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

Abstract

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.airway development | tracheomalacia T he trachea and the main bronchi, here collectively termed the upper airways, are essential conduits of air before and after gas exchange (1, 2). Normal upper airways offer the correct balance of elasticity and rigidity, which together maintain air pressure and prevent airway collapse. The elasticity is primarily provided by smooth muscle (SM) and the rigidity by cartilage. The balance is achieved via a precise juxtaposition of these tissues that together encircle and support the endoderm-derived upper airway epithelium. Clinical evidence suggests that malformation of the SM or cartilage leads to respiratory deficiencies. For example, patients with tracheomalacia and bronchomalacia exhibit airway collapse upon exhalation as a result of softened and disorganized cartilage (3). This collapse causes breathing difficulties that contribute to sleep apnea, respiratory infections, and possibly sudden infant death (4). Despite the importance of a proper SM and cartilage balance, how it is achieved during development is not understood.In mouse upper airways, SM is localized to the dorsal side of the trachea and the medial (inner) sides of the main bronchi. The lower conducting airways lack cartilage and instead SM surrounds the circumference of the epithelial tube. Data suggest that airway SM develops from fibroblast growth factor 10 (Fgf10)-expressing cells found in the distal mesenchyme (5). As the epithelial buds elongate, the Fgf10-expressing cells are thought to translocate proximally along the airway and begin to express SM-specific genes, including SM α-actin-2 (Acta2, also known as SM actin), one of the earliest markers of SM. Additionally, a recent study has u...

“Endodermal Wnt signaling is required for tracheal cartilage formation”

“…In peripheral lung, Wnt7b and Wnt2 play essential roles in establishment of vascular and airway smooth muscle (Cohen et al, 2009; Goss et al, 2011). In our model, deletion of Wls from the epithelium of the trachea resulted in expansion of muscle layer into the ventral aspect of the trachea findings not seen in other experimental models of tracheomalacia (Hines et al, 2013; Turcatel et al, 2013). Our studies showed that deletion of Sox9 from tracheal mesenchyme using the Col2a1Cre mice caused severe loss of cartilage; however, expansion of αSMA staining into the ventral side of the trachea was not observed (Fig.6).…”

“…A recent study proposed that the precise apposition of chondroblasts and myoblasts establishes the boundaries between cartilage and muscle, thus generating a structure needed for rigidity and flexibility characteristic of the trachea (Hines et al 2013). It is presently unclear how dorsal-ventral patterning of developing tracheal mesenchyme is established.…”

“…Mesenchymal cells derived from the splanchnic mesoderm, positioned ventral to the developing tracheal tube express the transcription factor Sox9 as early as E9 (Elluru and Whitsett, 2004; Hines et al, 2013). Sox9 is a key regulator of cartilage and bone development (Akiyama, 2008; Akiyama et al, 2002) and its role in tracheal cartilage has been demonstrated (Arora et al, 2012; Elluru and Whitsett, 2004; Hines et al, 2013; Park et al, 2010; Turcatel et al, 2013). Signaling mechanisms controlling the initial specification of tracheal cartilage progenitors are poorly defined; however, a number of transcription factors, receptors and ligands that influence Sox9 expression in the mesenchyme of the trachea have been identified.…”

Ezh2 represses the basal cell lineage during lung endoderm development

“…Together, these data indicate that Ezh2 regulates the temporal and spatial patterning of the basal cell lineage in the mouse lung. A recent report showed that Trp63+ basal cells were enriched on the side of the trachea that developed cartilaginous rings during development (Hines et al, 2013). Loss of cartilage in the lung mesenchyme leads to a loss of Trp63+ basal cells, suggesting an important mesenchymal-epithelial cross-talk that regulates basal cell development.…”

Localized Smooth Muscle Differentiation Is Essential for Epithelial Bifurcation during Branching Morphogenesis of the Mammalian Lung

“…Importantly, any perturbation in the stereotyped pattern of smooth muscle differentiation also prevented terminal bifurcation of the epithelium. Since transgenic knockout of smooth muscle is embryonic lethal (Hines et al, 2013), organ-specific genetic modulations will be required for definitive understanding of the role for visceral smooth muscle in epithelial morphogenesis.…”