Lung morphogenesis is disrupted in Celsr1Crsh and Vangl2Lp mutants
We examined lungs taken from two mouse mutants which carry loss of function mutations affecting the following proteins: Celsr1 (crash
) and Vangl2 (loop-tail
). Briefly, we observed smaller, misshapen lobes and fewer airways with narrow or absent lumina in both Celsr1Crsh
Specifically, macroscopic analysis of Celsr1Crsh and Vangl2Lp homozygotes revealed that mutant lungs were smaller than wild-type littermates, most strikingly, the topology of the lungs was often highly disturbed suggesting that in the absence of Celsr1 and Vangl2, the lung lobes were not able to attain their normal shape (Fig. A–C). Analysis of sections of E14.5 Celsr1Crsh and Vangl2Lp homozygous mutant lung stained with H&E (Fig. E and F) revealed changes to the structure of the epithelium from that observed in wild-type lung (Fig. D). During normal lung development, airway lumina are initially very narrow and are surrounded by multilayered/pseudostratified type epithelium. As development proceeds, the lumina widen and epithelial thickness subsequently decreases. In wild-type lung sections at E14.5, most airways contained clearly visible lumina surrounded by a single layer of uniformly aligned columnar epithelium (Fig. D and insert). However, in both Celsr1Crsh and Vangl2Lp homozygous lungs the majority of lumina were considerably narrower and the luminal space often contained cells (Fig. E, F and inserts). In addition, the surrounding epithelial cells were frequently multilayered and/or disorganized and not aligned uniformly. Although the phenotypes were broadly similar in both mutants, it was notable that Celsr1Crsh airways were frequently narrower and cells appeared more densely packed than those in Vangl2Lp.
Figure 1. Disruption of Celsr1 or Vangl2 causes lung morphogenesis defects. Analysis of E18.5 separated mouse embryonic lung lobes (A–C) reveals visibly misshapen lung lobes of Celsr1Crsh (B) and Vangl2Lp (C) lungs compared with wild-type (A). H&E (more ...)
At E18.5 hypoplasia was evident in mutant lungs (Fig. H and I) compared with wild-type (Fig. G). Moreover, lungs appeared to lack septation and had thickened interstitial mesenchyme. Quantification of these mutant lungs revealed significantly reduced numbers of airways compared with wild-type at E14.5 and E18.5 (P and Q) as well as decreased width of airways (Fig. R).
To further highlight the disordered cellular arrangements in the mutant lungs, we immunostained E14.5 lung sections using a pan-cytokeratin antibody to mark epithelial cells and DAPI to highlight all cell nuclei. Wild-type epithelial cells were readily distinguishable from mesenchyme by both cytokeratin and DAPI labelling. The majority of wild-type airways consisted of an open lumen surrounded by a single layer of uniformly aligned nuclei (Fig. J and M). In contrast in both mutants, the epithelial cells appeared highly disorganized and randomly orientated (Fig. K, L, N, O) with either small or no lumina (see inserts in Fig. K and L compared with J). It was often not possible to distinguish epithelial airways from surrounding mesenchyme by DAPI labelling. Celsr1Crsh airways appeared more severely affected than Vangl2Lp.
