1. Cell Biology
  2. Stem Cells and Regenerative Medicine

Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells

  1. Huachao Huang
  2. Yinshan Fang
  3. Ming Jiang
  4. Yihan Zhang
  5. Jana Biermann
  6. Johannes C Melms
  7. Jennifer A Danielsson
  8. Ying Yang
  9. Li Qiang
  10. Jia Liu
  11. Yiwu Zhou
  12. Manli Wang
  13. Zhihong Hu
  14. Timothy C Wang
  15. Anjali Saqi
  16. Jie Sun
  17. Ichiro Matsumoto
  18. Wellington V Cardoso
  19. Charles W Emala
  20. Jian Zhu
  21. Benjamin Izar
  22. Hongmei Mou  Is a corresponding author
  23. Jianwen Que  Is a corresponding author
  1. Columbia Center for Human Development, Department of Medicine, Columbia University Medical Center, United States
  2. Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, United States
  3. Institute of Genetics, the Children's Hospital, Zhejiang University School of Medicine, China
  4. Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, Harvard Medical School, United States
  5. Division of Hematology/Oncology, Department of Medicine, Columbia University Irving Medical Center, United States
  6. Columbia Center for Translational Immunology, Columbia University Irving Medical Center, United States
  7. Department of Anesthesiology, Columbia University Irving Medical Center, United States
  8. Program in Epithelial Biology, Stanford University School of Medicine, United States
  9. Department of Pathology & Cell Biology, Columbia University Medical Center, United States
  10. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, China
  11. Department of Forensic Medicine, Tongji Medical College of Huazhong University of Science and Technology, China
  12. Carter Immunology Center, the University of Virginia, United States
  13. Monell Chemical Senses Center, United States
  14. Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Columbia University Medical Center, United States
  15. Department of Pathology, Ohio State University College of Medicine, United States
https://doi.org/10.7554/eLife.78217
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Cite this article
  1. Huachao Huang
  2. Yinshan Fang
  3. Ming Jiang
  4. Yihan Zhang
  5. Jana Biermann
  6. Johannes C Melms
  7. Jennifer A Danielsson
  8. Ying Yang
  9. Li Qiang
  10. Jia Liu
  11. Yiwu Zhou
  12. Manli Wang
  13. Zhihong Hu
  14. Timothy C Wang
  15. Anjali Saqi
  16. Jie Sun
  17. Ichiro Matsumoto
  18. Wellington V Cardoso
  19. Charles W Emala
  20. Jian Zhu
  21. Benjamin Izar
  22. Hongmei Mou
  23. Jianwen Que
(2022)
Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells
eLife 11:e78217.
https://doi.org/10.7554/eLife.78217
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8 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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Tuft cells in homeostasis and their expansion in response to severe injuries.

(A) Tuft cells are present in the distal airways at P0, P10, P20 but not P56. n=4 for each group. (B) Quantification of tuft cells from panel (A). (C) Tuft cells expand in the large airway and are ectopically present in the terminal airways following viral infection. (D) Tuft cells expand in the airways following naphthalene and bleomycin challenge and are ectopically present in parenchyma following bleomycin challenge. Scale bars, 50 μm.

Figure 1—figure supplement 1
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Ectopic tuft cells appear after viral infection.

(A) Time course analysis of ectopic tuft cells in the lung parenchyma following viral infection. (B) Quantification of tuft cells in ectopic basal cells areas of lung parenchyma. Scale bars, 50 μm.

Figure 2 with 1 supplement
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Tuft cells in the parenchyma are derived from ectopic basal cells (EBCs) following H1N1 PR8 viral infection.

(A) Pou2f3CreERT2;R26Ai14 mouse line specifically labels Dclk1+ tuft cells in the large airways at homeostasis. (B) Lineage tracing of Pou2f3+ cells in the parenchyma after PR8 virus infection. Arrow indicates a tdT+Dclk1- cell. (C) Lineage labeled tuft cells do not contribute to other cell lineages, including Scgb1a1+ club cells, FoxJ1+ ciliated cells and Clca3+ goblet cells. (D) Lineage tracing confirms that tuft cells are derived from EBCs in the lung parenchyma of Trp63CreERT2;R26Ai14 mice following PR8 infection. (E) Lineage tracing shows about 80% of tuft cells are derived from EBCs in the lung parenchyma of KRT5-CreERT2;R26Ai14 mice following PR8 infection. Data represent mean ± s.e.m. Scale bars, 20 μm (A) and 50 μm (B to G).

Figure 2—figure supplement 1
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Tuft cells in the lung parenchyma are derived from ectopic basal cells (EBCs).

