Identification of downstream genetic pathways of Tbx1 in the second heart field

Abstract

Tbx1, a T-box transcription factor, and an important gene for velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) in humans, causes outflow tract (OFT) heart defects when inactivated in the mouse. Tbx1 is expressed in the second heart field (SHF) and is required in this tissue for OFT development. To identify Tbx1 regulated genetic pathways in the SHF, we performed gene expression profiling of the caudal pharyngeal region in Tbx1^-/- and wild type embryos. Isl1, a key marker for the SHF, as well as Hod and Nkx2-6, were downregulated in Tbx1^-/- mutants, while genes required for cardiac morphogenesis, such as Raldh2, Gata4, and Tbx5, as well as a subset of muscle contractile genes, signifying myocardial differentiation, were ectopically expressed. Pan-mesodermal ablation of Tbx1 resulted in similar gene expression changes, suggesting cell-autonomous roles of Tbx1 in regulating these genes. Opposite expression changes concomitant with SHF-derived cardiac defects occurred in TBX1 gain-of-function mutants, indicating that appropriate levels of Tbx1 are required for heart development. When taken together, our studies show that Tbx1 acts upstream in a genetic network that positively regulates SHF cell proliferation and negatively regulates differentiation, cell-autonomously in the caudal pharyngeal region.

INTRODUCTION

The development of the cardiac outflow tract (OFT) is vulnerable to genetic and environmental insults resulting in morphological defects of the heart. OFT anomalies, including abnormal alignment or septation, account for ~30% of all cases of congenital heart disease (Kelly and Buckingham, 2002), some of which co-occur in genetic syndromes. Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) (DiGeorge, 1965; Shprintzen et al., 1978) is associated with OFT defects due to de novo hemizygous 1.5-3 million base pair (Mb) 22q11.2 deletions (Morrow et al., 1995; Shaikh et al., 2000). Studies in the mouse have demonstrated that Tbx1, a member of T-box containing transcription factor family present within the 1.5 Mb 22q11.2 region, is required for OFT development (Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher et al., 2001). Functional mutations in TBX1 have been found in rare non-deleted patients with the syndrome as well (Paylor et al., 2006; Stoller and Epstein, 2005; Yagi et al., 2003).

Tbx1 is expressed in the non-neural crest derived mesoderm of the caudal pharyngeal region, which is part of the second heart field (SHF) (Hu et al., 2004; Xu et al., 2004). Unlike the first heart field (FHF) cells, which differentiate at the cardiac crescent stage and give rise to the atria and left ventricle of the heart, SHF cells differentiate later and give rise to the OFT, right ventricle and part of the atria (reviewed by Buckingham et al., 2005). Inactivation of Isl1, a LIM homeodomain transcription factor expressed in the SHF, resulted in failed formation of the OFT, right ventricle and most of the atria due to failed proliferation and migration of this population of cells (Cai et al., 2003). Thus, the SHF, via Isl1, contributes significantly to heart development.

Lineage tracing-experiments showed that the progeny of Tbx1-expressing cells in the SHF contribute to the OFT and right ventricle. In Tbx1^-/- embryos, although Tbx1-traced cells are present normally in the SHF, the contribution to the OFT is lower than in wild type embryos (Xu et al., 2004). Cell proliferation is also modestly reduced in the SHF of Tbx1^-/- embryos (Xu et al., 2004; Zhang et al., 2006). Tissue-specific inactivation of Tbx1 in the SHF by Nkx2-5-Cre (Xu et al., 2004) or Mesp1-Cre (Zhang et al., 2006) resulted in a persistent truncus arteriosus (PTA) and ventricular septal defect (VSD), same as those in Tbx1^-/- embryos.

Two fibroblast growth factor (Fgf) genes expressed in the SHF, Fgf8 and Fgf10 are hypothesized to act downstream of Tbx1 in cardiovascular development (Hu et al., 2004; Vitelli et al., 2002b). Both genes are downregulated in the SHF of Tbx1^-/- mutants, but surprisingly, Fgf10^-/- embryos do not have OFT defects (Marguerie et al., 2006) nor did Tbx1/Fgf8/Fgf10 triple heterozygous mice (Aggarwal et al., 2006), indicating that other genes are responsible for these defects. Recently, Pitx2 was shown to act downstream of Tbx1 in the early SHF, but Tbx1/Pitx2 double heterozygosity affected only OFT alignment but not septation, and it occurred with reduced penetrance (Nowotschin et al., 2006).

It has been difficult to identify genes downstream of Tbx1 in the SHF. Here, we report a systematic genome-wide search for genes regulated directly or indirectly by Tbx1 in the caudal pharyngeal region containing the SHF. Our data show that global or pan-mesodermal inactivation of Tbx1 disrupts a network of genes maintaining cell proliferation in the SHF and causes ectopic expression of known genes required for differentiation, whereas overexpression causes opposite expression changes of a subset of these genes. These findings for the first time delineate a potential molecular network downstream of Tbx1.

MATERIALS AND METHODS

Mouse mutants

Generation of Tbx1^+/-, BAC transgenic and Tbx1^flox/- mice have been described previously (Arnold et al., 2006; Merscher et al., 2001). T-Cre transgenic mice (Perantoni et al., 2005) were obtained from Dr. Mark Lewandoski. A ROSA26 reporter strain (Soriano, 1999) was obtained from Jackson Laboratories. Tbx1^+/- and BAC transgenic mice are congenic in the FVB genetic background. ROSA26 mice were maintained in the C57BL6 background. All the other strains were maintained in a mixed C57BL6/129 background.

Mouse embryo dissections

Mouse embryos in the FVB background at E8.75, E9.75 and E10.75 were isolated in cold PBS. Somite pairs were counted to define the stages: 10-12 pairs of somites were defined as E8.75; 23-25 pairs of somites were defined as E9.75 and 35-37 pairs of somites were defined as E10.75. The caudal pharyngeal region without the first pharyngeal arch and the neural tube was dissected and placed in RNAlater RNA stabilization reagent (QIAGEN) at 4 °C.

Total RNA preparation

To obtain enough RNA for the microarray hybridization experiments, dissected pharyngeal tissue from 5-10 embryos were pooled. The tissue was homogenized in Buffer RLT (QIAGEN). Total RNA was isolated with either RNeasy Mini Kit (QIAGEN) (for E9.75 and E10.75 tissue) or the RNeasy Micro Kit (QIAGEN) (for E8.75 tissue) according to the manufacturer’s protocol. Quality and quantity of total RNA was determined using an Agilent 2100 Bioanalyzer (Agilent) and a ND-1000 Spectrophotometer (NanoDrop) respectively.