Cell differentiation, proliferation and apoptosis are not affected in Celsr1Crsh and Vangl2Lp lungs
To begin to determine the cause of the lung tissue defects in Celsr1Crsh
mutants we first looked for evidence of changes in cell differentiation, proliferation and apoptosis. To determine whether epithelial cell differentiation was affected in the mutants, we performed immunohistochemistry on E18.5 Crsh/Crsh
(Fig. B and E) and Lp/Lp
(Fig. C and F) mutant lungs using α-smooth muscle actin, to highlight smooth muscle cells surrounding proximal airways, and pro-surfactant protein C, a marker of alveolar type II cells in distal airways. We found no major changes in expression of these markers compared with wild-type littermates (Fig. A and D). However, the disrupted tissue architecture in Celsr1Crsh
made detailed comparison of Pro-SpC staining in wild-type and mutant lungs difficult. To circumvent this issue, we compared the percentage of Pro-SpC positive cells in wild-type, Celsr1Crsh
lungs at E18.5 and no significant difference was observed (Fig. G). Comparison of a further two cell type specific markers (CC-10 for Clara cells and Aquaporin-5 for alveolar type I cells) also showed no obvious difference between wild-type and mutants (data not shown). We then compared the percentage of proliferating cells in E14.5 wild-type and mutant lung sections to determine whether the reduced number of airways was due to lack of tissue growth. Importantly, there was a comparable percentage of proliferating cells in wild-type, Celsr1Crsh
(Supplementary Material, Fig. S1A–C and H
). Similarly, no change in the level of apoptosis was observed in Celsr1Crsh
homozygous lungs (Supplementary Material, Fig. S1D–F
). We also saw no changes in proliferation or apoptosis in either mutant at E11.5 when mutants are morphologically indistinct from wild-type (Supplementary Material, Fig. S1G and I
). The absence of changes in cell differentiation, proliferation or apoptosis suggests that the cellular disorganization observed in Celsr1Crsh
mutants is caused by impaired cell and tissue morphogenesis. Taking this data into account, we hypothesize that the smaller size of mutant lungs is due to the reduced width and number of lumina and increased compaction of the tissue.
Figure 2. Immunohistochemical analysis of lung epithelial cell differentiation in Celsr1Crsh and Vangl2Lp compared with wild-type. Comparison of E18.5 control lung sections (A, D) with Celsr1Crsh (B, E), and Vangl2Lp (C, F) showed no apparent alteration in the (more ...)
Branching morphogenesis defects in Celsr1Crsh and Vangl2Lp ex vivo lung culture
The predominant driver of lung morphogenesis prior to E16.5 is branching morphogenesis, the process whereby the simple epithelial tube splits into numerous smaller tubes to form the vast number of airways. As airway number is significantly reduced at E14.5 in the mutant lungs, we hypothesized that PCP function is required for lung bud branching. Homozygous Celsr1Crsh
mutant embryos do not exhibit developmental delay as assessed by limb development, however, the rostrocaudal axis is markedly shorter due to disruption of axial convergent extension (31
). The shortened axis likely results in reduced intrathoracic space, which can affect lung development (33
). To circumvent space restriction, we used ex vivo
culture of E11.5 lungs from intercrosses between Celsr1Crsh
heterozygotes and examined homozygous mutant and wild-type lungs at 0 and 48 h. Bud number in wild-type and mutant lungs was the same at the beginning of branching morphogenesis, t
= 0 (Fig. A–C). However, after 48 h in culture the mutant lungs were smaller with significantly fewer terminal buds than wild-type littermates resulting in a simpler epithelial tree structure (Fig. D–F and G). Moreover, terminal buds were significantly enlarged (dashed lines in Fig. E and F compared with Fig. D, quantification in Fig. H). These results indicate that Celsr1
are required for normal lung branching morphogenesis and disrupted branching in mutant lungs is not a consequence of space restriction.
Figure 3. Ex vivo lung cultures reveal that mutations in Celsr1 and Vangl2 phenocopy treatment with Rho kinase inhibitor and Rho kinase activation partially rescues the branching defect in Celsr1Crsh. E11.5 left lung lobes from wild-type (A, D); Vangl2Lp (B, E (more ...)
