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.
- Sandra J Shefelbine
- Eun Kyung Jeon
- Eun Kyung Jeon
- James R Monaghan
- James R Monaghan
- Ester Comellas
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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.
- Tatiana Sandoval-Guzman
- Received: June 24, 2020
- Accepted: October 13, 2020
- Accepted Manuscript published: October 14, 2020 (version 1)
© 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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