Target preparation

For total RNA isolated from E9.75 or E10.75 tissue, target preparation was carried out according to Affymetrix’s One-Cycle Eukaryotic Target Labeling Assay. Specifically, using One-Cycle cDNA Synthesis Kit (Affymetrix), 1-5 μg total RNA was first reverse transcribed to obtain first-strand cDNA and then second-stranded cDNA was synthesized in an RNase H-mediated reaction. The double-stranded cDNA was purified with the Sample Cleanup Module (Affymetrix) and this served as a template in the subsequent in vitro transcription (IVT) reaction using the IVT Labeling Kit (Affymetrix). The resulting biotinylated cRNA targets were then cleaned up and fragmented with Sample Cleanup Module (Affymetrix).

For the E8.75 tissue, the biotinylated single-strand cDNA targets were amplified from 20-50 nanograms (ng) starting total RNA using The Ovation Biotin RNA Amplification and Labeling System (NuGEN).

Microarray processing

A total of 20 μg fragmented, biotin labeled cRNA (for E9.75 or E10.75 tissue) or 2.2μg cDNA (for E8.75 tissue) from the last step was hybridized to the GeneChip Test3 array (Affymetrix) to test the quality of the labeled target. Targets which passed the test were then hybridized to the GeneChip Mouse Genome 430A 2.0 Arrays (Affymetrix). Hybridization, washing, staining and scanning was performed in the AECOM Affymetrix Facility according to the Affymetrix manual.

Data analysis

GeneChip data were analyzed with ArrayAssist Lite (Stratagene). Briefly, the original Affymetrix GeneChip CEL files generated by the AECOM Affymetrix Facility were imported into ArrayAssist Lite and a master data table based on them was created by GC-RMA. Then the data was converted to logarithmic scale and the significance analysis was performed using two classes, unpaired t-test with a cut-off of fold change >2 and p-value <0.05.

Quantitative RT-PCR (qRT-PCR)

To obtain enough total RNA and minimize the variability of gene expression in individual embryos, each sample contained microdissected tissue from two to five embryos. 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). All values were normalized to the level of Gapdh, which was used as an internal control in each sample. For each experimental group, three to five samples were tested and the average of them was calculated. Statistical significance between different groups was calculated by unpaired Student’s t-test. Gene-specific primer pairs used for PCR amplification are listed in Table S4.

In situ hybridization (ISH)

Digoxigenin-labeled RNA probes for Tbx1 (Nowotschin et al., 2006), Tbx5 (Chapman et al., 1996), Gata4, Isl1 (Cai et al., 2003), and Nkx2-5 (Lyons et al., 1995) were prepared by standard methods (Roche). All the other probes were amplified by PCR from either mouse genomic DNA or cDNA, using the primers listed in Table S5. All forward primers contained T3 polymerase priming sequence and all reverse primers contained T7 polymerase priming sequence. The PCR product was purified by the PCR Purification Kit (Qiagen), and anti-sense RNA was in vitro transcribed and labeled with the T7 RNA polymerase (Roche) and DIG RNA Labeling Mix (Roche), using the Digoxigenin labeling method. Digoxigenin-labeled RNA probes were purified with mini Quick Spin RNA Columns (Roche). Whole mount in situ hybridization was performed as previously described (Nowotschin et al., 2006). To generate histological sections, embryos following whole mount in situ hybridization were dehydrated through a graded ethanol series (70% EtOH for 5 minutes, 95% EtOH for 10 minutes, 2 times 100% EtOH for 5 minutes, 100% EtOH for 10 minutes, 2 times Xylene for 15 minutes), embedded in paraffin wax and sectioned at a thickness of 10 μm in a microtome.

Histology

Mouse embryos were dissected in PBS (Mediatech) and fixed in 10% neutral buffered Formalin solution (Sigma) overnight. They were dehydrated through graded ethanol, embedded in paraffin and sectioned (7-10 μm) using a microtome.

In silico promoter analysis

The annotated mouse genomic sequences for Raldh2, Hod, and Nkx2-6 were examined for regions conserved among mammalian species (July 2007 Assembly; UCSC Genome Browser http://genome.ucsc.edu/). Most conserved blocks were located within 10 kb upstream or downstream of exons of the three. We searched for T-box binding sequences within each of the gene loci. The consensus sequences were fully conserved in mouse, rat, and human genomic DNA.

RESULTS

Expression profiling of the caudal pharyngeal apparatus in Tbx1^-/- mutants

To identify genes that act downstream of Tbx1 in the SHF, the caudal pharyngeal region was microdissected at E8.75 (10-12 somites), E9.75 (23-25 somites) and E10.75 (35-37 somites; see Fig. S1A in the supplementary material), in both wild type and Tbx1^-/- embryos. The three stages, coincident with highest Tbx1 expression levels, should provide a complete set of Tbx1-dependent genes. By quantitative measurements, we found the morphology of caudal pharyngeal region of Tbx1^-/- embryos was grossly normal at E8.75, though morphological defects due to hypoplasia of the caudal pharyngeal arches were observed at E9.75 and E10.75 (see Fig. S2 in the supplementary material). Labeled targets from three independent pools of RNA samples were hybridized to Affymetrix arrays, containing over 14,000 known mouse genes. With the criteria of fold change >2 and p-value < 0.05, 120, 89, and 113 probe sets were identified for stages of E8.75, E9.75, and E10.75 respectively (see Tables S1, S2, S3 in the supplementary material). The candidates included genes of all classes (Gene Ontology categories, GO; see Fig. S1B in the supplementary material). This analysis revealed some notable trends including a decrease in expression of genes encoding signaling molecules and increase in expression of genes encoding structural proteins, over time (see Fig. S1B in the supplementary material). Visible inspection of the gene lists (see Tables S1, S2, S3 in the supplementary material) revealed a strikingly large set of genes required for heart development altered in the mutant embryos, despite the fact that the heart was removed from the microdissected tissue (see Fig. S1A in the supplementary material).

