Development of Vessels, Airways and Cartilage Rings

Tbx4 and Tbx5 are two closely related genes that belong to the T-box family of transcription factor genes. Loss of Tbx4 results in absence of chorio-allantoic fusion and a failure of formation of the primary vascular plexus of the allantois leading to embryonic death at E10.5. Using a candidate gene approach we identified a number of genes downstream of Tbx4 in the allantois including, extracellular matrix molecules Vcan, Has2, Itgα5; transcription factors Snai1 and Twist, and signaling molecules Bmp2, Bmp7, Notch2, Jag1 and Wnt2In addition, we show that the canonical Wnt signaling pathway contributes to the vessel-forming potential of the allantois. Ex vivo, the Tbx4 mutant phenotype can be rescued using agonists of the Wnt signaling pathway and an inhibitor of the canonical Wnt signaling pathway phenocopies the Tbx4mutant phenotype in wildtype allantoises. In vivo, Tbx4 and Wnt2 double heterozygous placentas show decreased vasculature suggesting interactions between Tbx4 and the canonical Wnt signaling pathway in the process of allantois-derived blood vessel formation. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing respiratory system. Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Although many genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. 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 in the epithelium, Bmp4 and Spry2, 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.