(A) Lineage tracing shows heterogeneity of tuft cells in the trachea of Pou2f3CreERT2;R26Ai14 mice. Note that some lineage labeled cells do not express Dclk1 (arrowhead in left panel). (B) Tuft cells do not proliferate when examined at days 15, 21, and 30 post-infection. (C) ~5% of tuft cells within EBCs co-express Krt5 and Dclk1. (D) Lineage labeled cells contribute to ~21% ectopic tuft cells in the lung parenchyma of Scgb1a1CreERT;R26Ai14 mice. Scale bars, 50 μm.

Figure 3
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EBCs likely give rise to tuft cells in the parenchyma of COVID-19 lungs.

(A) SARS-CoV-2 infection causes the loss of club and ciliated cells (arrows in a), exposing the underlying basal cells (B) in the small airways. Note the detached basal cells (C). (B) EBCs proliferate in the parenchyma of COVID-19 lungs. H&E staining shows the presence of EBC clusters in COVID-19 lungs. (C) Representative clusters of EBCs are present in COVID-19 lungs. (D) Tuft cells within EBCs express both KRT5 and the tuft cell marker POU2F3. (E) Solitary tuft cells without KRT5 expression are present in the airways of COVID-19 lung. Abbreviation: bv, blood vessel. Scale bars, 100 μm (A, B and C) and 50 μm (D and E).

Figure 4 with 1 supplement
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Wnt inhibition promotes EBC differentiation into tuft cells while Notch inhibition has an opposite effect.

(A) Treatment with the WNT signaling inhibitor Wnt-C59 promotes tuft cell differentiation of Dclk1-GFP basal cells in ALI culture. (B) Wnt-C59 treatment increases the number of tuft cells in the lung parenchyma following viral infection. (C) Treatment with the Notch signaling inhibitor DBZ completely blocks tuft cell differentiation of Dclk1-GFP basal cells in ALI culture. (D) Daily injection of DBZ following PR8 infection dramatically reduces tuft cell derivation in the lung parenchyma. n=4 per group. Data represent mean ± s.e.m. *p<0.05, ***p<0.001; statistical analysis by unpaired two-tailed Student’s t-test. Scale bars, 50 μm.

Figure 4—figure supplement 1
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Isolation and expansion of Krt5+ EBCs from the lungs of Trp63CreERT2; R26Ai14 mice that were infected with PR8 virus.

(A) Schematic illustration depicts the methodology. (B) Lineage labeled EBCs are expanded and characterized. (C) Quantification of Dclk1+ cells derived from EBCs at various conditions (Vehicle, 2 μM CHIR99021, 5 μM IWR-1, and 2 μM DBZ) in air-liquid interface (ALI) culture. (D) Ablation of Notch effector Rbpjk in EBCs blocks differentiation of tuft cells from EBCs. Data represent mean ± s.e.m. * p<0.05, ** pP<0.01, *** pP<0.001; statistical analysis by unpaired two-tailed Student’s t-test. Scale bars, 50 μm.

Figure 5 with 1 supplement
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Increased generation of Trp63CreERT2 labeled alveolar epithelium in Trpm5 null mutants following PR8 infection.

(A) Trpm5 deletion leads to the increased presence of tdT+SftpC+Krt5- cells in the lung parenchyma at 60 dpi. (B) Increased tdT-labeled AT1 cells in the mutant lungs as compared to controls at 60 dpi. (C) Whole lung airway resistance improves in Trpm5-/- mice following viral infection (left panel). Central airway resistance is also reduced following viral infection (right panel). n=7 for WT group, n=9 for Trpm5-/- group. Data represent mean ± s.e.m. *p<0.05, **p<0.01; statistical analysis by unpaired two-tailed Student’s t-test. Scale bars, 100 μm (A), 20 µm (B).

Figure 5—figure supplement 1
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Increased Trp63CreERT2 labeled AT1 and AT2 cells in virus-infected Trpm5-/- mice.

(A) Quantification of tuft cells within EBCs areas established in the lung parenchyma of Trpm5+/- and Trpm5-/- mutants at 30 dpi. (B) Extensive Krt5+ EBCs in the lung parenchyma of controls (Trp63CreERT2;Trpm5+/-;R26Ai14) and mutants (Trp63CreERT2;Trpm5-/-;R26Ai14) at 60 dpi. (C) Clusters of lineage-labeled AT2 cells. Note some lineage-labeled cells are SftpC- (arrowheads). Also note the same image in Figure 5A. (D) Staining with Abca3 confirms the presence of lineage labeled AT2 cells in Trp63CreERT2;Trpm5-/-;R26Ai14 mutants at 60 dpi. (E) Lineage tracing confirms that the presence of lineage labeled AT1 (Hopx+) in the parenchyma of Trp63CreERT2;Trpm5-/-;R26Ai14 mutants. Data represent mean ± s.e.m. ns: not significant; statistical analysis by unpaired two-tailed Student’s t-test. Scale bars, 100 µm (B), 50 μm (C and D) and 20 µm (E).