The number of genes shared by different stages is summarized in a Venn diagram (see Fig. S1C in the supplementary material). Comparison of the gene lists revealed that some are shared by E8.75 and E9.75 or E9.75 and E10.75, but only two genes, Raldh2 (retinaldehyde dehydrogenase type 2, also named Aldh1a2) and Cpa1 (carboxypeptidase A1) were shared among all three stages, indicating a highly dynamic gene expression profile regulated by Tbx1 during pharyngeal development. Morphology alterations at E9.75 and E10.75 could also have accounted for some of the gene expression changes.

Validation of selected candidate genes

Many of the genes found have important functions in cardiac development, but were not previously linked to the SHF or Tbx1. Genes with potential direct relevance to the SHF are listed in Table 1. Some of these genes were chosen for further validation by qRT-PCR and ISH (Table 1). Three new pools of wild type and Tbx1^-/- embryo tissue were tested for each stage by qRT-PCR and p-values were calculated. Unlike the microarray experiments, all the samples for qRT-PCR analysis were prepared in the same way, making it possible to directly compare expression levels of candidate genes at all three stages. In both qRT-PCR and ISH experiments, Nkx2-5 served as a negative control because of its unchanged expression in the SHF of Tbx1 null embryos, while Raldh2 served as a positive control because its ectopic expression in the pharyngeal apparatus of Tbx1^-/- embryos has been reported at later stages (Guris et al., 2006; Ivins et al., 2005). As a prelude to ISH studies, the expression pattern of Tbx1 was assessed in detail and compared with those of selected genes. Co-expression with Tbx1 in the SHF was a determining factor for further analysis at multiple stages.

Table 1.

Selected Tbx1-dependent genes with a potential role in the SHF

			E8.75		E9.75		E10.75
Gene symbol	Probe set ID	GenBank ID	Fold change	p-value	Fold change	p-value	Fold change	p-value
Transcriptional regulation
Ankrd1	1420992_at	AK009959	2.01	0.231	-1.87	0.025	2.81	0.012
Gata4a,b	1418863_at	NM_008092	4.48	0.144	3.53	0.013	5.51	0.001
Gata5	1450126_at	BB447551	2.09	0.164	1.59	0.039	3.54	0.002
Gata6	1425463_at	BM214048	-1.07	0.778	1.57	0.077	2.30	0.014
	1425464_at	BM214048	1.38	0.020	1.76	0.003	2.75	0.003
Hoda,b	1428662_a_at	AK009007	-2.11	0.000	-1.63	0.015	1.00	0.990
Irx2	1426298_at	AF295369	-2.26	0.030	1.10	0.446	-1.05	0.705
Isl1a,b	1422720_at	BQ176915	-2.40	0.001	1.02	0.896	1.07	0.165
Myocda	1425978_at	AF384055	1.01	0.954	1.17	0.674	4.34	0.003
Nkx2-6a,b	1452018_at	AF045150	-4.24	0.004	-3.29	0.005	-1.47	0.026
Sox7a	1416564_at	NM_011446	2.19	0.009	1.15	0.370	1.80	0.055
T	1419304_at	NM_009309	1.47	0.459	2.24	0.029	1.19	0.303
Tbx5a,b	1425695_at	AI425735	2.59	0.161	3.50	0.001	3.57	0.001
Tbx18	1449871_at	NM_023814	1.84	0.108	3.08	0.045	2.48	0.002
Zfpm2a	1449314_at	NM_011766	4.47	0.103	2.73	0.003	3.54	0.005
Signaling
Bmp6a	1450759_at	NM_007556	-1.01	0.952	-2.06	0.006	1.28	0.185
Dkk1	1420360_at	NM_010051	-1.52	0.177	-2.10	0.002	-1.46	0.015
Wnt2	1449425_at	BC026373	3.33	0.061	5.01	0.001	5.39	0.006
Wnt11	1450772_at	NM_009519	1.00	0.650	1.25	0.311	2.25	0.016
Muscle contraction
Actc1a	1415927_at	NM_009608	3.01	0.251	1.60	0.021	2.72	0.007
Actg2a	1422340_a_at	NM_009610	1.16	0.256	1.19	0.206	2.32	0.024
Actn2	1448327_at	NM_033268	4.27	0.178	-1.46	0.157	2.97	0.012
Cnn1a	1417917_at	NM_009922	1.13	0.686	1.33	0.087	2.13	0.024
Mybpc3	1418551_at	NM_008653	2.00	0.308	1.05	0.778	2.29	0.006
Myh6a,b	1448826_at	BB481540	3.89	0.271	4.81	0.001	13.53	0.000
Myl4	1422580_at	NM_010858	2.36	0.222	1.05	0.858	7.08	0.004
Myl7a	1449071_at	NM_022879	1.80	0.256	1.17	0.327	7.23	0.002
Myom1	1420693_at	NM_010867	1.42	0.497	-1.58	0.006	3.12	0.013
Tnnc1	1418370_at	NM_009393	2.36	0.200	-1.64	0.017	4.13	0.000
Tnni1	1450813_a_at	NM_021467	1.34	0.333	1.20	0.212	3.17	0.006
Tnnt2	1424967_x_at	L47552	1.87	0.279	1.27	0.162	5.85	0.001
Ttn	1427446_s_at	BC025840	2.77	0.322	1.31	0.082	2.45	0.049
Metabolism
Raldh2a,b	1422789_at	NM_009022	2.45	0.035	4.70	0.016	2.11	0.005
Transport
Cmya1	1419220_at	NM_011724	1.22	0.724	-2.20	0.003	1.12	0.551

Fold change is the average of three replicates. Negative numbers mean downregulation in Tbx1 null embryos and positive numbers mean upregulation. p-value is calculated by Student’s t-test. The value is presented as 0.000 when it is less than 0.0005.

^avalidated by qRT-PCR (p<0.05).

^bvalidated by ISH.

