Establishment of smooth muscle and cartilage juxtaposition in the developing mouse upper airways.

In the trachea and bronchi of the mouse, airway smooth muscle (SM) and cartilage are localized to complementary domains surrounding the airway epithelium. Proper juxtaposition of these tissues ensures a balance of elasticity and rigidity that is critical for effective air passage. It is unknown how this tissue complementation is established during development. Here we dissect the developmental relationship between these tissues by genetically disrupting SM formation (through Srf inactivation) or cartilage formation (through Sox9 inactivation) and assessing the impact on the remaining lineage. We found that, in the trachea and main bronchi, loss of SM or cartilage resulted in an increase in cell number of the remaining lineage, namely the cartilage or SM, respectively. However, only in the main bronchi, but not in the trachea, did the loss of SM or cartilage lead to a circumferential expansion of the remaining cartilage or SM domain, respectively. In addition to SM defects, cartilage-deficient tracheas displayed epithelial phenotypes, including decreased basal cell number, precocious club cell differentiation, and increased secretoglobin expression. These findings together delineate the mechanisms through which a cell-autonomous disruption of one structural tissue can have widespread consequences on upper airway function.

Smooth Muscle Differentiation Is Essential for Airway Size, Tracheal Cartilage Segmentation, but Dispensable for Epithelial Branching.

Airway smooth muscle is best known for its role as an airway constrictor in diseases such as asthma. However, its function in lung development is debated. A prevalent model, supported by in vitro data, posits that airway smooth muscle promotes lung branching through peristalsis and pushing intraluminal fluid to branching tips. Here, we test this model in vivo by inactivating Myocardin, which prevented airway smooth muscle differentiation. We found that Myocardin mutants show normal branching, despite the absence of peristalsis. In contrast, tracheal cartilage, vasculature, and neural innervation patterns were all disrupted. Furthermore, airway diameter is reduced in the mutant, counter to the expectation that the absence of smooth muscle constriction would lead to a more relaxed and thereby wider airway. These findings together demonstrate that during development, while airway smooth muscle is dispensable for epithelial branching, it is integral for building the tracheal architecture and promoting airway growth.

Crouzon syndrome mouse model exhibits cartilage hyperproliferation and defective segmentation in the developing trachea.

Crouzon syndrome is the result of a gain-of-function point mutation in FGFR2. Mimicking the human mutation, a mouse model of Crouzon syndrome (Fgfr2342Y) recapitulates patient deformities, including failed tracheal cartilage segmentation, resulting in a cartilaginous sleeve in the homozygous mutants. We found that the Fgfr2C342Y/C342Y mutants exhibited an increase in chondrocytes prior to segmentation. This increase is due at least in part to over proliferation. Genetic ablation of chondrocytes in the mutant led to restoration of segmentation in the lateral but not central portion of the trachea. These results suggest that in the Fgfr2C342Y/C342Y mutants, increased cartilage cell proliferation precedes and contributes to the disruption of cartilage segmentation in the developing trachea.