Wnt5a–Vangl1/2 signaling regulates the position and direction of lung branching through the cytoskeleton and focal adhesions

Lung branching morphogenesis requires reciprocal interactions between the epithelium and mesenchyme. How the lung branches are generated at a defined location and projected toward a specific direction remains a major unresolved issue. In this study, we investigated the function of Wnt signaling in lung branching in mice. We discovered that Wnt5a in both the epithelium and the mesenchyme plays an essential role in controlling the position and direction of lung branching. The Wnt5a signal is mediated by Vangl1/2 to trigger a cascade of noncanonical or planar cell polarity (PCP) signaling. In response to noncanonical Wnt signaling, lung cells undergo cytoskeletal reorganization and change focal adhesions. Perturbed focal adhesions in lung explants are associated with defective branching. Moreover, we observed changes in the shape and orientation of the epithelial sheet and the underlying mesenchymal layer in regions of defective branching in the mutant lungs. Thus, PCP signaling helps define the position and orientation of the lung branches. We propose that mechanical force induced by noncanonical Wnt signaling mediates a coordinated alteration in the shape and orientation of a group of epithelial and mesenchymal cells. These results provide a new framework for understanding the molecular mechanisms by which a stereotypic branching pattern is generated.

Our analysis of Wnt5a and Vangl1/2 mutant lungs from 11.5 dpc and onward clearly showed a defect in the position and direction of branching. To further understand how Wnt5a-Vangl/2 signaling controls this process, we traced lung development in control and Vangl1/2 mutant lungs from 11.0-11.5 dpc. We found that the epithelium where RCr/RMd and L.L1/L.L2 emerge underwent coordinated morphological changes in control lungs  in the revision). The lumen was enlarged first. Rudiments of RCr/RMd and L.L1/L.L2 were then formed. Meanwhile, the mesenchyme appeared to "push down" the epithelium between the two future branches. Finally, RCr/RMd and L.L1/L.L2 emerged at the defined position and direction.
Wnt5a-Vangl1/2 signaling is known to control the cytoskeleton as shown in previous studies and focal adhesions in our current work. We thus speculate that the mechanical force between cells is affected in the absence of Wnt5a-Vangl1//2 signaling. This could change the overall shape and orientation of the epithelial sheet. As a result, the relative position and direction of RCr/RMd and L.L1/L.L2 are affected (Fig. 2D'-2G' in the revision). A model that illustrates this notion is depicted in Fig. 4Z. We have clarified this point in the revision.
2) Since Wnt5a deletion in either mesenchyme or epithelium appears to have the same result, and since p-FAK is active in both compartments in wild type lungs and reduced in both compartments in mutant lungs and with FAK inhibitor, it is not clear whether the phenotype can be ascribed to defective cytoskeletal control in the epithelial or mesenchymal compartment. In the Wnt5a knockout lung at E12.5, it even looks like p-FAK is only depleted in the mesenchyme ( Figure S5 P,T), while the model suggests epithelial loss drives the phenotype. In all stains, there still appears to be p-FAK staining present in knockout lungs, so it is not clear that its absence underlies the phenotype.
We have added data to show that removal of Vangl1/2 in the lung epithelium by Sox9-Cre and in the mesenchyme by Dermo1-Cre does not lead to apparent branching defects ( Fig. S5N-Q in the revision). This is in contrast to the branching defects observed in Vangl1/2 null lungs. Barring the onset and efficiency of these two Cre lines, the simplest interpretation is that coordination of Vangl1/2 signaling (hence the downstream effectors) in both the epithelium and mesenchyme contribute to lung branching.
p-FAK levels are reduced (but not absent) in both the epithelium and mesenchyme of Wnt5a or Vangl1/2 mutant lungs ( Fig. 3S, 3T, 3V, 3W in the revision) or in Vangl1/2-deficient cell lines. Moreover, lung explants treated with FAK inhibitors are associated with reduced p-FAK levels and branching defects. This suggests that p-FAK is one of the many downstream effectors of Wnt5a-Vangl1/2 signaling and can contribute to lung branching. However, effectors other than p-FAK also contribute to lung branching. These points have been clarified in the revision.
3) There is incomplete penetrance in some genotypes which is not addressed. Perhaps the authors could examine p-FAK signaling in unaffected versus affected lungs from the same litter to see if this correlates, which would support their claim that defective focal adhesions are responsible for their phenotype.
Reduction in p-FAK levels and severity of branching in lung explants treated with FAK inhibitors correlate with the concentration of FAK inhibitors (Fig. S12 in the revision). However, it is difficult to establish such a correlation in the lungs. Nevertheless, p-FAK is reduced in all Vangl1/2 knockout lungs even though their phenotypes are milder than those in Wnt5a knockout lungs. This suggests that p-FAK mediates important aspects of the phenotypes. The more severe phenotypes in Wnt5a mutant lungs compared to Vangl1/2 knockout lungs could be due to additional targets of Wnt5a. Incomplete penetrance suggests the involvement of multiple pathways, which requires future investigations. In addition, certain branches are preferentially affected. This raises that possibility that different pathway combinations are employed at distinct locations. We have discussed this point in the revision. Figure 1, J-L and Figure 2, P-R).

