In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

  1. Li He  Is a corresponding author
  2. Richard Binari
  3. Jiuhong Huang
  4. Julia Falo-Sanjuan
  5. Norbert Perrimon  Is a corresponding author
  1. Harvard Medical School, United States
  2. Chongqing University of Arts and Sciences, China
  3. Tufts University, United States

Abstract

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

Data availability

All essential data are provided in the supplementary materials. All the reagents created by this study (plasmids and transgenic flies) will be donated to public domains including Addgene and Bloomington Stock Center.

Article and author information

Author details

  1. Li He

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    Li_He@hms.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2155-606X
  2. Richard Binari

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Jiuhong Huang

    International Academy of Targeted Therapeutics and Innovation, Chongqing University of Arts and Sciences, Chongqing, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Julia Falo-Sanjuan

    School of Graduate Biomedical Sciences, Tufts University, Medford, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Norbert Perrimon

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    perrimon@receptor.med.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7542-472X

Funding

National Institute of General Medical Sciences

  • Norbert Perrimon

Damon Runyon Cancer Research Foundation

  • Li He

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

Reviewing Editor

  1. Hugo J Bellen, Baylor College of Medicine, United States

Version history

  1. Received: February 22, 2019
  2. Accepted: May 28, 2019
  3. Accepted Manuscript published: May 29, 2019 (version 1)
  4. Version of Record published: July 26, 2019 (version 2)

Copyright

© 2019, 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

  • 20,583
    views
  • 2,241
    downloads
  • 58
    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. Li He
  2. Richard Binari
  3. Jiuhong Huang
  4. Julia Falo-Sanjuan
  5. Norbert Perrimon
(2019)
In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer
eLife 8:e46181.
https://doi.org/10.7554/eLife.46181

Share this article

https://doi.org/10.7554/eLife.46181

Further reading

    1. Developmental Biology
    Phuong-Khanh Nguyen, Louise Y Cheng
    Research Article Updated

    The brain is consisted of diverse neurons arising from a limited number of neural stem cells. Drosophila neural stem cells called neuroblasts (NBs) produces specific neural lineages of various lineage sizes depending on their location in the brain. In the Drosophila visual processing centre - the optic lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise to neurons and glia cells of the medulla cortex. The timing and the mechanisms responsible for the cessation of medulla NBs are so far not known. In this study, we show that the termination of medulla NBs during early pupal development is determined by the exhaustion of the NE stem cell pool. Hence, altering NE-NB transition during larval neurogenesis disrupts the timely termination of medulla NBs. Medulla NBs terminate neurogenesis via a combination of apoptosis, terminal symmetric division via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm); however, these processes occur independently of each other. We also show that temporal progression of the medulla NBs is mostly not required for their termination. As the Drosophila OL shares a similar mode of division with mammalian neurogenesis, understanding when and how these progenitors cease proliferation during development can have important implications for mammalian brain size determination and regulation of its overall function.

    1. Developmental Biology
    2. Neuroscience
    Amy R Poe, Lucy Zhu ... Matthew S Kayser
    Research Article

    Sleep and feeding patterns lack strong daily rhythms during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. In Drosophila, circadian sleep patterns are initiated with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. Here, we identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. We find that in the 2nd instar larval period (L2), sleep and feeding are spread across the day; these behaviors become organized into daily patterns by the 3rd instar larval stage (L3). Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, we demonstrate that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, our data suggest that changes to energetic demands in developing organisms trigger the formation of sleep-circadian circuits and behaviors.