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One of the most fascinating questions in the field of neurobiology is to understand how neuronal connections are properly formed. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. Most of these guidance factors are regulated by heparan sulfate proteoglycans (HSPGs), a family of cell-surface and extracellular core proteins with attached heparan sulfate (HS) glycosaminoglycans. The unique diversity and structural complexity of HS sugar chains, as well as the variety of core proteins, have been proposed to generate a complex "sugar code" essential for brain wiring. While the functions of HSPGs have been well characterized in C. elegans or Drosophila, relatively little is known about their roles in nervous system development in vertebrates. In this chapter, we describe the advantages and the different methods available to study the roles of HSPGs in axon guidance directly in vivo in zebrafish. We provide protocols for visualizing axons in vivo, including precise dye labeling and time-lapse imaging, and for disturbing the functions of HS-modifying enzymes and core proteins, including morpholino, DNA, or RNA injections.

Analyzing the Role of Heparan Sulfate Proteoglycans in Axon Guidance In Vivo in Zebrafish