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rabbit monoclonal anti lyz2  (Abcam)


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    Abcam rabbit monoclonal anti lyz2
    ( A ) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also – . ( B ) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice ( n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also – . ( C ) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment ( n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also – . ( D ) Lung sections of GFP;CCSP.CRE mice ( n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also – . CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; <t>LYZ2,</t> lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.
    Rabbit Monoclonal Anti Lyz2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 719 article reviews
    rabbit monoclonal anti lyz2 - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "Club cells form lung adenocarcinomas and maintain the alveoli of adult mice"

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    Journal: eLife

    doi: 10.7554/eLife.45571

    ( A ) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also – . ( B ) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice ( n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also – . ( C ) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment ( n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also – . ( D ) Lung sections of GFP;CCSP.CRE mice ( n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also – . CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.
    Figure Legend Snippet: ( A ) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also – . ( B ) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice ( n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also – . ( C ) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment ( n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also – . ( D ) Lung sections of GFP;CCSP.CRE mice ( n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also – . CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.

    Techniques Used: Labeling, Marker, Staining

    TUBA1A, Tubulin alpha 1a or acetylated tubulin; KRT5, Keratin 5; FOXJ1, Forkhead box J1; CCSP, Secretoglobin, family 1A, member 1 (uteroglobin) or Clara cell secretory protein or Clara cell 10 KDa protein; SFTPC, Surfactant protein C; LYZ2, Lysozyme 2; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages.
    Figure Legend Snippet: TUBA1A, Tubulin alpha 1a or acetylated tubulin; KRT5, Keratin 5; FOXJ1, Forkhead box J1; CCSP, Secretoglobin, family 1A, member 1 (uteroglobin) or Clara cell secretory protein or Clara cell 10 KDa protein; SFTPC, Surfactant protein C; LYZ2, Lysozyme 2; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages.

    Techniques Used:

    CRE, causes recombination; TOMATO, tdTomato; GFP, green fluorescent protein; CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; SOX2, sex determining region Y (SRY)-box 2; VAV, Vav Guanine Nucleotide Exchange Factor 1; NES, nestin; JAX, Jackson Laboratories; EMMA, European Mutant Mouse Archive; MGI, Mouse Genome Informatics; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages; ΒM, bone marrow (myeloid) cells. Symbols indicate: - (white), no genetic labeling; + (magenta), complete genetic labeling; ± (blue), partial genetic labeling.
    Figure Legend Snippet: CRE, causes recombination; TOMATO, tdTomato; GFP, green fluorescent protein; CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; SOX2, sex determining region Y (SRY)-box 2; VAV, Vav Guanine Nucleotide Exchange Factor 1; NES, nestin; JAX, Jackson Laboratories; EMMA, European Mutant Mouse Archive; MGI, Mouse Genome Informatics; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages; ΒM, bone marrow (myeloid) cells. Symbols indicate: - (white), no genetic labeling; + (magenta), complete genetic labeling; ± (blue), partial genetic labeling.

    Techniques Used: Mutagenesis, Labeling

    Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage-labeled mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; TUBA1A, acetylated tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.
    Figure Legend Snippet: Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage-labeled mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; TUBA1A, acetylated tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

    Techniques Used: Marker, Staining, Labeling, Expressing

    Representative photographs (top row) and green epifluorescence images (second row), as well as merged fluorescent microscopic images of lung sections for nuclear Hoechst33258 stain, endogenous TOMATO, and endogenous GFP (bottom three rows), of tumor-bearing lungs from genetically marked mice employed in these studies (described in detail in ) at six months after initiation of ten weekly intraperitoneal urethane injections ( n = 30, 22, 18, and 20/strain, respectively). b, bronchi. Top two rows: arrows indicate lung tumors. Bottom three rows: white arrows indicate GFP-labeled cells in apparently non-affected alveolar areas of GFP;CCSP.CRE mice; green arrow indicates rare GFP+ cell in non-affected central airway of GFP;LYZ2.CRE mouse. Note the absence of GFP-labeling of lung tumors in TOMATO mice, the complete GFP-labeling in GFP;CCSP.CRE and GFP;SFTPC.CRE mice, and the partial GFP-labeling in GFP;LYZ2.CRE mice.
    Figure Legend Snippet: Representative photographs (top row) and green epifluorescence images (second row), as well as merged fluorescent microscopic images of lung sections for nuclear Hoechst33258 stain, endogenous TOMATO, and endogenous GFP (bottom three rows), of tumor-bearing lungs from genetically marked mice employed in these studies (described in detail in ) at six months after initiation of ten weekly intraperitoneal urethane injections ( n = 30, 22, 18, and 20/strain, respectively). b, bronchi. Top two rows: arrows indicate lung tumors. Bottom three rows: white arrows indicate GFP-labeled cells in apparently non-affected alveolar areas of GFP;CCSP.CRE mice; green arrow indicates rare GFP+ cell in non-affected central airway of GFP;LYZ2.CRE mouse. Note the absence of GFP-labeling of lung tumors in TOMATO mice, the complete GFP-labeling in GFP;CCSP.CRE and GFP;SFTPC.CRE mice, and the partial GFP-labeling in GFP;LYZ2.CRE mice.

    Techniques Used: Staining, Labeling

    Lineage marker protein-stained lung adenocarcinomas (dashed outlines) from genetically marked mice ( n = 10/group). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2.
    Figure Legend Snippet: Lineage marker protein-stained lung adenocarcinomas (dashed outlines) from genetically marked mice ( n = 10/group). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

    Techniques Used: Marker, Staining, Labeling, Expressing

    Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage reporter mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.
    Figure Legend Snippet: Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage reporter mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

    Techniques Used: Marker, Staining, Expressing, Labeling

    Representative merged fluorescent microscopic images of lineage marker protein-stained lung tumors (dashed outlines) from genetically marked mice (FVB background) at six months after a single intraperitoneal urethane injection ( n ≥ 10/strain). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2.
    Figure Legend Snippet: Representative merged fluorescent microscopic images of lineage marker protein-stained lung tumors (dashed outlines) from genetically marked mice (FVB background) at six months after a single intraperitoneal urethane injection ( n ≥ 10/strain). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

