Next, we determined whether ebax-1 functions in neurons that express guidance receptors or surrounding tissues that secrete guidance cues. We performed mosaic analysis in unc-6; ebax-1 and unc-40; ebax-1 double mutants coexpressing a rescuing transgene Pebax-1::EBAX-1 and a coinjection

marker Psur-5::SUR-5::mCherry that labels nuclei of cells carrying the transgenes ( Yochem et al., 1998). click here For simplicity of quantification, we focused on AVM as ebax-1 is exclusively involved in the slt-1/sax-3 pathway during AVM axon guidance. ebax-1; unc-6 or ebax-1; unc-40 double mutant animals universally expressing the transgenes showed full rescue of AVM axon guidance defects ( Figure 2J). We then identified animals specifically losing

the transgenes either in the AB lineage-derived cells (mainly neurons) or in the P1 lineage-derived cells (mainly nonneuronal cells) and scored them for AVM guidance defects. AB-loss animals showed the same severity of axon guidance defects as ebax-1; unc-6 or ebax-1; unc-40 double mutant animals. In contrast, the guidance defects in P1-loss animals were rescued by neuron-restricted expression of EBAX-1 ( Figure 2J). PLX3397 Therefore, we conclude that the cell-autonomous expression of EBAX-1 in neurons is important for regulating AVM axon guidance. Next, we addressed whether EBAX-1’s interactions with Elongins and cullin 2 are important for its function in AVM axon guidance. We expressed EBAX-1 mutants (ΔBox, M1, and M2; Figure 3A) deficient in interactions with ELC-1 and/or CUL-2 in the ebax-1(ju699);

unc-6(ev400) and unc-40(e1430); ebax-1(ju699) backgrounds and examined their activity on rescuing AVM guidance defects. All mutants showed similar expression as wild-type proteins ( Figure S3; data not shown). Strikingly, these EBAX-1 mutant proteins completely lost rescuing activity ( Figures 3B and 3C), indicating that the in vivo second function of EBAX-1 requires EBAX-1 to interact with ELC-1 and CUL-2. We then asked whether other components of the BC box-type Cullin-RING ligase are directly involved in axon guidance. Because Elongin B and Elongin C are also components of cullin 5-containing CRLs (Hua and Vierstra, 2011), we decided to address this question by examining the necessity of CUL-2/cullin 2 in AVM axon guidance. cul-2 was specifically knocked down in touch neurons of unc-6 mutants by coexpressing Pmec-7-driven cul-2 sense and antisense RNAs ( Najarro et al., 2012). Touch neuron-specific cul-2 RNA interference (RNAi) resulted in a significant enhancement of AVM guidance defects. In contrast, expression of sense RNA alone had no effect ( Figure 3D). Taken together, these results demonstrate that the EBAX-1-containing CRL is critical for AVM axon guidance in vivo. In our functional rescue experiments, we found that the SWIM domain was also important for the function of EBAX-1 in AVM guidance (Figure 3C).