Journal
ELIFE
Volume 9, Issue -, Pages -Publisher
ELIFE SCIENCES PUBLICATIONS LTD
DOI: 10.7554/eLife.60354
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Funding
- National Science Foundation [1727518, 1656429, 1558017]
- Northeastern University
- European Commission [MSCA-GF 841047]
- Division Of Integrative Organismal Systems
- Direct For Biological Sciences [1558017, 1656429] Funding Source: National Science Foundation
- Div Of Civil, Mechanical, & Manufact Inn
- Directorate For Engineering [1727518] Funding Source: National Science Foundation
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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.
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