    Techniques Used: Marker, Staining, Injection, Labeling, Expressing

    ( A ) DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Summary of duplexed digital droplet PCR (ddPCR) results using primers and probes specific for the Rosa mT and the Kras WT sequences. Note that all cell types equally suffer initial Kras Q61R mutations, but only GFP-labeled cells of GFP;CCSP.CRE mice (i.e. airway cells) maintain the Kras Q61R mutation after two weeks. See also . Data are shown as violin plot. P , overall probability, two-way ANOVA. ***: p<0.001 compared with all other groups, Bonferroni post-tests. ( B ) Representative high-resolution micro-computed tomography (μCT) lung sections (top) and three-dimensional reconstructions (bottom) from urethane-treated FVB mice six months after treatment ( n = 10). Note lung tumors attached to (green arrows) or contained within (blue arrows) the airways, as well as lung tumors with no obvious link to a bronchus (red arrows). ( C ) Summary of results from μCT (data from ) and pathology (data from ) shown as violin plot. P , probability, two-way ANOVA.*, ***, and ****: p<0.05, p<0.001, and p<0.0001, respectively, compared with airway-attached tumors, Bonferroni post-tests. Shown are also Spearman’s correlation coefficient ( ρ ) and probability ( P ) for correlation of μCT and pathology results. 10.7554/eLife.45571.031 Figure 2—source data 1. Quantification of Kras mutant droplets in duplexed digital droplet PCR (ddPCR). 10.7554/eLife.45571.032 Figure 2—source data 2. Quantification of tumor airway link.
    Figure Legend Snippet: ( A ) DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Summary of duplexed digital droplet PCR (ddPCR) results using primers and probes specific for the Rosa mT and the Kras WT sequences. Note that all cell types equally suffer initial Kras Q61R mutations, but only GFP-labeled cells of GFP;CCSP.CRE mice (i.e. airway cells) maintain the Kras Q61R mutation after two weeks. See also . Data are shown as violin plot. P , overall probability, two-way ANOVA. ***: p<0.001 compared with all other groups, Bonferroni post-tests. ( B ) Representative high-resolution micro-computed tomography (μCT) lung sections (top) and three-dimensional reconstructions (bottom) from urethane-treated FVB mice six months after treatment ( n = 10). Note lung tumors attached to (green arrows) or contained within (blue arrows) the airways, as well as lung tumors with no obvious link to a bronchus (red arrows). ( C ) Summary of results from μCT (data from ) and pathology (data from ) shown as violin plot. P , probability, two-way ANOVA.*, ***, and ****: p<0.05, p<0.001, and p<0.0001, respectively, compared with airway-attached tumors, Bonferroni post-tests. Shown are also Spearman’s correlation coefficient ( ρ ) and probability ( P ) for correlation of μCT and pathology results. 10.7554/eLife.45571.031 Figure 2—source data 1. Quantification of Kras mutant droplets in duplexed digital droplet PCR (ddPCR). 10.7554/eLife.45571.032 Figure 2—source data 2. Quantification of tumor airway link.

    Techniques Used: Labeling, Mutagenesis, Micro-CT

    DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Representative gating strategy of digital droplet PCR (ddPCR) using primers and probes specific for the Rosa mT and the Kras WT sequences. Dashed outlines indicated GFP+ Kras Q61R + droplet gates.
    Figure Legend Snippet: DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Representative gating strategy of digital droplet PCR (ddPCR) using primers and probes specific for the Rosa mT and the Kras WT sequences. Dashed outlines indicated GFP+ Kras Q61R + droplet gates.

    Techniques Used:

