously shown that the two hypomorphic alleles, Tbx1Neo2 and Tbx1Neo have uniformly low levels of expression across embryonic tissues. We harvested embryos at E9.5 and tested the Tbx1 mRNA dosage in whole embryo RNA, using real time quantitative PCR (qRT–PCR). Comparisons were made between somite-matched embryos (Fig. 1B). Results indicated that Tbx1 expression level gradually decreases from wild-type embryos (1.0±0.066), to null (Tbx1–/–) embryos (0.002±0.001) with intermediate levels represented by Tbx1Neo2/+ (0.705±0.051), Tbx1Neo/+ (0.533±0.025), Tbx1+/– (0.499±0.033), Tbx1Neo2/Neo2 (0.34±0.015), Tbx1Neo2/Neo (0.185±0.029), Tbx1Neo2/- (0.154±0.017), Tbx1Neo/Neo (0.041±0.009) embryos. These data indicate that Tbx1Neo is a ‘weak’ hypomorphic allele with an expression level around 2–3% of the wild-type allele. Consistently with this finding, phenotypic analysis demonstrated that Tbx1Neo/Neo embryos were nearly identical to Tbx1 null embryos (15). However, remarkably, such a low level of Tbx1 expression was sufficient to support palatogenesis, which is affected in null embryos, and to improve the alignment of the cardiac outflow tract (OFT) in some embryos (15). In contrast, the Tbx1Neo2 allele expressed 15–20% of the wild-type mRNA level. Both hypomorphic alleles were generated by inserting a PGKneo cassette into intron 5 (15,16), but in opposite orientation (Fig. 1A). Although by in situ hybridization and immunohistochemistry we could not detect tissue-specific differences in expression of the two hypomorphic alleles (15,16), we cannot exclude small, transient differences that could be missed by the methods applied here.


Figure 1
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  		Figure 1. Tbx1mRNA dosage in E9.5 embryos with different genotypes. (A) Schematic drawings of the Tbx1 alleles used in this study. e – exon and green triangle – loxP site. The small black arrows indicate the positions of primers used for quantitative real time PCR. (B) Relative expression level of Tbx1 associated with the genotypes studied (wild-type level = 1). At least three embryos per genotype were assayed. Error bars indicate standard error.

 
We also evaluated the Tbx1 expression level in Dp1/Dp1 homozygous mutants. Dp1 is a targeted chromosomal duplication that includes the Tbx1 gene as well as other genes located in the interval between genes Es2el and Ufd1l (17). Dp1/Dp1 mice have four copies of the endogenous Tbx1 gene and, therefore, are expected to have twice the amount of Tbx1 mRNA compared to wild-type. Our data showed that Tbx1 expression level in Dp1/Dp1 embryos is 1.79±0.053, i.e. slightly lower than expected.

In summary, the genotypes generated allowed us to obtain different levels of Tbx1 mRNA in vivo, ranging from 179 to 0% of the wild-type level.

A sharp biphasic viability curve is associated witha linear decrease in Tbx1 mRNA dosage curve
One of the most striking phenotypic variations in our Tbx1 genotype series is post-natal viability. All heterozygous mutants (Tbx1Neo2/+, Tbx1Neo/+ and Tbx1–/+) had high viability and were fertile [the Tbx1–/+ viability is ~96% in the genetic background tested (18)]. The low level of neonatal lethality in these mutants is caused by interrupted aortic arch type-B (IAA-B), which is incompatible with post-natal life (19). In contrast, all the homozygous and compound heterozygous mutants in the genotype series [with the exception of Dp1/Dp1 animals which are viable and fertile (17)] were associated with 100% lethality in the neonatal period. Most interestingly, Tbx1Neo2/Neo2 animals (34% wild-type Tbx1 dosage level) did not exhibit the cardiac OFT defects previously thought to kill Tbx1–/– animals (3,5). Tbx1Neo2/Neo2 neonates were able to breathe and feed but they all died within 1–2 days. The incidence of IAA-B (13%) in these animals does not explain the complete loss of viability. Thus, an mRNA dosage reduction between 50 and 34% triggers the failure of a yet to be identified vital function (Fig. 2A). There is no obvious correlate of this phenomenon in the human syndrome.


