Background

Understanding tracheal formation is a fundamental goal in the field of pulmonary development and disease. The trachea consists of mesoderm-derived smooth muscle (SM), cartilage, and connective tissue, as well as endoderm-derived epithelium (Brand-Saberi and Schafer, 2014). SM is positioned dorsally to control tracheal contraction, whereas the cartilage rings are located ventrally to prevent airway collapse (Hines et al., 2013; Yin et al., 2018 and 2019).

In humans, defects in the formation of the tracheal tube have been reported to lead to tracheomalacia, tracheostenosis, or complete tracheal ring deformity, which are characterized by a deficiency of the supporting cartilage or narrowing of the tracheal lumen and may lead to respiratory distress and death (Landing and Dixon, 1979; Fraga et al., 2016; Sinner et al., 2019).

In mice, tracheal SM differentiation starts by embryonic day 11.5 (E11.5) with the appearance of α-smooth muscle actin (αSMA) positive cells at the dorsal side (Hines et al., 2013; Yin et al., 2018). Newly differentiated tracheal SM cells exhibit round shapes and are not well organized (Yin et al., 2018 and 2019). They progressively develop into spindle-shaped cells that circumferentially align the tube (Yin et al., 2018 and 2019). During tracheal elongation, differentiated SM cells proliferate with increased area of SM stripes from E11.5 to P0 (Yin et al., 2018).

Tracheal cartilage development initiates as early as E9 (Elluru and Whitsett, 2004). Uncondensed SOX9⁺ mesenchymal cells appear and are restricted to the ventral trachea as early as E10.5 (Hines et al., 2013). Both Sox9 mRNA levels and the number of SOX9⁺ mesenchymal cells increase at E12.5 (Hines et al., 2013). By E13.5, SOX9⁺ mesenchymal cells condense to resemble cartilaginous rings with the number of 11–13. The number of C-shaped rings appears to be unchanged, whereas both the distance between rings and ring width increase during tracheal elongation (Yin et al., 2019). By E15.5, SOX9⁺ mesenchymal cells start differentiating into chondrocytes characterized by positive alcian blue staining, as well as aggrecan and type II collagen expression (Park et al., 2010; Yin et al., 2019).

Analysis of tracheal formation has mostly focused on cell condensation, differentiation, proliferation, and apoptosis by using tissue sections (Park et al., 2010; Hines et al., 2013; Lin et al., 2014). These studies have provided information on cell behavior in two-dimensional views, allowing for a better understanding of the cellular and molecular mechanisms underlying tracheal tubulogenesis. The method we present here for whole-mount immunostaining and imaging overcomes some of their limitations; for example, the precise sites of the three-dimensional trachea are not properly located. Thus, it is presently unclear how cells are organized in the whole trachea. Another not fully investigated question is the characteristics of SM cells. Although SM cell differentiation and proliferation have been tested (Hines et al., 2013; Gerhardt et al., 2018), cell alignment, polarization, and changes in cell shape in tracheal development remain largely unknown. To analyze tissue structure and cell organization, we provide a detailed protocol for examining SM cell organization and changes in cell shape as well as mesenchymal cell condensation, an important process during tracheal cartilage formation (Yin et al., 2019).