We investigated whether Tbx1 , the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3 , both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1 +/- ;Foxi3 +/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1 Cre and Sox17 2A-iCre/+ null mutant embryos. While Tbx1 Cre/+ ;Foxi3 f/f embryos had absent thymus and parathyroid glands, Foxi3 -/- and Sox17 2A-iCre/+ ;Foxi3 f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1 Cre/+ ;Foxi3 f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3 -/- , Sox17 2A-iCre/+ ;Foxi3 f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1 , Hes1 , and Hey1 , suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.

The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/β-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in β-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of β-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, β-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.

Non-allelic homologous recombination events on chromosome 22q11.2 during meiosis can result in either the deletion (22q11.2DS) or duplication (22q11.2DupS) syndrome. Although the spectrum and frequency of congenital heart disease (CHD) are known for 22q11.2DS, there is less known for 22q11.2DupS. We now evaluated cardiac phenotypes in 235 subjects with 22q11.2DupS including 102 subjects we collected and 133 subjects that were previously reported as a confirmation and found 25% have CHD, mostly affecting the cardiac outflow tract (OFT). Previous studies have shown that global loss or gain of function (LOF; GOF) of mouse Tbx1, encoding a T-box transcription factor mapping to the region of synteny to 22q11.2, results in similar OFT defects. To further evaluate Tbx1 function in the progenitor cells forming the cardiac OFT, termed the anterior heart field, Tbx1 was overexpressed using the Mef2c-AHF-Cre driver (Tbx1 GOF). Here we found that all resulting conditional GOF embryos had a persistent truncus arteriosus (PTA), similar to what was previously reported for conditional Tbx1 LOF mutant embryos. To understand the basis for the PTA in the conditional GOF embryos, we found that proliferation in the Mef2c-AHF-Cre lineage cells before migrating to the heart, was reduced and critical genes were oppositely changed in this tissue in Tbx1 GOF embryos versus conditional LOF embryos. These results suggest that a major function of TBX1 in the AHF is to maintain the normal balance of expression of key cardiac developmental genes required to form the aorta and pulmonary trunk, which is disrupted in 22q11.2DS and 22q11.2DupS.

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SummaryThe mechanisms required for segmentation of the pharyngeal apparatus to individual arches are not precisely delineated in mammalian species. Here, using conditional mutagenesis, we found that two transcription factor genes, Tbx1, the gene for 22q11.2 deletion syndrome and Foxi3, genetically interact in the third pharyngeal pouch endoderm for thymus and parathyroid gland development. We found that Tbx1 is autonomously required for the endoderm to form a temporary multilayered epithelium while invaginating. E-cadherin for adherens junctions remains expressed and cells in the apical boundary express ZO-1. Foxi3 is required autonomously to modulate proliferation and promote later restoration of the endoderm to a monolayer once the epithelia meet after invagination. Completion of this process cooccurs with expression of Alcam needed to stabilize adherens junctions and extracellular, Fibronectin. These processes are required in the third pharyngeal pouch to form the thymus and parathyroid glands, disrupted in 22q11.2 deletion syndrome patients.