Letter

Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung

  • Nature volume 528, pages 127131 (03 December 2015)
  • doi:10.1038/nature15715
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Abstract

Prevailing dogma holds that cell–cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed1. Mucociliary clearance2,3 in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor4,5,6,7,8. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations9,10, cells of the homeostatic adult airway epithelium are long-lived11,12,13, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation14. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation15. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the human JAG1–Fab complex have been deposited in the Protein Data Bank under accession code 5BO1

References

  1. 1.

    , & Stem cells living with a Notch. Development 140, 689–704 (2013)

  2. 2.

    & Local mucociliary defence mechanisms. Paediatr. Respir. Rev. 1, 27–34 (2000)

  3. 3.

    et al. Muc5b is required for airway defence. Nature 505, 412–416 (2014)

  4. 4.

    , , , & Jagged1 is the major regulator of Notch-dependent cell fate in proximal airways. Dev. Dyn. 242, 678–686 (2013)

  5. 5.

    , , & Different assemblies of Notch receptors coordinate the distribution of the major bronchial Clara, ciliated and neuroendocrine cells. Development 139, 4365–4373 (2012)

  6. 6.

    et al. Notch-dependent differentiation of adult airway basal stem cells. Cell Stem Cell 8, 639–648 (2011)

  7. 7.

    et al. Injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations. Cell Stem Cell 16, 184–197 (2015)

  8. 8.

    et al. Notch3-Jagged signaling controls the pool of undifferentiated airway progenitors. Development 142, 258–267 (2015)

  9. 9.

    & Lung regeneration: mechanisms, applications and emerging stem cell populations. Nature Med. 20, 822–832 (2014)

  10. 10.

    et al. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123–138 (2014)

  11. 11.

    & Ciliated epithelial cell lifespan in the mouse trachea and lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L231–L234 (2008)

  12. 12.

    & Epithelial progenitor cells in lung development, maintenance, repair, and disease. Annu. Rev. Cell Dev. Biol. 27, 493–512 (2011)

  13. 13.

    et al. Clonal dynamics reveal two distinct populations of basal cells in slow-turnover airway epithelium. Cell Rep . 12, 90–101 (2015)

  14. 14.

    & Epithelial stem cells of the lung: privileged few or opportunities for many? Development 133, 2455–2465 (2006)

  15. 15.

    , & Targeting mucus hypersecretion: New therapeutic opportunities for COPD? Drugs 74, 1073–1089 (2014)

  16. 16.

    et al. Structural basis for Notch1 engagement of Delta-like 4. Science 347, 847–853 (2015)

  17. 17.

    et al. A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition. Nature Struct. Mol. Biol . 15, 849–857 (2008)

  18. 18.

    et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121, 823–835 (2005)

  19. 19.

    et al. Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways. Proc. Natl Acad. Sci. USA 109, 12592–12597 (2012)

  20. 20.

    et al. Therapeutic antibody targeting of individual Notch receptors. Nature 464, 1052–1057 (2010)

  21. 21.

    et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neurosci. 13, 133–140 (2010)

  22. 22.

    et al. Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination. Proc. Natl Acad. Sci. USA 110, 15770–15775 (2013)

  23. 23.

    et al. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production. J. Clin. Invest. 119, 2914–2924 (2009)

  24. 24.

    , , , & Ciliated cells of pseudostratified airway epithelium do not become mucous cells after ovalbumin challenge. Am. J. Respir. Cell Mol. Biol. 48, 364–373 (2013)

  25. 25.

    , & Understanding asthma using animal models. Allergy Asthma Immunol. Res. 1, 10–18 (2009)

  26. 26.

    & Mouse models of allergic asthma: acute and chronic allergen challenge. Dis. Model. Mech . 1, 213–220 (2008)

  27. 27.

    , , , & NOTCH1 is required for regeneration of Clara cells during repair of airway injury. Stem Cells 30, 946–955 (2012)

  28. 28.

    , & Evidence for Scgb1a1+ cells in the generation of p63+ cells in the damaged lung parenchyma. Am. J. Respir. Cell Mol. Biol. 50, 595–604 (2014)

  29. 29.

    et al. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell 4, 525–534 (2009)

  30. 30.

    et al. Parent stem cells can serve as niches for their daughter cells. Nature 523, 597–601 (2015)

  31. 31.

    , & Bivalent antibody phage display mimics natural immunoglobulin. J. Immunol. Methods 284, 119–132 (2004)

  32. 32.

    et al. Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library. J. Mol. Biol. 366, 815–829 (2007)

  33. 33.

    et al. Differential effects of targeting Notch receptors in a mouse model of liver cancer. Hepatology 61, 942–952 (2015)

  34. 34.

    et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D 67, 293–302 (2011)

  35. 35.

    Collaborative Computational Project, Number 4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  36. 36.

    et al. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

  37. 37.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  38. 38.

    et al. BUSTER version 2.11.2 (2011)

  39. 39.

    , & Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993)

  40. 40.

    et al. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  41. 41.

    The PyMOL Molecular Graphics System version 1.2r3pre edn

  42. 42.

    , & Imaging cleared intact biological systems at a cellular level by 3DISCO. J. Vis. Exp . 89, 51382 (2014)

  43. 43.

