TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5-3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.     

Fgf8 expression in the Tbx1 domain causes skeletal abnormalities and modifies the aortic arch but not the outflow tract phenotype of Tbx1 mutants

Fgf8 and Tbx1 have been shown to interact in patterning the aortic arch, and both genes are required in formation and growth of the outflow tract of the heart. However, the nature of the interaction of the two genes is unclear. We have utilized a novel Tbx1(Fgf8) allele which drives Fgf8 expression in Tbx1-positive cells and an inducible Cre-LoxP recombination system to address the role of Fgf8 in Tbx1 positive cells in modulating cardiovascular development. Results support a requirement of Fgf8 in Tbx1 expressing cells to finely control patterning of the aortic arch and great arteries specifically during the pharyngeal arch artery remodeling process and indicate that the endoderm is the most likely site of this interaction. Furthermore, our data suggest that Fgf8 and Tbx1 play independent roles in regulating outflow tract development. This finding is clinically relevant since TBX1 is the candidate for DGS/VCFS, characterized clinically by variable expressivity and reduced penetrance of cardiovascular defects; Fgf8 gene variants may provide molecular clues to this variability.     

Isolation and characterization of a novel gene containing WD40 repeats from the region deleted in velo-cardio-facial/DiGeorge syndrome on chromosome 22q11

Three congenital disorders, cat-eye syndrome (CES), der(22) syndrome, and velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), result from tetrasomy, trisomy, and monosomy, respectively, of part of 22q11. They share a 1.5-Mb region of overlap, which contains 24 known genes. Although the region has been sequenced and extensively analyzed, it is expected to contain additional genes, which have thus far escaped identification. To understand completely the molecular etiology of VCFS/DGS, der(22) syndrome, and CES, it is essential to isolate all genes in the interval. We have identified and characterized a novel human gene, located within the 1.5-Mb region deleted in VCFS/DGS, trisomic in der(22) syndrome and tetrasomic in CES. The deduced amino acid sequence of the human gene and its mouse homologue contain several WD40 repeats, but lack homology to known proteins. We termed this gene WDR14 (WD40 repeat-containing gene deleted in VCFS). It is expressed in a variety of human and mouse adult and fetal tissues with substantial expression levels in the adult thymus, an organ hypoplastic in VCFS/DGS.     

22q11 deletions in isolated and syndromic patients with tetralogy of Fallot

Tetralogy of Fallot (TF) is a congenital conotruncal heart defect commonly found in DiGeorge (DGS) and velo-cardio-facial (VCFS) syndromes. The deletion of chromosome 22q11 (de122q11) is a well established cause of DGS and VCFS, and it has been demonstrated also in sporadic or familial cases of TF. In order to investigate the prevalence of de122q11 in patients with TF, we analyzed the DNA of 137 consecutive patients with syndromic and isolated TF, using the HD7k probe, which detects hemizygosity for the D22S134 locus. De122q11 has been detected in 11/26 (42%) syndromic patients. Evidence for hemizygosity was obtained in all patients with DGS and in 8/15 patients with VCFS. None of the 107 patients with isolated TF had de122q11. Our experience suggests that children with TF and de122q11 always present major or minor extracardiac anomalies. These features, including subtle facial dysmorphisms, should be checked routinely in patients with TF and other conotruncal heart defects.     

Multiple roles and interactions of tbx4 and tbx5 in development of the respiratory system

Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.     

The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro.

Hox 7.1 is a murine homeobox-containing gene expressed in a range of neural-crest-derived tissues and areas of putative epithelial-mesenchymal interactions during embryogenesis. We have examined the expression of Hox 7.1 during craniofacial development in the mouse embryo between days 8 and 16 of development. Whereas facial expression at day 10 of gestation is broadly localised in the neural-crest-derived mesenchyme of the medial nasal, lateral nasal, maxillary and mandibular processes, by day 12 expression is restricted to the mesenchyme immediately surrounding the developing tooth germs in the maxillary and mandibular processes. Hox 7.1 expression in the mesenchyme of the dental papilla and follicle is maximal at the cap stage of development and progressively declines in the bell stage prior to differentiation of odontoblasts and ameloblasts. Hox 7.1 expression in tooth germs is independent of overall embryonic stage of development but is dependent on stage of development of the individual tooth. Similar patterns of transient Hox 7.1 expression can also be detected in tooth germs in vitro in organ cultures of day 11 first branchial arch explants cultured for up to 7 days. Hox 7.1 is also expressed early in development (days 10/11) in the epithelium of the developing anterior pituitary (Rathke's pouch), the connective tissue capsule and meninges of the developing brain, and specific regions of neuroepithelium in the developing brain.     

