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cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

Bird AD, Flecknoe SJ, Tan KH, Olsson PF, Antony N, Mantamadiotis T, Mollard R, Hooper SB, Cole TJ - PLoS ONE (2011)

Bottom Line: Creb1(-/-) lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/-) lung on a Crem(-/-) genetic background.Cell differentiation of E17.5 Creb1(-/-) lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells.Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia. daniel.bird@monash.edu

ABSTRACT
The cAMP response element binding protein 1 (Creb1) transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/-) fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/-) fetal mice during development. Lungs of Creb1(-/-) fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/-) lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/-) lung on a Crem(-/-) genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/-) lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara), and neuroendocrine cells showed delayed onset of expression in the Creb1(-/-) lung. Finally, gene expression analyses of the E17.5 Creb1(-/-) lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

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Gene expression analysis of highly-differential Creb1 microarray lung gene targets.qPCR analysis of mRNA levels for down-regulated microarray gene targets: Chi3l1, Lyz1/2, and Lcn2 in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (A). qPCR analysis of mRNA levels for up-regulated microarray gene targets: Hist2h3c1, Hist1h3g, and D6Mm5e in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (B). Error bars represent SEM. Asterisk (*) indicates p<0.05 and ** indicates p<0.001. White bars: Wildtype, Black bars: Creb1−/−.
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pone-0017843-g009: Gene expression analysis of highly-differential Creb1 microarray lung gene targets.qPCR analysis of mRNA levels for down-regulated microarray gene targets: Chi3l1, Lyz1/2, and Lcn2 in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (A). qPCR analysis of mRNA levels for up-regulated microarray gene targets: Hist2h3c1, Hist1h3g, and D6Mm5e in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (B). Error bars represent SEM. Asterisk (*) indicates p<0.05 and ** indicates p<0.001. White bars: Wildtype, Black bars: Creb1−/−.

Mentions: In order to validate our microarray data, we further investigated differential gene expression for the three most-highly down-regulated (Chi3l1, Lyz1 and Lcn2) and up-regulated (Hist2h3c1, Hist1h3g and D6Mm5e) gene targets on our list in the lung of E17.5 Creb1−/− mice using qPCR (n = 4). Levels of Chi3l1 and Lcn2 mRNAs were reduced 12.5 fold (p<0.001) and 2.9 fold (p<0.05) in respectively (Fig. 9A). Due to very high sequence identity between Lyz1 and Lyz2 in mice, qPCR primers recognised Lyz2 as well as Lyz1, and we detected a 2.6 fold (p<0.05) reduction in Lyz1/2 levels in the Creb1−/− lung (Fig. 8A). Levels of Hist2h3c1 mRNA were increased 4.0 fold in the Creb1−/− lung, but did not quite reach statistical significance (p = 0.09), while Hist1h3g and D6Mm5e mRNA levels increased 5.0 (p<0.05) and 6.5 fold (p<0.05) respectively, in the Creb1−/− lung (Fig. 9B). Taken together, these data support the validity of our microarray analysis.


cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

Bird AD, Flecknoe SJ, Tan KH, Olsson PF, Antony N, Mantamadiotis T, Mollard R, Hooper SB, Cole TJ - PLoS ONE (2011)

Gene expression analysis of highly-differential Creb1 microarray lung gene targets.qPCR analysis of mRNA levels for down-regulated microarray gene targets: Chi3l1, Lyz1/2, and Lcn2 in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (A). qPCR analysis of mRNA levels for up-regulated microarray gene targets: Hist2h3c1, Hist1h3g, and D6Mm5e in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (B). Error bars represent SEM. Asterisk (*) indicates p<0.05 and ** indicates p<0.001. White bars: Wildtype, Black bars: Creb1−/−.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050929&req=5

pone-0017843-g009: Gene expression analysis of highly-differential Creb1 microarray lung gene targets.qPCR analysis of mRNA levels for down-regulated microarray gene targets: Chi3l1, Lyz1/2, and Lcn2 in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (A). qPCR analysis of mRNA levels for up-regulated microarray gene targets: Hist2h3c1, Hist1h3g, and D6Mm5e in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4) (B). Error bars represent SEM. Asterisk (*) indicates p<0.05 and ** indicates p<0.001. White bars: Wildtype, Black bars: Creb1−/−.
Mentions: In order to validate our microarray data, we further investigated differential gene expression for the three most-highly down-regulated (Chi3l1, Lyz1 and Lcn2) and up-regulated (Hist2h3c1, Hist1h3g and D6Mm5e) gene targets on our list in the lung of E17.5 Creb1−/− mice using qPCR (n = 4). Levels of Chi3l1 and Lcn2 mRNAs were reduced 12.5 fold (p<0.001) and 2.9 fold (p<0.05) in respectively (Fig. 9A). Due to very high sequence identity between Lyz1 and Lyz2 in mice, qPCR primers recognised Lyz2 as well as Lyz1, and we detected a 2.6 fold (p<0.05) reduction in Lyz1/2 levels in the Creb1−/− lung (Fig. 8A). Levels of Hist2h3c1 mRNA were increased 4.0 fold in the Creb1−/− lung, but did not quite reach statistical significance (p = 0.09), while Hist1h3g and D6Mm5e mRNA levels increased 5.0 (p<0.05) and 6.5 fold (p<0.05) respectively, in the Creb1−/− lung (Fig. 9B). Taken together, these data support the validity of our microarray analysis.

Bottom Line: Creb1(-/-) lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/-) lung on a Crem(-/-) genetic background.Cell differentiation of E17.5 Creb1(-/-) lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells.Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia. daniel.bird@monash.edu

ABSTRACT
The cAMP response element binding protein 1 (Creb1) transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/-) fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/-) fetal mice during development. Lungs of Creb1(-/-) fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/-) lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/-) lung on a Crem(-/-) genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/-) lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara), and neuroendocrine cells showed delayed onset of expression in the Creb1(-/-) lung. Finally, gene expression analyses of the E17.5 Creb1(-/-) lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

Show MeSH
Related in: MedlinePlus