Figure 2
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  		Figure 2. Graphical representation of phenotypic characteristics as a function of Tbx1mRNA dosage. (A) Incidence of neonatal lethality as a function of Tbx1 mRNA dosage. (B) Severity of aortic arch defects at E18.5. (C) Severity of OFT defects. (D) OFT phenotype variability associated with the genotypes indicated. See text for details on the scoring systems.

 
Aortic arch patterning and thymic defects correlatewell with Tbx1 mRNA levels
The phenotypes associated with the genotypes Dp1/Dp1, Tbx1Neo/+, Tbx1+/–, Tbx1Neo2/-, Tbx1Neo/Neo and Tbx1–/– have been described (3–5,15–17,19) and confirmed in this study. Here, we summarize their major defects in Table 1, together with those seen in Tbx1Neo2/+, Tbx1Neo2/Neo2 and Tbx1Neo2/Neo, which are presented here. Tbx1Neo2/+, Tbx1Neo/+ and Tbx1+/– animals had slightly smaller thymus than wild-type littermates (Fig. 3B) and aortic arch artery abnormalities derived from developmental defects of the fourth pharyngeal arch arteries, i.e. aberrant origin of the right subclavian artery (Fig. 4B), right aortic arch (Fig. 4C), IAA-B, or high aortic arch. The penetrance of both groups of defects increased with reduced Tbx1 dosage, as expected (Fig. 2B). In Tbx1Neo2/+ E18.5 embryos (n = 27), the penetrance of thymic and aortic arch artery defects was 29 and 11%, respectively, compared to 41 and 38% in Tbx1+/– animals (n = 29). In Tbx1Neo2/Neo2 embryos (n = 15), in which Tbx1 dosage was reduced to 34% of the wild-type level, the thymic hypoplasia was more severe. These animals presented with the same type of aortic arch artery defects as seen in heterozygous mutants, but the overall penetrance increased to 100%. In addition, the majority of individuals (11 out of 15) had bilateral defects derived from hypoplasia or aplasia of both fourth pharyngeal arch arteries in early embryonic development (Table 2). Further reduction of Tbx1 mRNA dosage in Tbx1Neo2/Neo embryos (~20% of wild-type), was associated with cardiac OFT defects. Plotting the severity of aortic arch artery phenotype (expressed as the number of arteries affected per embryo) versus Tbx1 mRNA dosage, results in an ‘S’ shaped curve that approximates a linear correlation within the range of 75–25% of mRNA level (Fig. 2B).


Figure 3
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  		Figure 3. Thymic morphology is sensitive to Tbx1 dosage.(A–E) Frontal views of the mediastinum of E18.5 embryos. (A) The thymus of wild-type embryos has two close and symmetric lobes. (B) In heterozygous mutants, lobes may be asymmetric, with one lobe slightly smaller. (C) In Tbx1Neo2/Neo2 embryos, the lobes are separate and smaller in size. (D) Most of the Tbx1Neo2/Neo embryos have no detectable thymus; some have very small remnants, as in this case (dotted lines). (E) No thymus is detectable in Tbx1Neo2/– embryos. T: Thymus.