Figure 6
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A complete loss of tuft cell does not increase the generation of Trp63CreERT2 labeled alveolar cells following PR8 infection.

(A) Extensive Krt5+SftpC- EBCs are present in the parenchyma of control (Trp63CreERT2;Pou2f3+/-;R26Ai14) and mutant (Trp63CreERT2;Pou2f3-/-;R26Ai14) lungs when examined at 60 dpi following PR8 infection. (B) Representative areas show Trp63CreERT2 labeled AT2 cells in both control and mutant lungs. Ns: not significant; statistical analysis by unpaired two-tailed Student’s t-test. Scale bar, 50 μm (A), 100 µm (B).

Author response image 1
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Trp63-CreERT2 lineage labeled cells in the airways but not alveoli when Tamoxifen was induced at day 5 and 7 after PR8 H1N1 viral infection. Trp63-CreERT2;R26-tdT mice were infected with PR8 at 250 pfu and Tmx were delivered at a dose of 0.25 mg/g bodyweight by oral gavage. Lung samples were collected and analyzed at 14 dpi. Stained antibodies are as indicated. Scale bar: 100 µm.

Author response image 2
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Representative area staining in Trpm5-/- mice at 30 dpi.

Area 1: Notch signaling is active (Hes1+, arrows) in pod cells following viral infection. Area 2: pod cells exhibit reduced Notch activities. Note few Hes1+ cells in pods (arrows). Scale bar: 50 µm.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)Trp63CreERT2Lee et al., 2014b; Lee et al., 2014a PMID:25210499Dr. Jianming Xu (Baylor College of Medicine)
Genetic reagent (Mus musculus)Tg(KRT5-CreERT2)Rock et al., 2009 PMID:19625615Dr. Brigid Hogan (Duke University); A transgenic mouse strain in which human KRT5 promoter drives CreERT2
Genetic reagent (Mus musculus)Scgb1a1CreERTRawlins et al., 2009
 PMID:19497281		MGI:3849566Dr. Brigid Hogan (Duke University)
Genetic reagent (Mus musculus)Pou2f3CreERT2McGinty et al., 2020 PMID:32160525MGI:6755141Dr. Jakob von Moltke (University of Washington)
Genetic reagent (Mus musculus)Tg(Trpm5-GFP)Clapp et al., 2006
 PMID:16573824		16573824Dr. Tod Clapp (Colorado State University)
Genetic reagent (Mus musculus)Trpm5-/-Damak et al., 2006
 PMID:16436689		Dr. Robert Margolskee (Mount Sinai)
Genetic reagent (Mus musculus)Pou2f3-/-Matsumoto et al., 2011
 PMID:21572433		MGI:5140071Dr. Keiko Abe (The University of Tokyo)
Genetic reagent (Mus musculus)Rbpjkloxp/loxpHan et al., 2002
 PMID:12039915		MGI:3583755Dr. Tasuku Honjo (Kyoto University)
Genetic reagent (Mus musculus)B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JMadisen et al., 2010
 PMID:20023653		MGI: 4436847Jackson Laboratories (#007914, R26Ai14)
AntibodyAnti-Dclk1
(Rabbit polyclonal)		Abcamab317041:200
AntibodyAnti-CC10 (E-11)
(Mouse monoclonal)		Santa Cruzsc-3659921:200; Scgb1a1
AntibodyAnti-SCGB1A1
(Rat monoclonal)		R&DMAB42181:500
AntibodyAnti-Krt5
(Chicken polyclonal)		BioLegend9059011:500
AntibodyAnti-Krt5
(Mouse monoclonal)		Abcamab171301:500
AntibodyAnti-Krt5
(Rabbit polyclonal)		Abcamab531211:500
AntibodyAnti-p63
(Rabbit polyclonal)		GenetexGTX1024251:200
AntibodyAnti-p63
(Mouse monoclonal)		Abcamab7351:200
AntibodyAnti-Acetylated tubulin
(Mouse monoclonal)		SigmaT74511:500
AntibodyAnti-TTF1
(Rabbit monoclonal)		Abcamab760131:500; Nkx2.1
AntibodyAnti-FOXA2
(Mouse monoclonal)		Abcamab607211:200
