SBECs are derived from Clara cells.

<p>A&B. Representative images of bronchioles of Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without (A) or with (B) tamoxifen treatment (no infection). Expression of EGFP (green) and tomato red (mT, red) are shown. Sections were counterstained with DAPI (blue). C&D. Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice were given tamoxifen, and then infected with influenza virus. Shown are representative images of lung sections analyzed for expression of EGFP (green) and tomato red (purple), and stained for pro-SPC (red) and Scgb1a1 (blue) at (C) 9 or (D) 21 dpi. E. Percentages (means ± S.E.) of Clara cells per bronchiole that express EGFP in Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without (black columns) or with (red columns) TMX treatment at various time-points post-infection. Data at each time-point were obtained by counting 5541 to 11012 Clara cells (Scgb1a1⁺ but pro-SPC^-) from at least 15 lung sections of 4–5 mice. F. Percentages (means ± S.E.) of SBECs that express EGFP in Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without (black columns) or with (red columns) TMX treatment at different dpi. G. Proportion of SBECs that are Scgb1a1⁺ (black columns) or Scgb1a1⁻ (red columns) in Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without or with TMX treatment at different dpi. Data for (F) and (G) were obtained by counting 490 to 1774 cells from at least 16 sections of 4–5 mice per time point. H. Percentages (means ± S.E.) of SBECs that express EGFP in Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without (black columns) or with (red columns) TMX treatment at different days after bleomycin treatment. I. Proportions (means ± S.E.) of SBECs that are Scgb1a1⁺ (black columns) or Scgb1a1⁻ (red columns) in Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without or with TMX treatment at different days post-bleomycin treatment. Data for (H) and (I) were obtained from counting 351 to 1323 SBECs (pro-SPC⁺) from at least 6 lung sections of at least 2 mice per time point. J. Representative image of Cyp2f2 (green), pro-SPC (red) and DAPI (blue) staining of lung sections of mice at 14 days post-bleomycin treatment. Scale bars: (A–D) 100 µm; (j) 50 µm.</p

Observation of EGFP-positive AT2s and AT1s in TMX-treated transgenic mice after bleomycin treatment.

<p><b>A–C.</b> Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice were given tamoxifen and then treated with bleomycin. Shown are representative images of lung sections analyzed for EGFP (green), pro-SPC (red) and Scgb1a1 (blue) at 21 days post-treatment. Tomato red is not shown. Higher magnification of boxed area in (B) is shown as (C). Arrows in (C) indicate EGFP-positive AT1-like cells. <b>D–F.</b> Representative images of confocal analysis for EGFP (D), PDPN (E) and merged (F) of lung sectons from TMX-treated transgenic mice at 21 days post-bleomycin treatment. Arrows in (D–F) indicate EGFP and PDPN double-positive AT1s. Scale bars: (A, B) 1000 µm; (C–F) 20 µm.</p

Clara cells give rise to pro-SPC⁺ cells in the ring structures.

<p>Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without TMX treatment (A&B) and with TMX treatment (C–G) were infected with influenza virus (A–E) or treated with bleomycin (F,G). (A–E) Shown are representative images of lung sections at 21 dpi analyzed for EGFP (green), pro-SPC (red), Scgb1a1 (blue), and tomato red (mT, purple). The white broken line in (A) and (C) demarcates the infiltrated area (to the left), and the normal area (to the right). Higher magnifications of the selected areas in (A) and (C) are shown as (B) and (D, E), respectively. The tomato red channel is shown in (A) and (B) but not in (C–E). (F,G) Representative images of lung sections at 21 day post bleomycin treatment analyzed for EGFP (green), pro-SPC (red) and tomato red (purple). Scale bars: (A,C) 200 µm; (F,G) 100 µm; (B,D,E) 20 µm.</p

SBECs differentiate from Scgb1a1⁺ to Scgb1a1⁻ phenotype during the course of lung damage repair.