Hod, Isl1, and Nkx2-6 are downregulated in the SHF of Tbx1^-/- embryos

Hod, also named Hop, encodes an unusual homeobox domain only protein expressed in the linear heart tube and SHF, required for forming the ventricular myocardium (Chen et al., 2002; Shin et al., 2002). We found that Hod was downregulated 2.11 fold at E8.75 by microarray analysis (Table 1), and confirmed by qRT-PCR analysis (Fig. 1A). ISH results from E8.5, E8.75, and E9.0 showed that Hod is expressed in both the pharyngeal arch core mesoderm and the SHF in wild type embryos (Fig. 1B,E,H), overlapping with Tbx1 expression (Fig. 1D,G). In contrast to the wild type embryos, Hod expression was not visible by whole mount ISH in Tbx1^-/- embryos at E8.5 (Fig. 1C). At E8.75, Hod expression was only observed in the pharyngeal arch core mesoderm, especially in the first arch mesoderm, but not in the SHF in Tbx1^-/- embryos (Fig. 1F). At E9.0, Tbx1^-/- embryos only showed Hod expression in the cranial part of SHF but not caudal part (Fig. 1I). Although there was a clear downregulation at E8.75, no significant difference in Hod expression between wild type and Tbx1^-/- embryos was observed after E9.75 by qRT-PCR (Fig. 1A). These results suggest that Tbx1 may be required for activation of Hod at the earliest stages of pharyngeal development, but not for its maintenance later on.

Fig. 1. Validation of altered expression for selected downregulated genes in the SHF of Tbx1^-/- embryos by qRT-PCR and ISH.

(A)Developmental time courses of the expression of Hod, Isl1, Nkx2-6, and Nkx2-5, which were generated from qRT-PCR data. For these and all time-course graphs in other figures: values are means of at least three samples and normalized by expression levels of wild type samples at E8.75. *p-value <0.05, **p-value<0.01 (calculated by two-tailed unpaired Student’s t-test). Error bars represent standard deviation. Note the significant decreased expression of Hod, Isl1, Nkx2-6 in Tbx1^-/- embryos at E8.75, while Nkx2-5 expression is not changed.

(B-I) Reduced Hod expression in the pharyngeal core mesoderm (white arrow) and SHF (black arrow) of Tbx1^-/- embryos (C,F,I) compared with wild type embryos (B,E,H) and its overlap with the Tbx1 expression domain in these regions of wild type embryos (D,G) at E8.5 (B,C), E8.75 (D-F), and E9.0 (G-I). B-F depict whole mount ISH analysis and G-I show sagittal sections cut from whole mount ISH stained embryos.

(J-O) Reduced Isl1 expression in the caudal part (black arrow) but not the cranial part (red arrow) of the SHF in Tbx1^-/- embryos (K,M,O) compared with wild type embryos (J,L,N) at E8.5 (J-M) and E9.0 (N,O). J-M are transverse sections and N, O are sagittal sections. Isl1 expression in the pharyngeal pouch endoderm (asterisk) also remains intact.

(P-S)Nkx2-6 expression in is absent in the caudal part of the SHF (arrow), while its expression in the pharyngeal pouch endoderm (asterisk) is still present in Tbx1^-/- embryos (Q) compared with wild type embryos (P) at E9.0. In contrast, Nkx2-5 expression has no difference in wild type (R) and Tbx1^-/- (S) embryos. All sections are sagittal.

Scale bars: 300 μm in B-I; 200 μm in J-S.

Isl1 is the key marker for the SHF and is required for OFT development (Cai et al., 2003). Inactivation of Isl1 results in major looping and OFT defects with missing segments of the heart (Cai et al., 2003). Isl1 expression was decreased 2.4 fold by microarray analysis and 2.08 fold by qRT-PCR in E8.75 Tbx1^-/- embryos (Table 1; Fig. 1A). ISH from E8.5 and E9.0 showed downregulation of Isl1 in the caudal part of the SHF of Tbx1^-/- embryos (Fig. 1J-O), suggesting precise spatial and temporal regulation by Tbx1. There was no change in the pattern of Isl1 expression in the pharyngeal endoderm in the same region, which is another domain of co-expression with Tbx1 (Fig. 1J-O).

The Nkx2-6 gene belongs to the same NK-2 homeobox gene family as Nkx2-5 and they have overlapping expression patterns in the pharyngeal endoderm and SHF (Biben et al., 1998; Nikolova et al., 1997). While Nkx2-5 was not changed, Nkx2-6 was significantly downregulated in Tbx1^-/- mutants at all three stages, with the greatest change at E8.75 (Table1), confirmed by qRT-PCR (Fig. 1A). ISH from E9.0 and E9.5 embryos showed that Nkx2-6 expression in the SHF was significantly reduced in Tbx1^-/- embryos, while its expression in the pharyngeal endoderm is unchanged (Fig. 1P,Q, and data not shown), similar to Isl1. Thus, Hod, Isl1, and Nkx2-6 were downregulated in the caudal SHF of Tbx1^-/- embryos at E8.5-E9.0. As expected, Nkx2-5 expression was not changed in the SHF (Fig. 1R,S).

Ectopic expression of Raldh2, Gata4 and Tbx5 in the SHF of Tbx1^-/- embryos

Numerous transcription factors form an interconnected network to regulate specification and myocardial differentiation of the heart (reviewed by Bruneau, 2002). Many of key genes in this network were ectopically expressed in Tbx1^-/- embryos (Table 1). In particular, three genes important for myocardial differentiation, Raldh2, Gata4, and Tbx5, showed increased expression at multiple stages by microarray (Table 1) and qRT-PCR analysis at E8.75 and later (Fig. 2A). To determine whether upregulation of these three genes is specific to the SHF, ISH experiments were performed.

Fig. 2. Validation of altered expression for selected upregulated genes in the SHF of Tbx1^-/-embryos by qRT-PCR and ISH.

(A) Developmental time courses of the expression of Raldh2, Gata4, and Tbx5, which were generated from qRT-PCR data. Note the significant increased expression of these three genes in Tbx1^-/- embryos at all three stages.

(B-F) Sagittal sectioned (C,D) or whole mount ISH (E,F) at E9.0 show expanded expression (arrow) of Raldh2 in the SHF of Tbx1^-/- embryos (D,F) compared with wild type embryos (C,E), which also overlap Tbx1 expression domain in the SHF of wild type embryos (B). Double-headed arrows in E and F indicate the relative distance between the cranial border of Raldh2 expression and the caudal border of the first arch.

(G-L) Sagittal sections from E9.0 whole mount ISH stained embryos show expression (arrow) of Gata4 (G,H), Tbx5 (I,J) and Hand1 (K,L) in the SHF of Tbx1^-/- embryos (H,J,L) compared with wild type embryos (G,I,K). Note the ectopic expression of Gata4 and Tbx5 but not Hand1 in Tbx1^-/- embryos.

