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Altered lung morphogenesis, epithelial cell differentiation and mechanics in mice deficient in the Wnt/β-catenin antagonist Chibby.

Love D, Li FQ, Burke MC, Cyge B, Ohmitsu M, Cabello J, Larson JE, Brody SL, Cohen JC, Takemaru K - PLoS ONE (2010)

Bottom Line: Notably, Cby protein is prominently associated with the centrosome/basal body microtubule structures in embryonic lung epithelial progenitor cells, and later enriches as discrete foci at the base of motile cilia in airway ciliated cells.At birth, Cby(-/-) lungs are grossly normal but spontaneously develop alveolar airspace enlargement with reduced proliferation and abnormal differentiation of lung epithelial cells, resulting in altered pulmonary function.Collectively, our results demonstrate that Cby facilitates proper postnatal lung development and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, New York, United States of America.

ABSTRACT
The canonical Wnt/β-catenin pathway plays crucial roles in various aspects of lung morphogenesis and regeneration/repair. Here, we examined the lung phenotype and function in mice lacking the Wnt/β-catenin antagonist Chibby (Cby). In support of its inhibitory role in canonical Wnt signaling, expression of β-catenin target genes is elevated in the Cby(-/-) lung. Notably, Cby protein is prominently associated with the centrosome/basal body microtubule structures in embryonic lung epithelial progenitor cells, and later enriches as discrete foci at the base of motile cilia in airway ciliated cells. At birth, Cby(-/-) lungs are grossly normal but spontaneously develop alveolar airspace enlargement with reduced proliferation and abnormal differentiation of lung epithelial cells, resulting in altered pulmonary function. Consistent with the Cby expression pattern, airway ciliated cells exhibit a marked paucity of motile cilia with apparent failure of basal body docking. Moreover, we demonstrate that Cby is a direct downstream target for the master ciliogenesis transcription factor Foxj1. Collectively, our results demonstrate that Cby facilitates proper postnatal lung development and function.

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Cby is a direct target for Foxj1.(A) Foxj1 is expressed in Cby−/− lungs. Airway sections from E15.5 embryos were immunostained with anti-Foxj1 antibody, followed by hematoxylin counterstain. Arrows indicate positive nuclear staining of Foxj1. Scale bar, 50 µm. (B) Foxj1 directly activates Cby expression. Sequence analysis revealed 6 putative Foxj1-binding sites within the 2-kb mouse Cby promoter region (ovals). A predicted TATA box (TATGAA) was found at -64. The start of a mouse Cby cDNA sequence (GenBank accession number NM_028634) was tentatively designated as +1. The 5′ promoter deletion constructs are also illustrated. For luciferase reporter assays, HEK293T cells were transfected with 100 ng of each Cby promoter construct with the indicated amounts of a Foxj1 plasmid. Luciferase activity was measured 24 h after transfection and normalized to Renilla luciferase activity used as an internal control. The basal luciferase value of each Cby promoter reporter was set as 1. Transfections were done in triplicate, and the means ± SD are shown.
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pone-0013600-g008: Cby is a direct target for Foxj1.(A) Foxj1 is expressed in Cby−/− lungs. Airway sections from E15.5 embryos were immunostained with anti-Foxj1 antibody, followed by hematoxylin counterstain. Arrows indicate positive nuclear staining of Foxj1. Scale bar, 50 µm. (B) Foxj1 directly activates Cby expression. Sequence analysis revealed 6 putative Foxj1-binding sites within the 2-kb mouse Cby promoter region (ovals). A predicted TATA box (TATGAA) was found at -64. The start of a mouse Cby cDNA sequence (GenBank accession number NM_028634) was tentatively designated as +1. The 5′ promoter deletion constructs are also illustrated. For luciferase reporter assays, HEK293T cells were transfected with 100 ng of each Cby promoter construct with the indicated amounts of a Foxj1 plasmid. Luciferase activity was measured 24 h after transfection and normalized to Renilla luciferase activity used as an internal control. The basal luciferase value of each Cby promoter reporter was set as 1. Transfections were done in triplicate, and the means ± SD are shown.

Mentions: The ciliary phenotypes of Cby−/− mice and high expression of Cby in ciliated cells are reminiscent of those associated with the master ciliogenesis transcription factor Foxj1. Foxj1 is expressed in respiratory ciliated cells, and drives the motile ciliogenesis program by directly stimulating expression of various ciliogenesis genes including dyneins [31], [32], [33], [34]. This prompted us to investigate whether Foxj1 is expressed in Cby−/− mice. In mouse lungs, expression of Foxj1 begins at E15.5, before the appearance of cilia, in differentiating ciliated cells [33]. As shown in Figure 8A, Foxj1 was detectable in the airway epithelial cell nuclei of E15.5 Cby−/− lungs. These results imply that Cby lies downstream of Foxj1 in ciliated cells.