    ( A ) Non-neoplastic alveolar regions from lung sections of aging GFP;CCSP.CRE mice (bottom right section is also SFTPC-immunostained) show increasing numbers of alveolar GFP-labeled cells with age (arrows). Green arrows: genetically GFP-labeled, SFTPC-immunoreactive airway cells in alveolus of 15-month-old GFP;CCSP.CRE mouse. ( B ) Data summary ( n = 5 mice/time-point) from ( A ) shown as violin plot. Color-coded boxes indicate time windows of experiments in ( C-H ). P , probability, one-way ANOVA. ns, ***, and ****: p>0.05, p<0.001, and p<0.0001, respectively, for comparison with time-point zero by Bonferroni post-tests. ( C ) SFTPC-immunostained lung sections of GFP;CCSP.CRE mice show accelerated increase of alveolar GFP-labeled SFTPC-immunoreactive airway cells after bleomycin treatment (arrows). See also and . ( D ) Data summary from ( C ) shown as violin plots ( n = 4 mice/time-point). P , probabilities, one-way ANOVA. ns, *, **, ***, and ****: p>0.05, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, for comparison with day zero by Bonferroni post-tests. ( E ) SFTPC-stained lung sections of GFP;CCSP.CRE mice at two months after perinatal exposure to 98% O 2 show enlarged alveoli (evident by increased mean linear intercept) enriched in GFP-labeled SFTPC-immunoreactive airway cells (arrows) compared with 21% O 2 . ( F ) Data summary from ( E ) shown as violin plots ( n = 6 mice/group). P , probabilities, t-test. ( G ) Lung sections (top) of GFP;CCSP.CRE mice ( n = 5 mice/group) show enrichment of alveoli in GFP-labeled cells post-naphthalene treatment (arrows). Lung sections (bottom) of GFP;LYZ2.CRE mice ( n = 5 mice/group) at six weeks post-naphthalene show no bronchial ( b ) GFP-labeled cells. See also and . ( H ) Data summary from ( G ) shown as violin plot ( n = 5 mice/time-point). P , probability, two-way ANOVA. ns and ****: p>0.05 and p<0.0001, respectively, for comparison with corn oil by Bonferroni post-tests. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.047 Figure 4—source data 1. Quantification of alveolar GFP+ cells in GFP;CCSP.CRE mice during aging. 10.7554/eLife.45571.048 Figure 4—source data 2. Quantification of SFTPC+ and GFP+ cells in GFP;CCSP.CRE mice after bleomycin treatment. 10.7554/eLife.45571.049 Figure 4—source data 3. Data of mean linear intercept and GFP+/SFTPC+cells in GFP;CCSP.CRE mice after hyperoxia treatment. 10.7554/eLife.45571.050 Figure 4—source data 4. Data of GFP+/SFTPC+ cells in GFP;CCSP.CRE and GFP;LYZ2.CRE mice after naphthalene treatment.
    Figure Legend Snippet: ( A ) Non-neoplastic alveolar regions from lung sections of aging GFP;CCSP.CRE mice (bottom right section is also SFTPC-immunostained) show increasing numbers of alveolar GFP-labeled cells with age (arrows). Green arrows: genetically GFP-labeled, SFTPC-immunoreactive airway cells in alveolus of 15-month-old GFP;CCSP.CRE mouse. ( B ) Data summary ( n = 5 mice/time-point) from ( A ) shown as violin plot. Color-coded boxes indicate time windows of experiments in ( C-H ). P , probability, one-way ANOVA. ns, ***, and ****: p>0.05, p<0.001, and p<0.0001, respectively, for comparison with time-point zero by Bonferroni post-tests. ( C ) SFTPC-immunostained lung sections of GFP;CCSP.CRE mice show accelerated increase of alveolar GFP-labeled SFTPC-immunoreactive airway cells after bleomycin treatment (arrows). See also and . ( D ) Data summary from ( C ) shown as violin plots ( n = 4 mice/time-point). P , probabilities, one-way ANOVA. ns, *, **, ***, and ****: p>0.05, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, for comparison with day zero by Bonferroni post-tests. ( E ) SFTPC-stained lung sections of GFP;CCSP.CRE mice at two months after perinatal exposure to 98% O 2 show enlarged alveoli (evident by increased mean linear intercept) enriched in GFP-labeled SFTPC-immunoreactive airway cells (arrows) compared with 21% O 2 . ( F ) Data summary from ( E ) shown as violin plots ( n = 6 mice/group). P , probabilities, t-test. ( G ) Lung sections (top) of GFP;CCSP.CRE mice ( n = 5 mice/group) show enrichment of alveoli in GFP-labeled cells post-naphthalene treatment (arrows). Lung sections (bottom) of GFP;LYZ2.CRE mice ( n = 5 mice/group) at six weeks post-naphthalene show no bronchial ( b ) GFP-labeled cells. See also and . ( H ) Data summary from ( G ) shown as violin plot ( n = 5 mice/time-point). P , probability, two-way ANOVA. ns and ****: p>0.05 and p<0.0001, respectively, for comparison with corn oil by Bonferroni post-tests. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.047 Figure 4—source data 1. Quantification of alveolar GFP+ cells in GFP;CCSP.CRE mice during aging. 10.7554/eLife.45571.048 Figure 4—source data 2. Quantification of SFTPC+ and GFP+ cells in GFP;CCSP.CRE mice after bleomycin treatment. 10.7554/eLife.45571.049 Figure 4—source data 3. Data of mean linear intercept and GFP+/SFTPC+cells in GFP;CCSP.CRE mice after hyperoxia treatment. 10.7554/eLife.45571.050 Figure 4—source data 4. Data of GFP+/SFTPC+ cells in GFP;CCSP.CRE and GFP;LYZ2.CRE mice after naphthalene treatment.

    Techniques Used: Labeling, Staining

    ( A ) Lineage marker-immunostained lung sections of 12-week-old GFP;CCSP.CRE;DTA and GFP;LYZ2.CRE;DTA mice ( n = 6/group) show increased bronchial and alveolar size and flat CCSP+ SFTPC+ LYZ2+ cells in the airways of GFP;CCSP.CRE;DTA mice (green arrows), and CCSP-SFTPC-LYZ2+ alveolar macrophages in the airspaces of GFP;LYZ2.CRE;DTA mice (blue arrows). See also and . ( B ) Hematoxylin and eosin-stained lung sections ( n = 6/group) from 12-week-old DTA (controls), CCSP.CRE;DTA (airway epithelial suicide model), and LYZ2.CRE;DTA (alveolar epithelial suicide model) mice. ( C ) Data summaries of mean linear intercept, bronchoalveolar lavage (BAL) myeloid cells, pressure-volume curves, airway resistance, and static compliance ( n = 6–10/group) from 12-week-old DTA, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice shown as violin plots. P , probabilities, one-way ANOVA. ns, **, and ***: p>0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Bonferroni post-tests. ( D ) Lung photographs of control, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice at six months into treatment with urethane started at six weeks of age. ( E ) Incidence table and data summaries of lung tumors from ( D ) (violin plots; n is given in table). P , probabilities, χ -test (table) and one-way ANOVA (graphs). ns, *, **, and ***: p>0.05, p<0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Fischer’s exact tests (table) or Bonferroni post-tests (graphs). a, alveoli; b, bronchi; ps, pleural space; v, vessel. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.056 Figure 5—source data 1. Quantifications of data shown in . 10.7554/eLife.45571.057 Figure 5—source data 2. Quantifications of data shown in .
    Figure Legend Snippet: ( A ) Lineage marker-immunostained lung sections of 12-week-old GFP;CCSP.CRE;DTA and GFP;LYZ2.CRE;DTA mice ( n = 6/group) show increased bronchial and alveolar size and flat CCSP+ SFTPC+ LYZ2+ cells in the airways of GFP;CCSP.CRE;DTA mice (green arrows), and CCSP-SFTPC-LYZ2+ alveolar macrophages in the airspaces of GFP;LYZ2.CRE;DTA mice (blue arrows). See also and . ( B ) Hematoxylin and eosin-stained lung sections ( n = 6/group) from 12-week-old DTA (controls), CCSP.CRE;DTA (airway epithelial suicide model), and LYZ2.CRE;DTA (alveolar epithelial suicide model) mice. ( C ) Data summaries of mean linear intercept, bronchoalveolar lavage (BAL) myeloid cells, pressure-volume curves, airway resistance, and static compliance ( n = 6–10/group) from 12-week-old DTA, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice shown as violin plots. P , probabilities, one-way ANOVA. ns, **, and ***: p>0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Bonferroni post-tests. ( D ) Lung photographs of control, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice at six months into treatment with urethane started at six weeks of age. ( E ) Incidence table and data summaries of lung tumors from ( D ) (violin plots; n is given in table). P , probabilities, χ -test (table) and one-way ANOVA (graphs). ns, *, **, and ***: p>0.05, p<0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Fischer’s exact tests (table) or Bonferroni post-tests (graphs). a, alveoli; b, bronchi; ps, pleural space; v, vessel. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.056 Figure 5—source data 1. Quantifications of data shown in . 10.7554/eLife.45571.057 Figure 5—source data 2. Quantifications of data shown in .

