DiGeorge syndrome is characterized by cardiovascular, thymus and parathyroid defects and craniofacial anomalies, and is usually caused by a heterozygous deletion of chromosomal region 22q11.2 (del22q11) (ref. 1). A targeted, heterozygous deletion, named Df(16)1, encompassing around 1 megabase of the homologous region in mouse causes cardiovascular abnormalities characteristic of the human disease. Here we have used a combination of chromosome engineering and P1 artificial chromosome transgenesis to localize the haploinsufficient gene in the region, Tbx1. We show that Tbx1, a member of the T-box transcription factor family, is required for normal development of the pharyngeal arch arteries in a gene dosage-dependent manner. Deletion of one copy of Tbx1 affects the development of the fourth pharyngeal arch arteries, whereas homozygous mutation severely disrupts the pharyngeal arch artery system. Our data show that haploinsufficiency of Tbx1 is sufficient to generate at least one important component of the DiGeorge syndrome phenotype in mice, and demonstrate the suitability of the mouse for the genetic dissection of microdeletion syndromes.

Cyclin-dependent kinases (Cdks} are positive regulators of cell proliferation, whereas Cdk inhibitors {CKIs) inhibit proliferation. We describe a new CKI, p57 v'w2, which is related to p21 cn'1 and p27 gxP1. p57 gn'2 is a potent, tight-binding inhibitor of several G~ cyclin/Cdk complexes, and its binding is cyclin dependent.Unlike CIP1, KIP2 is not regulated by p53. Overexpression of p57 gn'e arrests cells in G~. p57 me2 proteins have a complex structure. Mouse p57/~n'2 consists of four structurally distinct domains: an amino-terminal Cdk inhibitory domain, a proline-rich domain, an acidic-repeat region, and a carboxy-terminal domain conserved with p27gn'L Human p57 ~2 appears to have conserved the amino-and carboxy-terminal domains but has replaced the internal regions with sequences containing proline-alanine repeats. In situ hybridization during mouse embryogenesis revealed that KIP2 mRNA displays a striking pattern of expression during development, showing high level expression in skeletal muscle, brain, heart, lungs, and eye. Most of the KIP2-expressing cells are terminally differentiated, suggesting that p57 ~n'2 is involved in decisions to exit the cell cycle during development and differentiation. Human KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it as a candidate tumor suppressor. The discovery of a new member of the p21 cn'l inhibitor family with novel structural features and expression patterns suggests a complex role for these proteins in cell cycle control and development.

Dysmorphogenesis of the cardiac outflow tract (OFT) causes many congenital heart defects, including those associated with DiGeorge syndrome. Genetic manipulation in the mouse and mutational analysis in patients have shown that Tbx1, a T-box transcription factor, has a key role in the pathogenesis of this syndrome. Here, we have dissected Tbx1 function during OFT development using genetically modified mice and tissue-specific deletion, and have defined a dual role for this protein in OFT morphogenesis. We show that Tbx1 regulates cell contribution to the OFT by supporting cell proliferation in the secondary heart field, a source of cells fated to the OFT. This process might be regulated in part by Fgf10, which we show for the first time to be a direct target of Tbx1 in vitro. We also show that Tbx1 expression is required in cells expressing Nkx2.5 for the formation of the aortopulmonary septum, which divides the aorta from the main pulmonary artery. These results explain why aortic arch patterning defects and OFT defects can occur independently in individuals with DiGeorge syndrome. Furthermore, our data link, for the first time, the function of the secondary heart field to congenital heart disease.Key words: Tbx1, Mouse, Outflow tract, DiGeorge syndrome SummaryTbx1 has a dual role in the morphogenesis of the cardiac outflow tract