Celsr1 and Vangl2 are required for all three modes of lung branching
Recent studies identified three modes of branching in mouse lung: domain branching, planar bifurcation and orthogonal bifurcation (34
). These three modes of branching are used repeatedly to form a stereotypical pattern of branches in the lung. Domain branching is particularly used during the early stages of branching and involves new bud formation at regular intervals both along the length and around the circumference of an existing branch creating a ‘bottle-brush’ type of structure. Planar and orthogonal bifurcation both involve a single bud tip separating into two distinct tips that are either within the same plane (planar) or at right angles (orthogonal) to the original end bud (see Metzger et al
for further details). To determine whether mutations in the PCP pathway affected all branching modes or whether PCP signalling is part of the molecular mechanisms controlling formation of branches by one particular mode, we immunostained wild-type E12.5 and E14.5 whole lungs with antibodies to Celsr1 or Vangl2 and counterstained with pan-cytokeratin to highlight epithelium (Fig. , yellow, red, white lines denote planar, domain and orthogonal branching, respectively). Laser scanning confocal examination of wild-type lungs revealed Celsr1 and Vangl2 expression in all three branch modes (Fig. B, E, F and H). In addition, despite the defects already described and in particular the decreased branch number, examples of all three modes of branching were visible in Celsr1Crsh
homozygous lungs (Fig. C, D and data not shown) providing further evidence that Celsr1
are part of a general mechanism governing bud/branch formation.
Figure 4. Celsr1 and Vangl2 are expressed in all modes of branching. Confocal images of wild-type E14.5 (A, B, G, H) and Celsr1Crsh (C, D) or E12.5 wild-type (E, F) whole lungs immunostained with anti-cytokeratin (A, C, D, G), anti-Celsr1 (B, E, F) or anti-Vangl2 (more ...)
Celsr1Crsh and Vangl2Lp mutant lungs phenocopy lungs treated with Rho kinase inhibitor and Rho activation partially rescues the branching defect in Crsh
Rho kinases are key downstream effectors of the PCP pathway. Previous studies have shown that Rho kinases are important mediators of cellular morphogenesis; they facilitate cytoskeletal remodelling, play an obligatory role in embryonic morphogenesis and are required for normal lung branching morphogenesis (10
). To investigate whether Rho kinase may be part of the downstream signalling pathway utilized by Celsr1
in lung development, we explanted lungs from wild-type mice and cultured them with Rho kinase inhibitor (Y27632) (32
). We observed a dose-dependent inhibition of branching morphogenesis and enlarged terminal buds (Fig. I and L) as reported previously (9
). Importantly the phenotype following Rho kinase inhibition is very similar to the phenotype of Celsr1Crsh
lungs both in culture and in vivo
. Moreover, common to both mutants and wild-type lungs treated with Y27632, direct disruption of the actin cytoskeleton with Cytochalasin D also resulted in fewer and broader buds (Fig. J and M). Neither Y27632 nor Cytochalasin D adversely affected cell survival, as assessed by comparing the number of fragmented nuclei in control and treated explants following DAPI staining of explants post-culture (Supplementary Material, Fig. S2A–C
). Notably, addition of the Rho activator CNF-1 (38
) to wild-type lung explants stimulated branching morphogenesis, resulting in an increased number of buds (Fig. N).
In a separate set of experiments, the addition of CNF-1 to Crsh/Crsh mutant lungs also led to an increase in bud numbers (Fig. K and N). The increase in bud numbers was greater in Crsh/Crsh lungs than in wild-type (24% increase in Crsh/Crsh compared with 16% increase in wild-type Fig. N) indicating that activating the Rho signalling pathway in mutant embryos is able to partially ameliorate the branching defect. These data are consistent with Rho kinase being a downstream effector of the PCP signalling pathway in lung development. Thus, we propose that the defective cellular organization in Celsr1Crsh and Vangl2Lp mutants results from disruption to Rho kinase function which likely leads to cytoskeletal defects, thus perturbing tissue structure.
