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

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.


Introduction
The formation of neural circuits during development depends on the guidance of growing axons to their proper synaptic targets. This process relies on the growth cone, a dynamic actin based structure present at the tip of a growing axon. Growth cones contain a dynamic lamellipodial body ringed by filopodial protrusions, both important in guiding the axon to its target destination [1][2][3][4]. Guidance receptors present on the leading edge of the growth cone sense and respond to various extracellular guidance cues, which attract or repel axons enabling them to reach their proper target destination [5,6].
In C. elegans, UNC-6/Netrin is secreted by the ventral cells and along with its receptors UNC-40 and UNC-5 is required for the dorsal ventral guidance of circumferential neurons and axons [8,13,14]. Previous studies of repelled VD growth cones in Netrin signaling mutants revealed a correlation between attractive axon guidance and stimulation of growth cone protrusion, and repulsive axon guidance and inhibition of growth cone protrusion [15]. For example, in unc-5 mutants, growth cones were larger and more protrusive, and often displayed little or no directed movement. This is consistent with observation that increased growth cone size was associated with decreased neurite growth length [16]. Conversely, constitutive activation of UNC-40/UNC-5 signaling in repelled VD growth cones led to smaller growth cones with severely reduced filopodial protrusion [15,17]. Thus, directed growth cone repulsion away from UNC-6/Netrin requires a balance of pro-and anti-protrusive activities of the receptors UNC-40 and UNC-40-UNC-5, respectively, in the same growth cone [15].
Mechanisms downstream of UNC-5 in axon repulsion are less well described, but the PH/MyTH4/FERM molecule MAX-1 and the SRC-1 tyrosine kinase have been implicated [24,25]. We delineated a new pathway downstream of UNC-5 required for its inhibitory effects on growth cone protrusion, involving the Rac GEF UNC-73/Trio, the Rac GTPases CED-10 and MIG-2, and the cytoskeletal-interacting molecule UNC-33/CRMP [17].
Collapsin response mediating proteins (CRMPs) were first identified as mediators of growth cone collapse in response to the Semaphorin family of guidance cues [26], and we have shown that UNC-33/CRMP inhibits growth cone protrusion in response to Netrin signaling [17].
This motivated us to consider other mediators of Semaphorin-induced growth cone collapse in Netrin signaling. In Drosophila, the large multidomain cytosolic protein MICAL (Molecule Interacting with CasL) is required for the repulsive motor axon guidance mediated by interaction of Semaphorin 1a and Plexin A [27,28]. MICAL proteins are a class of flavoprotein monooxygenase enzymes that bind flavin adenine dinucleotide (FAD) and use the cofactor nicotinamide dinucleotide phosphate (NADPH) to facilitate oxidation-reduction (Redox) reactions [27]. MICAL regulates actin disassembly and growth cone collapse in response to semaphorin via direct redox interaction with F-actin [29,30]. MICAL molecules from Drosophila to vertebrates have a conserved domain organization: and N-terminal flavin-adenine dinucleotide (FAD)-binding monooxygenase domain, followed by a calponin homology (CH) domain, a LIM domain, a proline-rich domain, and a coiled-coil ERM α-like motif [27,31].
The C. elegans genome does not encode for a MICAL-like molecule with the conserved domain organization described above. However, it does contain five flavin monooxygenase (fmo) genes similar to the Flavin monooxygenase domain of MICAL: fmo-1, fmo-2, fmo-3, fmo-4 and fmo-5 [32]. Like MICAL, the C. elegans FMO molecules contain an N-terminal FAD binding domain and a C-terminal NADP or NADPH binding domain [27,32]. The C. elegans gene most similar to the non-FMO portion of MICAL is the Eps-15 homology domain binding protein EHBP-1 [33], which contains a CH domain as does MICAL.
In this work, we test the roles of the C. elegans FMOs and EHBP-1 in Netrin-mediated axon guidance and growth cone protrusion. We find that fmo-1, fmo-4, fmo-5 and ehbp-1 mutants display pathfinding defects of the dorsally-directed VD/DD motor neuron axons that are repelled by UNC-6/Netrin, and that they interact genetically with unc-40 and unc-5. We also find that VD growth cones in these mutants display increased filopodial protrusion, similar to mutants in repulsive UNC-6/Netrin signaling (e.g. unc-5 mutants), and that transgenic expression of FMO-5 inhibits growth cone protrusion, similar to constitutively-activated UNC-40 and UNC-5. We also show that FMO-1, FMO-4, FMO-5 and EHBP-1 are required for the growth cone inhibitory effects of activated UNC-5, UNC-40, and the Rac GTPases CED-10 and MIG-2. Together, these genetic analyses suggest that FMO-1, FMO-4, FMO-5, and EHBP-1 normally restrict growth cone protrusion, and that they might do so in UNC-6/Netrin-mediated growth cone repulsion.
fmo-3(gk184651) was a G to A substitution in the 3' splice site of intron 6. ehbp-1(ok2140) is a 1,369-bp deletion that removed all of exon 5 and 6.
The 19 D-class motor neurons cell bodies reside in the ventral nerve cord. They extend axons anteriorly and then dorsally to form a commissure, which normally extend straight dorsally to the dorsal nerve cord (Figure 2 and Figure 3B) On the right side of wild-type animals, an average of 16 commissures were observed, due to the fasciculation of some processes as a single commissure ( Figure 2C and Materials and Methods). fmo-1,4 and 5 and ehbp-1 mutants showed significant defects in VD/DD axon pathfinding, including ectopic axon branching and wandering (~3-5%; see Materials and Methods and Figure 3A, C and D). fmo-2 and fmo-3 mutations showed no significant defects compared to wild-type ( Figure 3A). Most double mutants showed no strong synergistic defects compared to the predicted additive effects of the single mutants ( Figure 3E). However, the fmo-2; fmo-3 and the fmo-2; fmo-4 double mutants showed significantly more defects compared to the predicted additive effects of the single mutants. The fmo-4; ehbp-1 double mutant displayed significantly reduced defects than either mutation alone. Lack of extensive phenotypic synergy suggests that the FMOs do not act redundantly, but rather that they might have discrete and complex roles in axon guidance, as evidenced by fmo-4; ehbp-1 mutual suppression.

