Chloride channels regulate differentiation and barrier functions of the mammalian airway

  1. Mu He  Is a corresponding author
  2. Bing Wu
  3. Wenlei Ye
  4. Daniel D Le
  5. Adriane W Sinclair
  6. Valeria Padovano
  7. Yuzhang Chen
  8. Ke-Xin Li
  9. Rene Sit
  10. Michelle Tan
  11. Michael J Caplan
  12. Norma Neff
  13. Yuh Nung Jan
  14. Spyros Darmanis  Is a corresponding author
  15. Lily Yeh Jan  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Chan Zuckerberg Biohub, United States
  3. Yale University School of Medicine, United States

Abstract

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/- mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.

Data availability

Sequencing reads and processed data in the format of gene-cell count tables are available from the Sequence Read Archive (SRA) (​SRA accession​: PRJNA548516).

The following data sets were generated

Article and author information

Author details

  1. Mu He

    Department of Physiology, University of California, San Francisco, San Francisco, United States
    For correspondence
    mu.he@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Bing Wu

    Chan Zuckerberg Biohub, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Wenlei Ye

    Department of Physiology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4694-1493
  4. Daniel D Le

    Chan Zuckerberg Biohub, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Adriane W Sinclair

    Division of Pediatric Urology, Department of Urology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Valeria Padovano

    Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0142-3385
  7. Yuzhang Chen

    Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Ke-Xin Li

    Department of Physiology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3879-294X
  9. Rene Sit

    Chan Zuckerberg Biohub, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Michelle Tan

    Chan Zuckerberg Biohub, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Michael J Caplan

    Department of Cellular and Molecular Physiology; Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Norma Neff

    Chan Zuckerberg Biohub, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Yuh Nung Jan

    Department of Physiology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1367-6299
  14. Spyros Darmanis

    Chan Zuckerberg Biohub, San Francisco, United States
    For correspondence
    spyros.darmanis@czbiohub.org
    Competing interests
    The authors declare that no competing interests exist.
  15. Lily Yeh Jan

    Department of Physiology, University of California, San Francisco, San Francisco, United States
    For correspondence
    Lily.Jan@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3938-8498

Funding

National Institute of Neurological Disorders and Stroke (RO1 NS069229)

  • Lily Yeh Jan

Eunice Kennedy Shriver National Institute of Child Health and Human Development (F32HD089639)

  • Mu He

Howard Hughes Medical Institute

  • Yuh Nung Jan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Edward E Morrisey, University of Pennsylvania, United States

Publication history

  1. Received: October 27, 2019
  2. Accepted: April 13, 2020
  3. Accepted Manuscript published: April 14, 2020 (version 1)
  4. Version of Record published: April 24, 2020 (version 2)

Copyright

© 2020, He et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,148
    Page views
  • 357
    Downloads
  • 9
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Mu He
  2. Bing Wu
  3. Wenlei Ye
  4. Daniel D Le
  5. Adriane W Sinclair
  6. Valeria Padovano
  7. Yuzhang Chen
  8. Ke-Xin Li
  9. Rene Sit
  10. Michelle Tan
  11. Michael J Caplan
  12. Norma Neff
  13. Yuh Nung Jan
  14. Spyros Darmanis
  15. Lily Yeh Jan
(2020)
Chloride channels regulate differentiation and barrier functions of the mammalian airway
eLife 9:e53085.
https://doi.org/10.7554/eLife.53085

Further reading

    1. Developmental Biology
    Hidenobu Miyazawa, Marteinn T Snaebjornsson ... Alexander Aulehla
    Research Article

    How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

    1. Developmental Biology
    2. Genetics and Genomics
    Janani Ramachandran, Weiqiang Zhou ... Steven A Vokes
    Research Article Updated

    The larynx enables speech while regulating swallowing and respiration. Larynx function hinges on the laryngeal epithelium which originates as part of the anterior foregut and undergoes extensive remodeling to separate from the esophagus and form vocal folds that interface with the adjacent trachea. Here we find that sonic hedgehog (SHH) is essential for epithelial integrity in the mouse larynx as well as the anterior foregut. During larynx-esophageal separation, low Shh expression marks specific domains of actively remodeling epithelium that undergo an epithelial-to-mesenchymal transition (EMT) characterized by the induction of N-Cadherin and movement of cells out of the epithelial layer. Consistent with a role for SHH signaling in regulating this process, Shh mutants undergo an abnormal EMT throughout the anterior foregut and larynx, marked by a cadherin switch, movement out of the epithelial layer and cell death. Unexpectedly, Shh mutant epithelial cells are replaced by a new population of FOXA2-negative cells that likely derive from adjacent pouch tissues and form a rudimentary epithelium. These findings have important implications for interpreting the etiology of HH-dependent birth defects within the foregut. We propose that SHH signaling has a default role in maintaining epithelial identity throughout the anterior foregut and that regionalized reductions in SHH trigger epithelial remodeling.