Mutations in Celsr1 and Vangl2 lead to disrupted cytoskeletal organization and disordered epithelial airways
To further investigate the hypothesis that Celsr1 and Vangl2 may influence tissue morphogenesis by affecting cytoskeletal organization, we analyzed the appearance of the actin-myosin cytoskeleton and adherens junctions in E14.5 lung tissue. Phalloidin staining of F-actin highlighted the disorder amongst cells in mutant tissue. In wild-type tissue, staining was visible around the entire circumference of cells, this was particularly evident in mesenchyme; in epithelial airways, a strong band of actin was visible surrounding the lumen (Fig. A). However, in Celsr1Crsh
lung tissue, actin was more discontinuous and diffuse around the circumference of many cells and we did not observe a distinct band of actin surrounding the narrower mutant lumina (Fig. B and C). At E18.5, phalloidin staining continued to reveal differences in the F-actin cytoskeleton of wild-type and mutant lung tissue. In wild-type lung, areas of focal enrichment of actin were observed within individual cells throughout the tissue, particularly in cells adjacent to airways (Fig. D). In both mutants, localized enrichment of actin was observed in some cells, however, this was either distributed evenly around the entire circumference of cells in Celsr1Crsh
(Fig. E) or was discontinuous and diffuse in Vangl2Lp
(Fig. F). Moreover, in both mutants, those cells which did display focal enrichment of actin were frequently not adjacent to ‘airways’. Levels of non-muscle myosin II, another critical component of the cytoskeleton which directly links the Rho signalling pathway with cytoskeletal dynamics (39
), were also perturbed and its spatial distribution altered at E14.5 in Celsr1Crsh
mutants (Fig. H and I; wild-type in G). In common with our earlier observations, both F-actin and myosin appeared considerably more disrupted in Celsr1Crsh
than in Vangl2Lp
. The cytoskeletal defects observed in Celsr1Crsh
lungs might be expected to cause disruption of adherens junctions, however, staining with anti-β-catenin antibody (Fig. J–L) showed no apparent changes in Celsr1Crsh
lungs indicating that adherens junctions were not grossly affected.
Figure 5. Mutant lungs display aberrant cell architecture, yet apical–basal polarity remains intact. Cryosections of E14.5 wild-type (A, D, G, J, M, P) Celsr1Crsh (B, E, H, K, N, Q) and Vangl2Lp (C, F, I, L, O, R). Rhodamine phalloidin staining of F-actin (more ...)
In light of the aberrant epithelial tube morphogenesis in Celsr1Crsh
lungs, it was important to determine whether apical–basal polarity was disrupted. Immunolabelling with ZO-2 and laminin, to highlight the apical and basal sides of airways, respectively (Fig. M–O), as well as with the apical membrane marker aPKCζ (Fig. P–R) and GM-130 (Supplementary Material, Fig. S3A–C
) to mark the polarized localization of the Golgi apparatus, showed no overt disruption to apical–basal polarity in either mutant. Some differences in the precise patterns of expression of markers was visible in mutants, however, this likely reflects the misalignment of cells, the narrowed lumina and the overall perturbation of tissue morphogenesis, rather than disrupted apical–basal polarity. Thus, mutations in Celsr1
lead to disruption of the cytoskeleton resulting in disordered epithelial airways.
Lung endoderm from Celsr1Crsh and Vangl2Lp mutants responds to the chemoattractant FGF10 but is unable to branch
We wished to determine whether the tissue morphogenesis defects observed in Celsr1
mutant lungs could reflect a defect in the response of mutant endoderm to a key signal for branching. To test this idea we exposed Celsr1Crsh
and wild-type lung epithelium denuded of mesenchyme to FGF10, which normally signals from the mesenchyme to direct lung branching. Both wild-type and mutant lungs responded to the FGF10 stimulus in terms of growth. However, whereas wild-type epithelium formed multiple long and narrow buds (Fig. A and B), in most cases, the mutant lung epithelium did not undergo any branching in response to FGF10 (Fig. C, D or rarely one or two short stumpy buds formed). Staining for phospho-ERK1/2, which is up-regulated in response to FGF10 in the lung (40
), revealed no significant difference in the number of positive cells in wild-type and mutant lung epithelium and this was confirmed by western blot (Fig. E and F). Together these results indicate that the FGF signalling pathway, at least that mediated by ERK1/2 activators, is unaffected in mutant lungs. Thus, normal morphogenetic movement within the epithelium is affected upon disruption of the PCP genes Celsr1
, rendering the epithelium almost incapable of branching in response to an FGF10 stimulus.
