Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we provide a missing link in understanding the modulation of UNC-6/netrin signaling in the extracellular milieu. We demonstrate that LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling, and may do this through interactions with the UNC-40/DCC netrin receptor. By combining loss-of-function and gain-of-function approaches, we demonstrate that LON-2/glypican functions in the attractive and repulsive UNC-6/netrin pathways, and guides AVM, the distal tip cells, and the motorneurons. We show that LON-2/glypican functions from the hypodermis, where it is expressed (1), to guide cells and axons. To directly test the associations between LON-2/glypican, UNC-6/netrin, and UNC-40/DCC, we have developed an S2 cell-based assay and demonstrate that LON-2/glypican specifically associates with cells expressing the UNC-40/DCC receptor. The association between LON-2/glypican and UNC-40/DCC-expressing cells occurs via the extracellular portion of UNC-40/DCC, independently of UNC-6/netrin binding. Furthermore, we show that the N-terminal globular region of LON-2/glypican, lacking the three HS chain attachment sites, is functional in UNC-6/netrin-mediated guidance. Our studies unravel a novel mechanism by which LON-2/glypican is produced by substrate hypodermal cells and is released from the membrane to associate with UNC-40/DCC-expressing cells and neurons, enabling the modulation of their responses to UNC-6/netrin during cell and axon migrations. Given the evolutionary conservation of both the UNC-6/netrin pathway components and of glypicans, and that synthesis of HS chains is required for mammalian axons to respond to netrin-1 in vitro (2,3), glypicans are likely to play a role in netrin-mediated axon pathfinding in mammals as well.
1. Gumienny et al., 2007
2. Matsumoto et al., 2007
3. Ogata-Iwao et al., 2011.
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