Tbx1 and Foxi3 genetically interact in the pharyngeal pouch endoderm in a mouse model for 22q11.2 deletion syndrome

Author summary The mechanisms required for segmentation of the pharyngeal apparatus (PA) to individual arches are not precisely delineated in mammalian species. Using mouse models, 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 during thymus and parathyroid gland development. When examining wildtype embryos, we found that each arch is surrounded by epithelial cells derived from the endoderm and ectoderm that undergo dynamic processes during PA segmentation. Invagination co-occurs with formation of multilayers of epithelia that become juxtaposed. The cells are then reorganized to form a dual-layer of tightly intercalated cells at the mature pouch-cleft junction. In Tbx1 and Foxi3 null mutant embryos, these processes are disrupted. Further, the endoderm cells form extensive multilayers in the region where cells should normally invaginate. When examining genes that may act downstream of Tbx1 and Foxi3 we found several including Notch pathway genes Jag1, Hes1, and Hey1 are downregulated in both mutant embryos. Together, we show that Tbx1 and Foxi3 are important for regulating PA segmentation through cellular and genetic mechanisms that may be critical in 22q11.2 deletion syndrome patients. 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.