Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Summary

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Abstract

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides^{1, 2}. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy³. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid⁴ and N-acetylgalactosamine^{5, 6}, in developing zebrafish and in other living organisms.

Protocol

1. Egg Collection and Dechorionation Collect and transfer zebrafish eggs to 35mm petri dish, remove as much water as possible and then add 1 mg/ml Pronease E in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2·2H2O, 0.33 mM MgSO4, pH = 7.4) to digest the chorion. After 3-5 minutes, merge the dish into a beaker filled with fish water (60 mg “instant Ocean” per liter distilled H2O), and gently transfer the eggs to the beaker and allow the eggs…

Discussion

Imaging biomolecules in vivo provides critical insights of their biological activities in their native environments. In this video, we demonstrate how the labeling of fucosylated glycans in the enveloping layer of zebrafish embryos is realized by microinjecting one-cell stage embryos with GDP-FucAl and a second-step fluorophore conjugation via BTTES-mediated biocompatible CuAAC³. Robust labeling can be achieved within 2-3 minutes, and the labeled glycans are detectable as early as 2.5 hpf. Importantl…

Materials

Name of the reagent	Company	Catalog number
Copper(II) sulfate pentahydrate	Sigma-Aldrich	203165
Alexa Fluor 488 azide	Invitrogen	A10266
dextran, Alexa Fluor 594	Invitrogen	D-22913
(+)-Sodium L-ascorbate	Sigma-Aldrich	A7631
Bathocuproinedisulfonic acid	Acros Organics	164060010
Glass bottom microwell dish	MatTek	P35G-1.5-14-C