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Wnt5a–Vangl1/2 signaling regulates the position and direction of lung branching through the cytoskeleton and focal adhesions [1]

['Kuan Zhang', 'Cardiovascular Research Institute', 'University Of California', 'San Francisco', 'California', 'United States Of America', 'Erica Yao', 'Ethan Chuang', 'Biao Chen', 'Evelyn Y. Chuang']

Date: 2022-08 

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.

Copyright: © 2022 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we have defined the role of epithelial and mesenchymal Wnt5a in controlling the position and direction of lung branching. A complete loss of Vangl1/2, the effectors of Wnt5a, resulted in similar albeit milder phenotypes than those due to Wnt5a removal. We discovered that cytoskeletal reorganization induced by PCP signaling leads to changes in focal adhesions required for branching. This is associated with alterations in the shape and orientation of the epithelial sheet and the underlying mesenchymal layer in regions of defective branching in the mutant lungs. Together, these novel findings reveal a molecular cascade that controls cellular properties required for branching morphogenesis.

Similarly, how the downstream effectors of Wnt5a control lung branching is unclear. In the literature, a hypomorphic (reduced function) allele of Vangl2, Vangl2 Lp (loop tail) [ 16 ], has been widely used. Vangl1 and Vangl2 encode the mammalian homologs of fly Van Gogh (Van)/strabismus and are absolutely required for PCP signaling [ 17 ]. Homozygous Vangl2 Lp/Lp mice die in utero due to an open neural tube. Analysis of Vangl2 Lp/Lp lungs revealed defective branching, resulting in fewer branches and narrow lumens [ 18 ]. Disrupted cytoskeletal organization was also observed in Vangl2 Lp/Lp lungs [ 18 ]. However, intact Vangl1 and residual Vangl2 activity in Vangl2 Lp/Lp mutants retained PCP signaling and prevented an accurate assessment of how PCP signaling promotes lung branching, especially during the early steps of branch formation. A complete loss of both Vangl1 and Vangl2 function is required to uncover the molecular mechanisms by which PCP signaling regulates branching. In addition, loss of PCP signaling in a select compartment (e.g., the lung epithelium or mesenchyme) through conditional gene inactivation is necessary to investigate PCP signaling in different niches.

Among the several Wnts that are expressed in the lung, Wnt5a is a prominent member of the noncanonical Wnt family [ 12 ]. Wnt5a is also capable of mediating canonical Wnt signaling [ 13 , 14 ]. The role of Wnt5a in lung branching has not been fully explored. A previous report on Wnt5a −/− mouse lungs primarily focused on later stages (e.g., 16.5 to 18.5 days postcoitus (dpc)) of lung development and concluded that Wnt5a controls distal lung morphogenesis [ 15 ]. Whether Wnt5a regulates early lung branching is unknown and the sources of Wnt5a in this process were not functionally defined.

The planar cell polarity (PCP) pathway is an evolutionarily conserved mechanism for orchestrating cell shape and motility during pattern formation [ 8 – 11 ]. PCP signaling has been broadly investigated. The major components of the PCP pathway are known, and their genetic interactions have been defined. The PCP pathway is the noncanonical branch of the Wnt pathway. Similar to the canonical Wnt pathway, PCP signaling is triggered by binding of the Wnt ligands to their cell surface receptors that include the Frizzled (FZ) receptors and ROR coreceptors. However, instead of controlling β-catenin levels as seen in the canonical Wnt pathway, PCP signaling utilizes multiple transmembrane and cytoplasmic proteins to regulate the actomyosin cytoskeleton. How changes in cellular properties induced by PCP signaling influence branching morphogenesis is a key unresolved question. Insight into this issue will offer a new framework for understanding branching morphogenesis.

Branching morphogenesis is a fundamental mechanism for pattern formation [ 1 , 2 ]. It is utilized by many organs and vasculature to generate a defined pattern required for tissue function. The lung, kidney, mammary gland, salivary gland, pancreas, and prostate are among the branching organs that have been extensively studied. For instance, many genes and pathways that control lung branching have been discovered [ 3 – 5 ]. However, we still lack a complete mechanistic understanding of how new lung branches are formed and extended in a spatially and temporally specific manner. In particular, the cellular and molecular basis of how lung epithelial cells undergo morphogenetic changes to produce a new branch remains underexplored [ 6 , 7 ].

