Netrin Participates in the Development of Retinotectal Synaptic Connectivity by Modulating Axon Arborization and Synapse Formation in the Developing Brain

Abstract

Netrin has been implicated in retinal ganglion cell (RGC) axon pathfinding in a number of species. In Xenopus laevis, RGC axons reaching their target in the optic tectum can be repelled by a netrin-1 gradient in vitro, suggesting that netrin may also function in wiring events that follow successful axon pathfinding. Here, we examined the contribution of netrin to RGC axon arborization and synapse formation at the target. Time-lapse confocal microscopy imaging of individual RGC axons coexpressing GFP-synaptobrevin and DsRed in the intact Xenopus brain demonstrated a role for deleted in colorectal cancer (DCC)-mediated netrin signaling. Microinjection of netrin-1 into the tectum induced a rapid and transient increase in presynaptic site addition that resulted in higher presynaptic site density over a 24 h observation period. Moreover, netrin induced dynamic axon branching, increasing branch addition and retraction; a behavior that ultimately increased total branch number. In contrast, microinjection of DCC function-blocking antibodies prevented the increase in presynaptic site number normally observed in control axons as well as the associated increase in branch number and axon arbor growth. Dynamic analysis of axon arbors demonstrated that the effects of anti-DCC on axon morphology and presynaptic connectivity were attributable to a specific decrease in new synapse and branch additions, without affecting the stability of existing synapses and branches. Together, these results indicate that, in the absence of DCC signaling, RGC axons fail to branch and differentiate, and support a novel role for netrin in later phases of retinotectal development.

Branch management: mechanisms of axon branching in the developing vertebrate CNS

“…Netrin-1 also shapes terminal arbors. In the frog optic tectum, injection of netrin-1 enhances development of terminal retinal arbors 37 by increasing the dynamic addition and retraction of branches, resulting in an increase in total branch number.…”

Netrin-1 in the developing enteric nervous system and colorectal cancer

“…Collectively, this indicates that netrin-1 can act as a cell survival factor for both non-neuronal cells and neurons. Netrin-1 has generally been associated with branching axons and synapse formation in the developing brain [75]. In the case of the adult brain, the limited distribution but ongoing expression of netrin-1 has been linked to axon regeneration, and it is proposed that the guidance role of netrin-1 for axon outgrowth shown in embryos may be recapitulated in the adult in response to injury [9].…”

The actin cytoskeleton in presynaptic assembly

“…Interestingly, however, axons may continue to arborize following synapse formation, and these secondary arbors often extend from existing presynaptic sites. [10][11][12] One hypothesis that could explain this relationship is that terminal arborization and presynaptic assembly employ shared actin-regulatory components. For example, it has been observed that the Netrin receptor, UNC-40/DCC, and downstream actin regulatory molecules are required locally both for the clustering of synaptic vesicles and for the extension of terminal arbors.…”