Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts

  1. Amrutha Kizhedathu
  2. Rose Sebastian Kunnappallil
  3. Archit V Bagul
  4. Puja Verma
  5. Arjun Guha  Is a corresponding author
  1. Institute for Stem Cell Biology and Regenerative Medicine (inStem), India
  2. National Center for Biological Sciences, India
  3. Institute of Genetics and Molecular and Cellular Biology, France
  4. Institute for Stem Cell Science and Regenerative Medicine, India

Abstract

Larval tracheae of Drosophila harbour progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. Here we investigate developmental regulation of Chk1 activation. We report that Wnt signaling is high in tracheoblasts and this is necessary for high levels of activated (phosphorylated) Chk1. We find that canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signalling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and are downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, we show that downregulation of Wnt signalling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signalling, driving tracheoblast proliferation.

Data availability

All data generated or analysed during this study are included in the manuscript.

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. Rose Sebastian Kunnappallil

    Neurobiology, National Center for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  3. Archit V Bagul

    Genetics and Molecular and Cellular Biology, Institute of Genetics and Molecular and Cellular Biology, Illkirch-Graffenstaden, France
    Competing interests
    The authors declare that no competing interests exist.
  4. Puja Verma

    Regulation of Cell Fate, Institute for Stem Cell Science and Regenerative Medicine, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  5. 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 (inStem Core Grant)

  • Arjun Guha

Department of Biotechnology , Ministry of Science and Technology (InStem Core Grant)

  • Arjun Guha

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

Reviewing Editor

  1. Amin S. Ghabrial, Columbia University, United States

Version history

  1. Received: March 20, 2020
  2. Accepted: August 29, 2020
  3. Accepted Manuscript published: September 2, 2020 (version 1)
  4. Accepted Manuscript updated: September 9, 2020 (version 2)
  5. Version of Record published: September 21, 2020 (version 3)
  6. Version of Record updated: September 24, 2020 (version 4)

Copyright

© 2020, 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.

Metrics

  • 1,213
    Page views
  • 112
    Downloads
  • 4
    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. Amrutha Kizhedathu
  2. Rose Sebastian Kunnappallil
  3. Archit V Bagul
  4. Puja Verma
  5. Arjun Guha
(2020)
Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts
eLife 9:e57056.
https://doi.org/10.7554/eLife.57056

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.

    1. Cell Biology
    2. Microbiology and Infectious Disease
    Valentin Wernet, Marius Kriegler ... Reinhard Fischer
    Research Article Updated

    Communication is crucial for organismic interactions, from bacteria, to fungi, to humans. Humans may use the visual sense to monitor the environment before starting acoustic interactions. In comparison, fungi, lacking a visual system, rely on a cell-to-cell dialogue based on secreted signaling molecules to coordinate cell fusion and establish hyphal networks. Within this dialogue, hyphae alternate between sending and receiving signals. This pattern can be visualized via the putative signaling protein Soft (SofT), and the mitogen-activated protein kinase MAK-2 (MakB) which are recruited in an alternating oscillatory manner to the respective cytoplasmic membrane or nuclei of interacting hyphae. Here, we show that signal oscillations already occur in single hyphae of Arthrobotrys flagrans in the absence of potential fusion partners (cell monologue). They were in the same phase as growth oscillations. In contrast to the anti-phasic oscillations observed during the cell dialogue, SofT and MakB displayed synchronized oscillations in phase during the monologue. Once two fusion partners came into each other’s vicinity, their oscillation frequencies slowed down (entrainment phase) and transit into anti-phasic synchronization of the two cells’ oscillations with frequencies of 104±28 s and 117±19 s, respectively. Single-cell oscillations, transient entrainment, and anti-phasic oscillations were reproduced by a mathematical model where nearby hyphae can absorb and secrete a limited molecular signaling component into a shared extracellular space. We show that intracellular Ca2+ concentrations oscillate in two approaching hyphae, and depletion of Ca2+ from the medium affected vesicle-driven extension of the hyphal tip, abolished the cell monologue and the anti-phasic synchronization of two hyphae. Our results suggest that single hyphae engage in a ‘monologue’ that may be used for exploration of the environment and can dynamically shift their extracellular signaling systems into a ‘dialogue’ to initiate hyphal fusion.