Figure 1.
Morphological analysis of lung development in lungs of Creb1−/− mice.
Haematoxylin and eosin-stained tissue sections from E15.5–E18.5 Creb1−/− and wildtype lungs (A–H). Lung morphology of Creb1−/− and wildtype fetal mice was similar at E15.5 (A, B) and E16.5 (C, D). At E17.5 proximal and distal airways of Creb1−/− mice failed to expand and showed compacted tissue morphology (F). Lungs of E18.5 Creb1−/− mice showed a comparable morphology to E17.5 Creb1−/− lungs (H). In comparison littermate controls at E17.5–E18.5 showed normal airway expansion (E, G). Lungs of E17.5 Creb1−/− mice were smaller than wildtype, though in approximate proportion to a reduced body size of Creb1−/− mice (I). Immunohistochemical analysis for the cell division marker Ki67 in lungs of E18.5 wildtype (J) and Creb1−/− (K) mice. Quantification of Ki67-positive and -negative cells (n = 3) in total lung showed a small increase in cell proliferation in lungs of Creb1−/− mice at E18.5 (L). TUNEL analysis for apoptotic nuclei showed very rare, but comparable numbers of apoptotic cells in E18.5 wildtype (M) and Creb1−/− (N) lungs (arrows indicate apoptotic nuclei). Error bars represent SEM. Asterisk (*) indicates p<0.05. Scale bars: A–H, 200 µm; I, 5 mm for whole pups and 1.5 mm for fetal lungs as shown; J,K, 100 µm; M,N, 200 µm; insets for M and N, 20 µm.
Figure 2.
Compensatory up-regulation of Crem does not alter Creb1−/− morphology in the developing lung.
qPCR analysis of Crem and Atf1 mRNA levels in the E17.5 Creb1−/− and wildtype lung (n = 4) (A). Haematoxylin and eosin-stained tissue sections from E17.5 lungs of Creb1+/+, Crem+/+ (B), Creb1+/+, Crem−/− (C), Creb1−/−, Crem+/− (D) and Creb1−/−, Crem−/− (E) mice. Lungs of E17.5 Creb1−/−, Crem−/− mice show no morphological differences as compared to Creb1−/−, Crem+/− mice. Error bars represent SEM. Asterisk (*) indicates p<0.05 and ** indicates p<0.001. White bars: Wildtype, Black bars: Creb1−/−. Scale bars: 100 µm.
Figure 3.
Creb1 is activated primarily in the distal lung epithelium during late gestation.
Immunohistochemistry for Creb1 (A,B) and pCreb1 (C,D) on tissue sections of E16.5 and E18.5 wildtype lungs. Creb1 expression was detected in most cells at E16.5 (A) and E18.5 (B), though a proportion of mesenchymal cells surrounding large airways were Creb1-negative (Black arrows). At E16.5, pCreb1 staining was detected sparsely in the mesenchyme and proximal epithelium but in most cells of the distal epithelium (C, white arrows). At E18.5, pCreb1 staining was found primarily in the mesenchyme surrounding large airways and also within saccular walls (D, white arrows). Scale bars: upper panels, 100 µm; magnified lower panels of proximal and distal lung, 25 µm.
Figure 4.
Vascular development is unaffected in lungs of Creb1−/− mice.
Immunohistochemistry for CD31 (A–F) and αSMA (G,H) in the lung of E16.5 to E18.5 Creb1−/− and wildtype mice. In wildtype mice, CD31 immunostaining shows a normal vascular structure with large blood vessels and in capillaries in the distal lung (A,C,E). Vascular structure in the lung of E16.5 Creb1−/− mice appears similar to E16.5 wildtype mice (B). An intact, although immature vasculature is seen in the lung of E17.5 (D) and E18.5 (F) Creb1−/− mice. A normal smooth muscle layer surrounding large blood vessels (arrows) was also detected in both E18.5 wildtype (G) and E18.5 Creb1−/− (H) mice. Scale bars: 100 µm; insets, 20 µm.
Figure 5.
Defective AEC differentiation and lamellar body development in lungs of Creb1−/− mice.
Tissue sections of E17.5 Creb1−/− and wildtype lungs were analysed for AEC proportions by electron microscopy. Representative electron micrographs show the ultrastructural appearance of undifferentiated, type-I, type-II and intermediate AECs in wildtype and Creb1−/− mice (A). Asterisks indicate cytoplasmic projections of the developing type-I AECs whereas arrows indicate lamellar bodies in type-II AECs. Note that the lamellar bodies in the type-II AEC from wildtype mice are more numerous and more mature in appearance when compared to lamellar bodies in Creb1−/− mice. Quantification of relative AEC proportions showed a statistically significant reduction in proportions of type-II and type-I AECs, together with a statistically significant increase in undifferentiated AECs (B). Quantification of lamellar body number showed a statistically significant reduction per cross sectional area of type-II AEC in E17.5 Creb1−/− (n = 4) lungs relative to wildtype (n = 5) (C). qPCR analysis of mRNA levels for Sftpa, Sftpb, Sftpc, Sftpd and also Abca3 and Aqp5 in the lung of E17.5 Creb1−/− mice relative to wildtype (n = 4). Error bars represent SEM. Asterisk (*) indicates p<0.05 and ** indicates p<0.001. White bars: Wildtype, Black bars: Creb1−/−. Scale bars: 2 µm.
Figure 6.
Sox9 and ProSPC are highly expressed in the distal lung epithelium of Creb1−/− mice.
Immunohistochemistry for the epithelial progenitor cell markers Sox9 (A,B) and ProSPC (C,D) in the lung of E17.5 Creb1−/− and wildtype mice. Sox9 was detected sporadically in distal epithelial cells of wildtype lungs (A), but was found within almost all cells of distal epithelial tubules in Creb1−/− lungs (B). In wildtype lungs, ProSPC was detected only in type-II AECs (C), however almost all cells of the distal epithelium showed strong ProSPC expression in Creb1−/− lungs (B). Scale bars: 100 µm.
Figure 7.
Sftpd is localised to the proximal airway epithelium, but is absent in the Creb1−/− lung.
Immunohistochemistry for Sftpd protein in the lung (A,B) and epidermis (C,D) of E17.5 Creb1−/− and wildtype mice. Sftpd was localised primarily to the conducting airway epithelium (enclosed area) in the lung of wildtype mice (A), but was absent in the lung of Creb1−/− mice (B). Strong Sftpd expression was also detected in the stratum spinosum epidermal layer of wildtype (C), but not Creb1−/− (D) mice. Scale bars: 100 µm.
Figure 8.
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.
Table 1.
Differentially expressed genes in the lung of E17.5 Creb1−/− mice identified by whole-genome microarray analysis.
Figure 9.
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−/−.