    Techniques Used: Marker, Staining

    Representative lung sections of 12-week-old GFP;CCSP.CRE, GFP;LYZ2.CRE, GFP;CCSP.CRE;DTA, and GFP;LYZ2.CRE;DTA mice ( n = 6/group). Shown are merges of Hoechst 33258-stained endogenous TOMATO- and GFP-labeling. Note increased bronchial ( b ) and alveolar ( a ) size, complete airway epithelial denudement, and prominent distortion of bronchial and alveolar structure of GFP;CCSP.CRE;DTA mice compared with other strains, mimicking chronic obstructive pulmonary disease. Note also the presence of some GFP-labeled alveolar macrophages in GFP;LYZ2.CRE;DTA mice (arrows). a, alveoli; b, bronchi.
    Figure Legend Snippet: Representative lung sections of 12-week-old GFP;CCSP.CRE, GFP;LYZ2.CRE, GFP;CCSP.CRE;DTA, and GFP;LYZ2.CRE;DTA mice ( n = 6/group). Shown are merges of Hoechst 33258-stained endogenous TOMATO- and GFP-labeling. Note increased bronchial ( b ) and alveolar ( a ) size, complete airway epithelial denudement, and prominent distortion of bronchial and alveolar structure of GFP;CCSP.CRE;DTA mice compared with other strains, mimicking chronic obstructive pulmonary disease. Note also the presence of some GFP-labeled alveolar macrophages in GFP;LYZ2.CRE;DTA mice (arrows). a, alveoli; b, bronchi.

    Techniques Used: Staining, Labeling

    ( A ) Our evidence supports the existence of distinct developmental ancestries for airway epithelial (AEC) and alveolar type II (ATII) cells, notwithstanding their common descent from an early (possibly Sftpc +) lung epithelial progenitor. The developmental airway lineage ( Scgb1a1+ Sftpc ±; green) gives rise to all types of airway cells, including club, ciliated, goblet, basal, and other cells, while the developmental ATII lineage ( Sftpc+ Lyz2 ±; red) gives rise to ATII cells before birth. These lineages appear to be segregated in the growing unaffected lung of the mouse till the age of six weeks, which roughly corresponds to a human age of six years, where cellular proliferation in the human lungs ceases. Thereafter, and likely due to the continuous exposure of the lungs to inhaled noxious agents, gradual expansion of Scgb1a1+ Sftpc ± marked cells ensues. Upon lung injury, this process is accelerated. Similarly, during carcinogenesis caused by chemical tobacco smoke carcinogens, Scgb1a1+ Sftpc ± marked cells expand and are ubiquitously present in peripheral lung adenocarcinomas. ( B ) Proposed neonatal proportions and postnatal dynamics of pulmonary epithelial cells during adulthood. Estimated proportions of lineage-marked cells at birth, based on flow cytometry and co-localization of proteinaceous and genetic cell marking. Lung lineages appear to be segregated in the growing lung till the age of full lung development (six weeks in mice and 6–8 years in humans) or till lung injury ensues. Schematic of proposed postnatal redistribution of marked cells in the adult lung. Upon injury, during multi-stage field carcinogenesis, or even during unchallenged aging, Scgb1a1 + marked cells appear in the distal alveolar regions, thereby maintaining lung structure and function. Bubble size indicates relative marked cell abundance. CCSP, Clara cell secretory protein; FOXJ1, forkhead box J1; KRT5, keratin 5; LYZ2, lysozyme 2; SFTPC, surfactant protein C; TUB1A1, acetylated α-tubulin.
    Figure Legend Snippet: ( A ) Our evidence supports the existence of distinct developmental ancestries for airway epithelial (AEC) and alveolar type II (ATII) cells, notwithstanding their common descent from an early (possibly Sftpc +) lung epithelial progenitor. The developmental airway lineage ( Scgb1a1+ Sftpc ±; green) gives rise to all types of airway cells, including club, ciliated, goblet, basal, and other cells, while the developmental ATII lineage ( Sftpc+ Lyz2 ±; red) gives rise to ATII cells before birth. These lineages appear to be segregated in the growing unaffected lung of the mouse till the age of six weeks, which roughly corresponds to a human age of six years, where cellular proliferation in the human lungs ceases. Thereafter, and likely due to the continuous exposure of the lungs to inhaled noxious agents, gradual expansion of Scgb1a1+ Sftpc ± marked cells ensues. Upon lung injury, this process is accelerated. Similarly, during carcinogenesis caused by chemical tobacco smoke carcinogens, Scgb1a1+ Sftpc ± marked cells expand and are ubiquitously present in peripheral lung adenocarcinomas. ( B ) Proposed neonatal proportions and postnatal dynamics of pulmonary epithelial cells during adulthood. Estimated proportions of lineage-marked cells at birth, based on flow cytometry and co-localization of proteinaceous and genetic cell marking. Lung lineages appear to be segregated in the growing lung till the age of full lung development (six weeks in mice and 6–8 years in humans) or till lung injury ensues. Schematic of proposed postnatal redistribution of marked cells in the adult lung. Upon injury, during multi-stage field carcinogenesis, or even during unchallenged aging, Scgb1a1 + marked cells appear in the distal alveolar regions, thereby maintaining lung structure and function. Bubble size indicates relative marked cell abundance. CCSP, Clara cell secretory protein; FOXJ1, forkhead box J1; KRT5, keratin 5; LYZ2, lysozyme 2; SFTPC, surfactant protein C; TUB1A1, acetylated α-tubulin.