fmo-1, fmo-4 and fmo-5 act cell-autonomously in the VD/DD neurons.
Expression of the fmo-1, fmo-4 and fmo-5 coding regions were driven in VD/DD motor neurons using the unc-25 promoter. Punc-25::fmo transgenes significantly rescued lateral midline crossing defects in fmo; unc-5(op468) and fmo; unc-5(e152) ( Figure 6). These data suggest that the axon defects observed in fmo mutants are due to mutation of the fmo genes themselves, and that fmo-1, 4, and 5 can act cell-autonomously in the VD/DD neurons in axon guidance.
Previous studies showed that fmo-1 and fmo-5 promoter regions were active in intestinal cells and the excretory gland cell, whereas the fmo-4 promoter was active in hypodermal cells, duct and pore cells [32,36]. ehbp-1 is expressed in all somatic cells including neurons [33].
In sum, previous expression studies combined with those described here suggest that fmo-1,4,5 and ehbp-1 are expressed in neurons, and that fmo-1,4, and 5 can act cell-autonomously in the VD/DD motor neurons in axon guidance.
Transgenic fmo-5 expression in unc-73(rh40) resulted in inhibited growth cone area and filopodial length compared to unc-73(rh40) alone, suggesting that FMO-5 can inhibit protrusion in the absence of UNC-73 Rac GEF activity. Transgenic fmo-5 expression inhibited growth cone size in unc-33(e204), but had a reduced capacity to inhibit filopodial protrusion (i.e. filopodial protrusion in unc-33(e204) with transgenic fmo-5 expression was reduced compared to unc-33 alone but was increased relative to fmo-5 transgenic expression alone) ( Figure 11E-I). In sum, these data indicate that that FMO-5 (and possibly FMO-1 and FMO-4) act downstream of the Rac GTPases MIG-2 and CED-10 in filopodial inhibition. FMO-5 might also act downstream of UNC-33, but the hybrid interaction of fmo-5 transgenic expression with unc-33(e204) mutants suggests that FMO-5 and UNC-33 might represent distinct pathways downstream of the Rac GTPases to inhibit filopodial protrusion.

