Department of Biology, Northeastern University, Boston, United States.
Department of Mathematics, Laboratori de Càlcul Numeric (LaCàN), Universitat Politècnica de Catalunya (UPC), Barcelona, Spain.
Elife. 2020 Oct 14;9:e60354. doi: 10.7554/eLife.60354.
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.
在体测量新生大分子合成对于理解细胞和组织如何在发育过程中进展以及对外界信号作出响应至关重要。在这里，我们通过体内注射炔烃或叠氮修饰的胸苷、尿苷、蛋氨酸和葡糖胺类似物，来标记 DNA、RNA、蛋白质和糖基化的新生合成。我们使用全器官点击化学荧光染色和光片荧光显微镜对再生墨西哥钝口螈（axolotl salamander）组织中的新生大分子合成进行了三维体积成像。我们还开发了一种图像处理管道，用于对感兴趣的形态区域和单个细胞进行分割和分类，并将其应用于再生肱骨。我们通过测量肢体去神经后 DNA 合成的变化来证明该方法对生物扰动的敏感性。该方法为在器官、细胞和分子组织水平上定量研究异质组织中的大分子合成提供了一种强大的手段。
