The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium

Abstract

To directly test the contribution of Scgb1a1(+) Clara cells to postnatal growth, homeostasis, and repair of lung epithelium, we generated a Scgb1a1-CreER "knockin" mouse for lineage-tracing these cells. Under all conditions tested, the majority of Clara cells in the bronchioles both self-renews and generates ciliated cells. In the trachea, Clara cells give rise to ciliated cells but do not self-renew extensively. Nevertheless, they can contribute to tracheal repair. In the postnatal mouse lung, it has been proposed that bronchioalveolar stem cells (BASCs) which coexpress Scgb1a1 (Secretoglobin1a1) and SftpC (Surfactant Protein C), contribute descendants to both bronchioles and alveoli. The putative BASCs were lineage labeled in our studies. However, we find no evidence for the function of a special BASC population during postnatal growth, adult homeostasis, or repair. Rather, our results support a model in which the trachea, bronchioles, and alveoli are maintained by distinct populations of epithelial progenitor cells.

Figure 1. Scgb1a1-CreER^TM mice show dose-sensitive activation of reporter gene expression in response to tamoxifen

A. Timecourse of tmx injections. Every adult mouse was injected on day 0 so 11 days always elapsed between the first tmx injection and sacrifice. B-D. Scgb1a1-CreER^TM;Rosa26R-eYFP adult lung sections. Green: anti-GFP, lineage label. Red: anti-pro-SpC, type 2 cells. Blue: anti-Scgb1a1, Clara cells. Note the increase in the number of lineage labeled cells with increasing tmx dose. Arrowheads = lineage labeled putative BASCs (always contiguous with bronchiolar epithelium). Arrows = lineage labeled type 2 cells. C. One. D. Two. E. Four tmx injections. E, F. Alveoli from 12 month old mouse. Green: anti-Scgb1a1. Red: anti-pro-SpC. F. Higher magnification of box in E. Note that Scgb1a1 and pro-SftpC are both present in many type 2 cells, in separate vesicles. Arrows = (Scgb1a1⁺, SftpC⁺) type 2 cells. The level of Scgb1a1 protein in type 2 cells is low and can only be detected by overexposing the bronchiolar Scgb1a1 signal. (Scale bars 100 μm B-E; 160 μm G.)

Figure 2. Clara cells are a self-renewing progenitor for postnatal growth of the bronchioles, but not the trachea

A-F. Scgb1a1-CreER^TM;Rosa26R-LacZ. G-L. Scgb1a1-CreER^TM;Rosa26R-eYFP. Embryos were exposed to tmx at E18.5. A-F. Wholemount lungs. A. P2. B, D. P3 weeks. C, E, F. P52 weeks (1 year). G-I. Lung sections. Green: anti-GFP, lineage label. Red: anti-pro-SpC, type 2 cells. Blue: anti-Scgb1a1, Clara cells. Arrowheads = lineage labeled type 2 cells. Arrows = lineage labeled putative BASCs. G. P2. H. P3 weeks. I. P52 weeks. Inset in H shows lineage labeled ciliated cells (arrowheads), but not neuroendocrine cells (arrow). Green: anti-GFP, lineage label. Red: anti-CGRP, neuroendocrine cells. Blue: anti-β-tubulin, ciliated cells. J-L. Trachea sections. Green: anti-GFP, lineage label. Red: anti-β-tubulin, ciliated cells. Blue: anti-Scgb1a1, Clara cells. Animals sacrificed at J. P2. K. P3 weeks. L. P52 weeks (1 year). M, N. Graphs to show mean percentage lineage labeled Clara, ciliated and type 2 cells following tmx exposure at E18.5. Error bars show s.e.m. M. Lung. N. Trachea. (Scale bars 2mm A; 1mm B, C; 200μm D-F; 50μm G-L.)

Figure 3. Clara cells are a self-renewing progenitor in the adult steady-state bronchioles, but not the trachea

A-I. Adult Scgb1a1-CreER^TM;Rosa26R-eYFP mice injected twice (E-G) or four times (A-D, H, I) with tmx or vehicle. A-F. Lung sections. Green: anti-GFP, lineage label. Red: anti-pro-SpC, type 2 cells. Blue: anti-Scgb1a1, Clara cells. Arrowheads = lineage labeled type 2 cells. Arrows = lineage labeled putative BASCs. A-C. 4x tmx followed by A. 4 days. B. 24 weeks (6 months). C. 52 weeks (1 year) chase. * = lineage labeled type 1 cells. D. Control plus four days chase. E, F. 2x tmx followed by E. 4 days. F. 52 weeks chase. G. Bronchiolar section, 2x tmx followed by 52 week chase. Green: anti-GFP, lineage label. Red: anti-CGRP, neuroendocrine cells. Blue: anti-β-tubulin, ciliated cells. Arrowheads = lineage labeled ciliated cells. Neuroendocrine cells are not linage-labeled. H, I. Tracheal sections. Green: anti-GFP, lineage label. Red: anti-Scgb1a1, Clara cells. Blue: anti-T1α, basal cells. 4x tmx followed by H. 4 days. I. 52 weeks chase. J, K. Graphs to show mean percentage lineage labeled Clara, ciliated and type 2 cells following 4x adult tmx injections. Error bars show s.e.m. J. Lung. K. Trachea. (Scale bars all 50 μm).

