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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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Delayed proximal epithelial cell differentiation in lungs of Creb1−/− fetal mice.Tissue sections from the lung of E16.5 and E18.5 Creb1−/− and wildtype were immunostained for Foxj1 (A–D), CC10 (E–H), and CGRP (I–L). At E16.5 Foxj1-positive cells were readily detected in conducting airway epithelium (A), but much less frequently in Creb1−/− lungs (C, arrowheads show two Foxj1-positive cells). Increased frequency of Foxj1-positive cells was detected in the lung of both E18.5 wildtype (B) and E18.5 Creb1−/− (D) mice. In wildtype lungs, Scgb1a1-positive cells were not detected at E16.5 (E,) but were common at E18.5 (F). In the lung of E18.5 Creb1−/− mice, Scgb1a1-positive cells were almost completely absent (H, arrowhead shows a solitary Scgb1a1-positive cell). Single CGRP-positive cells were detected at E16.5 in wildtype (I, arrowheads show CGRP-positive cells), but not Creb1−/− (K) lungs. At E18.5 Clusters of CGRP-positive cells were then detected in both wildtype and Creb1−/− lungs (J,L, arrowheads show CGRP-positive cell clusters). qPCR analysis of mRNA levels for Foxj1, Scgb1a1 and Calca in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4)(M). Error bars represent SEM. Asterisk (*) indicates p<0.05. White bars: Wildtype, Black bars: Creb1−/−. Scale bars: A–L, 50 µm.
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pone-0017843-g008: Delayed proximal epithelial cell differentiation in lungs of Creb1−/− fetal mice.Tissue sections from the lung of E16.5 and E18.5 Creb1−/− and wildtype were immunostained for Foxj1 (A–D), CC10 (E–H), and CGRP (I–L). At E16.5 Foxj1-positive cells were readily detected in conducting airway epithelium (A), but much less frequently in Creb1−/− lungs (C, arrowheads show two Foxj1-positive cells). Increased frequency of Foxj1-positive cells was detected in the lung of both E18.5 wildtype (B) and E18.5 Creb1−/− (D) mice. In wildtype lungs, Scgb1a1-positive cells were not detected at E16.5 (E,) but were common at E18.5 (F). In the lung of E18.5 Creb1−/− mice, Scgb1a1-positive cells were almost completely absent (H, arrowhead shows a solitary Scgb1a1-positive cell). Single CGRP-positive cells were detected at E16.5 in wildtype (I, arrowheads show CGRP-positive cells), but not Creb1−/− (K) lungs. At E18.5 Clusters of CGRP-positive cells were then detected in both wildtype and Creb1−/− lungs (J,L, arrowheads show CGRP-positive cell clusters). qPCR analysis of mRNA levels for Foxj1, Scgb1a1 and Calca in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4)(M). Error bars represent SEM. Asterisk (*) indicates p<0.05. White bars: Wildtype, Black bars: Creb1−/−. Scale bars: A–L, 50 µm.

Mentions: Cell differentiation within the proximal airway epithelium was also examined in the lung of Creb1−/− fetal mice, using several specific immunomarkers of proximal airway epithelial cells. Scgb1a1 (also known as CC10, CCSP) is a marker of non-ciliated secretory Clara cells, Foxj1 (also known as HFH-4) is a marker of ciliated epithelial cells, and calcitonin gene related peptide (CGRP, also known as CALCA) is a marker of neuroepithelial cells. Using immunohistochemistry, lungs of E16.5 and E18.5 wildtype and Creb1−/− mice were analysed for expression of these markers as an indicator of proximal epithelial differentiation. Ciliated cells in the developing mouse lung first express Foxj1 at approximately E16.5 [28], and in lungs from E16.5 wildtype mice we detected a proportion of Foxj1-positive cells in the conducting epithelium (Fig. 8A) which increased in frequency at E18.5 (Fig. 8B). In the lung of Creb1−/− mice however, very few Foxj1-positive cells were detected at E16.5 (Fig. 8C), but were found at an increased frequency at E18.5 (Fig. 8D).