Results

Global inactivation of Wnt5a perturbs the position and direction of early lung branching To search for signals that trigger PCP signaling and lung branching, we tested the function of Wnt5a and examined branching in lungs of Wnt5a−/− mice especially at the early stages of lung development. The null allele of Wnt5a (Wnt5a−) was derived from the floxed allele of Wnt5a (Wnt5af) [19] by Sox2-Cre [20]. We found that defective lung branching was already apparent in Wnt5a−/− lungs at 11.5 dpc (Fig 1A–1C). The most striking feature of Wnt5a−/− lungs was the loss of proper position and orientation of the lung buds when the initial pattern was being generated. As lung development proceeded, the well-established programs that are dubbed domain branching and planar and orthogonal bifurcation were also impaired. The phenotype was completely penetrant. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Wnt5a controls the position and direction of lung branching. (A-I) Ventral (A, B, D, E, G, H) and dorsal (C, F, I) views of dissected lungs from control and Wnt5a−/− mouse embryos at the developmental stages indicated. E-Cad marked epithelial cells. (J) Quantification of the ratio of the distance of RMd–RCr to the distance of RMd–bifurcation (mean value ± SEM, unpaired Student t test, n = 17 pairs). (K) Quantification of the angle (in degrees) between RMd and RCd branches (mean value ± SEM, unpaired Student t test, n = 17 pairs). (L) Quantification of the angle (in degrees) between RAc and RCd branches (mean value ± SEM, unpaired Student t test, n = 17 pairs). (M-R) Immunostaining of lung sections collected from control and Wnt5a−/− at 12.5 dpc. (S) Schematic diagram of the position and direction of lung branches in wild-type mice at 11.5 dpc. (T) Quantification of the cell proliferation rate in the epithelium of control and Wnt5a−/− lungs at 12.5 dpc (mean value ± SEM, unpaired Student t test, n = 3 pairs). The rate of epithelial cell proliferation was calculated as the ratio of the number of EdU+ epithelial cells (EdU+E-Cad+) to the number of epithelial cells (E-Cad+). (U-B’) Ventral views of dissected lungs from control, Wnt5af/f; Sox9Cre/+ and Wnt5af/f; Dermo1Cre/+ embryos at the developmental stages indicated. (***) p < 0.001; ns, not significant. The underlying data for Fig 1J, 1K, 1L and 1T and the exact P values can be found in S1 Data. (Scale bars: A-F, 0.5 mm; G-I, 1 mm; M-R, 25 μm; U, V, Y, Z, 0.5 mm; W, X, A’, B’, 1 mm.) dpc, days postcoitus; RAc, right accessary; RCd, right caudal; RCr, right cranial; RMd, right middle. https://doi.org/10.1371/journal.pbio.3001759.g001 In control lungs at 11.5 dpc, the 5 main branches designated as the right cranial (RCr), right middle (RMd), right caudal (RCd), right accessary (RAc), and left (L) branch were already fully separated (Fig 1A and 1S) [21]. Branching from these 5 branches would give rise to the 5 lobes (the cranial, middle, caudal, accessary lobes of the right lung, and a single left lobe of the left lung) in adult mice. RCr emerged at a more proximal position to that of RMd and RAc, which were at a similar axial level at this stage. In Wnt5a-deficient lungs, the distance between the RCr and RMd branches was shortened due to the abnormal appearance of RCr at the axial level of RMd/RAc at 11.5 dpc (Fig 1B and 1C). To quantify the defects of RCr/RMd in Wnt5a−/− lungs, we measured the relative position of RCr and RMd (Fig 1J). We first determined the distance between RMd and RCr (D RMd–RCr ) and the distance between RMd and the bifurcation point (from the trachea) (D RMd–bifurcation ), respectively, and calculated their ratio (R RCr–RMd ). The relative distance (R RCr–RMd ) between RCr and RMd was reduced in Wnt5a−/− lungs. The direction of the RMd and RAc branches relative to the RCd branch was also altered at 11.5 dpc. By contrast, the direction of RCr and left L1 (L.L1) was unaltered. We measured the angle between RMd and RCd (θ RMd–RCd ) and the angle between RCd and RAc (θ RCd–RAc ) (Fig 1K and 1L). In the absence of Wnt5a, θ RMd–RCd was increased in 75% and reduced in 25% of the mutant lungs. θ RCd–RAc was reduced in Wnt5a−/− lungs. In approximately 25% of Wnt5a-deficient lungs, the distance between the left L1 (L.L1) and L2 (L.L2) branches was reduced at 11.5 dpc. Moreover, we observed a complex change in the position and direction of branches derived from RCr and L.L1 in Wnt5a mutant lungs. The founder branch for RCr and L.L1 were initially produced at the correct position and orientation and bifurcated to form the longitudinal and lateral branches. At 12.5 dpc, the daughter branches of RCr and L.L1 displayed defects in the position and direction where they branched. While the lateral branch from RCr and L.L1 ramified to form the main growth axis of the cranial and left lobes in control lungs, respectively, it was the longitudinal branch of RCr and L.L1 in Wnt5a−/− lungs that dominated the main growth axis of the corresponding lobe. Daughter branches extended from the 5 main branches and subsequent branches also exhibited defective branching (Figs 1D–1F and 1G–1I and S1). At 12.5 dpc, the overall branching pattern of Wnt5a−/− lungs had diverged significantly from that in wild-type lungs. Together, these results suggest that Wnt5a signaling controls the position and direction of early lung branching. We noticed a shortened trachea in Wnt5a−/− lungs and wondered whether reduced cell proliferation in lung epithelial cells could be related to the branching defects. Interestingly, loss of Wnt5a did not perturb proliferation of lung epithelial cells. No difference in EdU+ epithelial cells between control and Wnt5a−/− lungs at 12.5 dpc was detected (Fig 1M–1R and 1T). This finding suggests that the primary defect in the absence of Wnt5a is likely changes in cellular organization.