    Techniques Used: Flow Cytometry


    Figure Legend Snippet:

    Techniques Used: Mutagenesis, Derivative Assay, Sequencing, Real-time Polymerase Chain Reaction, Software, Microarray, Expressing



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    ( A ) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also – . ( B ) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice ( n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also – . ( C ) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment ( n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also – . ( D ) Lung sections of GFP;CCSP.CRE mice ( n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also – . CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; <t>LYZ2,</t> lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.
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    ( A ) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also – . ( B ) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice ( n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also – . ( C ) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment ( n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also – . ( D ) Lung sections of GFP;CCSP.CRE mice ( n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also – . CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: ( A ) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also – . ( B ) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice ( n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also – . ( C ) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment ( n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also – . ( D ) Lung sections of GFP;CCSP.CRE mice ( n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also – . CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.

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    Techniques: Labeling, Marker, Staining

    TUBA1A, Tubulin alpha 1a or acetylated tubulin; KRT5, Keratin 5; FOXJ1, Forkhead box J1; CCSP, Secretoglobin, family 1A, member 1 (uteroglobin) or Clara cell secretory protein or Clara cell 10 KDa protein; SFTPC, Surfactant protein C; LYZ2, Lysozyme 2; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: TUBA1A, Tubulin alpha 1a or acetylated tubulin; KRT5, Keratin 5; FOXJ1, Forkhead box J1; CCSP, Secretoglobin, family 1A, member 1 (uteroglobin) or Clara cell secretory protein or Clara cell 10 KDa protein; SFTPC, Surfactant protein C; LYZ2, Lysozyme 2; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages.

    Article Snippet: Antibody , rabbit monoclonal anti-LYZ2 , Abcam , Cat. #: ab108508; RRID: AB_10861277 , IF (1:50).

    Techniques:

    CRE, causes recombination; TOMATO, tdTomato; GFP, green fluorescent protein; CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; SOX2, sex determining region Y (SRY)-box 2; VAV, Vav Guanine Nucleotide Exchange Factor 1; NES, nestin; JAX, Jackson Laboratories; EMMA, European Mutant Mouse Archive; MGI, Mouse Genome Informatics; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages; ΒM, bone marrow (myeloid) cells. Symbols indicate: - (white), no genetic labeling; + (magenta), complete genetic labeling; ± (blue), partial genetic labeling.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: CRE, causes recombination; TOMATO, tdTomato; GFP, green fluorescent protein; CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; SOX2, sex determining region Y (SRY)-box 2; VAV, Vav Guanine Nucleotide Exchange Factor 1; NES, nestin; JAX, Jackson Laboratories; EMMA, European Mutant Mouse Archive; MGI, Mouse Genome Informatics; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages; ΒM, bone marrow (myeloid) cells. Symbols indicate: - (white), no genetic labeling; + (magenta), complete genetic labeling; ± (blue), partial genetic labeling.

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    Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage-labeled mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; TUBA1A, acetylated tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage-labeled mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; TUBA1A, acetylated tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

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    Representative photographs (top row) and green epifluorescence images (second row), as well as merged fluorescent microscopic images of lung sections for nuclear Hoechst33258 stain, endogenous TOMATO, and endogenous GFP (bottom three rows), of tumor-bearing lungs from genetically marked mice employed in these studies (described in detail in ) at six months after initiation of ten weekly intraperitoneal urethane injections ( n = 30, 22, 18, and 20/strain, respectively). b, bronchi. Top two rows: arrows indicate lung tumors. Bottom three rows: white arrows indicate GFP-labeled cells in apparently non-affected alveolar areas of GFP;CCSP.CRE mice; green arrow indicates rare GFP+ cell in non-affected central airway of GFP;LYZ2.CRE mouse. Note the absence of GFP-labeling of lung tumors in TOMATO mice, the complete GFP-labeling in GFP;CCSP.CRE and GFP;SFTPC.CRE mice, and the partial GFP-labeling in GFP;LYZ2.CRE mice.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: Representative photographs (top row) and green epifluorescence images (second row), as well as merged fluorescent microscopic images of lung sections for nuclear Hoechst33258 stain, endogenous TOMATO, and endogenous GFP (bottom three rows), of tumor-bearing lungs from genetically marked mice employed in these studies (described in detail in ) at six months after initiation of ten weekly intraperitoneal urethane injections ( n = 30, 22, 18, and 20/strain, respectively). b, bronchi. Top two rows: arrows indicate lung tumors. Bottom three rows: white arrows indicate GFP-labeled cells in apparently non-affected alveolar areas of GFP;CCSP.CRE mice; green arrow indicates rare GFP+ cell in non-affected central airway of GFP;LYZ2.CRE mouse. Note the absence of GFP-labeling of lung tumors in TOMATO mice, the complete GFP-labeling in GFP;CCSP.CRE and GFP;SFTPC.CRE mice, and the partial GFP-labeling in GFP;LYZ2.CRE mice.

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    Lineage marker protein-stained lung adenocarcinomas (dashed outlines) from genetically marked mice ( n = 10/group). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

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    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: Lineage marker protein-stained lung adenocarcinomas (dashed outlines) from genetically marked mice ( n = 10/group). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

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    Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage reporter mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage reporter mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

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    Representative merged fluorescent microscopic images of lineage marker protein-stained lung tumors (dashed outlines) from genetically marked mice (FVB background) at six months after a single intraperitoneal urethane injection ( n ≥ 10/strain). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

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    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: Representative merged fluorescent microscopic images of lineage marker protein-stained lung tumors (dashed outlines) from genetically marked mice (FVB background) at six months after a single intraperitoneal urethane injection ( n ≥ 10/strain). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

