Ager-CreERT2: A New Genetic Tool for Studying Lung Alveolar Development, Homeostasis, and Repair

Abstract

The alveolar region of the lung is composed of two major epithelial cell types: cuboidal alveolar type 2 cells (AT2 cells), which produce surfactant proteins, and large, thin, alveolar type 1 cells (AT1 cells), specialized for efficient gas exchange. AT1 cells cover more than 95% of the alveolar surface and constitute a major barrier to the entry of pathogenic agents. Relatively few genetic tools are available for studying the development of AT1 cells, the function of genes expressed in them, and the effect of specifically killing them in vivo in the adult lung. One distinguishing feature of AT1 cells is the high level of expression of the gene Ager, encoding the advanced glycation endproduct-specific receptor, a member of the immunoglobulin superfamily of cell surface receptors. In this paper, we report the generation of a novel Ager-CreER^T2 allele in which Cre recombinase is inserted into the first coding exon of the endogenous gene. After treatment with tamoxifen the allele enables Ager⁺ progenitor cells to be efficiently lineage labeled during late embryonic development and AT1 cells to be killed in the adult lung using a Rosa26-diphtheria toxin A allele. Significantly, adult mice in which approximately 50% of the AT1 cells are killed survive the loss; repair is associated with increased proliferation of SFTPC⁺ (surfactant protein C-positive) AT2 cells and the upregulation of Ager expression. The Ager-CreER^T2 allele thus expands the repertoire of genetic tools for studying AT1 turnover, physiology, and repair.

Keywords: Ager; Cre recombinase; lung; type 1 alveolar epithelial cells; type 2 alveolar stem cells.

Figure 1.

Generation of Ager-CreER^T2 knock-in mice. (A) Schematic diagram of the strategy for generating the Ager-CreER^T2 allele. (B) Correct targeting of embryonic stem cells was confirmed using Southern blot analysis. AflII enzyme was used to generate 4.8-kb wild-type (WT) and 3.5-kb mutant DNA fragments. (C) Immunohistochemistry shows that the AT1 (alveolar type 1) markers AGER (advanced glycation endproduct–specific receptor) and HOPX (HOP homeobox) label Ager-CreER^T2 adult lung tissue. However, AGER is not detected in the homozygous Ager-CreER^T2/CreER^T2 lung. Scale bars = 100 μm. (D) qPCR analysis reveals that Ager transcript levels are significantly reduced in homozygous mouse lung, whereas levels of Hopx transcript are not affected. The mRNA expression levels of Ager-CreER^T2/+ and Ager-CreER^T2/CreER^T2 samples were normalized to Ager^+/+. n = 3 animals. Data shown as mean ± SD. *P < 0.05. HSV-TK = herpes simplex virus thymidine kinase; n.s. = not significant; polyA = polynucleotide adenylyltransferase; UTR = untranslated region.

Figure 2.

Ager-CreER^T2; Rosa26-tdTm lineage labels Ager⁺ epithelial progenitors. (A) Ager-CreER^T2; Rosa26-tdTm embryos were exposed to tamoxifen (50 μg/g) at Embryonic Day (E)16.5, and lungs were fixed and analyzed by immunohistochemistry at E17.5 (n = 3). All panels are images from the same section that was stained with antibodies to RFP (red fluorescent protein), AGER, and SFTPC (surfactant protein C). Of AGER⁺ AT1 cells, 18.32 ± 6.70% were lineage labeled. Of the lineage-positive cells, 96.16 ± 1.03% are AGER⁺, and 3.84 ± 1.03% are SFTPC⁺. (B) A higher dose of tamoxifen (100 μg/g) was given at E16.5, and lungs were fixed and analyzed at E18.5. Of HOPX⁺ AT1 cells, 52.37 ± 4.14% were labeled. At this dose, the majority of Tomato⁺ (tdTomato-positive) cells are still HOPX⁺ (97.78 ± 1.16%). However, a few Tomato⁺ cells are SFTPC⁺ (2.19 ± 1.13%) (arrow). The two left panels are images of the same section stained with antibodies to RFP and HOPX. The far right panel is an adjacent section stained with antibodies to RFP and SFTPC. Scale bars = 100 μm.