Loss of Wnt5a in either the lung mesenchyme or epithelium impairs branching morphogenesis Wnt5a is expressed in both the lung epithelium and mesenchyme. To explore how Wnt5a in different niches controls lung branching, we selectively removed Wnt5a in the lung mesenchyme with the expectation that mesenchymal Wnt5a would trigger in part epithelial PCP signaling. We produced Wnt5af/f; Dermo1Cre/+ mice in which Wnt5a was specifically eliminated in the lung mesenchyme by Dermo1-Cre [22]. Wnt5af/f; Dermo1Cre/+ mice died soon after birth. Their lungs appeared compact compared to wild-type controls at 18.5 dpc and postnatal (p) day 0. The phenotype was highly penetrant and Wnt5af/f; Dermo1Cre/+ mice exhibited branching defects. To further test this idea, we inspected Wnt5af/f; Dermo1Cre/+ lungs at different stages of lung development. We found that the early branching defects in Wnt5af/f; Dermo1Cre/+ lungs (Fig 1U–1X) were similar to those in Wnt5a−/− lungs described above. We also tested whether Wnt5a functions in the lung epithelium to control branching. To this end, we produced Wnt5af/f; ShhCre/+ mice. Unexpectedly, these animals were fully viable and, besides a mild digit phenotype, could not be distinguished from their wild-type littermates. To exclude the possibility that Shh-Cre [23] was inefficient in deleting Wnt5a, we generated Wnt5af/f; Sox9Cre/+ mice. While Sox9-Cre [24] is activated a few days later than Shh-Cre, we suspect that Sox9-Cre could be more effective than Shh-Cre in removing Wnt5a in the lung epithelium. Branching in Wnt5af/f; Sox9Cre/+ lungs appeared normal at 11.5 dpc. Approximately 40% of Wnt5af/f; Sox9Cre/+ mice exhibited lung defects (Fig 1Y–1B’) similar to but milder than those in Wnt5a−/− lungs at 12.5 dpc. However, the branching defects in most Wnt5af/f; Sox9Cre/+ lungs were not apparent until 13.5 dpc, reflecting the onset of Sox9-Cre expression at or after 11.5 dpc. Approximately 70% of Wnt5af/f; Sox9Cre/+ mice displayed lung branching defects at 13.5 dpc, which were restricted to the lineage branches from RCr, RMd, and L.L1. Defective branching in the RCr, RMd, and L.L1 lineages did not emerge from their daughter branches, but from the subsequent secondary or tertiary branches. As a result, axis extension was only partially affected and the growth axis of the lobes was preserved. Cell proliferation was unaltered in Wnt5af/f; Sox9Cre/+ lungs at 13.5 dpc or Wnt5af/f; Dermo1Cre/+ at 12.5 dpc (S2 Fig). Together, these results suggest that noncanonical Wnt signaling operates in both lung epithelium and mesenchyme to coordinate lung branching.