<p>A. Percentages (means ± S.E.) of bronchioles in the infiltrated area containing Scgb1a1⁺ (black bars) and Scgb1a1⁻ (red bars) SBECs. B. Proportions of Scgb1a1⁺ and Scgb1a1⁻ SBECs in a given lung section at the indicated dpi. Results are expressed as means ± S.E. C. Percentages (means ± S.E.) of bronchioles containing SBECs at the indicated days post-bleomycin treatment. D. Proportions of Scgb1a1⁺ and Scgb1a1⁻ SBECs in a given lung section at the indicated days post-bleomycin treatment. Results are expressed as means ± S.E. The numbers in (A–D) indicate the number of SBECs, bronchioles, lung sections, and mice from which the data were obtained.</p

Induction of EGFP-positive AT2s in transgenic mice without TMX treatment.

<p><b>A&B.</b> Representative images of bronchioles of Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without (A) or with (B) TMX treatment. Expression of EGFP (green) and tomato red (mT, red) are depicted. Sections were counterstained with DAPI (blue). <b>C.</b> Percentages (mean ± S.E.) of Clara cells per bronchiole that expressed EGFP in Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without or with TMX treatment. <b>D–G.</b> Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice without TMX treatment were treated with bleomycin (D) or infected with influenza virus (F). Shown are representative images of lung sections analyzed for expression of EGFP (green) and tomato red (purple), and stained for pro-SPC (red) and Scgb1a1 (blue) at 21 days post-bleomycin treatment (D) or 17 days post-infection (F). Higher magnification images of boxed areas in (D) and (F) are shown as (E) and (G), respectively. White broken line in (F) demarcates the infiltrated area (left) from the normal area (right) of the lung. Scale bars: (A, B, D, F) 100 µm; (E, G) 50 µm.</p

SBECs are induced following severe alveolar damage.

<p>A–C. Representative Scgb1a1 (green), pro-SPC (red) and DAPI (blue) staining of lung sections of (A) uninfected mice or (C) influenza virus-infected mice at 14 dpi. Higher magnification of the selected area in (A) is shown as (B). The white broken line in (C) demarcates the infiltrated area (to the right) and the normal area (to the left). D&E. Higher magnification of the selected area #1 in (C) either (D) without or (E) with the pro-SPC channel. Arrows indicate pro-SPC single-positive cells. F&G. Higher magnification of the selected area #2 in (C) either (F) without or (G) with the pro-SPC channel. Arrows indicate Scgb1a1 and pro-SPC double-positive cells. H-O. Representative confocal images of Scgb1a1 (green), pro-SPC (red) and DAPI (blue) staining of lung tissue sections of mice at 14 dpi. Higher magnifications of the boxed areas in H and L are shown as I–K and M-O, respectively. Scgb1a1⁺ (H–K) and Scgb1a1⁻ (L–O) SBECs are shown. P. Representative Scgb1a1 (green), pro-SPC (red) and DAPI (blue) staining of lung sections of mice at day 7 post-bleomycin treatment. High magnification of the boxed area is depicted at the upper-right corner. Scale bars: (A,C,P) 500 µm; (H,L) 100 µm; (B) 50 µm; (D–G, I–K, M–O) 20 µm.</p

Repair of pulmonary damage exhibits features similar to embryonic lung development.

<p>A–C. Representative images of pro-SPC (red) and DAPI (blue) staining of a fetal lung at gestational day 15. Higher maganification images of the boxed areas in (A) are shown in (B) and (C). (B) The cuboidal shape of pro-SPC⁺ cells and (C) the tip of a developing bronchiole are evident. D. Representative image of immunofluorescent staining of clusterin (red) and DAPI (blue) in fetal lung sections at gestational day 16. E&F. Representative images of immunofluorescent staining of clusterin (red) and DAPI (blue) in the lung sections of adult mice (E) without infection or (F) at 9 dpi. G&H. Co-staining of clusterin (red), pro-SPC (green) and DAPI (blue) in the lung sections of mice (G) 9 or (H) 15 dpi. Arrows point to clusterin-positive SBECs in a bronchiole (G) or in a ring structure (H). Scale bars: (A, D) 100 µm; (B,H) 20 µm; (C,G) 50 µm; (E,F) 500 µm.</p

Observation of EGFP-positive AT2s and AT1s in TMX-treated transgenic mice after influenza virus infection. A–E.