Scale bars: 200 μm in B-F and K-L; 150 μm in G-J

Radlh2 is an enzyme required for the synthesis of retinoic acid (RA), one of the earliest morphogens needed for the specification of linear heart tube (reviewed by Xavier-Neto et al., 2001). Previous studies of Tbx1 mouse mutants showed ectopic expression of Raldh2 in the pharyngeal mesoderm of Tbx1^-/- embryos after E9.5, but it was not known whether the upregulation was due to gene activation or morphological alterations in these later stages (Guris et al., 2006; Ivins et al., 2005). ISH analysis showed that Raldh2 was expressed in the caudal part of the SHF at E9.0 in wild type embryos (Fig. 2C,E). In contrast, Raldh2 expression was expanded cranially at E9.0 in Tbx1^-/- embryos (Fig. 2D,F). Since there was no detectable morphological difference between wild type and Tbx1^-/- embryos at E9.0 (compare Fig. 2C and 2D, and also see Fig. S2 in the supplementary material), this indicates that ectopic expression of Raldh2 is likely a direct result of the Tbx1 null mutation and not secondary to morphological defects.

Gata4 is one of the earliest transcription factors expressed in the heart and is needed for activation of many myocardial differentiation genes (reviewed by Pikkarainen et al., 2004). There is a low level of Gata4 expression in the SHF of wild type embryos at E9.0-E9.5 (Fig. 2G and data not shown), while in Tbx1^-/- embryos, ectopic expression was found throughout the SHF region at E9.0 and more restricted to the cranial part of the SHF at E9.5 (Fig. 2H and data not shown).

Tbx5 haploinsufficiency is responsible for human Holt-Oram syndrome (Bruneau et al., 2001). Although Tbx5^-/- mice have atrial and left ventricular defects, the OFT and right ventricle are normal, indicating that Tbx5 is not required for the SHF development (Bruneau et al., 2001). ISH experiments also showed that Tbx5 was expressed very weakly in the SHF of wild type embryos (Fig. 2I), but was expressed ectopically in the caudal SHF of Tbx1^-/- embryos at E9.0 and E9.5 (Fig. 2J and data not shown), and as for the other genes, in regions containing Tbx1 expressing cells (Fig. 2B). In contrast, Hand1, another gene expressed in the heart tube, did not show any ectopic expression in the SHF of Tbx1^-/- embryos (Fig. 2K,L).

Ectopic expression of myogenic structural genes in the SHF

As the myocardial progenitor cells differentiate to cardiac muscle, specific muscle structural genes are expressed, such as myosins, actins, troponins, calponin, actinin and titin (reviewed by Franco et al., 1998). Since the heart itself was removed from the dissected tissues used for microarray analysis, increased expression of a large group of muscle contractile genes found by microarray in the homozygous null mutants, especially at E10.75, was unexpected (Table 1). We tested three of them, Actc1 (alpha cardiac actin), Myh6 (α-MHC, alpha myosin heavy chain), and Myl7 (Mlc2a, atrial myosin light chain 2) by qRT-PCR. The results showed increased expression of Actc1, Myh6, and Myl7 in Tbx1^-/- embryos at the three stages examined, with the greatest changes detected at E8.75 (Fig. 3A). Differences in the ability to detect increased expression earlier in development by qRT-PCR rather than by microarray reflect enhanced sensitivity of the qRT-PCR approach.

Fig. 3. Validation of altered expression for selected muscle structural genes in the SHF of Tbx1^-/- embryos by qRT-PCR and ISH.

(A)Developmental time courses of the expression of muscle structural genes Actc1, Myh6, and Myl7, which were generated from qRT-PCR data. Note the significant increased expression of these genes in Tbx1^-/- embryos at all three stages.

(B-C) Sagittal sections from E9.0 whole mount ISH stained embryos show increased expression of Myh6 (arrow) in the SHF of Tbx1^-/- embryos (C) compared with wild type embryos (B).

Scale bars: 150 μm.

ISH experiments were performed for Myh6 at E9.0 and increased expression of Myh6 was detected in the SHF of Tbx1^-/- embryos (Figure 3C). These results demonstrate muscle differentiation marker genes were ectopically expressed in the SHF of Tbx1^-/-embryos.

Gene expression changes are cell autonomous

Tbx1 is expressed in both pharyngeal endoderm and mesoderm and both expression domains seem important for OFT development (Arnold et al., 2006; Zhang et al., 2006). Thus it is important to determine whether Tbx1 expression in the SHF plays a cell-autonomous role in regulating above downstream genes in the SHF. For this question, we ablated Tbx1 by crossing Tbx1^flox/- mice (Arnold et al., 2006) with pan-mesodermal expressing T-Cre transgenic mice (Perantoni et al., 2005). Recombination occurred in mesoderm derived tissues including the SHF but not in the pharyngeal endoderm (see Fig. S3A in the supplementary material). We then assessed Tbx1 expression in conditional null mutant embryos at E9.5 (see Fig. S3C in the supplementary material) and E10.5 (data not shown) to determine whether it was properly inactivated. We found that Tbx1 was expressed normally in the pharyngeal endoderm as expected, and its expression was reduced in the mesodermal tissues but not eliminated (see Figure S3C in the supplementary material), indicating partial ablation. The embryos had hypoplastic 2nd, 3rd, and 4th pharyngeal arches (compare Fig. S3B with S3C in the supplementary material). These morphological defects were less severe than those in Tbx1^-/- and Mesp1-Cre-mediated conditional mutants, which completely lacked the caudal pharyngeal arches (Liao et al., 2004; Zhang et al., 2006). However, as in the Tbx1^-/- and Mesp1-Cre conditional mutants, all T-Cre-mediated conditional mutant embryos died at birth with a PTA and VSD (see Fig. S3D-G in the supplementary material), indicating that the Tbx1 expression level was below the dosage threshold needed for normal outflow tract development in the pharyngeal mesoderm.

We examined the expression patterns of Isl1, Nkx2-6, Raldh2, Tbx5 and Gata4 in the SHF of these embryos at E9.0 or E9.5. As before, we found Isl1 and Nkx2-6 were downregulated (Fig. 4A-D), while Raldh2, Tbx5 and Gata4 were upregulated (Fig. 4E-J). Our data suggests that alteration of expression of these genes in the SHF is cell autonomous.