    & Culture and differentiation of mouse tracheal epithelial cells. Methods Mol. Biol. 945, 123–143 (2012)

  44. 44.

    et al. Automatic nuclei segmentation in H&E stained breast cancer histopathology images. PLoS ONE 8, e70221 (2013)

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Acknowledgements

The authors thank E. Jackson and R. Pattni for work generating the Scgb1a1-ERT2GNE mice; L.Nguyen, L. Orellana, P. Grigg and the Genentech Transgenic Technology Laboratories and Research Support Facility for technical assistance with mouse strains and colonies; F. Chu, L. Rangell, S. Chalasani, C. Jones III and C. Espiritu for cell staining; A. Ertürk, C. Chalouni, S. Gierke, M. Gonzalez-Edick and the Genentech Center for Advanced Light Microscopy (CALM) for imaging; C. K. Poon for cytokine measurements; S. P. Tsai and M. Dostalek for pharmacokinetic analyses; T. Hagenbeek for help with the immune cell studies. Use of the Stanford Synchrotron Radiation Lightsource SSRL 12-12 at Stanford Linear Accelerator Center National Accelerator Laboratory is supported by the US Department of Energy (DOE), DOE Office of Biological and Environmental Research, National Institutes of Health, and National Institute of General Medical Sciences. The contents of this publication are the responsibility of the authors and do not necessarily represent the views of NIH or NIGMS.

Author information

Author notes

    • Christian Siltanen
    •  & Jackson Egen

    Present addresses: Department of Biomedical Engineering, University of California, Davis, 451 E. Health Sciences Drive, Davis, California 95616, USA (C.S.); Amgen, Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, USA (J.E.).

Affiliations

  1. Department of Discovery Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Daniel Lafkas
    • , Amy Shelton
    • , Christian Siltanen
    •  & Christian W. Siebel
  2. Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Cecilia Chiu
    • , Yongmei Chen
    • , Scott S. Stawicki
    •  & Yan Wu
  3. Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Gladys de Leon Boenig
    •  & Jian Payandeh
  4. Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Mike Reichelt
    • , Jeffrey Eastham-Anderson
    • , Cary Austin
    •  & John B. Lowe
  5. Department of Translational Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Meijuan Zhou
    • , Xiumin Wu
    •  & Wyne P. Lee
  6. Department of Discovery Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Heather Moore
    •  & Jackson Egen
  7. Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Meron Roose-Girma
    •  & Søren Warming
  8. Departments of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA

    • Yvonne Chinn
    •  & Julie Q. Hang

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Contributions

D.L. performed experiments and analysed data of Figs 2, 3, 4 and Extended Data Fig. 4, 5, 6, 7, 8, 9, 10, A.S. performed reporter assays for Fig. 1 and experiments for Extended Data Fig. 2c and mouse studies for Extended Data Fig. 4b, G.d.L.B. purified antibody fragments and crystallized the JAG1–Fab complex, Y. Chen and S.S.S performed affinity maturation and characterization of antibodies, C.C., S.W. and Y.W. generated the phage display antibodies and performed the in vitro binding experiments and affinity maturation, M.R. performed all electron microscopy studies, C.S. performed the cilia functionality studies, M.R.-G. and S.W. designed the targeting vector and supervised the generation of the Scgb1a1-CreERT2GNE mouse line, M.Z. and X.Wu performed the ovalbumin studies, J.E.-A. performed all quantifications of immunofluorescence staining, H.M. performed qPCR and analysis of whole lungs, Y.Chinn and J.Q.H. assisted with anti-JAG1 development and expressed and purified JAG1 protein; W.P.L. helped design and supervise the ovalbumin study, C.A. analysed tissue sections from the ovalbumin study, J.E. contributed to the design of qPCR and ovalbumin studies as well as contributing intellectually, J.P. solved and analysed the structure, and made the structural figures in Fig. 1 and Extended Data Fig. 3, J.B.L. analysed the histology of all lung and skin samples except samples from the ovalbumin study. C.W.S. supervised the experiments and wrote the paper with D.L.

Competing interests

D.L., A.S., C.C., G.d.L.B., Y.Chen S.S.S., M.R., M.Z., J.E.-A., H.M., W.P.L., Y.Chinn, J.Q.H., C.A., J.E., Y.W., J.P., J.B.L. and C.W.S. are or were employed by Genentech Inc., which has commercial interests in some of the molecules described.

Corresponding author

Correspondence to Christian W. Siebel.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the image of the entire Western blot film used to compile Extended Data Figure 2c.

Excel files

  1. 1.

    Supplementary Data

    This file contains a spreadsheet showing the raw data used for quantifications in Fig. 2, 3 and Extended Data Fig. 6.

Videos

  1. 1.

    Control for cilia motility

    The video shows single tracheal cells from Scgb1a1-CreERT2GNE /Rosa26-lsl-tdTomato mice induced with four doses of 200mg kg-1 tamoxifen and treated with a single dose of control antibody for six days, one week after the last Tamoxifen injection. CC10-traced cells appear white. At least two ciliated cells (control, not labeled white and, thus, not converted from club cells following JAG blockade) with motile cilia are visible (right side). A tdTomato-positive (white) club cell (no cilia) is visible on the left.

  2. 2.

    Cilia on transdifferentiated cells are motile

    The video shows single tracheal cells from Scgb1a1-CreERT2GNE /Rosa26-lsl-tdTomato mice induced with four doses of 200mg kg-1 Tamoxifen and treated with a single dose of with anti-JAG1.b70 plus anti-JAG2.b33 for six days, one week after the last Tamoxifen injection. At least two ciliated cells (control, not labeled white and, thus, not converted from club cells following JAG blockade) with motile cilia are visible (middle). A tdTomato-positive cell (white), marking a daughter cell derived from the club cell lineage, has beating cilia (middle), supporting the notion that an induced ciliated cell functions normally, similar to the control (unlabelled) ciliated cells.

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