Developing axons are led by growth cones, which are dynamic actin-based structures that sense and respond to extracellular guidance cues, driving the forward motion of the axon towards the target of migration. The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5/UNC-40 heterodimer, inhibits growth cone protrusion (Norris et al., 2011). Expression of an activated MYR::UNC-40 construct in the repelled VD axons constitutively inhibits protrusion. We used MYR::UNC-40 to delineate a pathway involving UNC-73/Trio, the Rac GTPases CED-10 and MIG-2, and the microtubule-interacting protein UNC-33/CRMP that inhibits growth cone protrusion downstream of MYR::UNC-40 (Norris et al., 2014). While CRMPs have been implicated in semaphorin-mediated collapse, our data suggest that they also control UNC-6/Netrin-mediated repulsion.In
unc-33 mutants, excess microtubule plus ends were observed in growth cones, correlating with excess growth cone protrusion. The MICAL proteins are flavin monooxygenases that cause actin depolymerization involved in growth cone collapse downstream of semaphorin (Hung et al., 2010). The C. elegans genome does not encode a single MICAL-like gene. However, it encodes five flavin monooxygenases (
fmo-1-5) (Petalcorin et al., 2005), similar to that found in MICAL, as well as an actin-interacting protein,
ehbp-1 (Shi et al., 2010), which is similar to the non-FMO portion of MICAL. Here we show that the
ehbp-1 and fmo genes may play a role in Netrin directed repulsive guidance mediated through UNC-40/DCC and UNC-5. A subset of the fmo genes and
ehbp-1 showed VD/DD axon guidance and branching defects and enhanced
unc-40 VD/DD guidance defects. When placed in a MYR::UNC-40 background these mutants partially suppressed myr::
unc-40. Further studies will be directed at determining if these genes act downstream of the Rac GTPases and UNC-73/Trio, if they are expressed in and act cell-autonomously in neurons to regulate axon guidance, and if they affect the actin cytoskeleton of the growth cone.In sum, we have defined a novel pathway that inhibits growth cone protrusion downstream of UNC-6/Netrin. While CRMP has been shown to mediate semaphorin induced growth cone collapse, our results implicate UNC-33/CRMP in UNC-6/Netrin repulsive axon guidance and that that the MICAL-like fmo genes and
ehbp-1 also act in inhibition, possibly via the actin cytoskeleton.