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First published online 3 July 2008
doi: 10.1242/dev.021964

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Kruppel-like factor 5 is required for perinatal lung morphogenesis and function

¹ Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
² Laboratory of Respiratory Disease, West China Hospital, Sichuan University, 37 Guo Xue Xiang, Chengdu, 610041, People's Republic of China.

View larger version (136K): [in this window] [in a new window]   Fig. 1. Immunohistochemical analysis of KLF5 in the developing mouse lung. (A) At E12.5, during the early pseudoglandular stage of development, KLF5 was detected in nuclei of epithelial cells in the proximal bronchial tubules (arrow). Staining was generally more intense along medial aspects of the tubules. ES, esophagus. Arrowhead indicates peripheral epithelial cells. (B) At E15.5, KLF5 was detected at highest levels in bronchiolar tubules (arrow); however, staining of subsets of cells lining the peripheral lung buds (arrowhead) was also noted. (C) At E18.5, KLF5 was present in both peripheral (arrowhead) and proximal (arrow) airways. (D) In mature lung, KLF5 was detected in nuclei of subsets of epithelial cells in both conducting airways (arrow) and alveolar regions (arrowhead). Insets are higher magnifications of the corresponding figures. (E-G) Dual immunolabeling for KLF5 (green nuclei) and (E) proSPC (red cytoplasm), (F) CCSP (red cytoplasm), and (G) FOXJ1 (red nuclei) was performed on E18.5 lung sections. KLF5 staining was observed in proSPC-positive (large arrow) and -negative (small arrow) cells (E), in CCSP-positive cells (large arrow, F), and in FOXJ1-negative cells (small arrow, G). Yellow signal is due to the autofluoresence of red blood cells, detectable in both channels. Scale bars: 100 µm.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Targeting of the Klf5 gene. Indicated are the structures of the endogenous Klf5 locus, the targeting vector, targeted allele, and recombined allele. Exons 1 to 4 are indicated by the boxes. The 5' and 3' probes used for Southern blot analysis are indicated.

View larger version (131K): [in this window] [in a new window]   Fig. 3. Deletion of Klf5 disrupts lung morphogenesis. (A-H) Lung sections were prepared from the fetus at E15.5 (A,B) and E18.5 (C-H), and were stained with a guinea pig anti-mouse KLF5 polyclonal antibody (A-F) or pancytokeratin (G,H), which outlined the epithelial cells. Nuclear staining of KLF5 was observed in epithelial cells lining the conducting and peripheral airways in control (A,C) and Klf5/+ (E) mice. Staining was absent or decreased in Klf5/ mice (B,D,F). Morphological changes in the lungs of Klf5/ mice were not observed at E15.5 (A versus B). At E18.5, lung morphology was markedly perturbed in Klf5/ mice (D,F,H), note the thickened walls of the mutant saccules, compared with Klf5+/+ (C,G) and Klf5/+ mice (E). Squamous epithelial cells (arrow) were rarely observed in the lungs of Klf5/ mice (H), compared with controls (G). Scale bars: 50 µm.

View larger version (132K): [in this window] [in a new window]   Fig. 4. Ultrastructural and biochemical analysis of lungs from Klf5/ mice. (A-D) Lungs from control (A,C) and Klf5/ (B,D) mice (approximately E18) were fixed and processed for electron microscopy as described in the Materials and methods. (B) Immature Type 2 cells (T2) with no lamellar bodies and abundant glycogen particles (g), distributed diffusely throughout the cytoplasm, were observed in the lungs of the Klf5/ mice. Lipofibroblasts (LF) were found ensheathing the undilated acinar tubules and buds in the periphery of the lung. (A) By comparison, T2 cells (T2) from control mice were more mature, exhibiting multiple lamellar bodies (arrows), smaller patches of glycogen (g), and increased amounts of rough endoplasmic reticulum. (D) In the Klf5/ mice, immature T2 cells (T2) with no lamellar bodies were observed lining the more dilated acinar tubules found in the central regions of the Klf5/ lungs. Note the prominent lipofibroblasts (LF) directly adjacent to these epithelial cells. (C) By contrast, differentiating T1 cells (T1) with thin cytoplasmic extensions (arrows) and centrally located glycogen patches (g) were found in the dilated alveolar saccules of control mice. Pulmonary capillaries filled with red blood cells (*) were found in close apposition to squamous type I cells (arrows). Fibroblasts (F) without lipid droplets were observed in the interior of the alveolar septa. (E) Whole lung homogenates were prepared at E18.5 and 100 µg protein used for western blot using antiserum against the mature SP-B peptide. Mature SP-B was decreased in the lungs of Klf5/ mice at E18.5, as assessed in four individual animals. Scale bars: 4 µm.

