UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans

Abstract

SUMMARYThe UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. We show that UNC-6/netrin, UNC-5 and UNC-40/DCC modulated the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants displayed increased protrusion in repelled growth cones, whereas loss-offunction unc-6 or unc-40 mutants caused decreased protrusion. In contrast to previous studies, our work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controlled polarity of growth cone protrusion and F-actin accumulation that correlated with attraction versus repulsion. However, filopodial dynamics were affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, we show here that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion.

ENU-3 is a new player in UNC-6/Netrin directed axon outgrowth and guidance in C. elegans

“…Therefore, in the absence of UNC-5, UNC-40 opposes axon outgrowth, probably because the UNC-40 homodimers are attracted towards ventral sources of UNC-6. This observation is consistent with the observation by Lundquist's group that the presence of UNC-40 increased the guidance defects in the VD and DD motor neuron axons in the absence of UNC-5 [26] . One possibility for a role for ENU-3 is that it favours the formation of UNC-5:UNC-40 heterodimers rather than UNC-40 homodimers, as they guide long-range migrations of motor neurons in both Drosophila and C. elegans [22,27] .…”

“…The cellular role of ENU-3 in growth cone outgrowth is not understood and it is not entirely clear where it fits with UNC-6 and its receptors. A study that looked at growth cone dynamics showed that lack of functional UNC-5 resulted in increased protrusion of growth cones in the VD and DD motor neurons while lack of either UNC-40 or UNC-6 resulted in decreased protrusion of the growth cones in the same neurons [26] . Lack of UNC-5 also caused an increase in the area of the growth cones and the duration of the filopodia.…”

Multiple cytoskeletal pathways and PI3K signaling mediate CDC-42-induced neuronal protrusion inC. elegans

“…The ectopic protrusion induced by CDC-42(G12V) resembles the protrusions normally found in growth cones of axons during their outgrowth. 13,55 Indeed, the pathways that we have identified acting downstream of CDC-42 in neuronal protrusion also affect axon guidance, including Arp2/3, UNC-115/abLIM, UNC-34/ Ena, 19,21 MIG-15/NIK, 18 and PI3K signaling. 32 Thus, pathways identified by suppression of CDC-42-induced ectopic protrusion might normally act to regulate protrusion in the growth cone during outgrowth.…”

“…There is evidence to suggest that both attractive and repulsive guidance cues affect protrusiveness of the filopodial and lamellipodial structures of the growth cone. 13 Proper issuance and interpretation of these cues are necessary for normal axon guidance. Disruptions in the proper extension of axons in the nervous system are associated with several neurological disorders including dyslexia and autism spectrum disorders.…”

A novel role for the DNA repair gene Rad51 in Netrin-1 signalling

“…Whereas Dcc typically has been reported to mediate the stimulatory effects of Netrin-1 on neuritogenesis. the Unc5 family has been reported to oppose this action262829. Using qPCR we measured relative mRNA levels of Unc5a-d and Dcc in cultured neurons following siRNA mediated knockdown of Rad51 (Fig.…”