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    ( A ) DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Summary of duplexed digital droplet PCR (ddPCR) results using primers and probes specific for the Rosa mT and the Kras WT sequences. Note that all cell types equally suffer initial Kras Q61R mutations, but only GFP-labeled cells of GFP;CCSP.CRE mice (i.e. airway cells) maintain the Kras Q61R mutation after two weeks. See also . Data are shown as violin plot. P , overall probability, two-way ANOVA. ***: p<0.001 compared with all other groups, Bonferroni post-tests. ( B ) Representative high-resolution micro-computed tomography (μCT) lung sections (top) and three-dimensional reconstructions (bottom) from urethane-treated FVB mice six months after treatment ( n = 10). Note lung tumors attached to (green arrows) or contained within (blue arrows) the airways, as well as lung tumors with no obvious link to a bronchus (red arrows). ( C ) Summary of results from μCT (data from ) and pathology (data from ) shown as violin plot. P , probability, two-way ANOVA.*, ***, and ****: p<0.05, p<0.001, and p<0.0001, respectively, compared with airway-attached tumors, Bonferroni post-tests. Shown are also Spearman’s correlation coefficient ( ρ ) and probability ( P ) for correlation of μCT and pathology results. 10.7554/eLife.45571.031 Figure 2—source data 1. Quantification of Kras mutant droplets in duplexed digital droplet PCR (ddPCR). 10.7554/eLife.45571.032 Figure 2—source data 2. Quantification of tumor airway link.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: ( A ) DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Summary of duplexed digital droplet PCR (ddPCR) results using primers and probes specific for the Rosa mT and the Kras WT sequences. Note that all cell types equally suffer initial Kras Q61R mutations, but only GFP-labeled cells of GFP;CCSP.CRE mice (i.e. airway cells) maintain the Kras Q61R mutation after two weeks. See also . Data are shown as violin plot. P , overall probability, two-way ANOVA. ***: p<0.001 compared with all other groups, Bonferroni post-tests. ( B ) Representative high-resolution micro-computed tomography (μCT) lung sections (top) and three-dimensional reconstructions (bottom) from urethane-treated FVB mice six months after treatment ( n = 10). Note lung tumors attached to (green arrows) or contained within (blue arrows) the airways, as well as lung tumors with no obvious link to a bronchus (red arrows). ( C ) Summary of results from μCT (data from ) and pathology (data from ) shown as violin plot. P , probability, two-way ANOVA.*, ***, and ****: p<0.05, p<0.001, and p<0.0001, respectively, compared with airway-attached tumors, Bonferroni post-tests. Shown are also Spearman’s correlation coefficient ( ρ ) and probability ( P ) for correlation of μCT and pathology results. 10.7554/eLife.45571.031 Figure 2—source data 1. Quantification of Kras mutant droplets in duplexed digital droplet PCR (ddPCR). 10.7554/eLife.45571.032 Figure 2—source data 2. Quantification of tumor airway link.

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    Techniques: Labeling, Mutagenesis, Micro-CT

    DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Representative gating strategy of digital droplet PCR (ddPCR) using primers and probes specific for the Rosa mT and the Kras WT sequences. Dashed outlines indicated GFP+ Kras Q61R + droplet gates.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Representative gating strategy of digital droplet PCR (ddPCR) using primers and probes specific for the Rosa mT and the Kras WT sequences. Dashed outlines indicated GFP+ Kras Q61R + droplet gates.

    Article Snippet: Antibody , rabbit monoclonal anti-LYZ2 , Abcam , Cat. #: ab108508; RRID: AB_10861277 , IF (1:50).

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    ( A ) Non-neoplastic alveolar regions from lung sections of aging GFP;CCSP.CRE mice (bottom right section is also SFTPC-immunostained) show increasing numbers of alveolar GFP-labeled cells with age (arrows). Green arrows: genetically GFP-labeled, SFTPC-immunoreactive airway cells in alveolus of 15-month-old GFP;CCSP.CRE mouse. ( B ) Data summary ( n = 5 mice/time-point) from ( A ) shown as violin plot. Color-coded boxes indicate time windows of experiments in ( C-H ). P , probability, one-way ANOVA. ns, ***, and ****: p>0.05, p<0.001, and p<0.0001, respectively, for comparison with time-point zero by Bonferroni post-tests. ( C ) SFTPC-immunostained lung sections of GFP;CCSP.CRE mice show accelerated increase of alveolar GFP-labeled SFTPC-immunoreactive airway cells after bleomycin treatment (arrows). See also and . ( D ) Data summary from ( C ) shown as violin plots ( n = 4 mice/time-point). P , probabilities, one-way ANOVA. ns, *, **, ***, and ****: p>0.05, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, for comparison with day zero by Bonferroni post-tests. ( E ) SFTPC-stained lung sections of GFP;CCSP.CRE mice at two months after perinatal exposure to 98% O 2 show enlarged alveoli (evident by increased mean linear intercept) enriched in GFP-labeled SFTPC-immunoreactive airway cells (arrows) compared with 21% O 2 . ( F ) Data summary from ( E ) shown as violin plots ( n = 6 mice/group). P , probabilities, t-test. ( G ) Lung sections (top) of GFP;CCSP.CRE mice ( n = 5 mice/group) show enrichment of alveoli in GFP-labeled cells post-naphthalene treatment (arrows). Lung sections (bottom) of GFP;LYZ2.CRE mice ( n = 5 mice/group) at six weeks post-naphthalene show no bronchial ( b ) GFP-labeled cells. See also and . ( H ) Data summary from ( G ) shown as violin plot ( n = 5 mice/time-point). P , probability, two-way ANOVA. ns and ****: p>0.05 and p<0.0001, respectively, for comparison with corn oil by Bonferroni post-tests. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.047 Figure 4—source data 1. Quantification of alveolar GFP+ cells in GFP;CCSP.CRE mice during aging. 10.7554/eLife.45571.048 Figure 4—source data 2. Quantification of SFTPC+ and GFP+ cells in GFP;CCSP.CRE mice after bleomycin treatment. 10.7554/eLife.45571.049 Figure 4—source data 3. Data of mean linear intercept and GFP+/SFTPC+cells in GFP;CCSP.CRE mice after hyperoxia treatment. 10.7554/eLife.45571.050 Figure 4—source data 4. Data of GFP+/SFTPC+ cells in GFP;CCSP.CRE and GFP;LYZ2.CRE mice after naphthalene treatment.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: ( A ) Non-neoplastic alveolar regions from lung sections of aging GFP;CCSP.CRE mice (bottom right section is also SFTPC-immunostained) show increasing numbers of alveolar GFP-labeled cells with age (arrows). Green arrows: genetically GFP-labeled, SFTPC-immunoreactive airway cells in alveolus of 15-month-old GFP;CCSP.CRE mouse. ( B ) Data summary ( n = 5 mice/time-point) from ( A ) shown as violin plot. Color-coded boxes indicate time windows of experiments in ( C-H ). P , probability, one-way ANOVA. ns, ***, and ****: p>0.05, p<0.001, and p<0.0001, respectively, for comparison with time-point zero by Bonferroni post-tests. ( C ) SFTPC-immunostained lung sections of GFP;CCSP.CRE mice show accelerated increase of alveolar GFP-labeled SFTPC-immunoreactive airway cells after bleomycin treatment (arrows). See also and . ( D ) Data summary from ( C ) shown as violin plots ( n = 4 mice/time-point). P , probabilities, one-way ANOVA. ns, *, **, ***, and ****: p>0.05, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, for comparison with day zero by Bonferroni post-tests. ( E ) SFTPC-stained lung sections of GFP;CCSP.CRE mice at two months after perinatal exposure to 98% O 2 show enlarged alveoli (evident by increased mean linear intercept) enriched in GFP-labeled SFTPC-immunoreactive airway cells (arrows) compared with 21% O 2 . ( F ) Data summary from ( E ) shown as violin plots ( n = 6 mice/group). P , probabilities, t-test. ( G ) Lung sections (top) of GFP;CCSP.CRE mice ( n = 5 mice/group) show enrichment of alveoli in GFP-labeled cells post-naphthalene treatment (arrows). Lung sections (bottom) of GFP;LYZ2.CRE mice ( n = 5 mice/group) at six weeks post-naphthalene show no bronchial ( b ) GFP-labeled cells. See also and . ( H ) Data summary from ( G ) shown as violin plot ( n = 5 mice/time-point). P , probability, two-way ANOVA. ns and ****: p>0.05 and p<0.0001, respectively, for comparison with corn oil by Bonferroni post-tests. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.047 Figure 4—source data 1. Quantification of alveolar GFP+ cells in GFP;CCSP.CRE mice during aging. 10.7554/eLife.45571.048 Figure 4—source data 2. Quantification of SFTPC+ and GFP+ cells in GFP;CCSP.CRE mice after bleomycin treatment. 10.7554/eLife.45571.049 Figure 4—source data 3. Data of mean linear intercept and GFP+/SFTPC+cells in GFP;CCSP.CRE mice after hyperoxia treatment. 10.7554/eLife.45571.050 Figure 4—source data 4. Data of GFP+/SFTPC+ cells in GFP;CCSP.CRE and GFP;LYZ2.CRE mice after naphthalene treatment.