AntibodyAnti-POU2F3
(Rabbit polyclonal)		SigmaHPA0196521:200
AntibodyAnti-ABCA3
(Rabbit polyclonal)		Seven HillsWRAB-705651:500
AntibodyAnti-Hop (E-1)
(Mouse monoclonal)		Santa Cruzsc-3987031:100; Hopx
AntibodyAnti- Prosurfactant Protein C
(Rabbit polyclonal)		Abcamab907161:500; SftpC
AntibodyAnti-Pro-SP-C
(Rabbit polyclonal)		Seven HillsWRAB-93371:1000
AntibodyAnti-tdTomato
(Goat polyclonal)		Biorbytorb1823971:1000
AntibodyAnti-Pdpn
(Syrian hamster monoclonal)		DSHB8.1.1 c1:500; T1α
AntibodyAnti- Green Fluorescent Protein
(Chicken polyclonal)		Fisher (Aves Lab)GFP10201:200; GFP
AntibodyAnti-Ki-67
(Rat monoclonal)		Invitrogen14-5698-821:50
AntibodyAlexa Fluor 488 Donkey Anti-Chicken
(Donkey polyclonal)		Jackson Immuno Research703-546-1551:500
AntibodyAlexa Fluor 488 Donkey Anti-Mouse
(Donkey polyclonal)		Jackson Immuno Research715-545-1511:500
AntibodyCy5 AffiniPure Donkey Anti-Rat IgG
(Donkey polyclonal)		Jackson Immuno Research712-175-1501:500
AntibodyCy3 AffiniPure Donkey Anti-Mouse IgM
(Donkey polyclonal)		Jackson Immuno Research715-165-1401:500
AntibodyAlexa Fluor 488 Donkey anti-Rat IgG
(Donkey polyclonal)		InvitrogenA212081:500
AntibodyAlexa Fluor 555 Donkey anti-Rabbit IgG
(Donkey polyclonal)		InvitrogenA315721:500
AntibodyAlexa Fluor 555 Donkey anti-Goat IgG
(Donkey polyclonal)		InvitrogenA214321:500
AntibodyAlexa Fluor 555 Donkey anti-Mouse IgG
(Donkey polyclonal)		InvitrogenA315701:500
AntibodyAlexa Fluor 647 Goat anti-Chicken IgG
(Goat polyclonal)		InvitrogenA214491:500
Chemical compound, drugDBZTocris4489
Chemical compound, drugWnt-C59Tocris5148
Chemical compound, drugCHIR99021Tocris4423
Chemical compound, drugIWR-1Tocris3532
Chemical compound, drugDorsomorphinSigmaP5499
Chemical compound, drugVerteporfinSigmaSML0534
Chemical compound, drugGDC-0449SelleckchemS1082
Chemical compound, drugDexamethasoneSigmaD2915
Chemical compound, drug3-Isobutyl-1-methylxanthine (IBMX)SigmaI5879
Peptide, recombinant proteinRecombinant Il-6Peprotech200–06
Peptide, recombinant proteinRecombinant Il-4Peprotech200–13
Peptide, recombinant proteinRecombinant Murine Il-13Peprotech210–13
Chemical compound, drug8-Bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP)SigmaB5386
Chemical compound, drugBleomycinFresenius Kabi63323013610
Chemical compound, drugNaphthaleneSigma-Aldrich84679
Commercial assay or kitSmall Airway Epithelial Cell MediumLonzaCC-3118
Commercial assay or kitSmall Airway Epithelial Cell MediumPromocell mediumC-21170
Commercial assay or kitComplete Pneumacult-ALI mediumStemCell Technology05001
OtherAntigen unmasking solution, Citric acid basedVector LaboratoriesH-3300Antigen retrieval buffer for immunostaining
OtherNormal donkey serumJackson Immuno Research017-000-121Blocking reagent for immunostaining

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  1. Huachao Huang
  2. Yinshan Fang
  3. Ming Jiang
  4. Yihan Zhang
  5. Jana Biermann
  6. Johannes C Melms
  7. Jennifer A Danielsson
  8. Ying Yang
  9. Li Qiang
  10. Jia Liu
  11. Yiwu Zhou
  12. Manli Wang
  13. Zhihong Hu
  14. Timothy C Wang
  15. Anjali Saqi
  16. Jie Sun
  17. Ichiro Matsumoto
  18. Wellington V Cardoso
  19. Charles W Emala
  20. Jian Zhu
  21. Benjamin Izar
  22. Hongmei Mou
  23. Jianwen Que
(2022)
Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells
eLife 11:e78217.
https://doi.org/10.7554/eLife.78217