Fig. 4. Partial inactivation of Tbx1 in mesoderm causes the same gene expression changes as those in Tbx1^-/- embryos.

Sagittal sectioned (A-D,G-J) or whole mount (E,F) ISH at E9.0 (A,B,E-H) or E9.5 (C,D,I,J) show the same expression changes (arrow) in Tbx1^flox/-;T-Cre embryos (B,D,F,H,J) compared with Tbx1^flox/+;T-Cre embryos (A,C,E,G,I) for Isl1 (A,B), Nkx2-6 (C,D), Raldh2 (E,F), Tbx5 (G,H), and Gata4 (I,J). Double-headed arrows indicate the relative distance between the cranial border of Raldh2 expression and the caudal border of the first arch.

Scale bars: 200 μm in A,B,E,F,G,H; 400 μm in C,D,I,J.

SHF-derived defects in TBX1 gain-of-function mutants

We previously generated bacterial artificial chromosome (BAC) transgenic mice overexpressing four transgenes including TBX1 (Funke et al., 2001; Merscher et al., 2001). These mice show reduced viability and malformations in the derivatives of the pharyngeal apparatus with variable expressivity. Most of these defects were rescued by crossing in the Tbx1 null allele, indicating the important role of Tbx1 dosage in the pharyngeal development (Liao et al., 2004). Occasionally intracardiac and OFT defects such as a VSD (Merscher et al., 2001), PTA, and a double outlet right ventricle (DORV) (Liao et al., 2004) were also found in these mice. Here we performed more detailed histological studies of the transgenic mice at E17.5 (n = 12) than was previously done (Merscher et al., 2001). The most common heart defect (5 of 12 embryos) was a thinning of the myocardial wall in the right ventricle, especially in the region juxtaposed to the interventricular septum (Fig. 5B,G,H). In the most severe cases, a muscular VSD was present (data not shown). A membranous VSD, typical in VCFS/DGS, occurred in 3 out of 12 embryos (Fig. 5E,G). Interestingly, it was always present together with a DORV, but not necessarily with myocardial hypoplasia (Fig. 5F,H). In contrast, none of wild type embryos we checked (n = 5) had any of these defects (Fig. 5A,C,D). Since SHF cells contribute to endothelial and myocardial components of the OFT, right ventricle and ventricular septum (Verzi et al., 2005), most of these defects could be due to the abnormal development of the SHF resulting from increased TBX1 dosage. We postulated that some of the genes found by microarray analysis may be altered in the transgenic embryos, and if so, it would further implicate their function in the SHF.

Fig. 5. SHF-derived cardiac defects and opposite gene expression changes in TBX1 gain-of-function mutants.

(A-H)SHF-derived defects in E17.5 BAC transgenic (TG) embryos overexpressing TBX1 shown by transverse sections. A, C, and D are wild type (WT) embryos. B shows the first BAC transgenic embryo with a thin myocardial wall of the right ventricle (boxed area and magnified in the insets at the top-right of A, B). E and F show ventricular septal defect (vsd) and double outlet right ventricle (dorv) but normal myocardium in the second BAC transgenic embryo. G and H show the third BAC transgenic embryo with ventricular septal defect (vsd), double outlet right ventricle (dorv), and right-ventricular myocardial hypoplasia (black arrow).

(I,J) Whole-mount ISH show Raldh2-expressing domain is reduced in the SHF of transgenic embryos (J) compared with wild type embryos (I) at E9.5. Double-headed arrows indicate the relative distance between the cranial border of expression and the caudal border of the first arch.

(K) qRT-PCR analysis of expressions of Nkx2-6, Raldh2, Tbx5, and Gata4 in BAC transgenic embryos at E8.75. Data displayed here is the mean of at least four independent samples and normalized by Gapdh expression. *p-value<0.01, calculated by two-tailed, unpaired Student’s t-test. The error bars indicate the standard deviation.

Scale bars: 1 mm in A-H and F-I; 200 μm in I,J.

Opposite gene expression changes in TBX1 gain-of-function mutants

To determine whether expression levels of the same genes that were altered in the Tbx1^-/- mutant were also changed in the TBX1 transgenic embryos, qRT-PCR studies were performed. Unlike Tbx1^-/- mice, TBX1 transgenic embryos do not have morphological changes in the pharyngeal apparatus (Liao et al., 2004), which makes them an ideal model to validate whether the expression changes observed in Tbx1^-/-embryos are morphology-independent. The expression levels of Hod, Isl1, Nkx2-6, Raldh2, Tbx5, Gata4, Actc1, and Myh6 in the caudal pharyngeal apparatus of E8.75 TBX1 transgenic embryos were assessed by qRT-PCR analysis. Among genes that were downregulated in Tbx1^-/- embryos, Nkx2-6 showed significantly increased expression in TBX1 transgenic embryos (Fig. 5K), while three upregulated genes, Raldh2, Tbx5, and Gata4 showed significantly decreased expression (Fig. 5K). There was a reduced area of Raldh2 expression in the SHF which was detected by ISH in transgenic embryos as compared to wild type embryos (Fig. 5I-J).

These results suggest that these four genes, Nkx2-6, Raldh2, Tbx5 and Gata4, are highly sensitive to altered Tbx1 dosage and some of them may contribute to the SHF-derived defects in the transgenic mice. On the other hand, reduction of expression of Tbx5 and Gata4, whose expression is already at low levels, may not directly cause the OFT defects present in the transgenic mice. However, these findings support our conclusion that most gene expression changes are a result of Tbx1 dosage alteration and not due to morphological differences. We did not find significant expression changes for the rest of the genes tested in TBX1 transgenic embryos (data not shown) indicating either that these genes are not dosage-sensitive, or their expression changes were not obvious in some transgenic embryos because the defects caused by increased Tbx1 dosage were milder than those in the null mutants.

