In ventrally guided AVM axons, SLT-1 signals play repulsive roles in development and regrowth ( Gabel et al., 2008 and Hao et al., 2001). In contrast, in PLM neurons SAX-3/Robo appears to switch from a growth-promoting

role during development to an inhibitory role in regrowth. Among the few genes with inhibitory effects on regrowth we focused on EFA-6, the C. elegans member of the EFA6 (Exchange Factor for Arf6) family. EFA6 proteins contain a variable N-terminal region, a Sec7 homology domain with GEF activity specific to ARF6 GTPases ( Franco et al., 1999), a pleckstrin homology (PH) domain, and a coiled-coil domain ( Figure S4A). C. elegans efa-6 mutants displayed mild PLM axon overshooting in development ( Figures S4B and S4C) and enhanced regrowth of PLM ( Figures 4A and 4B). Cell-type specific transgenic expression of EFA-6 from pan-neural or touch neuron-specific promoters, but not from a muscle-specific promoter, rescued efa-6 developmental FG-4592 mouse defects ( Figure S4C) and inhibited PLM regrowth after axotomy both in efa-6(lf) ( Figure 4E) and efa-6(+) backgrounds (data not shown; see also Figure 5), indicating EFA-6 acts cell autonomously

and that PLM regrowth is sensitive Selleckchem Epacadostat to EFA-6 levels. In contrast to slt-1 or sax-3, efa-6 mutants displayed enhanced regrowth during the 0–6 hr period (Figures 1D and 4C), implying EFA-6 acts early in regrowth. Furthermore, heat shock induced EFA-6 MYO10 overexpression 1 hr before axotomy inhibited PLM regrowth, whereas induction earlier or later had little effect ( Figure 4D). To investigate the mechanism underlying EFA-6 function, we next examined arf-6(lf) mutants. arf-6(lf) mutants displayed modestly increased regrowth and did not further enhance efa-6(lf) in regrowth ( Figure 4E). However, EFA-6-overexpressing transgenes potently inhibited regrowth in arf-6(lf) backgrounds ( Figure 4E), suggesting EFA-6 acts on regrowth independent of ARF-6. To dissect which functional domains of EFA-6 were important in axon regrowth we expressed mutant

EFA-6 lacking either the Sec7 domain, the PH domain, or the C-terminal coiled coil domain ( Table S2). Each of these “gain-of-function” transgenes rescued efa-6 developmental overgrowth ( Figure S4B) and inhibited regrowth, as did constructs in which the conserved catalytic residue of the Sec7 domain was mutated (E447K). In contrast, expression of an EFA-6 variant lacking the N terminus did not block PLM regrowth ( Figure 4E). As overexpression of EFA-6 might affect nonphysiological pathways, we made single-copy insertion transgenes expressing full length EFA-6 or the E447K mutant and found that both rescued efa-6 developmental and regrowth phenotypes ( Figure 4F), suggesting a GEF-independent role for EFA-6 in inhibiting regrowth. Our recent studies on C. elegans embryos indicate that EFA-6 regulates microtubule (MT) growth by promoting MT catastrophe ( O’Rourke et al., 2010).