Figure 3.

Ager-CreER^T2 efficiently and specifically lineage labels adult AT1 (alveolar type 1) cells using the Rosa26-tdTm reporter. (A) Adult Ager-CreER^T2; Rosa26-tdTm mice (n = 3) were treated with four doses of tamoxifen (Tmx; 0.2 mg/g) to lineage label Ager⁺ cells. After 4 days, Tomato⁺ cells were widely distributed in the alveolar region but not in the bronchioles (br). (B) Higher magnification shows that tdTomato signal colocalizes with AGER. (C) A small percentage (1.04 ± 0.57%) of SFTPC⁺ AT2 cells were also labeled (arrow). The inset shows the boxed region with SFTPC signal only. (D and D′) Immunohistochemistry of the same section with antibodies to RFP/tdTomato and HOPX. Quantification based on colocalization of tdTomato signal with HOPX nuclear staining (arrowheads) shows that 56.87 ± 3.63% of HOPX⁺ AT1 cells were lineage labeled. All non–lineage-labeled HOPX⁺ cells are marked with arrows. (E–H) Lineage labeling with different concentrations of Tmx. (E) Of AT1 cells, 0.12 ± 0.03% were tdTomato⁺ in lungs without Tmx. (F) Four doses of 10 μg/g Tmx labels 9.52 ± 0.98% of AT1 cells. (G) One dose of 0.2 mg/g Tmx labels 21.85 ± 1.18% of AT1 cells. (H) Four doses of 0.2 mg/g Tmx label 56.87 ± 3.63% of AT1 cells. Scale bars = 100 μm in A and D, 20 μm in B, and 50 μm in C and E–H.

Figure 4.

Specific in vivo ablation of AT1 cells. (A) Immunofluorescence reveals lineage-labeled HOPX⁺ AT1 cells in Ager-CreER^T2; Rosa26-tdTm (control) and Ager-CreER^T2; Rosa26-tdTm/Rosa26-DTA (DTA) (n = 4) lungs 4 days after Tmx treatment. Quantification in F shows that HOPX⁺ cells make up 8.35 ± 0.66% of total DAPI⁺ cells in the control lung, whereas this proportion is reduced to 3.78 ± 0.26% four days after Tmx treatment. By 14 days, the proportion is restored to normal (6.75 ± 1.15% in control and 6.53 ± 0.46% in DTA lungs). (B) Immunofluorescence shows increased numbers of CD11b⁺ cells after ablation of about half of the AT1 cells (4 d after last dose of Tmx). (C) Histochemistry also shows more immune cells present in the DTA lung at this time, but no grossly abnormal phenotype. (D and F) Control and DTA mice were exposed to 5-ethynyl-2′-deoxyuridine (EdU) for 3 hours before being killed, 4 days after last Tmx treatment. Boxed region in upper panels is shown at higher magnification below. In Ager-CreER^T2; Rosa26-tdTm control lungs (left panels), only about 1.53 ± 0.45% of AT2 cells proliferate (EdU⁺). By contrast, the number significantly increases to 21.15 ± 3.98% in Ager-CreER^T2; Rosa26-tdTm/Rosa26-DTA lungs (white arrowheads). SFTPC⁺ cells showing elongated morphology are labeled with yellow arrowheads. (E) Analysis of lungs from Ager-CreER^T2/H2b:Venus and Ager-CreER^T2/H2b:Venus; Rosa26-DTA mice 4 days after Tmx treatment reveals that 50.17 ± 2.13% of SFTPC⁺ cells express Ager-H2B:Venus following loss of AT1 cells, compared with 5.70 ± 1.87% in control. SFTPC⁺ Ager-H2B:Venus⁺ cells are labeled with white arrowheads. Scale bars = 100 μm in A and C and 50 μm in B, D, and E. Data shown are mean ± SD. *P < 0.05; **P < 0.01.