Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 2010 Jun 8;107(23):10360-5. doi: 10.1073/pnas.0912081107. Epub 2010 May 20.
Developmental events can be monitored at the cellular and molecular levels by using noninvasive imaging techniques. Among the biomolecules that might be targeted for imaging analysis, glycans occupy a privileged position by virtue of their primary location on the cell surface. We previously described a chemical method to image glycans during zebrafish larval development; however, we were unable to detect glycans during the first 24 hours of embryogenesis, a very dynamic period in development. Here we report an approach to the imaging of glycans that enables their visualization in the enveloping layer during the early stages of zebrafish embryogenesis. We microinjected embryos with azidosugars at the one-cell stage, allowed the zebrafish to develop, and detected the metabolically labeled glycans with copper-free click chemistry. Mucin-type O-glycans could be imaged as early as 7 hours postfertilization, during the gastrula stage of development. Additionally, we used a nonmetabolic approach to label sialylated glycans with an independent chemistry, enabling the simultaneous imaging of these two distinct classes of glycans. Imaging analysis of glycan trafficking revealed dramatic reorganization of glycans on the second time scale, including rapid migration to the cleavage furrow of mitotic cells. These studies yield insight into the biosynthesis and dynamics of glycans in the enveloping layer during embryogenesis and provide a platform for imaging other biomolecular targets by microinjection of appropriately functionalized biosynthetic precursors.
可以使用非侵入性成像技术在细胞和分子水平上监测发育事件。在可能成为成像分析目标的生物分子中，糖链因其位于细胞表面的主要位置而占据有利地位。我们之前描述了一种在斑马鱼幼虫发育过程中对糖链进行成像的化学方法；然而，我们无法在胚胎发生的前 24 小时内检测到糖链，这是发育过程中非常活跃的时期。在这里，我们报告了一种对糖链进行成像的方法，该方法能够在斑马鱼胚胎发生的早期阶段在包被层中可视化糖链。我们在一细胞期将叠氮糖微注射到胚胎中，让斑马鱼发育，并使用无铜点击化学检测代谢标记的糖链。在受精后 7 小时，即胚胎发育的原肠胚期，就可以对粘蛋白型 O-糖链进行成像。此外，我们使用非代谢方法用另一种化学方法对唾液酸化糖链进行标记，从而能够同时对这两种不同类型的糖链进行成像。糖链运输的成像分析揭示了糖链在第二个时间尺度上的剧烈重排，包括快速迁移到有丝分裂细胞的分裂沟。这些研究深入了解了胚胎发生过程中包被层中糖链的生物合成和动态变化，并为通过适当功能化的合成前体的微注射对其他生物分子靶标进行成像提供了平台。
