1. Cell Biology
  2. Genetics and Genomics

Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster

  1. Peter S Johnstone
  2. Maite Ogueta
  3. Olga Akay
  4. Inan Top
  5. Sheyum Syed
  6. Ralf Stanewsky
  7. Deniz Top  Is a corresponding author
  1. Dalhousie University, Canada
  2. Westfälische Wilhelms University, Germany
  3. You.i Labs Inc, Canada
  4. University of Miami, United States
https://doi.org/10.7554/eLife.77029
Share this article
Cite this article
  1. Peter S Johnstone
  2. Maite Ogueta
  3. Olga Akay
  4. Inan Top
  5. Sheyum Syed
  6. Ralf Stanewsky
  7. Deniz Top
(2022)
Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster
eLife 11:e77029.
https://doi.org/10.7554/eLife.77029
  2. Download BibTeX
  3. Download .RIS

Abstract

Circadian clocks are highly conserved transcriptional regulators that control ~24-hour oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence, or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. We found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. We also demonstrate that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. Our results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo.

Data availability

The codes used in data analysis can be found at https://github.com/deniztop/LABLAll data points used in generating the figures can be found at Dryad

The following data sets were generated

Article and author information

Author details

  1. Peter S Johnstone

    Department of Pediatrics, Dalhousie University, Halifax, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9421-811X

  2. Maite Ogueta

    Institute of Neuro- and Behavioral Biology, Westfälische Wilhelms University, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.

  3. Olga Akay

    Department of Pediatrics, Dalhousie University, Halifax, Canada
    Competing interests
    The authors declare that no competing interests exist.

  4. Inan Top

    You.i Labs Inc, Ottawa, Canada
    Competing interests
    The authors declare that no competing interests exist.

  5. Sheyum Syed

    Department of Physics, University of Miami, Miami, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Ralf Stanewsky

    Institute of Neuro- and Behavioral Biology, Westfälische Wilhelms University, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8238-6864

  7. Deniz Top

    Department of Pediatrics, Dalhousie University, Halifax, Canada
    For correspondence
    dtop@dal.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1042-8460

Funding

Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-06101)

  • Deniz Top

National Science Foundation (IOS 1656603)

  • Sheyum Syed

Deutsche Forschungsgemeinschaft (INST 211/835-1 FUGG)

  • Ralf Stanewsky

Deutsche Forschungsgemeinschaft (STA421/7-1)

  • Ralf Stanewsky

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

Reviewing Editor

  1. Kristin Tessmar-Raible, University of Vienna, Austria

Version history

  1. Preprint posted: January 12, 2022 (view preprint)
  2. Received: January 13, 2022
  3. Accepted: September 30, 2022
  4. Accepted Manuscript published: October 3, 2022 (version 1)
  5. Version of Record published: November 14, 2022 (version 2)

Copyright

© 2022, Johnstone 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

  • 1,554
    views
  • 414
    downloads
  • 2
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Peter S Johnstone
  2. Maite Ogueta
  3. Olga Akay
  4. Inan Top
  5. Sheyum Syed
  6. Ralf Stanewsky
  7. Deniz Top
(2022)
Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster
eLife 11:e77029.
https://doi.org/10.7554/eLife.77029

Share this article

https://doi.org/10.7554/eLife.77029

Categories and tags

Research organism

Further reading

    1. Cell Biology
    2. Chromosomes and Gene Expression

    Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis

    Lucie Crhak Khaitova, Pavlina Mikulkova ... Karel Riha
    Research Article

    Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

    1. Cell Biology

    High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

    Wan-ping Yang, Mei-qi Li ... Qian-qian Luo
    Research Article

    High-altitude polycythemia (HAPC) affects individuals living at high altitudes, characterized by increased red blood cells (RBCs) production in response to hypoxic conditions. The exact mechanisms behind HAPC are not fully understood. We utilized a mouse model exposed to hypobaric hypoxia (HH), replicating the environmental conditions experienced at 6000 m above sea level, coupled with in vitro analysis of primary splenic macrophages under 1% O2 to investigate these mechanisms. Our findings indicate that HH significantly boosts erythropoiesis, leading to erythrocytosis and splenic changes, including initial contraction to splenomegaly over 14 days. A notable decrease in red pulp macrophages (RPMs) in the spleen, essential for RBCs processing, was observed, correlating with increased iron release and signs of ferroptosis. Prolonged exposure to hypoxia further exacerbated these effects, mirrored in human peripheral blood mononuclear cells. Single-cell sequencing showed a marked reduction in macrophage populations, affecting the spleen’s ability to clear RBCs and contributing to splenomegaly. Our findings suggest splenic ferroptosis contributes to decreased RPMs, affecting erythrophagocytosis and potentially fostering continuous RBCs production in HAPC. These insights could guide the development of targeted therapies for HAPC, emphasizing the importance of splenic macrophages in disease pathology.

    1. Cell Biology

    Golgi Apparatus: Making progress

    Jurgen Denecke
    Insight

    Mapping proteins in and associated with the Golgi apparatus reveals how this cellular compartment emerges in budding yeast and progresses over time.