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)

Delayed proximal epithelial cell differentiation in lungs of Creb1−/− fetal mice.Tissue sections from the lung of E16.5 and E18.5 Creb1−/− and wildtype were immunostained for Foxj1 (A–D), CC10 (E–H), and CGRP (I–L). At E16.5 Foxj1-positive cells were readily detected in conducting airway epithelium (A), but much less frequently in Creb1−/− lungs (C, arrowheads show two Foxj1-positive cells). Increased frequency of Foxj1-positive cells was detected in the lung of both E18.5 wildtype (B) and E18.5 Creb1−/− (D) mice. In wildtype lungs, Scgb1a1-positive cells were not detected at E16.5 (E,) but were common at E18.5 (F). In the lung of E18.5 Creb1−/− mice, Scgb1a1-positive cells were almost completely absent (H, arrowhead shows a solitary Scgb1a1-positive cell). Single CGRP-positive cells were detected at E16.5 in wildtype (I, arrowheads show CGRP-positive cells), but not Creb1−/− (K) lungs. At E18.5 Clusters of CGRP-positive cells were then detected in both wildtype and Creb1−/− lungs (J,L, arrowheads show CGRP-positive cell clusters). qPCR analysis of mRNA levels for Foxj1, Scgb1a1 and Calca in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4)(M). Error bars represent SEM. Asterisk (*) indicates p<0.05. White bars: Wildtype, Black bars: Creb1−/−. Scale bars: A–L, 50 µm.
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pone-0017843-g008: Delayed proximal epithelial cell differentiation in lungs of Creb1−/− fetal mice.Tissue sections from the lung of E16.5 and E18.5 Creb1−/− and wildtype were immunostained for Foxj1 (A–D), CC10 (E–H), and CGRP (I–L). At E16.5 Foxj1-positive cells were readily detected in conducting airway epithelium (A), but much less frequently in Creb1−/− lungs (C, arrowheads show two Foxj1-positive cells). Increased frequency of Foxj1-positive cells was detected in the lung of both E18.5 wildtype (B) and E18.5 Creb1−/− (D) mice. In wildtype lungs, Scgb1a1-positive cells were not detected at E16.5 (E,) but were common at E18.5 (F). In the lung of E18.5 Creb1−/− mice, Scgb1a1-positive cells were almost completely absent (H, arrowhead shows a solitary Scgb1a1-positive cell). Single CGRP-positive cells were detected at E16.5 in wildtype (I, arrowheads show CGRP-positive cells), but not Creb1−/− (K) lungs. At E18.5 Clusters of CGRP-positive cells were then detected in both wildtype and Creb1−/− lungs (J,L, arrowheads show CGRP-positive cell clusters). qPCR analysis of mRNA levels for Foxj1, Scgb1a1 and Calca in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4)(M). Error bars represent SEM. Asterisk (*) indicates p<0.05. White bars: Wildtype, Black bars: Creb1−/−. Scale bars: A–L, 50 µm.
Mentions: Cell differentiation within the proximal airway epithelium was also examined in the lung of Creb1−/− fetal mice, using several specific immunomarkers of proximal airway epithelial cells. Scgb1a1 (also known as CC10, CCSP) is a marker of non-ciliated secretory Clara cells, Foxj1 (also known as HFH-4) is a marker of ciliated epithelial cells, and calcitonin gene related peptide (CGRP, also known as CALCA) is a marker of neuroepithelial cells. Using immunohistochemistry, lungs of E16.5 and E18.5 wildtype and Creb1−/− mice were analysed for expression of these markers as an indicator of proximal epithelial differentiation. Ciliated cells in the developing mouse lung first express Foxj1 at approximately E16.5 [28], and in lungs from E16.5 wildtype mice we detected a proportion of Foxj1-positive cells in the conducting epithelium (Fig. 8A) which increased in frequency at E18.5 (Fig. 8B). In the lung of Creb1−/− mice however, very few Foxj1-positive cells were detected at E16.5 (Fig. 8C), but were found at an increased frequency at E18.5 (Fig. 8D).

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