C. elegans GLP-1/Notch activates transcription in a probability gradient across the germline stem cell pool

  1. ChangHwan Lee
  2. Erika B Sorensen
  3. Tina R Lynch
  4. Judith Kimble  Is a corresponding author
  1. Howard Hughes Medical Institute, University of Wisconsin-Madison, United States
  2. Wabash College, United States
  3. University of Wisconsin-Madison, United States

Abstract

C. elegans Notch signaling maintains a pool of germline stem cells within their single-celled mesenchymal niche. Here we investigate the Notch transcriptional response in germline stem cells using single-molecule fluorescence in situ hybridization coupled with automated, high-throughput quantitation. This approach allows us to distinguish Notch-dependent nascent transcripts in the nucleus from mature mRNAs in the cytoplasm. We find that Notch-dependent active transcription sites occur in a probabilistic fashion and, unexpectedly, do so in a steep gradient across the stem cell pool. Yet these graded nuclear sites create a nearly uniform field of mRNAs that extends beyond the region of transcriptional activation. Therefore, active transcription sites provide a precise view of where the Notch-dependent transcriptional complex is productively engaged. Our findings offer a new window into the Notch transcriptional response and demonstrate the importance of assaying nascent transcripts at active transcription sites as a readout for canonical signaling.

Article and author information

Author details

  1. ChangHwan Lee

    Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Erika B Sorensen

    Department of Biology, Wabash College, Crawfordsville, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Tina R Lynch

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Judith Kimble

    Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, United States
    For correspondence
    jekimble@wisc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5622-2073

Funding

American Cancer Society (PF-14-147-01-DDC)

  • Erika B Sorensen

Howard Hughes Medical Institute

  • Judith Kimble

American Cancer Society (PF-14-147-01-DDC)

  • Erika B Sorensen

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

Reviewing Editor

  1. Janet Rossant, University of Toronto, Canada

Version history

  1. Received: June 1, 2016
  2. Accepted: October 4, 2016
  3. Accepted Manuscript published: October 5, 2016 (version 1)
  4. Version of Record published: November 3, 2016 (version 2)
  5. Version of Record updated: August 10, 2017 (version 3)

Copyright

© 2016, Lee 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

  • 4,252
    Page views
  • 670
    Downloads
  • 53
    Citations

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

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. ChangHwan Lee
  2. Erika B Sorensen
  3. Tina R Lynch
  4. Judith Kimble
(2016)
C. elegans GLP-1/Notch activates transcription in a probability gradient across the germline stem cell pool
eLife 5:e18370.
https://doi.org/10.7554/eLife.18370

Further reading

    1. Cell Biology
    2. Developmental Biology
    Simon Schneider, Andjela Kovacevic ... Hubert Schorle
    Research Article

    Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Bronwyn A Lucas, Benjamin A Himes, Nikolaus Grigorieff
    Research Advance

    Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.