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. 2013 Nov 26;110(48):19444-9.
doi: 10.1073/pnas.1313223110. Epub 2013 Nov 11.

Establishment of Smooth Muscle and Cartilage Juxtaposition in the Developing Mouse Upper Airways

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Free PMC article

Establishment of Smooth Muscle and Cartilage Juxtaposition in the Developing Mouse Upper Airways

Elizabeth A Hines et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Keywords: airway development; tracheomalacia.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Temporal and spatial relationship of SM and cartilage in WT embryonic trachea and bronchi. (A, B, E, F, I, and J) RNA in situ hybridization of lungs for Acta2 or Sox9 at indicated stages. Solid lines indicate approximate planes of tracheal transverse sections in C, G, and K; dashed lines indicate the approximate planes of bronchial transverse sections in D, H, and L. (C, D, G, H, K, and L) Immunofluorescence staining for ACTA2 (red), SOX9 (green), and nuclei (DAPI; blue) of transverse sections of trachea or main bronchi at indicated stages. White arrowheads indicate the pulmonary artery. ca., carina.
Fig. 2.
Fig. 2.
Inactivation of Srf resulted in SM loss and cartilage increase in the upper airways. (A and B) Acta2 RNA in situ hybridization in E12.5 lungs. (CF) Immunostaining for ACTA2 (red), SOX9 (green), and nuclei (DAPI, blue) on transverse sections of E12.5 trachea or main bronchi. Arrowheads in D mark the limit of SOX9+ airway coverage. (GJ) Graphs of control and Foxg1cre;Srf cell counts for SOX9+ cells in the trachea (G) and main bronchi (I), and the percentage of the airway epithelium circumference covered by SOX9+ cells in the trachea (H) and main bronchi (J). All graphs were made by using three independent mutant and control data points and averages are shown with SEM. P values are indicated.
Fig. 3.
Fig. 3.
Inactivation of Sox9 resulted in cartilage loss and SM increase in the upper airways. (A and B) Alcian blue staining of E17.5 tracheas. (C and D) Col2a1 RNA in situ hybridization of E14.5 lungs. Arrows indicate the trachea, and arrowheads indicate the main bronchi. (EH) Immunostaining for ACTA2 (red), SOX9 (green), and nuclei (DAPI, blue) of transverse sections of E12.5 trachea or main bronchi. (IL) Graphs of control and Foxg1cre;Sox9 cell counts for ACTA2+ cells in the trachea (I) and main bronchi (K) and the percentage of the airway epithelium circumference covered by ACTA2+ cells in the trachea (J) and main bronchi (L). All graphs were made by using three independent mutant and control data points, and averages are shown with SEM. P values are indicated.
Fig. 4.
Fig. 4.
Inactivation of Sox9 resulted in decreased tracheal basal cell density. (AF) Immunostaining for markers as indicated on E17.5 trachea transverse sections. Sections at middle height trachea level are shown as representative examples. Solid brackets indicate dorsal regions magnified in A′–F′. Dashed brackets indicate ventral regions magnified in A′′–F′′. (G) Basal cell density (ratio of P63+ cells to total luminal cells) was compared between control and Foxg1cre;Sox9 tracheas at high, middle, and low heights as well as an overall average (Avg.) across the trachea. (H) In control tissue, basal cell density on the cartilage side and SM side were compared with the overall basal cell density in Foxg1cre;Sox9 tracheas across high, middle, and low heights as well as an overall average (Avg.). For each level, graphs were made by using four independent mutant and control data points. For the overall average, all 12 independent high, middle, and low data points were averaged. All graphs are shown with SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). Dr, dorsal; Vn, ventral.
Fig. 5.
Fig. 5.
Inactivation of Sox9 did not affect differentiated cell number but did lead to increased club cell gene expression. (AD) Immunostaining for TUBA1A (red), SCGB1A1 (green), and nuclei (DAPI, blue) on middle height transverse sections of tracheas at indicated stages. Red signal in the mesenchyme is not specific to ciliated cells. (E and F) Quantification of SCGB1A1+ cells (E) and TUBA1A+ cells (F) as a percentage of total luminal epithelial cells at high, middle, and low tracheal heights, and an overall average (Avg.). For each level, graphs were made by using four independent mutant and control data points. For the overall average, all 12 independent high, middle, and low data points were averaged. All cell count graphs are shown with SEM. No statistically significant differences were observed between mutant and control. (G) qRT-PCR of Scgb1a1 expression in E18.5 Foxg1cre;Sox9 tracheas compared with controls, shown with SD (*P = 0.02).
Fig. 6.
Fig. 6.
A model summarizing distinct relationships between SM and cartilage in the developing upper airways. (Center) Whole-mount WT E12.5 upper airway stained with ACTA2 (red) and SOX9 (green). Codependent relationship between SM and cartilage (Left) with regard to cell number in the trachea (Upper) and bronchi (Lower) and airway coverage (Right) in the bronchi (Lower), and (Upper) independent relationship between SM and cartilage with regard to airway coverage in the trachea.

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