Tbx20 regulation of cardiac cell proliferation and lineage specialization during embryonic and fetal development in vivo

TBX20 gain-of-function mutations in humans are associated with congenital heart malformations and myocardial defects. However the effects of increased Tbx20 function during cardiac chamber development and maturation have not been reported previously. CAG-CAT-Tbx20 transgenic mice were generated for Cre-dependent induction of Tbx20 in myocardial lineages in the developing heart. βMHCCre-mediated overexpression of Tbx20 in fetal ventricular cardiomyocytes results in increased thickness of compact myocardium, induction of cardiomyocyte proliferation, and increased expression of Bmp10 and pSmad1/5/8 at embryonic day (E) 14.5. βMHCCre-mediated Tbx20 overexpression also leads to increased expression of cardiac conduction system (CCS) genes Tbx5, Cx40, and Cx43 throughout the ventricular myocardium. In contrast, Nkx2.5Cre mediated overexpression of Tbx20 in the embryonic heart results in reduced cardiomyocyte proliferation, increased expression of a cell cycle inhibitor, p21(CIP1), and decreased expression of Tbx2, Tbx5, and N-myc1 at E9.5, concomitant with decreased phospho-ERK1/2 expression. Together, these analyses demonstrate that Tbx20 differentially regulates cell proliferation and cardiac lineage specification in embryonic versus fetal cardiomyocytes. Induction of pSmad1/5/8 at E14.5 and inhibition of dpERK expression at E9.5 are consistent with selective Tbx20 regulation of these pathways in association with stage-specific effects on cardiomyocyte proliferation. Together, these in vivo data support distinct functions for Tbx20 in regulation of cardiomyocyte lineage maturation and cell proliferation at embryonic and fetal stages of heart development.     

T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis

Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. We provide evidence that Tbx2 is a direct target for repression by Tbx20 in developing heart. We have also found that Tbx2 directly binds to the Nmyc1 promoter in developing heart, and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. Our data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. We present a model whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, we have found that Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development.     

Endothelial neuropilin disruption in mice causes DiGeorge syndrome-like malformations via mechanisms distinct to those caused by loss of Tbx1

The spectrum of human congenital malformations known as DiGeorge syndrome (DGS) is replicated in mice by mutation of Tbx1. Vegfa has been proposed as a modifier of DGS, based in part on the occurrence of comparable phenotypes in Tbx1 and Vegfa mutant mice. Many additional genes have been shown to cause DGS-like phenotypes in mice when mutated; these generally intersect in some manner with Tbx1, and therefore impact the same developmental processes in which Tbx1 itself is involved. In this study, using Tie2Cre, we show that endothelial-specific mutation of the gene encoding the VEGFA coreceptor neuropilin-1 (Nrp1) also replicates the most prominent terminal phenotypes that typify DGS. However, the developmental etiologies of these defects are fundamentally different from those caused by absence of TBX1. In Tie2Cre/Nrp1 mutants, initial pharyngeal organization is normal but subsequent pharyngeal organ growth is impaired, second heart field differentiation is normal but cardiac outflow tract cushion organization is distorted, neural crest cell migration is normal, and palatal mesenchyme proliferation is impaired with no change in apoptosis. Our results demonstrate that impairment of VEGF-dependent endothelial pathways leads to a spectrum of DiGeorge syndrome-type malformations, through processes that are distinguishable from those controlled by Tbx1.     

Cell proliferation during formation of the embryonic facial primordia.