Figure 6. Mutant lung endoderm responds to FGF10 but is unable to undergo branching. (A, B) Wild-type lung endoderm branches in response to 400 ng/ml FGF10 (mean bud number = 11, n = 6). (C, D) mutant lungs do not branch in response to FGF10 (mean bud number Crsh (more ...)
Celsr1 and Vangl2 proteins are spatially restricted and differentially expressed in lung epithelia
To provide additional mechanistic insight into how Celsr1 and Vangl2 proteins regulate tissue morphogenesis during lung branching, we next examined their expression patterns in E11.5 and E14.5 lung sections by immunohistochemistry. Studies in other tissues and organisms have shown that these proteins often co-localize at cell membranes and are thought to form a multi-protein complex (20
). In the lung we did observe co-localization in some regions of lung epithelia but we also noted some differences in the spatial expression of Celsr1 and Vangl2.
At E11.5 at the onset of branching of the secondary buds, Celsr1 expression was mainly restricted to lung epithelium and staining was enriched in the basal membranes as well as towards the more apical side of airways (Fig. A). Double immunostaining of Celsr1 with laminin revealed co-localization indicating that Celsr1 was also present in the basement membrane (Fig. A–C). This surprising result was confirmed by comparison of the Celsr1 and laminin double labelling with that for laminin and the basolateral membrane marker, β-catenin; no co-localization of β-catenin and laminin was observed since these two proteins are expressed in different compartments (compare Supplementary Material, Fig. S3D–F with H–J
). We also noted that basement membrane Celsr1 staining frequently was not evenly distributed around the entire airway and instead was localized to the basement membrane on one side or a portion of the airway, rather than being evenly distributed around it. Interestingly, laminin shares this uneven, differential distribution around the basal side of airways which results from thinning or discontinuity of the basement membrane at the epithelial/mesenchymal interface in regions of active bud outgrowth (42
). The co-localization of Celsr1 with laminin in the basement membrane indicated that Celsr1 associates with areas of morphogenetic stability such as clefts (42
Figure 7. Celsr1 and Vangl2 proteins are spatially restricted and differentially expressed in lung epithelia. (A, B) E11.5 transverse cryosections of lungs immunostained with antibodies against Celsr1 (A) and Vangl2 (B). (C–F) Immunostaining of transverse (more ...)
Figure 8. Differential expression of Celsr1 and Vangl2 is observed in branching lung endoderm explants and morpholino knockdown highlights a role for Celsr1 in bifurcation. Double-labelling of wild-type E14.5 cryosections with Celsr1 (A, C) and laminin (B, C) antibodies. (more ...)
Punctate Vangl2 staining was observed in both epithelium and mesenchyme, though expression in the epithelium was stronger. In the epithelium, Vangl2 staining was more evenly distributed around cell membranes than Celsr1, however, in many airways, we observed an enrichment of Vangl2 expression in cells surrounding the lumen (Fig. B), in agreement with a previous study (43
At E14.5 the patterns of Celsr1 (Fig. C) and Vangl2 (Fig. E) were similar to E11.5, but the enrichment of Celsr1 towards the luminal side of airways was more predominant. Immunostaining of E14.5 lung sections from homozygous mutants of Celsr1Crsh with anti-Celsr1 (Fig. D) and Vangl2Lp with anti-Vangl2 (Fig. F) antibodies revealed a dramatic reduction in protein levels in mutant lung tissue relative to wild-type. This demonstrates the specificity of the antibodies and provides supportive evidence that these mutations represent loss of function alleles.
