Wnt ligand/Frizzled 2 receptor signaling regulates tube shape and branch-point formation in the lung through control of epithelial cell shape

Abstract

Changing the morphology of a simple epithelial tube to form a highly ramified branching network requires changes in cell behavior that lead to tissue-wide changes in organ shape. How epithelial cells in branched organs modulate their shape and behavior to promote bending and sculpting of the epithelial sheet is not well understood, and the mechanisms underlying this process remain obscure. We show that the Wnt receptor Frizzled 2 (Fzd2) is required for domain branch formation during the initial establishment of the respiratory tree. Live imaging and transcriptome analysis of lung-branching morphogenesis demonstrate that Fzd2 promotes changes in epithelial cell length and shape. These changes in cell morphology deform the developing epithelial tube to generate and maintain new domain branches. Fzd2 controls branch formation and the shape of the epithelial tube by regulating Rho signaling and by the localization of phospho-myosin light chain 2, in turn controlling the changes in the shape of epithelial cells during morphogenesis. This study demonstrates the importance of Wnt/Fzd2 signaling in promoting and maintaining changes in epithelial cell shape that affect development of a branching network.

Fig. 1.

Loss of Fzd2 in developing lung epithelium leads to formation of cysts in distal airways and defective branching morphogenesis. (A and B) Expression of Fzd2 in Shh^cre:Fzd2^flox/flox mutants is decreased specifically in the developing lung epithelium at E12.5 (arrows and dotted outlines). (C and D) At E14.5, Shh^cre:Fzd2^flox/flox mutants exhibit cysts in the distal airway region of the whole-mount lung (C, arrows) and in H+E-stained histological tissue sections (D, arrows). Controls in all experiments are Shh^cre mice. (E–N) Whole-mount immunohistochemistry using vimentin to mark mesenchyme (red) and E-cadherin to mark epithelium (cyan) was used to characterize branching morphogenesis in control (E–I) and Shh^cre:Fzd2^flox/flox mutants (J–N) from approximately E10.5 (36 somites) through E13.5 (60 somites). The development of cysts in the distal regions of Shh^cre:Fzd2^flox/flox mutants correlates with a decrease in formation of domain branches (I and N, arrows). (O) The deficiency in domain branch formation is apparent by the 57–60 somite stage, as enumerated in O. (Scale bars: 500 μm.)

Fig. 2.

Fzd2 regulates the components of the signaling niche that controls branching morphogenesis. (A) Microarray analysis reveals disruption in the expression of multiple signaling factors, including Fgf10, that are important for branching morphogenesis. (B) qPCR analysis of multiple components of the branching signaling niche reveals increased expression in Fgf10 and Bmp4 and decreased expression of Fgfr2 and Shh. (C) Diagram showing how the Fgf10, Bmp4, and Shh pathways are thought to interact to control branching morphogenesis; Fgf10 induces the outgrowth of a new bud, and Bmp4 and Shh inhibit Fgf10 activity to form a cleft, leading to bifurcation and two new bud points. (D–K) In situ hybridization showing expanded and increased Fgf10 expression, denoted by the bracket in H as compared to the arrows in D, increased Bmp4 expression, and decreased Fgfr2 and Shh expression in control and Shh^cre:Fzd2^flox/flox mutants). (Scale bars: 100 μm.)

Fig. 3.

Real-time imaging and single-cell analysis show the cellular and morphological changes required for new branch formation. (A and B) Real-time imaging of control and Shh^cre:Fzd2^flox/flox mutants ex vivo shows that although control lungs form new domain branch points through extension at predictable sites of bud formation, Shh^cre:Fzd2^flox/flox mutant lung epithelium expands without bending of the epithelium, leading to a wider airway tube. (C) At sites of new domain branch-point formation, the epithelium first thickens (C, i), then buckles at a distinct point (C, ii), and then buds and extends away from the founder epithelial tube (C, iii); the epithelial tube constricts at the base of this new branch as extension proceeds (C, iv). (D) Using the Shh^creERT2:R26R^mTmG reporter to mark individual epithelial cells, we found that epithelial cells at sites of new branch points have a broad basal surface and an elongated and narrow apical region. Conversely, epithelial cells at the growing tip are more columnar and shorter with fairly equivalent apical and basal surface areas. (E) Diagram showing how the epithelial sheet that composes the developing lung airways deforms at sites of new branch-point formation: Epithelial cells lengthen along the apical–basal axis, creating a bend in the epithelial sheet. This process results in the formation of new bud or branch point in the epithelial tube. As the new bud extends, the cells in the bud tip once again adopt a short columnar morphology.

Fig. 4.

Loss of Fzd2 causes defects in epithelial cell shape. (A–C) The average apical surface area was increased in Shh^cre:Fzd2^flox/flox mutant lung epithelial cells as measured by outlining the apical surface delineated by E-cadherin immunostaining. (The apical region is diagrammed in G). (D–F) The epithelial thickening observed in control lungs at sites of new branch-point formation was absent in Shh^cre:Fzd2^flox/flox mutant lungs. (G) To visualize control and Shh^cre:Fzd2^flox/flox mutant lung epithelial cells individually, confocal images of E-cadherin–stained epithelium were analyzed using EDGE (44) as diagrammed. (H and I) Representative images of individual tracings of five control (H) and Shh^cre:Fzd2^flox/flox mutant (I) lung epithelial cells are shown. (J and K) Although overall cell volume was not changed (J), apical–basal cell length (K) was reduced in Shh^cre:Fzd2^flox/flox mutants. n.s., not significant. **P < 0.001.

Fig. 5.

Changes in the RhoA/pMLC2 pathway cause branching defects and cystic lungs. (A) Components of the noncanonical Wnt signaling pathway have been shown to affect changes in cytoskeletal behavior via the Rho pathway. (B) qPCR data showing decreased levels of Celsr1 and Arhgef19 expression in Shh^cre:Fzd2^flox/flox mutant lungs at E11.5. (C and D) Expression of pMLC2 in Shh^cre (C, ii) and Shh^cre:Fzd2^flox/flox (D, ii) mutant lungs. (E) Expression of pMLC2 is quantitatively decreased in Shh^cre:Fzd2^flox/flox mutant lungs. (F) Quantification of domain branch numbers in lung explants treated with ML7 or fasudil. (G–J) E-cadherin whole-mount immunostaining of Shh^cre or Shh^cre:Fzd2^flox/flox mutant lung explants treated with DMSO (G and H), ML7 (I), or fasudil (J). Lower panels (G, i–J, i) show cell outlines after each treatment regime. (K and K, i). Automated tile-scanning was required to capture the entire lung in the fasudil treated Shh^cre sample (J), and the dashed line indicates the region stitched by the Zeiss software due to the size of these control lung explants. n = 9–12 lungs treated per condition. (Scale bars: 10 μm.) *P < 0.05, **P < 0.01.