Altered lung morphogenesis, epithelial cell differentiation and mechanics in mice deficient in the Wnt/β-catenin antagonist Chibby.

Love D, Li FQ, Burke MC, Cyge B, Ohmitsu M, Cabello J, Larson JE, Brody SL, Cohen JC, Takemaru K - PLoS ONE (2010)

Cby is a direct target for Foxj1.(A) Foxj1 is expressed in Cby−/− lungs. Airway sections from E15.5 embryos were immunostained with anti-Foxj1 antibody, followed by hematoxylin counterstain. Arrows indicate positive nuclear staining of Foxj1. Scale bar, 50 µm. (B) Foxj1 directly activates Cby expression. Sequence analysis revealed 6 putative Foxj1-binding sites within the 2-kb mouse Cby promoter region (ovals). A predicted TATA box (TATGAA) was found at -64. The start of a mouse Cby cDNA sequence (GenBank accession number NM_028634) was tentatively designated as +1. The 5′ promoter deletion constructs are also illustrated. For luciferase reporter assays, HEK293T cells were transfected with 100 ng of each Cby promoter construct with the indicated amounts of a Foxj1 plasmid. Luciferase activity was measured 24 h after transfection and normalized to Renilla luciferase activity used as an internal control. The basal luciferase value of each Cby promoter reporter was set as 1. Transfections were done in triplicate, and the means ± SD are shown.
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Related In: Results  -  Collection

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pone-0013600-g008: Cby is a direct target for Foxj1.(A) Foxj1 is expressed in Cby−/− lungs. Airway sections from E15.5 embryos were immunostained with anti-Foxj1 antibody, followed by hematoxylin counterstain. Arrows indicate positive nuclear staining of Foxj1. Scale bar, 50 µm. (B) Foxj1 directly activates Cby expression. Sequence analysis revealed 6 putative Foxj1-binding sites within the 2-kb mouse Cby promoter region (ovals). A predicted TATA box (TATGAA) was found at -64. The start of a mouse Cby cDNA sequence (GenBank accession number NM_028634) was tentatively designated as +1. The 5′ promoter deletion constructs are also illustrated. For luciferase reporter assays, HEK293T cells were transfected with 100 ng of each Cby promoter construct with the indicated amounts of a Foxj1 plasmid. Luciferase activity was measured 24 h after transfection and normalized to Renilla luciferase activity used as an internal control. The basal luciferase value of each Cby promoter reporter was set as 1. Transfections were done in triplicate, and the means ± SD are shown.
Mentions: The ciliary phenotypes of Cby−/− mice and high expression of Cby in ciliated cells are reminiscent of those associated with the master ciliogenesis transcription factor Foxj1. Foxj1 is expressed in respiratory ciliated cells, and drives the motile ciliogenesis program by directly stimulating expression of various ciliogenesis genes including dyneins [31], [32], [33], [34]. This prompted us to investigate whether Foxj1 is expressed in Cby−/− mice. In mouse lungs, expression of Foxj1 begins at E15.5, before the appearance of cilia, in differentiating ciliated cells [33]. As shown in Figure 8A, Foxj1 was detectable in the airway epithelial cell nuclei of E15.5 Cby−/− lungs. These results imply that Cby lies downstream of Foxj1 in ciliated cells.

Bottom Line: Notably, Cby protein is prominently associated with the centrosome/basal body microtubule structures in embryonic lung epithelial progenitor cells, and later enriches as discrete foci at the base of motile cilia in airway ciliated cells.At birth, Cby(-/-) lungs are grossly normal but spontaneously develop alveolar airspace enlargement with reduced proliferation and abnormal differentiation of lung epithelial cells, resulting in altered pulmonary function.Collectively, our results demonstrate that Cby facilitates proper postnatal lung development and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, New York, United States of America.

ABSTRACT
The canonical Wnt/β-catenin pathway plays crucial roles in various aspects of lung morphogenesis and regeneration/repair. Here, we examined the lung phenotype and function in mice lacking the Wnt/β-catenin antagonist Chibby (Cby). In support of its inhibitory role in canonical Wnt signaling, expression of β-catenin target genes is elevated in the Cby(-/-) lung. Notably, Cby protein is prominently associated with the centrosome/basal body microtubule structures in embryonic lung epithelial progenitor cells, and later enriches as discrete foci at the base of motile cilia in airway ciliated cells. At birth, Cby(-/-) lungs are grossly normal but spontaneously develop alveolar airspace enlargement with reduced proliferation and abnormal differentiation of lung epithelial cells, resulting in altered pulmonary function. Consistent with the Cby expression pattern, airway ciliated cells exhibit a marked paucity of motile cilia with apparent failure of basal body docking. Moreover, we demonstrate that Cby is a direct downstream target for the master ciliogenesis transcription factor Foxj1. Collectively, our results demonstrate that Cby facilitates proper postnatal lung development and function.

Show MeSH
Related in: MedlinePlus