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

Abstract

The airway epithelium develops into a tree-like structure via branching morphogenesis. Here, we show a critical role for localized differentiation of airway smooth muscle during epithelial bifurcation in the embryonic mouse lung. We found that during terminal bifurcation, changes in the geometry of nascent buds coincided with patterned smooth muscle differentiation. Evaluating spatiotemporal dynamics of α-smooth muscle actin (αSMA) in reporter mice revealed that αSMA-expressing cells appear at the basal surface of the future epithelial cleft prior to bifurcation and then increase in density as they wrap around the bifurcating bud. Disrupting this stereotyped pattern of smooth muscle differentiation prevents terminal bifurcation. Our results reveal stereotyped differentiation of airway smooth muscle adjacent to nascent epithelial buds and suggest that localized smooth muscle wrapping at the cleft site is required for terminal bifurcation during airway branching morphogenesis.

Figure 1. Smooth muscle differentiation is required for airway epithelial bifurcation

(A) At E12, the embryonic mouse lung has two left (L) buds, and the right cranial (Cr) lobe has started to bifurcate. Dotted line indicates the airway epithelium, which is surrounded by mesenchyme. (B) Snapshots from time-lapse movies. Scale bar, 100 µm. (C) Morphometric parameters used to quantify the kinematics of terminal bifurcation. (D) Airway smooth muscle wraps around the bifurcating neck. Scale bar, 50 µm. (E–F) Smooth muscle differentiation is inhibited using nifedipine (10 µM). Shown are staining and quantitative RT-PCR analysis of the smooth muscle markers αSMA (acta2), calponin-1, smooth muscle myosin heavy chain (smMHC), transgelin (tagln; SM22α), and the transcription factor SRF. Scale bar, 50 µm. (G) Branching morphogenesis was quantified as number of terminal buds after drug treatment. Shown are mean ± s.d. for n ≥ 9 for each condition; * P < 0.05; ** P < 0.01. (See also Figure S1 and Movie S1 and S2)

Figure 2. Smooth muscle appears at cleft sites prior to terminal bifurcation

(A) Snapshots from time-lapse movie of αSMA-RFP lung explant. Kymograph shows the temporal sequence of αSMA expression from regions indicated in the yellow inset (12 hr). Airway epithelium outlined by dotted red line. Scale bar, 100 µm. (B) Quantification of morphometric parameters and αSMA intensity as a function of time. Yellow shaded region indicates the duration of αSMA appearance at the bud tip prior to bifurcation (arrows on top indicate the timing of first appearance of αSMA (left yellow arrow) and terminal bifurcation (right yellow arrow)). αSMARFP intensity was measured along the perimeter of the bud tip. (C) Quantification of time-lapse movies shows average duration of appearance of αSMA-positive cells (8.6 ± 1.9 hr) prior to the bifurcation. (D) Immunostained buds before and after the terminal bifurcation. Scale bar, 50 µm. (E) Schematic representation of smooth muscle localization during terminal bifurcation of the airway epithelium. (See also Figure S2 and Movie S3)

Figure 3. Pharmacologically disrupting patterned smooth muscle differentiation blocks terminal bifurcation

(A) Lung explants treated with SU5402 (5 µM), SAG (1 µg/ml), cyclopamine (1 µM), or nifedipine (10 µM). SU5402 was added after 24 hours of treatment with nifedipine for the “nifedipine + SU5402” condition. Fixed lungs were stained for E-cadherin and αSMA. Scale bars, 100 µm. Disrupting the pattern of smooth muscle differentiation (B) disrupts terminal bifurcation and (C) induces epithelial buckling. (**P< 0.01) (D) Snapshots from time-lapse movies of αSMA-RFP lung explants treated with SU5402, SAG, or cyclopamine. Scale bar, 100 µm. (E) Quantification of αSMA intensity and epithelial length around the perimeter of the bud from time-lapse movies in (D). (See also Figure S3 and Movie S4)

Figure 4. Specifically targeting the airway smooth muscle blocks terminal bifurcation

(A) Lung explants transduced with AdGFP, AdSRF, or AdshSRF. Fixed lungs were stained for E-cadherin and αSMA. Red arrow indicates bifurcation; gray arrowheads indicate buckling. Scale bars, 100 µm. (B) Relative transcript levels for markers of smooth muscle differentiation in explants transduced with AdSRF or AdshSRF. Shown are mean ± s.d. for 3 independent experiments. (***P<0.001) Disrupting SRF levels (C) disrupts terminal bifurcation and (D) induces epithelial buckling. (**P<0.01) (E) Brightfield images of lung explant before and after dissecting off the mesenchyme from a stage 3 bud. Graph indicates depth of cleft before and after surgical removal of the smooth muscle. Shown are mean ± s.e.m. for 5 independent experiments. (*P<0.05) (See also Figure S4)