<p>Representative images of lung sections of TMX-treated Scgb1a1-CreER:ACTB-mT-EGFP transgenic mice after (at 21 dpi) (A–C) and before (D–E) influenza infection. Expression of EGFP (green), tomato red (purple) and pro-SPC (red) are analyzed in (A, B, D, E). Higher magnification images of selected areas in (A) and (D) are shown as (B) and (E), respectively. White broken line in (A) demarcates the infiltrated area (below) and normal area (above). Expression of EGFP (green) and PDPN (red) with DAPI (blue) staining are analyzed in (C). Arrows in (E) indicate EGFP and PDPN double-positive cells. Scale bars: (A, D) 500 µm; (B, E) 100 µm; (C) 20 µm.</p

Damage and repair of alveolar epithelia following bleomycin treatment or influenza virus infection. A.

<p>Representative lung sections of untreated C57BL/6 (B6) mice displaying Scgb1a1 (green), pro-SPC (red) and DAPI (blue) staining. Higher magnification of the boxed area is shown at the upper-right corner. <b>B–D.</b> Representative images of PDPN (green), pro-SPC (red) and DAPI (blue) staining of lung sections of untreated B6 mice (B); or mice at 7 days post-treatment with bleomycin (C); or mice at 11 days post-infection with influenza virus (D). <b>E&F.</b> Representative images of Scgb1a1(green), pro-SPC (red) and DAPI (blue) staining of lung sections of B6 mice at 21 days post-bleomycin treatment. Higher magnification of the selected area is shown as (F). White broken line in (E) demarcates the damaged area (left) from the normal area (right) of the lung. <b>G–I.</b> Representative images of PDPN (green), pro-SPC (red) and DAPI (blue) staining of lung sections of B6 mice at 21 days post-bleomycin treatment (G), or at 21 days after virus infection (H). Higher magnification of the selected area in (H) is shown as (I). <b>J–K.</b> Representative images of pro-SPC (red in J) or Scgb1a1 (red in K), BrdU (green) and DAPI (blue) staining of lung sections of B6 mice at 19 days (J) or 9 days (K) after virus infection, arrows indicate pro-SPC and BrdU double positive (J) or Scgb1a1 and BrdU double positive (K) cells. Note, BrdU-positive cells that are outside of the bronchiole in (K) are negative for pro-SPC. Scale bars: (A) 500 µm; (B, F, I–K) 50 µm; (C, D, G) 100 µm; (E) 1000 µm; (H) 200 µm. G and I not labeled.</p

Scgb1a1⁻ SBECs likely give rise to the pro-SPC⁺ ring structures and clusters in the damaged parenchyma.

<p>A–C. Representative images of pro-SPC (red), Scgb1a1 (green) and DAPI (blue) staining of lung sections of B6 mice at 17 dpi. Higher magnification of the selected areas #1 and #2 in (A) are shown as (B) and (C), respectively. The white broken line in (A) demarcates the infiltrated area (to the left) and the normal area (to the right). D&E. Representative images of Scgb1a1 (green), pro-SPC (red) and DAPI (blue) staining of lung sections of B6 mice at 21 days post-bleomycin treatment. High magnification of the boxed area in (E) is depicted at the upper-right corner. Circles in (B–D) point to pro-SPC⁺ ring structures. F. Frequency of ring structures in the infiltrated areas of the lung at different dpi. Data (means ± S.E.) at each time-point were obtained from 7 to 15 lung sections of 3 to 8 mice. Scale bars: (A) 1000 µm; (B,C) 100 µm; (E) 200 µm; (D) 50 µm.</p