 


Figure 4
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  		Figure 4. Great artery patterning phenotype and Tbx1dosage. (A–I) Frontal views of great arteries of E18.5 embryos after removal of the thymus. (A) A wild-type embryo shown with the normal positions of aorta ‘A', pulmonary trunk ‘P', right and left subclavian arteries (RSA and LSA) and right and left common carotid arteries (RCA and LCA). (B) Tbx1+/– mutant with cervical RSA (arrowhead), a defect due to failure of the right fourth pharyngeal arch artery in earlier development. (C) Tbx1Neo2/Neo2 mutant with retroesophageal RSA (arrowhead) and IAA-B (asterisk), indicating that both fourth pharyngeal arch arteries have failed. (D–F) Tbx1Neo2/Neo mutants with different types of OFT defects, the embryo in D had VSD (not shown) but normally separated aorta and pulmonary trunk. The embryo in E exhibits an alignment defect, note the aorta and pulmonary trunk positioned side-by-side. The embryo in F exhibits normal separation of the distal portion of the aorta and pulmonary trunk, but the two arteries merge proximally to the heart into a single vessel (arrowhead). (G–I) Tbx1Neo2/– mutants with incomplete separation of great arteries (G and H), or with complete truncus arteriosus ‘T'.

 


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  		Table 1. Tbx1 dosage-dependent phenotype

 


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  		Table 2. Aortic arch artery patterning defects in E18.5 mutant embryos

 
Thymic morphology was a sensitive indicator of dosage. Normally, wild-type E18.5 embryos have two adjacent, symmetric thymic lobes located cranially to the atria (Fig. 3A). Tbx1 heterozygous mutants had mild or very mild hypoplasia of one thymic lobe (Fig. 3B), while homozygous mutants had no thymus at all (3,5). In Tbx1Neo2/Neo2 embryos (n = 15), the majority had two severely hypoplastic and well-separated lobes (Fig. 3C). Occasionally (n = 3), they presented with a single severely hypoplastic and ectopic lobe located near the thyroid gland (data not shown). The majority (Nine out of 13) of Tbx1Neo2/Neo embryos had thymic aplasia, similar to Tbx1Neo2/- embryos (Fig. 3E). However, four out of 13 Tbx1Neo2/Neo embryos had two very small, normally positioned thymic lobes (Fig. 3D). In summary, the thymic phenotype, similar to the aortic arch phenotype varied over a broad range of Tbx1 mRNA dosage (from 70 to 20% of the wild type level), and showed increasing severity in parallel with dosage reduction.

Most of the abnormal phenotype of Tbx1 mutants derived from development defects of the pharyngeal apparatus (PA) and the gross morphology of this structure at E10.5 also reflected mRNA dosage. In Tbx1Neo2/Neo2 the PA was mildly affected, exhibiting absence of the fourth pharyngeal arch arteries and hypoplastic fourth pharyngeal arches (data not shown). Tbx1Neo2/- embryos had a more severe PA phenotype, very similar to that of Tbx1–/– embryos, but in contrast to these, one of the two sixth pharyngeal arch arteries was sometimes present, and there were rudimentary fourth pouches (16), which are not present in Tbx1–/– embryos. The PA phenotype of Tbx1Neo/Neo could not be distinguished from the Tbx1–/– phenotype (15).

Outflow tract development has a low Tbx1dosage threshold
Some of the most severe defects in Tbx1–/– mutants (and in DGS patients) are OFT abnormalities (3,5,19). These defects appeared only at a dosage range of <20% (i.e. from Tbx1Neo2/Neo to Tbx1–/– genotypes, Table 1). At this range, OFT defects ranged from complete truncus arteriosus communis (TAC) to ventricular septum defect (VSD). To better describe this phenotype, we categorized the defects into four different types, in the order of increasing severity.

Type I: Perimembranous VSD. The aorta and pulmonary trunk are completely septated and the aorta is correctly aligned with the left ventricle, but the lower conal region is abnormal resulting in an interventricular communication in the subvalvular (membranous) region (Figs 4D, 5B).

Type II: Double outlet right ventricle (DORV). The ascending aorta and the pulmonary trunk are separated but are positioned side-by-side and both connect to the right ventricle (Figs 4E, 5C). This defect also includes a VSD.

Type III: Incomplete TAC. The distal segments of the aorta and pulmonary trunk are septated but the proximal segments merge together to form a common arterial trunk (Figs 4F–H, 5D). This defect is also associated with a VSD (Fig. 5D).


