Flavin Monooxygenases Regulate C. elegans Axon Guidance and Growth Cone Protrusion with UNC-6/Netrin signaling and Rac GTPases

Abstract The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, 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. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5/UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies, we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance. Author Summary Molecular mechanisms of axon repulsion mediated by UNC-6/Netrin are not well understood. Inhibition of growth cone lamellipodial and filopodial protrusion is critical to repulsive axon guidance. Previous work identified a novel pathway involving Rac GTPases and the cytoskeletal interacting molecule UNC-33/CRMP required for UNC-6/Netrin-mediated inhibition of growth cone protrusion. In other systems, CRMP mediates growth cone collapse in response to semaphorin. Here we demonstrate a novel role of flavoprotein monooxygenases (FMOs) in repulsive axon guidance and inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling and Rac GTPases. In Drosophila and vertebrates, the multidomain MICAL FMO mediates semaphorin-dependent growth cone collapse by direct oxidation and depolymerization of F-actin. The C. elegans genome does not encode a multidomain MICAL-like molecule, and we speculate that the C. elegans FMOs might have an equivalent role downstream of UNC-6/Netrin signaling. Indeed, we show that EHBP-1, similar to the non-FMO portion of MICAL, also controls repulsive axon guidance and growth cone inhibition, suggesting that in C. elegans, the functions of the multidomain MICAL molecule might be distributed across different molecules. In sum, we show conservation of function of molecules involved in semaphorin growth cone collapse with inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling.