Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

  1. Amrutha Kizhedathu
  2. Piyush Chhajed
  3. Lahari Yeramala
  4. Deblina Sain Basu
  5. Tina Mukherjee
  6. Kutti R Vinothkumar
  7. Arjun Guha  Is a corresponding author
  1. Institute for Stem Cell Biology and Regenerative Medicine (inStem), India
  2. National Centre for Biological Sciences, India

Abstract

Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H2O2 generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/ Claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of H2O2 in minutes. The findings presented reveal that H2O2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1,2,3,4

Article and author information

Author details

  1. Amrutha Kizhedathu

    Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  2. Piyush Chhajed

    Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  3. Lahari Yeramala

    National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  4. Deblina Sain Basu

    Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  5. Tina Mukherjee

    Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3776-5536
  6. Kutti R Vinothkumar

    National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  7. Arjun Guha

    Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India
    For correspondence
    arjung@instem.res.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3753-1484

Funding

Department of Biotechnology, Ministry of Science and Technology, India (inStem Core Funds)

  • Arjun Guha

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

Copyright

© 2021, Kizhedathu 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.

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  1. Amrutha Kizhedathu
  2. Piyush Chhajed
  3. Lahari Yeramala
  4. Deblina Sain Basu
  5. Tina Mukherjee
  6. Kutti R Vinothkumar
  7. Arjun Guha
(2021)
Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts
eLife 10:e68636.
https://doi.org/10.7554/eLife.68636

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https://doi.org/10.7554/eLife.68636

Further reading

    1. Developmental Biology
    Amrutha Kizhedathu, Archit V Bagul, Arjun Guha
    Research Article Updated

    Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. Here we investigate the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). We report that tracheoblasts pause in G2 for ~48–56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). We find that mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24–32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. We propose that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.

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    Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate transporter SLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here, Fgf23 knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name: Bglap, protein name). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lower Slc34a2 and higher Bglap/Slc34a1 (protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lower SLC34A2 and a subpopulation of germ cells express phosphate transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbance in vitro using calcium or phosphate treatment induced deposition of calcium phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.