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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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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 staining of sections of E14.5 Celsr1Crsh (E) and Vangl2Lp (F) lungs compared with wild-type (D). (P) There was a significant reduction in the number of epithelial branches in both mutants at E14.5: wild-type 44.42, ±2.43, n = 12; Celsr1Crsh 20.00, ±1.3, n = 12; Vangl2Lp 18.58, ±1.48, n=12. H&E sections from a minimum of three mutants for each genotype were used for counts. Airway lumina were narrow or absent in many Crsh/Crsh and Lp/Lp airways and epithelium had a multilayered morphology, in contrast to the wider lumina surrounded by single layered epithelium that predominates in wild-type. H&E staining of sections of E18.5 wild-type (G), Celsr1Crsh (H), Vangl2Lp (I) lungs. Control lung sections display typical saccular structure and evidence of septation (arrows in G). In contrast, mutant lung sections (H, I) show no evidence of septation. Number (Q) and width (R) of airways is dramatically reduced at E18.5. Number or width of airways was determined by counting airways visible in a complete section of E18.5 lungs from a minimum of two separate embryos (Q) wild-type 90.89, ±3.97; Celsr1Crsh 52.22, ±3.37; Vangl2Lp 69.11, ±3.43, n = 9 for each genotype (R) wild-type 12.13, ±0.05, Celsr1Crsh 4.21, ±0.03, Vangl2Lp 4.63, ±0.03, n = 10 for each genotype. E14.5 cryosections immunostained for expression of pan-cytokeratin (J–L, inserts show airways at higher magnification), corresponding sections to (J–L) counterstained with DAPI (M–O), dashed lines outline cytokeratin positive cells (as seen in J–L) difficult to distinguish from surrounding cells in Celsr1Crsh (N) and Vangl2Lp (O), airways are easily visualized in wild-type (M). Scale bars: (A–C) 62.5 µM; (D–F) 50 µM; (G–O) 12.5 µM, inserts in (D–F); (J–L) 5 µM; *P < 0.05.
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DDQ104F1: 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 staining of sections of E14.5 Celsr1Crsh (E) and Vangl2Lp (F) lungs compared with wild-type (D). (P) There was a significant reduction in the number of epithelial branches in both mutants at E14.5: wild-type 44.42, ±2.43, n = 12; Celsr1Crsh 20.00, ±1.3, n = 12; Vangl2Lp 18.58, ±1.48, n=12. H&E sections from a minimum of three mutants for each genotype were used for counts. Airway lumina were narrow or absent in many Crsh/Crsh and Lp/Lp airways and epithelium had a multilayered morphology, in contrast to the wider lumina surrounded by single layered epithelium that predominates in wild-type. H&E staining of sections of E18.5 wild-type (G), Celsr1Crsh (H), Vangl2Lp (I) lungs. Control lung sections display typical saccular structure and evidence of septation (arrows in G). In contrast, mutant lung sections (H, I) show no evidence of septation. Number (Q) and width (R) of airways is dramatically reduced at E18.5. Number or width of airways was determined by counting airways visible in a complete section of E18.5 lungs from a minimum of two separate embryos (Q) wild-type 90.89, ±3.97; Celsr1Crsh 52.22, ±3.37; Vangl2Lp 69.11, ±3.43, n = 9 for each genotype (R) wild-type 12.13, ±0.05, Celsr1Crsh 4.21, ±0.03, Vangl2Lp 4.63, ±0.03, n = 10 for each genotype. E14.5 cryosections immunostained for expression of pan-cytokeratin (J–L, inserts show airways at higher magnification), corresponding sections to (J–L) counterstained with DAPI (M–O), dashed lines outline cytokeratin positive cells (as seen in J–L) difficult to distinguish from surrounding cells in Celsr1Crsh (N) and Vangl2Lp (O), airways are easily visualized in wild-type (M). Scale bars: (A–C) 62.5 µM; (D–F) 50 µM; (G–O) 12.5 µM, inserts in (D–F); (J–L) 5 µM; *P < 0.05.

Mentions: 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. 1A–C). Analysis of sections of E14.5 Celsr1Crsh and Vangl2Lp homozygous mutant lung stained with H&E (Fig. 1E and F) revealed changes to the structure of the epithelium from that observed in wild-type lung (Fig. 1D). 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. 1D 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. 1E, 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.