Figure 4. Scgb1a1⁺ bronchiolar cells, including putative BASCs, do not contribute to the alveolar compartment following O₂-induced alveolar injury

Adult wildtype C57Bl/6 (A-C) or Scgb1a1-CreER^TM;Rosa26R-eYFP (D-G) lung sections. A. 48 hours post-O₂, hemotoxylin and eosin. Note extensive inflammatory cells around BADJ and terminal bronchioles, consistent with injury and repair. B, C. Black: anti-BrdU, S-phase cells. B. Control, 48 hours post-room air. C. 48 hours post-O₂. Note extensive proliferation in terminal bronchioles and alveoli. D-G. Green: anti-GFP, lineage label. Red: anti-pro-SpC, type 2 cells. Arrowheads = lineage labeled type 2 cells. Arrows = lineage labeled type 1 cells. D, E. 4x tmx. D. Control, room air plus 3 weeks recovery. (Dotted box represents the alveolar area around each BADJ scored for presence/absence of lineage labeled cells in H.) E. 56 hours O₂ plus 3 weeks recovery. Inset shows lineage labeled type 1 cells at higher magnification. F, G. 2x tmx. F. Control, room air plus 3 weeks recovery. G. 56 hours O₂ plus 3 weeks recovery. No lineage labeled type 1 cells were found. H. Bar graph showing percentage of alveolar regions adjacent to a BADJ which contain lineage labeled cells in control (blue bars) and recovered (red bars) animals in four independent O₂ experiments. Error bars show s.e.m. Means in experiments 1 and 2 are statistically different in a two-tailed t-test. (Scale bars 500μm A-C; 1mm D-G).

Figure 5. Tracheal Clara cells have greater proliferative and differentiative potential following injury than at steady-state

A-I. Scgb1a1-CreER^TM;Rosa26R-LacZ trachea, 4x tmx. Blue: X-gal staining, lineage label. A-E. Ventral half of bisected wholemount trachea. A. No injury control. B, C. 24 hours post-SO₂ showing variable levels of Clara cell survival. D, E. 2 weeks post-SO₂. F-I. Tracheal sections. F, G. 24 hours post-SO₂. Black: anti-BrdU S-phase cells. F. Surviving lineage labeled Clara cells can enter the cell cycle. G. The majority of proliferating cells are not lineage labeled. H, I. Brown: anti-β-tubulin, ciliated cells. H. Control plus 2 weeks. Lineage labeled cells are in small groups. I. 2 weeks post-SO₂. Large patches of lineage labeled cells, including both ciliated and Clara cells, are present. J, J'. Scgb1a1-CreER^TM;Rosa26R-CAG-fGFP tracheal sections 2 weeks post-SO₂. Green: anti-GFP, lineage label. Red: anti-T1α, basal cells. Two optical sections from a confocal Z-stack are shown. Arrow = lineage labeled basal cell in which co-localization of the membrane-associated lineage label and membrane-associated T1α is visible in every optical section. Arrowheads = basal cells which appear to be lineage labeled in some optical sections only; these were scored as not lineage labeled. (Scale bars 0.5mm A; 20 μm F-J.)

Figure 6. Scgb1a1⁺ cells proliferate during postnatal growth of both the bronchioles and the trachea

A-D. Scgb1a1-CreER^TM;Rosa26R-eYFP wholemount lungs exposed to 2.5μg/gram tmx at E18.5. Green: anti-GFP, lineage label. Red: anti-Scgb1a1, Clara cells. Blue: anti-acetylated tubulin, ciliated cells. A, B. Trachea. A. P2. B. P3 weeks. C, D. Bronchioles. C. P2. D. P3 weeks. E, F. Bar graphs showing percentage of different sized clones at P2 (blue bars) and P3 weeks (red bars). E. Trachea. F. Bronchioles. G, H. Bar graphs showing cellular composition of the lineage labeled clones at P2 and P3 weeks. Blue, ratio of Clara : ciliated cells ≤ 1, clones consist mostly of ciliated cells. Red, ratio of Clara : ciliated cells > 1, clones consist mostly of Clara cells. G. Trachea. H. Bronchioles. (Scale bars all 100 μm.)