Figure 5
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  		Figure 5. Histological sections showing the intracardiac phenotype of E18.5 embryos. (A) A Tbx1Neo2/Neo2 embryo shown as a normal control. (B) Heart from a Tbx1Neo2/Neo embryo with VSD. (C) Heart from a Tbx1Neo2/– embryo with DORV. The inset ‘C'' shows a VSD in the same heart. (D) Heart from a Tbx1Neo2/– embryo, note the merging of the aorta and pulmonary trunk to form a TAC ‘T' connected to the right ventricle. Also note the presence of a VSD (arrowhead). ‘A': aorta; ‘P': pulmonary trunk; ‘T': truncus arteriosus; ‘RV': right ventricle; ‘LV': left ventricle.

 
Type IV: Complete TAC. No separation between the aorta and pulmonary trunk (Fig. 4I). It includes a VSD. This defect is present in all the Tbx1–/– mutants (5,19).

Based on this classification, we could distinguish the OFT phenotypes associated with the various genotypes of Tbx1Neo2 and Tbx1Neo combined mutants (mRNA dosage range from 18 to 4%) (Table 3). Tbx1Neo2/- embryos have more severe OFT defects than Tbx1Neo2/Neo embryos. More than 90% of Tbx1Neo2/- embryos had Type III or IV defects characterized by (complete or incomplete) truncus. In contrast, more than 60% of Tbx1Neo2/Neo embryos had completely separated great arteries. The mean difference in Tbx1 mRNA level between Tbx1Neo2/Neo and Tbx1Neo2/- mice was only 3.1%. Even considering the differences among individual embryos (rather than mean values), the maximum difference was only 8% (the highest value in Tbx1Neo2/Neo embryos was 22% and the lowest in Tbx1Neo2/- embryos was 14%). Surprisingly, the OFT phenotype varied from normal septation to complete TAC over a very small range of mRNA dosage. To generate an approximate measure of OFT phenotypic severity, we assigned a score (1–4 according to the types of defects described above) to each embryo and then calculated the average score per genotype. Values were plotted against mRNA dosage (Fig. 2C). The resulting curve had a very steep ‘S’ shape, considerably steeper than the one for aortic arch defects (Fig. 2B).


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  		Table 3. Cardiac outflow tract phenotype variation in E18.5 embryos

 
In addition to severity, OFT phenotype variability presented a surprising trend. Generally, we observed very limited variability in animals with the same genotype, with one notable exception (Table 3). The Tbx1Neo2/Neo group was the only one to include individuals with all four types of defects as well as individuals with a normal OFT. Fig. 2D shows a graphical representation of the variability, where 1 indicates no variability and 2–5 indicate the number of different OFT phenotypes observed among embryos with the same genotype, including a normal phenotype. These data suggest that phenotypic variability is linked to mRNA dosage, being highest at a threshold level beyond which the likelihood of normal development becomes very low. The phenotype of Tbx1Neo2/Neo mutants is the one that most closely resembles that of patients as they have a variable OFT phenotype, ranging from normal to complete TAC.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Using combinations of Tbx1 alleles, we were able to test the phenotypic consequences of Tbx1 mRNA dosage reduction over an extensive and finely tuned range. As expected, the general trend is that penetrance and expressivity increase as the dosage decreases. However, the dynamic phenotypic response to dosage is complex with different trends for different phenotypes, indicating that different developmental processes have different sensitivities to Tbx1 mRNA dosage.