4) In several important experiments, there was no statistical analysis performed (for example
We have added statistical analysis to Fig. 1J, 1K, 1L and Fig. 2P, 2Q, 2R.

Minor criticisms: 1) Data that is bi-modally distributed could be analyzed using a bi-modal statistical approach.
We have changed the way bi-modally distributed data are analyzed in Fig. 1K. Figure 3T the authors should use a one-way ANOVA.

2) In some cases the wrong statistical test is applied. For example, in
We have used one-ANOVA for determining the statistical significance between three or more independent groups, such as that in Fig. 3T (now Fig. 3C' in the revision) and others.

Rev. 2: Zhang et al. investigated the mechanisms controlling the position and direction of lung branching in mice focusing on the early developmental stages. Using complex knockout studies, the authors show that Wnt5a controls branching morphogenesis via non-canonical PCP signaling, specifically Vangl 1/2. Moreover, the authors show that Foxa 2 is also involved in branching regulation. Interestingly, the authors show that perturbation of PCP proteins leads to
changes in cellular organization and, specifically, the focal adhesion regulated by FAK. While I find the results highly relevant and that they can advance our understanding of lung morphogenesis, there are significant points that require to be addressed before this work is published. See below: 1. The authors claim that upon PCP perturbation, cytoskeletal components and mechanical cellular properties are impaired. However, the manuscript does not provide any detailed characterization of cellular morphology, measurements of cellular actomyosin properties, or ECM characterization in vivo. Without any more detailed cellular description, the model presented in Figure 4 T, U does not represent the manuscript's findings.
We have added data to show that the levels of phosphorylated Cofilin (p-Cofilin) were increased in Vangl1/2-deficient lungs (Fig. S10 in the revision). This suggests that the assembly and disassembly of actin filaments regulated by Cofilin are affected in the mutant lungs.
Finally, we have modified the model in Fig. 4T and 4U (now Fig. 4Z) to more accurately reflect the data presented in this study.

The authors should improve the quantification of p-FAK in vivo. As in point above, imaging at the cellular and sub-cellular levels would be essential to understand how p-FAK distribution changes.
We have included images at a higher resolution to better visualize the distribution and intensity of FAK (Fig. 3M, 3N, 3P, 3Q in the revision) and p-FAK (Fig. 3S, 3T, 3V, 3W in the revision). FAK and p-FAK was widely expressed in lung epithelial cells and the signal was concentrated along the apical and basal surface. The p-FAK signal was reduced but the subcellular distribution of p-FAK was unaltered in Vangl1/2-deficient lungs. The intensity of FAK and p-FAK was quantified (Fig. 3K in the revision).
Western blot analysis for p-FAK in lysates from Wnt5a mutant lungs, Vangl1/2-deficient lungs and Vangl1/2-deficient cells is provided in Fig. S9 in the revision.

It is unclear what the function of Foxa2 is in terms of regulating the morphology of brunching. Again, cellular imaging could help to distinguish between the role of Vangl and Foxa2
Loss of Foxa1 and Foxa2 transcription factors has been reported to result in defective branching (Wan et al. J Biol Chem 2005 PMID: 15668254). The more severe branching defects in Wnt5a mutant lungs than Vangl1/2 lungs suggest that Wnt5a has additional targets besides Vangl1/2. Our transcript analysis of mutant lungs implicates Foxa2 as one of the potential targets of Wnt5a. The role of nuclear Foxa2 in epithelial cells to mediate Wnt5a function in lung branching requires future investigation. VANGL2 is expressed in all lung cells and is concentrated at the apical region of epithelial cells, where VANGL2 expression colocalizes with p-FAK and the actin cytoskeleton (Fig. S5A-5M). We have clarified this point in the revision.

For FAK inhibitor experiments, please provide quantification of p-FAK reduction. Also, I can not find the figure of western blot mentioned in the text. Do levels of E-Cadhering also change upon FAK inhibition?
We have provided quantification of p-FAK reduction in lung explants treated with FKA inhibitor (Fig. S12 in the revision).
As mentioned above, Western blot analysis for p-FAK in lysates from Wnt5a mutant lungs, Vangl1/2-deficient lungs and Vangl1/2-deficient cells is provided in Fig. S9 in the revision. E-Cad levels were reduced upon FAK inhibition. However, reduction in E-Cad levels was similar in lung explants treated with different concentrations of PF-573228.