Inactivation of Tbx 1 in the pharyngeal endoderm results in 22 q 11 DS malformations

INTRODUCTION 22q11DS is the most common microdeletion syndrome in humans, occurring in 1/4000 live births (Burn and Goodship, 1996). Most patients have craniofacial anomalies, thymus gland hypoplasia or aplasia, hypocalcaemia and cardiac outflow tract defects (DiGeorge, 1965; Shprintzen et al., 1978). The affected tissues and organs derive from the pharyngeal apparatus, a transient embryonic structure lateral to the developing head. In mammalian species, the pharyngeal apparatus is segmented into five bilaterally symmetrical arches by the juxtaposition of the pharyngeal endoderm (PE) and ectodermal clefts. Neural crest cells (NCCs) migrate from the rhombomeres of the neural tube through the splanchnic mesenchyme to surround the core mesoderm of the pharyngeal arches (Bockman et al., 1989; Le Lievre and Le Douarin, 1975). Tissue interactions between the mesenchyme and the epithelia, including the cleft ectoderm and pouch endoderm, are required to form all of the derivatives of pharyngeal arches (Graham, 2003). Numerous studies have emphasized the role of NCCs in the patterning of the pharyngeal arches (PAs). Classic ablation studies in chick have demonstrated that NCC removal results in abnormal development of the pharyngeal apparatus (Bockman et al., 1990; Kirby et al., 1983; Kirby and Waldo, 1990). More specifically, transplantation studies in chick embryos have shown that three different streams of NCCs migrate into the pharyngeal apparatus. Each stream provides the individual arches with a distinct identity (Noden, 1983). However, more recent experiments have demonstrated that individual arches can be formed and regionalized in the absence of NCCs (Veitch et al., 1999). Consequently, the role of all three germ layers in pharyngeal patterning has been reassessed, and it is now becoming clear that the PE may play a central role in the development of the pharyngeal apparatus. Part of its role may be in forming pouches, thereby segmenting the pharyngeal apparatus, while its other function may be in providing specific signals to surrounding tissues (Graham, 2003; Piotrowski et al., 2003; Piotrowski and Nusslein-Volhard, 2000). For example, it has been shown in chick embryos that NCCs respond to patterning cues produced by the PE, and that PE ablation at early developmental stages results in the loss of NCC-derived skeletal elements (Couly et al., 2002). However, most of the PE-specific signals required for patterning remain elusive. Evidence pointing to the essential role of endodermal cells in patterning comes from Tbx1 inactivation studies in the mouse. TBX1, a member of the T-box containing family of transcription factors, is the candidate gene most likely to be responsible for the pharyngeal arch-derived defects observed in individuals with 22q11DS (Jerome and Papaioannou, 2001; Lindsay and Baldini, 2001; Merscher et al., 2001). Throughout murine embryogenesis, Tbx1 is expressed dynamically in multiple adjacent tissues relevant to the pharyngeal malformations in 22q11DS individuals. During early embryonic development (E8.5-E9.5) Tbx1 is expressed in the splanchnic mesoderm ventral to the developing pharyngeal apparatus (Chapman et al., 1996). Later (E9.5-E11.5), Tbx1 is expressed in both the PE and the core mesoderm (Chapman et al., 1996; Vitelli et al., 2002a; Yamagishi et al., 2003). However, Tbx1 is not expressed in the NCC mesenchyme, despite the fact NCC ablation in the chick phenocopies the Tbx1 homozygous null mutant phenotype (Bockman et al., 1990; Kirby et al., 1983; Kirby and Waldo, 1990). This suggests non cell-autonomous roles for the gene (Bockman and Kirby, 1989; Vitelli et al., 2002a; Xu et al., 2004). Further support for indirect roles of Tbx1 in NCC development derives from studies of Tbx1 null mutations in mice. Tbx1 heterozygous mutant mice survive in relatively normal Mendelian Inactivation of Tbx1 in the pharyngeal endoderm results in 22q11DS malformations