DISCUSSION

Tbx1, the VCFS/DGS gene, is highly expressed in the SHF during embryonic development and Tbx1^-/- embryos have major OFT defects. In this study, we dissected the caudal pharyngeal region containing the SHF from wild type and Tbx1^-/- embryos to identify genetic pathways downstream of Tbx1 required for heart development. We identified a number of critical genes with altered expression in the SHF; most of them have not been previously linked to the SHF or Tbx1. What was particularly striking was a downregulation of Isl1, Hod and Nkx2-6, among others, coupled with ectopic expression of genes that promote differentiation, including Raldh2, Gata4, Tbx5, as well as muscle contractile genes in Tbx1^-/- mutants. In addition, the temporal pattern of expression of these genes indicates that Tbx1 regulates SHF development no later than E8.75, consistent with results from a study in which Tbx1 was ablated at different time points in development. It was shown that OFT defects in Tbx1 mutants occurred at E8.5-E9.5 (Xu et al., 2005). Recently, Tbx1 was found to function cell autonomously in the SHF for OFT development (Zhang et al., 2006). Similar gene expression changes were observed when a Tbx1 conditional allele was inactivated using a pan-mesodermal driver of Cre recombinase, T-Cre. In addition, opposite gene expression changes caused by overexpression of TBX1, associated with heart malformations support these findings. Our data for the first time provides a set of genes downstream of Tbx1 required for the morphogenesis of the cardiac outflow tract.

Tbx1 regulates the balance between myocardial proliferation and differentiation in the SHF through Isl1, Hod and Nkx2.6

Although both FHF and SHF cells are derived from a common pool of mesodermal progenitors in the early embryo, they obtain different cell fates and contribute to distinct regions of the heart during later development (Meilhac et al., 2004). For example, FHF cells start to differentiate into myocardial cells at the cardiac crescent stage, while SHF cells maintain an undifferentiated precursor state until they migrate into the heart (Cai et al., 2003). Therefore, it is important to maintain a proliferating as well as undifferentiated progenitor cell pool in the SHF for appropriate OFT development. Although reduced cell proliferation together with decreased cell contribution to the OFT has been found in Tbx1^-/- embryos (Xu et al., 2004; Zhang et al., 2006), the molecular mechanisms have remained elusive. Here we found that Isl1, Hod and Nkx2.6 are co-expressed with Tbx1 in the SHF and are downregulated in Tbx1^-/- embryos. These three genes, particularly the SHF marker, Isl1, have key roles in the development of the SHF. Inactivation of Isl1 results in failed OFT and right ventricle formation as well as reduced posterior atrial development (Cai et al., 2003). It is expressed strongly in SHF precursor cells, required for cell proliferation and is downregulated when cells differentiate into myoblasts (Cai et al., 2003). It has been hypothesized that Isl1 may act upstream of Tbx1, since Tbx1^-/- mutants have only OFT defects (Kelly, 2005). However, it is not unusual for transcription factors to have important functions at more than one stage of development, regulated by different mechanisms. Our data suggests that Tbx1 regulates Isl1 expression levels from E8.5 to E9.0, when the OFT and heart chambers are forming. Based upon the known function of Isl1 in promoting proliferation and perhaps also suppressing differentiation, we hypothesize that Tbx1 maintains Isl1 function in the SHF during OFT development as shown in the model in Figure 6.

Fig. 6. Model for the regulation of SHF development by Tbx1 through a genetic Network.

Tbx1 maintains the balance between proliferation and differentiation in the SHF by activating Isl1, Nkx2-6, and Hod, while at the same time inhibiting Raldh2-Gata4/Tbx5-Actc1/Myh6/Myl7 genetic pathway.

In addition to Isl1, two other genes, Hod and Nkx2-6, co-expressed with Tbx1 in the SHF, were downregulated in Tbx1^-/- embryos. Hod encodes an unusual homeodomain protein that functions at multiple stages in heart development. Some Hod^-/- embryos have extremely thin ventricular walls (Chen et al., 2002; Shin et al., 2002). In addition to a possible role in promoting proliferation in the SHF, Hod interacts with serum response factor (SRF) to inhibit differentiation by preventing SRF-dependent transcription of muscle structural genes, such as Actc1, which was upregulated in Tbx1^-/- embryos (Chen et al., 2002; Shin et al., 2002). Inactivation of Tbx1 causes delayed onset of Hod expression in the SHF, indicating Tbx1 may promote cell proliferation and inhibit differentiation through Hod (Figure 6). Hod is a direct downstream target gene of Nkx2-5 (Chen et al., 2002; Shin et al., 2002). We found that Nkx2-5 expression was unaltered in Tbx1^-/- embryos. This indicates that Tbx1 regulates Hod independent of its regulation by Nkx2-5. Of interest, we found another member of Nk-2 gene family was downregulated in Tbx1^-/- embryos.

Nkx2-6 belongs to the same Nk-2 family of homeobox transcription factors as Nkx2-5. However, unlike Nkx2-5 null mutants, which have arrested heart formation at the looping stage (Lyons et al., 1995), homozygous Nkx2-6 mutant mice are viable and normal (Tanaka et al., 2000). Interestingly, Nkx2-5/Nkx2-6 double-mutants show some severe pharyngeal and cardiac defects which were not found in any single mutants, indicating a functional overlap between these two genes (Tanaka et al., 2001). In addition, the strong conservation in the homeodomain between Nkx2-5 and Nkx2-6, as well as expression overlap between them in the SHF also suggests molecular and functional redundancy. Since Nkx2-5 acts in a genetic pathway upstream of Hod (Chen et al., 2002; Shin et al., 2002), and more recently Nkx2-1, another Nk-2 gene was also found to directly regulate Hod expression in the lung (Yin et al., 2006), it is possible that Tbx1 may regulate Hod through Nkx2-6 in the SHF. This hypothesis is also supported by the similar expression dynamics and pattern between Nkx2-6 and Hod in both wild type and Tbx1^-/- embryos revealed by qRT-PCR and ISH analysis. The role of Nkx2-6 in OFT development is further supported by the finding that an NKX2-6 mutation associates with a family with PTA (Heathcote et al., 2005).

Thus, Isl1, Hod and Nkx2-6 are important genes for the normal development of the OFT, but besides Isl1, they did not have defined roles in the SHF, especially downstream of Tbx1. Our data suggests that Tbx1 supports expression of these genes to maintain the balance between proliferation and differentiation of progenitor cells in the SHF (Fig. 6).