Manipulation of the Tbx1 dosage to build a better model of the human disease
Tbx1 heterozygous deletion in mice produces a mild version of the human syndrome, with only a limited range of phenotypic features, mainly aortic arch patterning defects and behavioral abnormalities. Breeding the mutation into different strains or into single gene mutant background such as Fgf8, Crkl and Pitx2 led to changes in penetrance of aortic arch defects, but did not expand the range of phenotypic features, which is very broad in patients. Intuitively, this species-specific difference should be resolvable by reducing the mRNA dosage from the 50% of heterozygous mutants to somewhere between 50 and 0%. Our data demonstrate that this is indeed true, but it is less straightforward than expected. We have examined in greater detail the cardiovascular phenotype and found that the aortic arch and OFT phenotypes have independent behavior, the former being very sensitive to Tbx1 mRNA dosage, the latter much less so. Thus, at a dosage when OFT defects start to appear, the aortic arch defects are close to the highest possible expressivity (i.e. both fourth pharyngeal arch arteries are defective). One implication is that in the mouse, unilateral aortic arch defects are virtually never associated with OFT defects; for example, IAA-B is almost never associated with VSD. In contrast, in patients, IAA-B is virtually always associated with VSD. If we consider OFT defects in isolation, the mouse response to ~20% Tbx1 mRNA dosage approximates the human response to 50% of TBX1, not only for the types of defects but also for the phenotypic variability. These characteristics make the Tbx1Neo2/Neo mutant a far better model than either the heterozygous or homozygous null mutants, as far as OFT defects are concerned.

The sharp biphasic viability response to gene dosage was surprising for two reasons, first because neonatal lethality occurred in the absence of lethal cardiovascular defects, second because it appeared with full penetrance in response to a modest reduction of mRNA dosage. In another study carried out using alleles of another T-box encoding gene, Tbx5, lethality appeared gradually with dosage reduction (20). A review of 22q11.2DS clinical data (21–25) could not identify any obvious lethal conditions that could constitute a correlate of this mouse phenotype (apart from heart defects, not present in this mutant, and severe immunodeficiency, which is associated with thymic aplasia, not a characteristic of this mutant). It is possible that the condition that causes loss of viability in Tbx1Neo2/Neo2 mice, yet to be identified, is also present in human patients but in a milder or sub-clinical form.

An alternative possibility is that the insertion of the PGKneo cassette into the Tbx1 locus affects a tissue-specific regulatory element of the Tbx1 gene or even of neighboring genes, so that when both alleles have the PGKneo insertion, homozygous loss of the putative regulatory element may cause the lethal phenotype independently from the overall Tbx1 mRNA dosage. This possibility, however, appears unlikely because (i) the intron into which the PGKneo cassette is inserted (identical point in Neo and Neo2 alleles) does not contain evolutionary conserved sequences, (ii) the excision of the PGKneo cassette restores normal expression of both alleles (15,16) and is not associated with any phenotypic abnormality at the homozygous state, even though a loxP site still disrupts the intronic sequence.

Phenotypic variability at the intersection of dosage, genetic makeup and chance
Phenotypic variability is a striking characteristic of the human syndrome (2,25–27), but the available mouse mutants model it very poorly or not at all. Possible explanations for the discrepancy are that in patients, genetic variants at the remaining TBX1 allele or at modifier loci may affect TBX1 mRNA dosage and/or interact with TBX1-dependent pathways. Our data underline the effect of dosage because the highest variability was associated with a specific dosage level at the border between those associated with consistently normal and consistently abnormal OFT phenotype. Presumably, this level of dosage (associated with the Tbx1Neo2/Neo genotype) supports a precarious balance between normal and abnormal development, which may be perturbed by environmental or stochastic events that would be insufficient to cause heart defects in wild-type organisms or even modify the heterozygous or homozygous mutant phenotype. Thus, the Tbx1Neo2/Neo genotype represents a genetic background that may be very sensitive to genetic and non-genetic factors affecting OFT development. In humans, heterozygous deletion may produce a similar effect making OFT developmental processes highly susceptible to events (and chance) that could tip the balance in one way or the other and thus generate variability. The finding of 22q11.2DS monozygotic twins with discordant phenotypes (28,29) suggests non-genetic contribution to variability. The Tbx1Neo2/Neo model could be used for sensitized screens to test the cardiovascular impact of environmental or genetic insults.