View larger version (88K): [in this window] [in a new window]   Fig. 5. KLF5 influences the differentiation of lung epithelium and mesenchyme. (A-L) Lung sections from Klf5/ mice and littermate controls were prepared at E18.5 and immunostained for CEBP (A,B), CCSP (C,D), FOXJ1 (E,F), SMA (G,H), PECAM (I,J) and VEGFR2 (K,L). Significantly decreased staining of CEBP (B) and CCSP (D) was detected in the airway epithelial cells, whereas no change in FOXJ1 staining was observed (F). The intensity and distribution of SMA staining were increased in the peripheral lung of the Klf5/ mice (H). Although staining for PECAM (J) and VEGFR2 (L) was readily detected, the pulmonary mesenchyme was thickened and the blood vessels were not found in close proximity to the adjacent epithelial cells. Insets are higher magnification views of the regions indicated by the arrows. Scale bar: 50 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Decreased Vegfa188, Vegfa120 and Aqp5 mRNA in lungs from Klf5/ mice. mRNAs from Klf5/ mice and littermate controls were isolated at E18.5. (A) RT-PCR was performed for β-actin, Klf5, Aqp5 and Vegfa. Vegfa isoforms were determined by using primers flanking exon 6 and exon 7 of Vegfa. (B-F) Densitometric quantitation of the PCR products, which were normalized to β-actin in each sample, revealed significantly decreased levels of Klf5 (B), Aqp5 (C), Vegfa188 (D) and Vegfa120 (F) mRNA, but not Vegfa164 mRNA (E) in the lungs of Klf5/ mice. The mean of the control was set to 1; relative mRNA levels are shown as mean±s.e.m. and were compared by a two-tailed Student's t-test (*P<0.05).

View larger version (37K): [in this window] [in a new window]   Fig. 7. Model of the paracrine signaling network influenced by deletion of Klf5. mRNAs that were significantly altered in lungs from Klf5/ mice were subjected to literature mining to identify potential functional associations and regulatory relationships using Pathway Studio 5.0 (Ariadne Genomics). KLF5 influenced the expression of genes previously associated with various signaling pathways, including those regulated by PDGF, FGF and TGFβ. mRNAs increased in Klf5/ mice are framed in red, those decreased are framed in green. Each line indicates a regulatory relationship between gene nodes based upon literature references. Regulatory relationships are denoted by line colors and patterns: purple lines represent binding; blue lines represent regulation of expression; the gray lines represent `regulation'; and arrows with a plus indicate positive regulation.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Transcriptional regulation of Vegfa and Tgfb promoter activity by KLF5. (A) JEG cells were transfected with promoter plasmid pGL2-Vegfa-Luc and increasing amounts of the expression plasmid pcDNA-Klf5. The plasmid pcDNA was used as an empty control. KLF5 increased the Vegfa promoter activity in a dose-responsive manner. (B) H441 cells were transfected with promoter plasmid pGL2-Vegfa-Luc and Klf5-targeting siRNA, with or without an HIF2 expression vector. A control negative siRNA was used as empty control. Inhibition of KLf5 expression moderately, but significantly inhibited Vegfa promoter activity. Klf5 siRNA significantly inhibited HIF2-dependent activity of the Vegfa promoter. (C) H441 cells were transfected with the promoter plasmid pGL3-3TP-Luc and the Klf5 expression vector, with or without TGFβ1 (2 ng/ml). Expression of Klf5 inhibited pGL3-3TP luciferase activity and inhibited TGFβ1-dependent activity of pGL3-3TP. The plasmid pcDNA was used as a control. (D) H441 cells were transfected with the promoter plasmid pGL3-3TP-Luc and Klf5-targeting siRNA, with or without TGFβ1 (2 ng/ml). A control negative siRNA was used as empty control. Inhibition of Klf5 expression moderately, but significantly enhanced 3TP promoter activity. Inhibition of Klf5 expression significantly enhanced TGFβ1-dependent activity of 3TP promoter (*P<0.05).

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