The spatial and temporal distribution of Celsr1 and Vangl2 in lung epithelium clearly overlaps in some regions, such as the apical membranes, but also differs in others, suggesting a differential role for these proteins in some aspects of lung development. This observation prompted us to examine whether Celsr1 was correctly localized in Vangl2Lp mutants and conversely whether Vangl2 was correctly localized in Celsr1Crsh mutants. In Lp/Lp lungs, the spatial distribution of Celsr1 did not appear to be altered (Fig. G). In contrast, we observed a loss of enrichment of Vangl2 in cells adjacent to the lumen in Crsh/Crsh lungs (Fig. H) suggesting that Celsr1 is required for this enrichment of Vangl2. Thus, despite differences in Celsr1 and Vangl2 expression patterns, mutations in Celsr1 affect the localization of Vangl2, suggesting an interaction between these proteins in lung.
Celsr1 and Vangl2 expression is enriched in highly specific regions of the branching lung epithelium
Lung branching morphogenesis is a complex 3D process that depends on interactions between the mesenchyme and the epithelium. However, it is possible to simplify this system by culture of the lung endoderm denuded of mesenchyme in the presence of FGF10 to induce branching. In this way, we can gain a better 3D view of the spatial expression of both Celsr1 and Vangl2 in the endoderm during the branching process. Specifically, wild-type lung endoderm explants denuded of mesenchyme were cultured with FGF10 to induce branching and subsequently immunostained for Celsr1 or Vangl2 and phalloidin to detect F-actin cytoskeleton (Fig. D–K). This 3D view of branching endoderm revealed dramatic differences in the expression patterns of Celsr1 and Vangl2. Low levels of both proteins were observed in membranes of epithelial cells, imaging through z-stacks of endoderm explants revealed clear enrichment of Vangl2 at the apical/luminal surface of epithelial buds (Figs F, G, J, K and H). Celsr1 was also enriched towards the luminal surface of buds but in addition, high levels of Celsr1 were detected in the basement membrane, in regions of restricted tissue growth, e.g. immediately adjacent to a bud or at sites of bud bifurcation (Figs D, E, H, I and E and F) These data confirm what we had previously observed both in lung sections (Fig. A–C and E) and with wholemount antibody staining of lungs (Fig. B, E, F, H) and strengthen the idea that the functions of Celsr1 and Vangl2 are not completely overlapping.
Morpholino knockdown reveals a role for Celsr1 in bud bifurcation
Given the intriguing expression pattern of Celsr1 in the basement membrane surrounding lung endoderm, we sought to understand more precisely how Celsr1
affects branching morphogenesis. To do this, we conducted time-lapse imaging of E11.5 wild-type lung explants in the presence of one of two Celsr1 morpholinos (MO) directed against different Celsr1 sites, or control morpholino. Identical results were obtained with both Celsr1 MOs. Knockdown efficiency was validated by immunostaining and western blotting (Supplementary Material, Fig. S4
Wild-type lungs cultured with control MO exhibited a reproducible branching pattern in culture. The process of branching began with a uniform increase in distal bud size followed by bifurcation (Fig. L–N). During bifurcation, the two sides of the bud branched and grew outwards, whereas cells in the middle were constrained and remained in place. In contrast, lungs cultured with Celsr1 MO formed greatly expanded ‘fat’ buds and bifurcation into two new buds did not occur (Fig. O–Q). In Figure L and O, a single bud is shown for both Control and Celsr1-MO at t = 0. In the control, a cleft forms by 200 min (Fig. M) and is in the same position and deeper by 360 min (Fig. N). At t = 0 in in the Celsr1 MO treated culture (Fig. O), a deformity in the epithelial bud is visible but this is not retained in the same position in the bud at 200 or 360 min (Fig. P and Q). Moreover, the deformity/apparent cleft does not deepen showing that this is not the beginning of bifurcation. Considerable movement of the epithelial sheet occurs in the Celsr1 MO treated explants resulting in a looser/uneven bud shape. In some buds, after considerable delay, multiple small new buds formed randomly in a non-stereotypical fashion. The absence of properly co-ordinated bifurcation of distal lung buds treated with Celsr1 MO coupled with our protein localization data indicates that Celsr1 is required for bifurcation during the branching process.