    Article Snippet: Antibody , rabbit monoclonal anti-LYZ2 , Abcam , Cat. #: ab108508; RRID: AB_10861277 , IF (1:50).

    Techniques: Labeling, Staining

    ( A ) Lineage marker-immunostained lung sections of 12-week-old GFP;CCSP.CRE;DTA and GFP;LYZ2.CRE;DTA mice ( n = 6/group) show increased bronchial and alveolar size and flat CCSP+ SFTPC+ LYZ2+ cells in the airways of GFP;CCSP.CRE;DTA mice (green arrows), and CCSP-SFTPC-LYZ2+ alveolar macrophages in the airspaces of GFP;LYZ2.CRE;DTA mice (blue arrows). See also and . ( B ) Hematoxylin and eosin-stained lung sections ( n = 6/group) from 12-week-old DTA (controls), CCSP.CRE;DTA (airway epithelial suicide model), and LYZ2.CRE;DTA (alveolar epithelial suicide model) mice. ( C ) Data summaries of mean linear intercept, bronchoalveolar lavage (BAL) myeloid cells, pressure-volume curves, airway resistance, and static compliance ( n = 6–10/group) from 12-week-old DTA, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice shown as violin plots. P , probabilities, one-way ANOVA. ns, **, and ***: p>0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Bonferroni post-tests. ( D ) Lung photographs of control, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice at six months into treatment with urethane started at six weeks of age. ( E ) Incidence table and data summaries of lung tumors from ( D ) (violin plots; n is given in table). P , probabilities, χ -test (table) and one-way ANOVA (graphs). ns, *, **, and ***: p>0.05, p<0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Fischer’s exact tests (table) or Bonferroni post-tests (graphs). a, alveoli; b, bronchi; ps, pleural space; v, vessel. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.056 Figure 5—source data 1. Quantifications of data shown in . 10.7554/eLife.45571.057 Figure 5—source data 2. Quantifications of data shown in .

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: ( A ) Lineage marker-immunostained lung sections of 12-week-old GFP;CCSP.CRE;DTA and GFP;LYZ2.CRE;DTA mice ( n = 6/group) show increased bronchial and alveolar size and flat CCSP+ SFTPC+ LYZ2+ cells in the airways of GFP;CCSP.CRE;DTA mice (green arrows), and CCSP-SFTPC-LYZ2+ alveolar macrophages in the airspaces of GFP;LYZ2.CRE;DTA mice (blue arrows). See also and . ( B ) Hematoxylin and eosin-stained lung sections ( n = 6/group) from 12-week-old DTA (controls), CCSP.CRE;DTA (airway epithelial suicide model), and LYZ2.CRE;DTA (alveolar epithelial suicide model) mice. ( C ) Data summaries of mean linear intercept, bronchoalveolar lavage (BAL) myeloid cells, pressure-volume curves, airway resistance, and static compliance ( n = 6–10/group) from 12-week-old DTA, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice shown as violin plots. P , probabilities, one-way ANOVA. ns, **, and ***: p>0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Bonferroni post-tests. ( D ) Lung photographs of control, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice at six months into treatment with urethane started at six weeks of age. ( E ) Incidence table and data summaries of lung tumors from ( D ) (violin plots; n is given in table). P , probabilities, χ -test (table) and one-way ANOVA (graphs). ns, *, **, and ***: p>0.05, p<0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Fischer’s exact tests (table) or Bonferroni post-tests (graphs). a, alveoli; b, bronchi; ps, pleural space; v, vessel. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.056 Figure 5—source data 1. Quantifications of data shown in . 10.7554/eLife.45571.057 Figure 5—source data 2. Quantifications of data shown in .

    Article Snippet: Antibody , rabbit monoclonal anti-LYZ2 , Abcam , Cat. #: ab108508; RRID: AB_10861277 , IF (1:50).

