UC Berkeley

Glycans are carbohydrate structures that cover the surfaces of all cells and mediate cell-cell interactions essential for the functioning of multicellular organisms. Glycans play roles in cell adhesion, signaling, and differentiation processes, particularly during embryonic development. A better understanding of these processes at the molecular level would benefit from the ability to visualize glycans in living organisms.

As post-translational modifications of proteins and head groups of lipids, glycans are not directly encoded in the genome. As a result, they cannot be labeled and imaged using the standard genetic techniques used to tag proteins with fluorescent probes. To enable imaging of glycans in living systems, we have developed a chemical reporter strategy in which glycans are tagged with a small reporter group that is not naturally found in biological systems. This can be achieved by supplying endogenous biosynthetic pathways with azide-labeled monosaccharides that become incorporated into cell-surface glycans. In a second step, the chemical reporter can be visualized by covalent ligation to a fluorescent probe.

This dissertation describes the development of chemical methods for imaging glycans during zebrafish embryogenesis. Zebrafish are popular vertebrate model organisms with transparent embryos that develop externally, characteristics that render them ideal for optical imaging. In Chapter 1 of this dissertation, I present an overview of the structures and functions of eukaryotic glycans and methods for imaging them in living systems. In Chapter 2, I discuss a method for visualizing mucin-type O-glycans during early embryogenesis by microinjecting zebrafish embryos at the one-cell stage with azide-functionalized precursor molecules. This method enabled imaging of glycans as early as 7 hours post-fertilization, during the gastrulation stage of development, and revealed dramatic trafficking of glycans to the cleavage furrow of dividing cells. In Chapters 3 and 4, I describe the extension of this metabolic labeling strategy toward fucosylated and sialylated glycans, respectively. Finally, in Chapter 5, I present a non-metabolic method for chemical labeling of sialylated glycans. This method enabled the simultaneous visualization of two distinct classes of glycans in live embryos.