Multiple Roles and Interactions of <em>Tbx4</em> and <em>Tbx5</em> in Development of the Respiratory System

Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, 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 Bmp4 and Spry2, in the epithelium 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.呼吸系统的正常发育对机体生存至关重要，且受多基因与信号通路的协同调控。Tbx4与Tbx5均在发育中肺与气管的间充质（mesenchyme）内表达；尽管已知上皮组织（epithelium）的发育需要多种基因参与，但目前仅间充质中的成纤维细胞生长因子（Fibroblast Growth Factors, FGFs）得到了较为充分的研究。本研究利用条件突变等位基因与两种不同的Cre重组酶（Cre recombinase）转基因品系，探究了Tbx4与Tbx5在肺与气管发育过程中的功能。Tbx5的缺失会导致器官培养模型中单侧肺芽特化丧失，且气管特化完全缺失。Tbx4与Tbx5双缺陷突变体在妊娠中期出现肺分支发育严重受限的表型。与该发育缺陷相一致，上皮组织中间充质标志物Wnt2、Fgf10，以及Fgf10的下游靶基因Bmp4与Spry2的表达均出现下调。当Tbx4与Tbx5完全缺失时，体外培养的肺分支发育会完全停滞。肺特异性Tbx4杂合子；Tbx5条件性敲除小鼠会因呼吸窘迫在出生后迅速死亡。这些新生幼鼠的肺体积偏小，且气管/支气管软骨环出现严重结构紊乱。作为软骨形成的主调控因子，Sox9在气管内的表达并无异常，但间充质细胞无法发生正常凝聚，因此幼鼠出生时无法正常发育为软骨。Tbx4；Tbx5双杂合突变体则表现为肺分支减少、气管软骨环数量降低，这提示二者存在遗传互作。最后，本研究证实Tbx4与Tbx5在肺生长及分支过程中与Fgf10存在调控互作，但在气管/支气管软骨发育过程中并无此类互作。