Global elimination of Vang1/2 exhibits branching defects, similar to but milder than those due to loss of Wnt5a To understand how the PCP pathway controls lung branching, we eliminated PCP signaling in the lung by generating mice deficient in both Vangl1 and Vangl2. To this end, we set up crosses between Vangl1gt/gt; Vangl2f/+; Sox2Cre/+ and Vangl1gt/gt; Vangl2f/f mice and collected embryos at different developmental stages (10.5 to 18.5 dpc). Vangl1gt is a gene-trapped allele [25] that leads to a complete loss of Vangl1 activity while a floxed (f) allele of Vangl2 (Vangl2f) [26] is converted into a null allele (Vangl2−) upon Cre expression. We focused on Vangl1gt/gt; Vangl2f/f; Sox2Cre/+ embryos (denoted as Vangl1gt/gt; Vangl2−/− in this study) that are deficient in both Vangl1 and Vangl2. Early ubiquitous expression of Sox2-Cre resulted in the production of Vangl2− from Vangl2f in all tissues (Fig 2A–2F). Of note, Vangl1gt/gt mice are viable and fertile without apparent phenotypes. We noticed that neither Vangl2−/− nor Vangl1gt/+; Vangl2−/− lungs displayed branching defects despite the fact that all of these embryos had an open neural tube. This is consistent with a functional redundancy between Vangl1 and Vangl2 during lung branching. Such a dose-dependent effect of Vangl1/2 levels on PCP signaling has been documented in several other tissues. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Vangl1/2 control the position and direction of lung branching. (A-F) Immunostaining of lung sections collected from control and Vangl1gt/gt; Vangl2−/− mice at 12.5 dpc. CATNB-labeled epithelial and mesenchymal cells. (G-O) Ventral (G, H, J, K, M, N) and dorsal (I, L, O) views of dissected lungs from control and Vangl1gt/gt; Vangl2−/− embryos at the developmental stages indicated. (P) Quantification of the ratio of the distance of RMd–RCr to the distance of RMd–bifurcation (mean value ± SEM, unpaired Student t test, n = 12 pairs). (Q) Quantification of the angle (in degrees) between RMd and RCd branches (mean value ± SEM, unpaired Student t test, n = 12 pairs). (R) Quantification of the angle (in degrees) between RAc and RCd branches (mean value ± SEM, unpaired Student t test, n = 12 pairs). (S-X) Immunostaining of lung sections collected from control and Vangl1gt/gt; Vangl2−/− mice at 12.5 dpc. (Y) Quantification of the cell proliferation rate in the epithelium of control and Vangl1gt/gt; Vangl2−/− lungs at 12.5 dpc (mean value ± SEM, unpaired Student t test, n = 3 pairs). The rate of epithelial cell proliferation was calculated as the ratio of the number of EdU+ epithelial cells (EdU+E-Cad+) to the number of epithelial cells (E-Cad+). (Z-C’) Ventral views of dissected lungs from ShhCre/+; ROSA26mTmG/+ embryos at the developmental stages indicated. Note that A’-C’ came from embryos within the same litter. Arrows point to enlarged lumen where RCr and RMd will emerge. (D’-G’) Ventral views of dissected lungs from control and Vangl1gt/gt; Vangl2−/− embryos within the same litter at approximately 11.25 dpc. Arrows point to changes in the overall shape and direction of the epithelial sheet where RCr and RMd will emerge in Vangl1gt/gt; Vangl2−/− lungs. (*) p < 0.05; (**) p < 0.01; ns, not significant. The underlying data for Fig 2P, 2Q, 2R and 2Y and the exact P values can be found in S1 Data. (Scale bars: A-F, 25 μm; G-O, 0.5 mm; S-X, 25 μm; Z-G’, 0.5 mm) dpc, days postcoitus; RAc, right accessary; RCd, right caudal; RCr, right cranial; RMd, right middle. https://doi.org/10.1371/journal.pbio.3001759.g002 Vangl1gt/gt; Vangl2−/− embryos exhibited