Nkx2-1 has long been known to be essential for proper respiratory development, as its loss in mice results in an unseparated trachea with underdeveloped cystic lungs. However, how NKX2-1 regulates distinct cell fates in the proximal airways is unknown. While Nkx2-1 has been extensively studied for its role in lung adenocarcinomas, few studies have described the effects of Nkx2-1 loss on the trachea beyond its role in early tracheal separation morphogenesis. In previously published work we have shown that the majority of genes differentially expressed in the trachea and esophagus, during early foregut development are not regulated by NKX2-1 and maintain their expected dorsoventral expression patterns in the unseparated foregut upon NKX2-1 loss. These results suggested that Nkx2-1 may not be a master regulator of early respiratory fate, but may instead regulate specific cell fate programs during tracheal development. To address this question, we examined E16.5 and E18.5 Nkx2-1Δ/Δ and control mouse tracheas by immunofluorescent staining for tracheal cell types. Secretory cells (MUC5AC), multiciliated cells (acetylated α-TUBULIN and FOXJ1), and Club cells (SCGB1A1) were all found in Nkx2-1Δ/Δ ventral foreguts by immunofluorescence. While all expected proximal airway cell types were present in Nkx2-1Δ/Δ tracheas, a disproportionate number of multiciliated cells were present in the Nkx2-1Δ/Δ foregut compared to control. This was not a consequence of effects on early respiratory induction, because tamoxifen-induced loss of NKX2-1 in Nkx2-1CreER/lox embryos at E12.5 also resulted in overabundance of FOXJ1 positive cells at E16.5. Based on these results, while we conclude that NKX2-1 is not required to define the early proximal respiratory epithelial lineage, it may instead be required for specification of proper proportions and distribution of respiratory cell types.