    Techniques: Marker, Staining

    Representative lung sections of 12-week-old GFP;CCSP.CRE, GFP;LYZ2.CRE, GFP;CCSP.CRE;DTA, and GFP;LYZ2.CRE;DTA mice ( n = 6/group). Shown are merges of Hoechst 33258-stained endogenous TOMATO- and GFP-labeling. Note increased bronchial ( b ) and alveolar ( a ) size, complete airway epithelial denudement, and prominent distortion of bronchial and alveolar structure of GFP;CCSP.CRE;DTA mice compared with other strains, mimicking chronic obstructive pulmonary disease. Note also the presence of some GFP-labeled alveolar macrophages in GFP;LYZ2.CRE;DTA mice (arrows). a, alveoli; b, bronchi.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: Representative lung sections of 12-week-old GFP;CCSP.CRE, GFP;LYZ2.CRE, GFP;CCSP.CRE;DTA, and GFP;LYZ2.CRE;DTA mice ( n = 6/group). Shown are merges of Hoechst 33258-stained endogenous TOMATO- and GFP-labeling. Note increased bronchial ( b ) and alveolar ( a ) size, complete airway epithelial denudement, and prominent distortion of bronchial and alveolar structure of GFP;CCSP.CRE;DTA mice compared with other strains, mimicking chronic obstructive pulmonary disease. Note also the presence of some GFP-labeled alveolar macrophages in GFP;LYZ2.CRE;DTA mice (arrows). a, alveoli; b, bronchi.

    Article Snippet: Antibody , rabbit monoclonal anti-LYZ2 , Abcam , Cat. #: ab108508; RRID: AB_10861277 , IF (1:50).

    Techniques: Staining, Labeling

    ( A ) Our evidence supports the existence of distinct developmental ancestries for airway epithelial (AEC) and alveolar type II (ATII) cells, notwithstanding their common descent from an early (possibly Sftpc +) lung epithelial progenitor. The developmental airway lineage ( Scgb1a1+ Sftpc ±; green) gives rise to all types of airway cells, including club, ciliated, goblet, basal, and other cells, while the developmental ATII lineage ( Sftpc+ Lyz2 ±; red) gives rise to ATII cells before birth. These lineages appear to be segregated in the growing unaffected lung of the mouse till the age of six weeks, which roughly corresponds to a human age of six years, where cellular proliferation in the human lungs ceases. Thereafter, and likely due to the continuous exposure of the lungs to inhaled noxious agents, gradual expansion of Scgb1a1+ Sftpc ± marked cells ensues. Upon lung injury, this process is accelerated. Similarly, during carcinogenesis caused by chemical tobacco smoke carcinogens, Scgb1a1+ Sftpc ± marked cells expand and are ubiquitously present in peripheral lung adenocarcinomas. ( B ) Proposed neonatal proportions and postnatal dynamics of pulmonary epithelial cells during adulthood. Estimated proportions of lineage-marked cells at birth, based on flow cytometry and co-localization of proteinaceous and genetic cell marking. Lung lineages appear to be segregated in the growing lung till the age of full lung development (six weeks in mice and 6–8 years in humans) or till lung injury ensues. Schematic of proposed postnatal redistribution of marked cells in the adult lung. Upon injury, during multi-stage field carcinogenesis, or even during unchallenged aging, Scgb1a1 + marked cells appear in the distal alveolar regions, thereby maintaining lung structure and function. Bubble size indicates relative marked cell abundance. CCSP, Clara cell secretory protein; FOXJ1, forkhead box J1; KRT5, keratin 5; LYZ2, lysozyme 2; SFTPC, surfactant protein C; TUB1A1, acetylated α-tubulin.

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet: ( A ) Our evidence supports the existence of distinct developmental ancestries for airway epithelial (AEC) and alveolar type II (ATII) cells, notwithstanding their common descent from an early (possibly Sftpc +) lung epithelial progenitor. The developmental airway lineage ( Scgb1a1+ Sftpc ±; green) gives rise to all types of airway cells, including club, ciliated, goblet, basal, and other cells, while the developmental ATII lineage ( Sftpc+ Lyz2 ±; red) gives rise to ATII cells before birth. These lineages appear to be segregated in the growing unaffected lung of the mouse till the age of six weeks, which roughly corresponds to a human age of six years, where cellular proliferation in the human lungs ceases. Thereafter, and likely due to the continuous exposure of the lungs to inhaled noxious agents, gradual expansion of Scgb1a1+ Sftpc ± marked cells ensues. Upon lung injury, this process is accelerated. Similarly, during carcinogenesis caused by chemical tobacco smoke carcinogens, Scgb1a1+ Sftpc ± marked cells expand and are ubiquitously present in peripheral lung adenocarcinomas. ( B ) Proposed neonatal proportions and postnatal dynamics of pulmonary epithelial cells during adulthood. Estimated proportions of lineage-marked cells at birth, based on flow cytometry and co-localization of proteinaceous and genetic cell marking. Lung lineages appear to be segregated in the growing lung till the age of full lung development (six weeks in mice and 6–8 years in humans) or till lung injury ensues. Schematic of proposed postnatal redistribution of marked cells in the adult lung. Upon injury, during multi-stage field carcinogenesis, or even during unchallenged aging, Scgb1a1 + marked cells appear in the distal alveolar regions, thereby maintaining lung structure and function. Bubble size indicates relative marked cell abundance. CCSP, Clara cell secretory protein; FOXJ1, forkhead box J1; KRT5, keratin 5; LYZ2, lysozyme 2; SFTPC, surfactant protein C; TUB1A1, acetylated α-tubulin.

    Article Snippet: Antibody , rabbit monoclonal anti-LYZ2 , Abcam , Cat. #: ab108508; RRID: AB_10861277 , IF (1:50).

    Techniques: Flow Cytometry

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet:

    Article Snippet: Antibody , rabbit monoclonal anti-LYZ2 , Abcam , Cat. #: ab108508; RRID: AB_10861277 , IF (1:50).

    Techniques: Mutagenesis, Derivative Assay, Sequencing, Real-time Polymerase Chain Reaction, Software, Microarray, Expressing

    Journal: eLife

    Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

    doi: 10.7554/eLife.45571

    Figure Lengend Snippet:

    Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

    Techniques: Mutagenesis, Derivative Assay, Sequencing, Real-time Polymerase Chain Reaction, Software, Microarray, Gene Expression