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)

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 staining of sections of E14.5 Celsr1Crsh (E) and Vangl2Lp (F) lungs compared with wild-type (D). (P) There was a significant reduction in the number of epithelial branches in both mutants at E14.5: wild-type 44.42, ±2.43, n = 12; Celsr1Crsh 20.00, ±1.3, n = 12; Vangl2Lp 18.58, ±1.48, n=12. H&E sections from a minimum of three mutants for each genotype were used for counts. Airway lumina were narrow or absent in many Crsh/Crsh and Lp/Lp airways and epithelium had a multilayered morphology, in contrast to the wider lumina surrounded by single layered epithelium that predominates in wild-type. H&E staining of sections of E18.5 wild-type (G), Celsr1Crsh (H), Vangl2Lp (I) lungs. Control lung sections display typical saccular structure and evidence of septation (arrows in G). In contrast, mutant lung sections (H, I) show no evidence of septation. Number (Q) and width (R) of airways is dramatically reduced at E18.5. Number or width of airways was determined by counting airways visible in a complete section of E18.5 lungs from a minimum of two separate embryos (Q) wild-type 90.89, ±3.97; Celsr1Crsh 52.22, ±3.37; Vangl2Lp 69.11, ±3.43, n = 9 for each genotype (R) wild-type 12.13, ±0.05, Celsr1Crsh 4.21, ±0.03, Vangl2Lp 4.63, ±0.03, n = 10 for each genotype. E14.5 cryosections immunostained for expression of pan-cytokeratin (J–L, inserts show airways at higher magnification), corresponding sections to (J–L) counterstained with DAPI (M–O), dashed lines outline cytokeratin positive cells (as seen in J–L) difficult to distinguish from surrounding cells in Celsr1Crsh (N) and Vangl2Lp (O), airways are easily visualized in wild-type (M). Scale bars: (A–C) 62.5 µM; (D–F) 50 µM; (G–O) 12.5 µM, inserts in (D–F); (J–L) 5 µM; *P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDQ104F1: 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 staining of sections of E14.5 Celsr1Crsh (E) and Vangl2Lp (F) lungs compared with wild-type (D). (P) There was a significant reduction in the number of epithelial branches in both mutants at E14.5: wild-type 44.42, ±2.43, n = 12; Celsr1Crsh 20.00, ±1.3, n = 12; Vangl2Lp 18.58, ±1.48, n=12. H&E sections from a minimum of three mutants for each genotype were used for counts. Airway lumina were narrow or absent in many Crsh/Crsh and Lp/Lp airways and epithelium had a multilayered morphology, in contrast to the wider lumina surrounded by single layered epithelium that predominates in wild-type. H&E staining of sections of E18.5 wild-type (G), Celsr1Crsh (H), Vangl2Lp (I) lungs. Control lung sections display typical saccular structure and evidence of septation (arrows in G). In contrast, mutant lung sections (H, I) show no evidence of septation. Number (Q) and width (R) of airways is dramatically reduced at E18.5. Number or width of airways was determined by counting airways visible in a complete section of E18.5 lungs from a minimum of two separate embryos (Q) wild-type 90.89, ±3.97; Celsr1Crsh 52.22, ±3.37; Vangl2Lp 69.11, ±3.43, n = 9 for each genotype (R) wild-type 12.13, ±0.05, Celsr1Crsh 4.21, ±0.03, Vangl2Lp 4.63, ±0.03, n = 10 for each genotype. E14.5 cryosections immunostained for expression of pan-cytokeratin (J–L, inserts show airways at higher magnification), corresponding sections to (J–L) counterstained with DAPI (M–O), dashed lines outline cytokeratin positive cells (as seen in J–L) difficult to distinguish from surrounding cells in Celsr1Crsh (N) and Vangl2Lp (O), airways are easily visualized in wild-type (M). Scale bars: (A–C) 62.5 µM; (D–F) 50 µM; (G–O) 12.5 µM, inserts in (D–F); (J–L) 5 µM; *P < 0.05.
Mentions: 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. 1A–C). Analysis of sections of E14.5 Celsr1Crsh and Vangl2Lp homozygous mutant lung stained with H&E (Fig. 1E and F) revealed changes to the structure of the epithelium from that observed in wild-type lung (Fig. 1D). 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. 1D 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. 1E, 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.

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