As loss of function in dlk-1 and

other regrowth-promoting genes results in similar phenotypes, we used a gain-of-function effect caused DAPT by overexpression of DLK-1 [dlk-1(++)] to address their order of activity. Overexpression of DLK-1 is sufficient to enhance PLM axon regeneration ( Yan et al., 2009). DLK-1 overexpression completely suppressed the regrowth defects of unc-51/Atg1 and unc-57/Endophilin mutants ( Figure 6E), consistent with DLK-1 acting downstream or in parallel to UNC-51/ATG1 and the SV endocytosis genes. Loss of function in RPM-1, a negative regulator of DLK-1, did not suppress unc-57/Endophilin regrowth defects (data not shown), consistent with previous findings that PLM regrows normally in rpm-1 mutants ( Yan et al., 2009). Among all double mutants tested, only efa-6(lf) suppressed regeneration defects ABT-263 datasheet of dlk-1 mutants

(Figures 5F and 6E). In efa-6 dlk-1 double mutants the proximal stumps of severed axons extended significantly further than in dlk-1(lf) although they did not form growth cones ( Figure 5G). efa-6 mutations also partially suppressed the regrowth defects of unc-26/Synaptojanin and unc-51/Atg1 mutants ( Figure 6E), consistent with EFA-6 acting downstream or in parallel to DLK-1, UNC-26, and UNC-51 in axon regrowth. Genes with inhibitory roles, such as slt-1 and efa-6, affect different stages of regrowth and therefore likely act in distinct pathways. To test whether elimination of multiple inhibitory pathways could further enhance regrowth relative to single mutants, we analyzed slt-1 efa-6 double mutants. We found that regrowth at the 24 hr time point was not further enhanced in slt-1 efa-6 double mutants compared with the highest single mutant ( Figure 6F). However, regrowth at 48 hr postaxotomy was significantly enhanced in efa-6 slt-1 double mutants compared with single mutants. Thus, the combined loss of two inhibitory pathways can result in further increases

in regrowth at later time points. Our results establish the feasibility of systematic genetic screening for axon regeneration phenotypes using genetically Ketanserin amenable model organisms. Our findings underscore the molecular complexity of axon regeneration and provide a genetic framework for a more comprehensive understanding of axonal repair and regrowth mechanisms. As a forward genetic phenotype-based screen in axon regeneration remains technically challenging, we have focused on systematic large-scale testing of conserved candidate genes. Our selection of candidates is by necessity biased, and we plan to expand the screen to reduce this bias. Nonetheless, our analysis supports the view that regenerative axon regrowth requires many genetic pathways in addition to those defined in developmental axon outgrowth, polarity, or guidance.