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The PCP genes Celsr1 and Vangl2 are required for normal lung branching morphogenesis.

Yates LL, Schnatwinkel C, Murdoch JN, Bogani D, Formstone CJ, Townsend S, Greenfield A, Niswander LA, Dean CH - Hum. Mol. Genet. (2010)

Bottom Line: Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis.We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway.Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Harwell, Oxfordshire OX11 0RD, UK.

ABSTRACT
The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.

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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. E11.5 lung endoderm explants cultured for 48 h in 400 ng/ml FGF10 and double labelled with phalloidin (H–K) and Celsr1 (D, E) or Vangl2 (F, G) antibodies. High levels of Celsr1 expression are present in regions of restricted tissue growth such as points of bifurcation (D, E, H, I). Vangl2 is most highly expressed at the luminal surface of outgrowing buds (F, G, J, K). E11.5 lung explants from β-actin promoter driven GFP embryos were cultured for 48 h in the presence of control (L–N) or Celsr1 (O–Q) morpholinos and subsequently imaged over a 24 h period. Images show three timepoints from this series. Scale bars: (A–C) 125 µM ×2 zoom (D, H, F, J) 125 µM ×2.7 zoom, (E, I) 125 µM ×10 zoom, (G, K) 125 µM ×8 zoom, (L–Q) 50 µM.
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DDQ104F8: 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. E11.5 lung endoderm explants cultured for 48 h in 400 ng/ml FGF10 and double labelled with phalloidin (H–K) and Celsr1 (D, E) or Vangl2 (F, G) antibodies. High levels of Celsr1 expression are present in regions of restricted tissue growth such as points of bifurcation (D, E, H, I). Vangl2 is most highly expressed at the luminal surface of outgrowing buds (F, G, J, K). E11.5 lung explants from β-actin promoter driven GFP embryos were cultured for 48 h in the presence of control (L–N) or Celsr1 (O–Q) morpholinos and subsequently imaged over a 24 h period. Images show three timepoints from this series. Scale bars: (A–C) 125 µM ×2 zoom (D, H, F, J) 125 µM ×2.7 zoom, (E, I) 125 µM ×10 zoom, (G, K) 125 µM ×8 zoom, (L–Q) 50 µM.

Mentions: 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. 7A). Double immunostaining of Celsr1 with laminin revealed co-localization indicating that Celsr1 was also present in the basement membrane (Fig. 8A–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).


The PCP genes Celsr1 and Vangl2 are required for normal lung branching morphogenesis.

Yates LL, Schnatwinkel C, Murdoch JN, Bogani D, Formstone CJ, Townsend S, Greenfield A, Niswander LA, Dean CH - Hum. Mol. Genet. (2010)

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. E11.5 lung endoderm explants cultured for 48 h in 400 ng/ml FGF10 and double labelled with phalloidin (H–K) and Celsr1 (D, E) or Vangl2 (F, G) antibodies. High levels of Celsr1 expression are present in regions of restricted tissue growth such as points of bifurcation (D, E, H, I). Vangl2 is most highly expressed at the luminal surface of outgrowing buds (F, G, J, K). E11.5 lung explants from β-actin promoter driven GFP embryos were cultured for 48 h in the presence of control (L–N) or Celsr1 (O–Q) morpholinos and subsequently imaged over a 24 h period. Images show three timepoints from this series. Scale bars: (A–C) 125 µM ×2 zoom (D, H, F, J) 125 µM ×2.7 zoom, (E, I) 125 µM ×10 zoom, (G, K) 125 µM ×8 zoom, (L–Q) 50 µM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC2865378&req=5

DDQ104F8: 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. E11.5 lung endoderm explants cultured for 48 h in 400 ng/ml FGF10 and double labelled with phalloidin (H–K) and Celsr1 (D, E) or Vangl2 (F, G) antibodies. High levels of Celsr1 expression are present in regions of restricted tissue growth such as points of bifurcation (D, E, H, I). Vangl2 is most highly expressed at the luminal surface of outgrowing buds (F, G, J, K). E11.5 lung explants from β-actin promoter driven GFP embryos were cultured for 48 h in the presence of control (L–N) or Celsr1 (O–Q) morpholinos and subsequently imaged over a 24 h period. Images show three timepoints from this series. Scale bars: (A–C) 125 µM ×2 zoom (D, H, F, J) 125 µM ×2.7 zoom, (E, I) 125 µM ×10 zoom, (G, K) 125 µM ×8 zoom, (L–Q) 50 µM.
Mentions: 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. 7A). Double immunostaining of Celsr1 with laminin revealed co-localization indicating that Celsr1 was also present in the basement membrane (Fig. 8A–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).

Bottom Line: Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis.We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway.Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Harwell, Oxfordshire OX11 0RD, UK.

ABSTRACT
The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus