Lung epithelial branching program antagonizes alveolar differentiation

Abstract

Mammalian organs, including the lung and kidney, often adopt a branched structure to achieve high efficiency and capacity of their physiological functions. Formation of a functional lung requires two developmental processes: branching morphogenesis, which builds a tree-like tubular network, and alveolar differentiation, which generates specialized epithelial cells for gas exchange. Much progress has been made to understand each of the two processes individually; however, it is not clear whether the two processes are coordinated and how they are deployed at the correct time and location. Here we show that an epithelial branching morphogenesis program antagonizes alveolar differentiation in the mouse lung. We find a negative correlation between branching morphogenesis and alveolar differentiation temporally, spatially, and evolutionarily. Gain-of-function experiments show that hyperactive small GTPase Kras expands the branching program and also suppresses molecular and cellular differentiation of alveolar cells. Loss-of-function experiments show that SRY-box containing gene 9 (Sox9) functions downstream of Fibroblast growth factor (Fgf)/Kras to promote branching and also suppresses premature initiation of alveolar differentiation. We thus propose that lung epithelial progenitors continuously balance between branching morphogenesis and alveolar differentiation, and such a balance is mediated by dual-function regulators, including Kras and Sox9. The resulting temporal delay of differentiation by the branching program may provide new insights to lung immaturity in preterm neonates and the increase in organ complexity during evolution.

Fig. 1.

Temporal (A), spatial (B), and evolutionary (C) negative correlations between branching morphogenesis and alveolar differentiation. (A) Time-course microarray expression profiling of FACS-purified distal lung epithelial cells. Gene expression values (log₂) in E14 samples are used as a baseline for comparison. Horizontal histograms for E15–E19 samples are the frequency distributions of average fold-changes for all genes; the units are shown as the number of genes (# genes). Alveolar markers are up-regulated (red), and branching (green) and cell cycle (blue) related genes are down-regulated over time. Down-regulated genes appear to have a smaller fold-change than up-regulated genes, possibly because of perdurance of transcripts. (B) Confocal images of immunostained E18 lung sections in areas where the airway lumen can be continuously traced from the proximal conducting airways (black/white long dashed lines) to the distal nonbranch tip (magenta) and branch tip (green) regions, as illustrated in the schematics. Branch tips are outlined with dashed lines. The boxed regions are enlarged as insets outlined in corresponding colors, showing a spatial negative correlation between branching and alveolar differentiation. AQP1 labels alveolar type I cells, the vasculature and the mesothelium, which are separated by dashed lines (Insets). There is a very low level of SOX9 expression in nonbranch tip epithelium, possibly because of protein perdurance. (Scale bars, 20 μm.) (C) Whole-mount in situ hybridization and immunostaining of Xenopus embryos of indicated stages (St) showing the absence of Sox9 expression and branching in the Xenopus lung. The lungs are indicated with dashed lines if stained or arrowheads if unstained. Open arrowheads indicate Sox9 expression in the pharyngeal arches. The two Xenopus Sox9 homologs (Sox9a and Sox9b) are 95% identical on the nucleotide level. OPT images of immunostained embryos (Left, Bottom) are shown as maximal intensity projection (Left) and sectional view (Right). (Scale bar, 200 μm.)

Fig. 2.

Hyperactive Kras suppresses alveolar differentiation. Confocal images of immunostained E19 lung sections from embryos of indicated genotypes and their respective littermate controls, showing that hyperactive Kras causes expansion of the SOX9⁺ branching region (A) and corresponding restriction of alveolar differentiation based on molecular markers (B) and cell morphology (C). Cre Recombination is induced at E15 to specifically target epithelial progenitors in late stage lungs. Shown are areas where the airway lumen can be continuously traced from the proximal conducting airways (black/white long dashed lines) to the distal nonbranch tip (magenta) and branch tip (green) regions, as illustrated in the schematics. The boxed regions are enlarged (Insets) outlined in corresponding colors. Dashed green boxes indicate low branching activity with few SOX9⁺ cells. GFP staining (C) allows visualization of the morphology of isolated alveolar type I (square brackets) and type II (arrowheads) cells by outlining the cell membrane; ECAD staining only labels cell junctions. Under our experimental conditions, the Rosa^mTmG allele is less sensitive to the Cre recombinase than the hyperactive Kras allele, and therefore labels a subset of Kras mutant cells. (Scale bars, 20 μm.)

Fig. 3.

