3D Visualization of Macromolecule Synthesis
- Northeastern University, United States
- Universitat Politècnica de Catalunya (UPC), Spain
- University of Technology of Compiègne, France
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.
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Author details
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.
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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3D Visualization of Macromolecule Synthesis
eLife 9:e60354.
https://doi.org/10.7554/eLife.60354