Tbx1 prevents premature differentiation in the SHF through the Raldh2 genetic pathway

Raldh2, Tbx5 and Gata4 play essential roles in regulating myocardial differentiation and patterning of the linear heart tube. In particular, Raldh2 is expressed in the posterior mesoderm precursors of the inflow part of the heart tube (Moss et al., 1998). Localized synthesis of retinoic acid (RA) by Raldh2 determines atrial cell fates, whereas ventricular cell fates occur in the absence of Raldh2 expression and RA signaling (reviewed by Xavier-Neto et al., 2001). Inhibition of endogenous RA synthesis represses expression of atrial-specific markers and results in an oversized ventricle and aplastic atria. Conversely, exogenous RA induces atrial-specific gene expression and produces a heart with hyperplastic atria but missing ventricles and OFT (Chazaud et al., 1999; Hochgreb et al., 2003; Xavier-Neto et al., 1999; Yutzey et al., 1994).

The two genes, Tbx5 and Gata4, physically interact to regulate transcription of downstream genes (Garg et al., 2003). Both are expressed in a posterior-to-anterior gradient in the linear heart tube during heart development (Bruneau et al., 1999; Molkentin et al., 1997) and both act downstream of Raldh2 (Kostetskii et al., 1999; Liberatore et al., 2000; Niederreither et al., 2001). In vitro experiments showed that Gata4 is necessary and sufficient to induce cardiac differentiation marked by a number of myocardial structural genes including several genes found in this study, Actc1, Myh6, Myl4, and Tnnc1 (Grepin et al., 1997; Grepin et al., 1995; Ip et al., 1994; Jiang and Evans, 1996; Molkentin et al., 1994; Sepulveda et al., 1998; Zeisberg et al., 2005). Similarly, Tbx5 overexpression in cell lines can also accelerate myocardial differentiation and induce Myh6 expression (Ching et al., 2005; Hiroi et al., 2001). The role of Tbx5 in anterior-posterior (AP) patterning of the heart is further suggested via mouse models. Tbx5^-/- mouse embryos have hypoplastic posterior heart structures including the left ventricle, atria, and inflow tract (Bruneau et al., 2001). Furthermore, ectopic overexpression of Tbx5 in both chick and mice results in loss of ventricular-specific gene expression and retardation of ventricular chamber morphogenesis (Liberatore et al., 2000; Takeuchi et al., 2003). Our data is consistent with Tbx1 acting to restrict myocardial differentiation in the SHF through a Raldh2-Tbx5/Gata4-Myh6/Myl7/Actc1 pathway, which are all ectopically expressed in the SHF of Tbx1^-/- embryos (Fig. 6).

Gene expression changes versus altered morphology

The caudal pharyngeal apparatus fails to develop in Tbx1^-/- embryos due to defects in neural crest migration (Moraes et al., 2005; Vitelli et al., 2002a). Seemingly, gene expression changes we found in Tbx1^-/- embryos could be caused by morphological defects in these embryos. There are several reasons why we believe that it is not the case. The first and most important is that Tbx1^-/- embryos at E8.75 did not yet have grossly detectable morphological defects though subtle changes in cell populations might have occurred. Second, if changes in gene expression were due to morphological defects in the caudal pharyngeal region of Tbx1^-/- embryos, we would expect to see a more general profile of genes with expression changes. However, according to our experimental results, the expression levels and patterns of many other SHF and myocardial marker genes such as Nkx2-5, Foxh1, Mef2c, and Hand1 (see Table S6 in the supplementary material) were not changed in Tbx1^-/- embryos, indicating very selective and specific effect. Third, Tbx1 is co-expressed with these genes in the SHF, indicating a possible role of Tbx1 in directly regulating transcription of a subset of these genes. Finally, we found the opposite expression changes for some of the same genes in BAC transgenic mice overexpressing TBX1, in the absence of morphological defects in the caudal pharyngeal apparatus. Thus, we believe that most of the changes observed at E8.75 and perhaps an important subset later, resulted from gene expression changes in the SHF cells and not due to gross alterations in cell populations.

We also noted some inconsistencies between our microarray data and a few previous reports about expression changes of Tbx1-regulated genes. For example, we previously showed that Pitx2 expression was downregulated while Isl1 expression did not change in the SHF of E8.0 Tbx1 -/- embryos (Nowotschin et al., 2006). Our microarray analysis found an increased level of Pitx2 expression at E9.75 (Table S2) which was not confirmed by qRT-PCR (data not shown). Decreased Isl1 expression at E8.75 (Table 1), might suggest different responses to Tbx1-regulation at different developmental stages. Another example is expression of Foxa2 and Fgf8, which was previously reported to be downregulated in the SHF of Tbx1 mutant embryos (Hu et al., 2004). Although none of them was found by our microarray experiments, qRT-PCR experiments for these two genes showed significant downregulation as previously reported (data not shown). This is why we validated all gene changes of interest by qRT-PCR and ISH studies.

Conclusions

In the model shown in Figure 6, we hypothesize that Tbx1 maintains the balance between the proliferation and differentiation of myocardial progenitor cells in the SHF by activating, either directly or indirectly, Isl1, Hod and Nkx2-6. A Raldh2-Tbx5/Gata4 genetic pathway, normally repressed by Tbx1, may also contribute to the role of Tbx1 in preventing premature differentiation of the SHF cells. Inactivation of Tbx1 in the pharyngeal mesoderm causes the abnormal expression of these genes, which further disrupts the appropriate proliferation-versus-differentiation balance required for normal contribution of SHF cells to the OFT myocardium, resulting in OFT defects. If the changes are cell autonomous, it is possible that some could be direct downstream transcriptional target genes of Tbx1. For example, Isl1, Gata4, and Tbx5 contain one or multiple T-box consensus binding sites in their promoter or enhancer regions (Cai et al., 2005; Heicklen-Klein and Evans, 2004; Sun et al., 2004). We also examined the DNA sequence surrounding the first exon of Raldh2 and found a conserved T-box consensus sequence present (see Fig. S4 in the supplementary material), suggesting that Tbx1 might be able to regulate Raldh2 expression levels directly.

Interestingly, for most of these genes, their expression changes more specifically occurred in the caudal/posterior part of the SHF region, which may be caused by stronger expression of Tbx1 itself in the caudal pharyngeal region (Fig. 2B). Another interpretation is that Tbx1 may play a role in regulating AP patterning of the SHF since some of these genes we found such as Raldh2, Tbx5, and Gata4 have well-known functions in regulating AP patterning of the heart tube, as we discussed above. Further detailed analysis will be necessary to address this question. In addition, characterization of additional genes found by microarray analysis would likely expand the genetic network described here. Finally, nucleotide variations in some of these genes might influence the expressivity of VCFS/DGS, thereby serving as genetic modifiers.