Cell proliferation of mesenchyme in the developing primary palate of the chick embryo was analyzed by tritiated thymidine autoradiography. Pulse labeling, repeated labeling, and label dilution techniques were employed to determine generation times, transit times, growth fractions, and other parameters of the cell cycle. In vivo and in vitro studies were performed to evaluate the role of tissue interactions during outgrowth of the facial primordia. These studies indicated that initially, during early stages of primary palate formation, virtually all mesenchymal cells are in the division cycle with relatively short generation times. As development proceeds, mesenchymal cell populations in the facial primordia, such as the maxillary process, retain cycle characteristics comparable to those of the progenitor cell populations. In regions adjacent to the facial primordia, such as the roof of the stomodeum, cell cycle times become more heterogeneous and result in removal of cells from rapidly cycling cell populations into subpopulations that are cycling more slowly and that, in some instances, become quiescent. Regional analysis of cell proliferation in the maxillary process indicated that growth rates of mesenchyme differ based on proximity to the overlying epithelium. Correlative in vitro studies of epithelial-mesenchymal separation and recombination experiments in organ culture revealed that the viability of mesenchyme was dependent on the presence of epithelium and that this effect was strongly stage-dependent. These and other results lead us to the conclusion that epithelial-mesenchymal interaction is significant to the maintenance of growth rates in the facial primordia and that the effects observed are mediated, at least in part, by developmental signals at the epithelial-mesenchymal interface.     

High-density single nucleotide polymorphism array analysis in patients with germline deletions of 22q11.2 and malignant rhabdoid tumor

Malignant rhabdoid tumors are highly aggressive neoplasms found primarily in infants and young children. The majority of rhabdoid tumors arise as a result of homozygous inactivating deletions or mutations of the INI1 gene located in chromosome band 22q11.2. Germline mutations of INI1 predispose to the development of rhabdoid tumors of the brain, kidney and extra-renal tissues, consistent with its function as a tumor suppressor gene. We now describe five patients with germline deletions in chromosome band 22q11.2 that included the INI1 gene locus, leading to the development of rhabdoid tumors. Two patients had phenotypic findings that were suggestive but not diagnostic for DiGeorge/Velocardiofacial syndrome (DGS/VCFS). The other three infants had highly aggressive disease with multiple tumors at the time of presentation. The extent of the deletions was determined by fluorescence in situ hybridization and high-density oligonucleotide based single nucleotide polymorphism arrays. The deletions in the two patients with features of DGS/VCFS were distal to the region typically deleted in patients with this genetic disorder. The three infants with multiple primary tumors had smaller but overlapping deletions, primarily involving INI1. The data suggest that the mechanisms underlying the deletions in these patients may be similar to those that lead to DGS/VCFS, as they also appear to be mediated by related, low copy repeats (LCRs) in 22q11.2. These are the first reported cases in which an association has been established between recurrent, interstitial deletions mediated by LCRs in 22q11.2 and a predisposition to cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

UFD1L and CDC45L: a role in DiGeorge syndrome and related phenotypes?

Molecular genetics is contributing to the understanding of normal and abnormal cardiovascular development and morphogenesis. Deletions of chromosome 22q11.2 have been associated with distinct phenotypes that result from a failure to form derivatives of third and fourth branchial arches, including DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS). The biochemical mechanisms underlying these phenotypes remain undetermined. A recent study provides new insight into the mechanism by which gene deletions produce the DGS and VCFS phenotypes.     

Genomic disorders on 22q11

The 22q11 region is involved in chromosomal rearrangements that lead to altered gene dosage, resulting in genomic disorders that are characterized by mental retardation and/or congenital malformations. Three such disorders-cat-eye syndrome (CES), der(22) syndrome, and velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS)-are associated with four, three, and one dose, respectively, of parts of 22q11. The critical region for CES lies centromeric to the deletion region of VCFS/DGS, although, in some cases, the extra material in CES extends across the VCFS/DGS region. The der(22) syndrome region overlaps both the CES region and the VCFS/DGS region. Molecular approaches have revealed a set of common chromosome breakpoints that are shared between the three disorders, implicating specific mechanisms that cause these rearrangements. Most VCFS/DGS and CES rearrangements are likely to occur by homologous recombination events between blocks of low-copy repeats (e.g., LCR22), whereas nonhomologous recombination mechanisms lead to the constitutional t(11;22) translocation. Meiotic nondisjunction events in carriers of the t(11;22) translocation can then lead to offspring with der(22) syndrome. The molecular basis of the clinical phenotype of these genomic disorders has also begun to be addressed. Analysis of both the genomic sequence for the 22q11 interval and the orthologous regions in the mouse has identified >24 genes that are shared between VCFS/DGS and der(22) syndrome and has identified 14 putative genes that are shared between CES and der(22) syndrome. The ability to manipulate the mouse genome aids in the identification of candidate genes in these three syndromes. Research on genomic disorders on 22q11 will continue to expand our knowledge of the mechanisms of chromosomal rearrangements and the molecular basis of their phenotypic consequences.