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by hypoplastic or absent clavicles, large fontanelles, dental anomalies and delayed skeletal development. The phenotype is suggestive of a generalized defect in ossification and is one of the most common skeletal dysplasias not associated with disproportionate stature. To date, no genetic determinants of ossification have been identified. CCD has been mapped to chromosome 6p21, where CBFA1, a gene encoding OSF2/CBFA1, a transcriptional activator of osteoblast differentiation, has been localized. Here, we describe two de novo missense mutations, Met175Arg and Ser191Asn, in the OSF2/CBFA1 gene in two patients with CCD. These two mutations result in substitution of highly conserved amino acids in the DNA-binding domain. DNA-binding studies with the mutant polypeptides show that these amino acid substitutions abolish the DNA-binding ability of OSF2/CBFA1 to its known target sequence. Concurrent studies show that heterozygous nonsense mutations in OSF2/CBFA1 also result in CCD, while mice homozygous for the osf2/cbfa1 mull allele exhibit a more severe lethal phenotype. Thus, these results together suggest that CCD is produced by haploinsufficiency of OSF2/CBFA1 and provide direct genetic evidence that the phenotype is secondary to an alteration of osteoblast differentiation.

TBX1 is the major candidate gene for DiGeorge syndrome (DGS). Mouse studies have shown that the Tbx1 gene is haploinsufficient, as expected for a DGS candidate gene, and that it is required for the development of pharyngeal arches and pouches, as predicted by the DGS clinical phenotype. However, a detailed analysis of the cardiovascular phenotype associated with Tbx1 mutations has not been reported. Here we show that Tbx1 deficiency causes a number of distinct vascular and heart defects, suggesting multiple roles in cardiovascular development - specifically formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Comparison of phenotype and gene expression using a Tbx1-lacZ reporter allele supports a cell-autonomous function in the growth of the pharyngeal apparatus, and a cell non-autonomous function in the growth and early remodeling of the pharyngeal arch arteries. Our data do not support a direct role of neural crest cells in the pathogenesis of the Tbx1 mutant phenotype; however, these cells, and the cranial nerves, are misdirected. We hypothesize that this is due to the lack of a guidance role from the pouch endoderm, which is missing in these mutants.

No abstract

The LIM-homeodomain protein Lmx1b plays a central role in dorso-ventral patterning of the vertebrate limb. Targeted disruption of Lmx1b results in skeletal defects including hypoplastic nails, absent patellae and a unique form of renal dysplasia (see accompanying manuscript by H. Chen et al.; ref. 2). These features are reminiscent of the dominantly inherited skeletal malformation nail patella syndrome (NPS). We show that LMX1B maps to the NPS locus and that three independent NPS patients carry de novo heterozygous mutations in this gene. Functional studies show that one of these mutations disrupts sequence-specific DNA binding, while the other two mutations result in premature termination of translation. These data demonstrate a unique role for LMX1B in renal development and in patterning of the skeletal system, and suggest that alteration of Lmx1b/LMX1B function in mice and humans results in similar phenotypes. Furthermore, we provide evidence for the first described mutations in a LIM-homeodomain protein which account for an inherited form of abnormal skeletal patterning and renal failure.

The heterozygous chromosome deletion within the band 22q11 (del22q11) is an important cause of congenital cardiovascular defects. It is the genetic basis of DiGeorge syndrome and causes the most common deletion syndrome in humans. Because the deleted region is largely conserved in the mouse, we were able to engineer a chromosome deletion (Df1) spanning a segment of the murine region homologous to the human deleted region. Here we describe heterozygously deleted (Df1/+) mice with cardiovascular abnormalities of the same type as those associated with del22q11; we have traced the embryological origin of these abnormalities to defective development of the fourth pharyngeal arch arteries. Genetic complementation of the deletion using a chromosome duplicated for the Df1 DNA segment corrects the heart defects, indicating that they are caused by reduced dosage of genes located within Df1. The Df1/+ mouse model reveals the pathogenic basis of the most clinically severe aspect of DiGeorge syndrome and uncovers a new mechanism leading to aortic arch abnormalities. These mutants represent a mouse model of a human deletion syndrome generated by chromosome engineering.