Imaging Tools for Probing Glycosaminoglycans In Vivo in Developing Zebrafish.

摘要

Glycosaminoglycans are linear polysaccharides that decorate the surfaces of all animal cells. The heterogeneity of the polysaccharide, especially the pattern of sulfation on the distal end of the chain, endows these glycans with diverse biological functions including essential roles in signaling during animal development.;Thus, glycosaminoglycans represent an extremely important and interesting class of glycans to probe using metabolic labeling in concert with bioorthogonal chemistry. This thesis describes the extension of the metabolic labeling strategy to glycosaminoglycans (GAGs). Prior to the work described herein, GAGs could not be metabolically labeled because xylose, the monosaccharide specific to these glycans, lacks a salvage or biosynthetic pathway capable of converting xylose derivatives, or other simple sugars, to the ultimate sugar donor UDP-xylose. In order to label GAGs, the UDP-xylose derivative itself must be delivered to cells. However, the charged nature of nucleotide sugars prevents passive diffusion through hydrophobic membranes, and eukaryotic cells lack a plasma membrane nucleotide sugar transporter to provide facilitated or active transport. Prior research in our lab, however, had demonstrated that unnatural nucleotide sugars could be delivered to cells by microinjection into the yolk sacs of developing zebrafish embryos. In addition to providing a tractable solution to deliver nucleotide sugars, zebrafish are a well-established vertebrate development model organism with excellent properties for molecular imaging.;It was hypothesized that GAGs could be metabolically labeled by microinjecting unnatural variants of UDP-xylose into zebrafish embryos. To this end, three analogs of UDP-xylose were synthesized, microinjected into zebrafish embryos, and analyzed for incorporation onto zebrafish embryo cell surfaces by reaction with fluorescent cyclooctyne probes. Two of the analogs, UDP-2-XylAz and UDP-3-XylAz, did not provide azide-dependent labeling. UDP-4-XylAz did provide azide-dependent labeling, and the azide replacing the C-4 hydroxyl group inhibits extension of the GAG polysaccharide beyond the initial xylose unit. Therefore, UDP-4-XylAz functions as a selective metabolic inhibitor of GAG biosynthesis. The phenotypic consequences of aberrant GAG production were ascertained and these results add to our understanding of the importance of GAGs during normal vertebrate development.