Tbx1 dosage and outflow tract development
The OFT phenotype associated with Tbx1 loss of function has at least two components, one related to a function in the second heart field (SHF) to promote or maintain proliferation of cardiac progenitors, and one related to migration/function of cardiac neural crest cells (CNCCs) (15,30). We postulated that the failure of the former is responsible for proximal septation defects (defects of separation of the OFT valves, and VSD), and the latter for distal septation defects (failure of separation of the aorta and pulmonary trunk). The function of Tbx1 in the second heart field depends on its expression in the mesoderm, while the effects on CNCCs, which are cell non-autonomous, depend on multiple expression domains (16). In this dosage study, we show that proximal septation defects (VSD and TAC with septated great arteries) occur at higher Tbx1 dosages than distal defects (lack of separation of aorta and pulmonary trunk). A possible interpretation of this differential sensitivity to dosage is that the function of Tbx1 in the second heart field (SHF) is more dosage sensitive than in CNCCs.

Dosage sensitivity and gene function
In this study, we have considered a limited set of clinically relevant phenotypic features to evaluate gene dosage sensitivity of the organism. These are complex features that involve multiple developmental pathways, but nevertheless, the data provide a macroscopic view within which future, detailed, and tissue-specific molecular studies can focus on. Future molecular analyses, including transcriptome and proteome analyses, could be integrated with the available information concerning tissue and time-specific effects of Tbx1 loss of function to reconstruct an integrated picture of the gene’s role in development. We showed that even at the gross phenotypic level, dosage response analysis could lead to novel and unexpected information, such as, for example, the discovery of a vital function of Tbx1 independent of heart development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Mouse lines and breeding
All the mouse mutant alleles used in this study have been reported previously: Tbx1– (3), Tbx1Neo (15) and Tbx1Neo2 (16). All lines were created in AB2.2 ES cells (129SvEv background) and backcrossed into the C57Bl/6 genetic background for at least two generations. Various homozygous and compound heterozygous mutants were generated by heterozygous mutant mating. Embryos were collected at E9.5 and E18.5, considering the day of observation of a vaginal plug to be E0.5. PCR strategies for mouse genotyping have been described in the original articles.

Dissection, imaging and histology
E9.5 embryos were dissected in di-ethylpolycarbonate-treated phosphate-buffered saline and then quickly frozen in liquid nitrogen until total RNA extraction. E18.5 embryos were dissected and photographed under a stereomicroscope. Hearts were isolated by manual dissection and embedded in paraffin. Sections were stained with Hematoxylin and Eosin.

RNA extraction, cDNA synthesis and quantification PCR
Whole embryos were homogenized in 1 ml TRIzol (Invitrogen) and extracted according to the manufactures instructions, cDNA was synthesized from 3 µg total RNA with random primers in a total volume of 50 µl, using High Capacity cDNA Archive kit (Applied Biosystem). Quantitative PCR was performed with 0.5 µl cDNA, MGB TaqMan probe (5'-CAATGGCCATATTATTCTC) and primers (F: 5'-CTGACCAATAACCTGCTGGATGA; R: 5'-GGCTGATATCTGTGCATGGAGTT). β-Actin was used as internal reference gene. Probe and primers set was commercially available from Applied Biosystem (Mm00607939_s1). PCR conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 1 min and 72°C for 30 s. Reactions were performed on Mx3000P system (Stratagene), using QuantiTect Probe PCR Master Mix (Qiagen). Relative quantification was calculated by {Delta}{Delta}Ct method incorporated in the Mx3000P software.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This work was funded in part by grants from the NIH (National Institutes of Health)-NHLBI and from the EC program AnEUploidy (to A.B.). A.B. is supported by the Telethon Foundation.


   ACKNOWLEDGEMENTS
 
We wish to thank Dr Elizabeth Illingworth for critical reading of the manuscript.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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