an open neural tube and a shortened axis and died shortly after birth as stated in prior publications [26]. At 18.5 dpc, their lungs appeared more compact than control lungs while the lumen diameter in the airways was reduced. This suggested defects in lung branching due to loss of PCP signaling. We examined lung branching in Vangl1gt/gt; Vangl2−/− lungs at earlier stages. The first sign of defective branching detected at 11.5 dpc was misplacement of lung buds (Fig 2G–2I), similar to those observed in Wnt5a−/− or Wnt5af/f; Dermo1Cre/+ lungs. However, subsequent branching defects in Vangl1gt/gt; Vangl2−/− lungs were not as pronounced (Figs 2J–2R and S3) as those in Wnt5a−/− or Wnt5af/f; Dermo1Cre/+ lungs. These results suggest that Vangl1/2 mediate Wnt5a signaling in controlling branching but Wnt5a has additional targets other than Vangl1/2. No difference in EdU+ epithelial cells between control and Vangl1gt/gt; Vangl2−/− lungs at 12.5 dpc was detected (Fig 2S–2Y), again supporting a primary defect in cellular organization. No apparent defects in smooth muscle cells or blood vessels were detected in Vangl1gt/gt; Vangl2−/− lungs (S4 Fig). To further understand how Wnt5a–Vangl1/2 signaling controls lung branching, we traced lung development in control and Vangl1/2 mutant lungs from 11.0 to 11.5 dpc. We found that the epithelium where RCr/RMd and L.L1/L.L2 emerge underwent coordinated morphological changes in control lungs (Fig 2Z–2C’). 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 2 future branches. Finally, RCr/RMd and L.L1/L.L2 emerged at the defined position and direction. We speculate that the mechanical force between cells is affected in the absence of Wnt5a–Vangl1/2 signaling. This could alter 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 were affected (Fig 2D’–2G’). Unlike Wnt5a, no apparent branching defects were observed in Vangl1gt/gt; Vangl2f/f; Sox9Cre/+ or Vangl1gt/gt; Vangl2f/f; Dermo1Cre/+ lungs (S5N–S5Q Fig). Although Sox9-Cre was expressed after early branching had initiated, epithelial Vangl2 was efficiently removed in Vangl2f/f; Sox9Cre/+ lungs by 14.5 dpc (S5A–S5M Fig) when active branching was proceeding. This suggests that coordination of Vangl1/2 signaling (hence the downstream effectors) in both the epithelium and mesenchyme contribute to lung branching.

Foxa2 participates in transducing the Wnt5a signal during lung branching The discrepancy in phenotypes between Wnt5a and Vangl1/2 mutant lungs prompted us to search for Wnt5a targets other than Vangl1/2. We performed qPCR analysis on control and Wnt5a-deficient lungs to identify these potential targets. We found that Foxa2 expression in the lung was significantly reduced in the absence of Wnt5a at 12.5 dpc (S6 Fig). We then tested if Wnt5a regulated Foxa2 expression through noncanonical or canonical pathways. Foxa2 expression was unaltered in Vangl1gt/gt; Vangl2−/− lungs by qPCR analysis (S6 Fig), suggesting that Foxa2 expression is not controlled by noncanonical Wnt5a signaling. Moreover, expression of β-catenin-related genes (such as Axin2 and Lef1) was reduced in Wnt5a−/− lungs (S6 Fig). These results support the notion that Wnt5a controls Foxa2 expression through the canonical pathway. Loss of Foxa1 and Foxa2 transcription factors has been reported to result in defective branching [27]. We speculate that Wnt5a coordinates lung branching by signaling through Vangl1/2, Foxa2, and other targets. The role of Foxa2 in mediating Wnt5a function in lung branching requires future investigation.

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