1. Developmental Biology
  2. Stem Cells and Regenerative Medicine
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3D Visualization of Macromolecule Synthesis

  1. Timothy J Duerr
  2. Ester Comellas
  3. Eun Kyung Jeon
  4. Johanna E Farkas
  5. Marylou Joetzjer
  6. Julien Garnier
  7. Sandra J Shefelbine
  8. James R Monaghan  Is a corresponding author
  1. Northeastern University, United States
  2. Universitat Politècnica de Catalunya (UPC), Spain
  3. University of Technology of Compiègne, France
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Cite this article as: eLife 2020;9:e60354 doi: 10.7554/eLife.60354

Abstract

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here, we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

Data availability

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

Article and author information

Author details

  1. Timothy J Duerr

    Department of Biology, Northeastern University, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4945-0294
  2. Ester Comellas

    Department of Mathematics, Universitat Politècnica de Catalunya (UPC), Barcelona, Spain
    Competing interests
    The authors declare that no competing interests exist.
  3. Eun Kyung Jeon

    Department of Biology, Northeastern University, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Johanna E Farkas

    Department of Biology, Northeastern University, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6540-7870
  5. Marylou Joetzjer

    Department of Biological Engineering, University of Technology of Compiègne, Compiègne, France
    Competing interests
    The authors declare that no competing interests exist.
  6. Julien Garnier

    Department of Biological Engineering, University of Technology of Compiègne, Compiègne, France
    Competing interests
    The authors declare that no competing interests exist.
  7. Sandra J Shefelbine

    Department of Mechanical and Industrial Engineering, Department of Bioengineering, Northeastern University, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. James R Monaghan

    Biology, Northeastern University, Boston, United States
    For correspondence
    j.monaghan@northeastern.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6689-6108

Funding

National Science Foundation (1727518)

  • Sandra J Shefelbine

Northeastern University Matz Scholarship

  • Eun Kyung Jeon

Northeastern University Undergraduate Research Fellowship

  • Eun Kyung Jeon

National Science Foundation (1656429)

  • James R Monaghan

National Science Foundation (1558017)

  • James R Monaghan

European Commission MSCA-GF (841047 CompLimb)

  • Ester Comellas

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

Ethics

Animal experimentation: Axolotls (Ambystoma mexicanum: d/d RRID Catalog #101L) were either bred in captivity at Northeastern University or purchased from the Ambystoma Genetic Stock Center at the University of Kentucky. Experiments were performed in accordance with Northeastern University Institutional Animal Care and Use Committee. Animals were grown to 4-6cm (Mean 5.3cm, SD 0.36) and 1-1.5g (Mean 1.3g, SD 0.19g) for use in all studies. For all experiments, animals were anesthetized by treatment of 0.01% benzocaine until visually immobilized.

Reviewing Editor

  1. Tatiana Sandoval-Guzman

Publication history

  1. Received: July 6, 2020
  2. Accepted: October 13, 2020
  3. Accepted Manuscript published: October 14, 2020 (version 1)
  4. Version of Record published: November 16, 2020 (version 2)

Copyright

© 2020, Duerr 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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