Epithelial deletion of Sox9 impairs branching morphogenesis. (A) OPT images of whole-mount lungs from littermate control (Left) and Sox9^CKO/CKO;Shh^Cre/+ mutant (Right) embryos at indicated embryonic stages, immunostained for SOX2, SOX9, and ECAD. The RCd.L3 branch lineages (2) in the boxed areas are enlarged and traced with dashed lines. SOX9 expression is lost before E12. Fewer branches form in the Sox9 mutant lungs as early as E13. Branches of the RCd.L3 branch lineages start to dilate at E13 in the Sox9 mutant. E, ECAD; S, SOX9. (Scale bar, 250 μm.) (B) OPT images of whole-mount immunostained lungs from E15 Sox9^CKO/CKO;Shh^Cre/+ mutant and littermate control embryos. Similar to SOX9, CLU is normally expressed in branch tips and this expression is lost in the Sox9 mutant. CLU is also expressed at a lower level in airway smooth muscles and this expression is not affected and becomes apparent in the Sox9 mutant. (Scale bar, 250 μm.) (C) Mosaic deletion of Sox9. Confocal images of branch tips of whole-mount immunostained lungs from E15 Sox9^CKO/CKO;Nkx2.1^CreER/+ mutant and littermate control embryos. The boxed regions are enlarged in subsequent panels. SOX9 is deleted in a subset of epithelial cells outlined with dashed lines and these Sox9 mutant cells do not express CLU, demonstrating a cell autonomous requirement of Sox9 for CLU expression (Fig. S7E). (Scale bars, 10 μm.)

Fig. 4.

Sox9 functions downstream of the Fgf/Kras branching signal. (A) OPT images of E13 whole-mount immunostained lungs from littermate control and Fgfr2^CKO/CKO;Shh^Cre/+ mutant embryos. The boxed areas are shown in a cross-section view. Arrowheads indicate that SOX9 expression in the branching epithelium is lost in the Fgfr2 mutant lung. Arrows indicate that SOX9 expression in the cartilage precursors is not affected. (Scale bar, 200 μm.) (B) OPT images of E13 whole-mount immunostained lungs from control, Sox9^CKO/CKO;Shh^Cre/+ mutant, Kras^LSL-G12D/+;Shh^Cre/+ mutant, and Kras^LSL-G12D/+;Sox9^CKO/CKO;Shh^Cre/+ mutant embryos. Regions of the tracheas (brackets) are shown in a cross-section view. Epithelial Sox9 deletion suppresses the overgrowth and ectopic tracheal branch phenotypes in the Kras mutant lung. We note that Sox9 and Kras double-mutant branches remain cystic, possibly because of the presence of the Sox9-independent branching program (Fig. S7 C and D). (Scale bars, 200 μm.)

Fig. 5.

Sox9 suppresses alveolar differentiation. (A) Average fold-change in gene expression on a log₂ scale comparing Sox9^CKO/CKO;Shh^Cre/+ mutant and littermate control lungs at indicated embryonic stages by microarray expression profiling. Genes up- and down-regulated in the Sox9 mutant lungs are shown in red and green, respectively. The diameters of the circles represent the differences in fold-change between two biological replicates. The numbers in parenthesis represent the rank orders of the genes based on fold-change at E15. Horizontal histograms represent the frequency distributions of fold-changes for all genes on the microarray at indicated embryonic stages; the units are shown as the number of genes (# genes). (B) Sftpb whole-mount in situ hybridization of littermate control and Sox9^CKO/CKO;Shh^Cre/+ mutant lungs at indicated embryonic stages. Sftpb is up-regulated in the branch tips (dashed lines) of the Sox9 mutant lung as early as E11 before appearance of any defects in branch morphology. (Scale bars, 200 μm.) (C) Confocal images of immunostained sections from E15 Sox9^CKO/CKO;Shh^Cre/+ mutant and littermate control lungs, showing up-regulation in SFTPB and LAMP3 protein expression in the branch tips (dashed lines) of Sox9 mutant lungs. LAMP3 is localized to intracellular vesicle-like structures (arrowheads). (Scale bar, 20 μm.) (D) The branching program antagonizes the alveolar differentiation program and this antagonization is mediated by dual-function regulators including Kras and Sox9 that promote branching and suppress alveolar differentiation. The branching program contains additional Sox9 and Fgf/Kras independent genes, including Bmp4 and Id2 (dashed arrows).