Discussion
Results here implicate the C. elegans flavoprotein monooxygenase molecules FMO-1, FMO-4 and FMO-5 in inhibition of growth cone protrusion via UNC-6/Netrin receptor signaling in repulsive axon guidance. The MICAL molecule found in vertebrates and Drosophila is a flavoprotein monooxygenase required for semaphorin-plexin mediated repulsive motor axon guidance [27,40]. MICAL is a multi-domain molecule that also includes a calponin homology (CH) domain, a LIM domain and multiple CC domains. No molecule encoded in the C. elegans genome has a similar multi-domain organization. However, the Eps-15 homology (EH) domain binding protein EHBP-1 is similar to the non-FMO portion of MICAL and contains a CH domain [33]. We show here that EHBP-1 also is also involved in inhibition of growth cone protrusion and axon guidance. Thus, while C. elegans does not have a multidomain MICAL-like molecule, it is possible that the functional equivalents are the FMOs and EHBP-1.
Possibly, fmo-2 and fmo-3 have roles in axon guidance that were not revealed by the mutations used.
Drosophila and vertebrate MICAL regulate actin cytoskeletal dynamics in both neuronal and non-neuronal processes through direct redox activity of the monooxygenase domain [27,30,[41][42][43][44][45]. In Drosophila, loss of MICAL showed abnormally shaped bristles with disorganized and larger F-actin bundles, whereas, overexpression of MICAL caused a rearrangement of F-actin into a complex meshwork of short actin filaments [29]. Here we show that loss of fmo-1, fmo-4, and fmo-5 resulted in longer filopodial protrusions in the VD motor neurons (Figure 7), suggesting that their normal role is to limit growth cone filopodial protrusion. Indeed, transgenic expression of wild-type FMO-5 resulted in VD growth cones with a marked decrease in growth cone filopodial protrusion ( Figure 11). Growth cone size was not affected in any loss-of-function mutation, but growth cone size was reduced by transgenic expression of wild-type FMO-5 ( Figure 11), suggesting a role of the FMO-5 in both filopodial protrusion and growth cone lamellipodial protrusion.
Previous studies have shown that Drosophila MICAL may require both its FMO and CH domain to induce cell morphological changes; however, mammalian MICAL in non-neuronal cell lines requires only its FAD domain suggesting a difference in the mechanism of action in these MICALs [29,46]. These data suggest that in some cases, the FMO domain is sufficient for the function of MICAL. Thus, single domain FMOs as in C. elegans could function despite lacking the multi-domain structure of MICAL. Loss of EHBP-1, which contains a CH domain and is similar to the non-FMO portion of MICAL (Figure 1), also resulted in VD/DD axon guidance defects, but did not significantly affect growth cone filopodial protrusion. EHBP-1 might act with the FMOs in axon guidance. Phenotypic differences could be due to EHBP-1dependent and independent events, or to the wild-type maternal contribution in ehbp-1 homozygous mutants derived from a heterozygous mother. It is also possible that EHBP-1 affects axon guidance independently of the FMOs. EHBP-1 is involved in Rab-dependent endosomal vesicle trafficking by bridging interaction of endosomal Rabs with the actin cytoskeleton [33,47]. MICAL has also been implicated in Rab-dependent endosomal biogenesis and trafficking [48][49][50], suggesting that FMO/EHBP-1 and MICALs might share common functions, although it remains to be determined if FMOs in C. elegans regulate endosomal trafficking.
MICAL has been shown to directly oxidize cysteine residues in F-actin, leading to actin depolymerization and growth cone collapse [29,30,51,52]. We speculate that FMO-1, FMO-4, and FMO-5 might act by a similar mechanism to inhibit growth cone filopodial protrusion. The role of EHBP-1 is less clear, but previous studies have shown that Drosophila MICAL might require both its FMO and CH domain to induce cell morphological changes [29]. Thus, in axon guidance, FMO-1, FMO-4, and FMO-5 might require the CH domain provided by EHBP-1 in some instances. Mammalian MICAL requires only the FMO domain [46], suggesting that in some cases the CH domain is not required and the FMO domain can act alone. Future studies will be directed at answering these questions.  elegans CRMP-like molecule UNC-33 is required in a pathway downstream of Rac GTPases for inhibition of growth cone protrusion in response to UNC-6/Netrin [17]. unc-33 loss-of-function mutants with FMO-5 transgenic expression displayed a mutually-suppressed phenotype. The excessively-long filopodial protrusions of unc-33 mutants were reduced to wild-type levels, but were significantly longer than in animals with FMO-5 transgenic expression, and the growth cone area was reduced to resemble FMO-5 transgenic expression alone ( Figure 11). This phenotype could be interpreted as FMO-5 acting upstream of UNC-33/CRMP (i.e. UNC-33/CRMP is required for the full effect on FMO-5 overexpression). Alternatively, this hybrid phenotype could be interpreted as FMO-5 and UNC-33/CRMP acting independently to inhibit protrusion.
One proposed mechanism of cytoskeletal regulation by MICAL is the production of the reactive oxygen species (ROS) H 2 O 2 by the FAD domain in the presence of NADPH [53]. Upon activation by Sema3A, MICALs generate H 2 O 2 , which can, via thioredoxin, promote phosphorylation of CRMP2 by glycogen synthase kinase-3, leading to microtubule growth cone collapse [54]. This is consistent with our genetic results suggesting that FMO-5 may function upstream of UNC-33/CRMP in modulating the cytoskeleton of the VD growth cones to inhibit growth cone filopodial protrusion. CRMPs have been shown coordinate both microtubules and actin in axon elongation and growth cone dynamics [55,56]. Thus, the FMOs have the potential to inhibit growth cone protrusion through direct oxidation of F-actin resulting in depolymerization, and through redox regulation of the activity of UNC-33/CRMP.

Genetic methods
Experiments were performed at 20°C using standard C. elegans techniques [57]. respectively. Expression analysis for fmo-5 was done by amplifying the entire genomic region of fmo-5 along with its endogenous promoter (1.2kb upstream) and fusing it to gfp followed by the 3' UTR of fmo-5.

Analysis of axon guidance defects
VD neurons were visualized with a Punc-25::gfp transgene, juIs76 [59], which is expressed in GABAergic neurons including the six DDs and 13 VDs, 18 of which extend commissures on the right side of the animal. The commissure on the left side (VD1) was not scored. In wild-type, an average of 16 of these 18 VD/DD commissures are apparent on the right side, due to fasciculation of some of the commissural processes ( Figure 2C). In some mutant backgrounds, fewer than 16 commissures were observed (e.g. unc-5). In these cases, only observable axons emanating from the ventral nerve cord were scored for axon guidance defects. VD/DD axon defects scored include axon guidance (termination before reaching the dorsal nerve cord or wandering at an angle greater than 45° before reaching the dorsal nerve cord), lateral midline crossing (axons that fail to extend dorsally past the lateral midline) and ectopic branching

Growth cone imaging
VD growth cones were imaged as previously described [15,22]. Briefly, animals at 16 h posthatching at 20°C were placed on a 2% agarose pad and paralyzed with 5mM sodium azide in M9 buffer, which was allowed to evaporate for 4 min before placing a coverslip over the sample. Some genotypes were slower to develop than others, so the 16 h time point was adjusted for each genotype. Growth cones were imaged with a Qimaging Rolera mGi camera on a Leica DM5500 microscope. Projections less than 0.5 µm in width emanating from the growth cone were scored as filopodia. Filopodia length and growth cone area were measured using ImageJ software. Significance of difference was determined a two-sided t-test with unequal variance.    3. Mutations in fmo-1, fmo-4, fmo-5 and ehbp-1