We used mCherry fluorescence to cut both NICD-GFP(+) and NICD-GFP(−) axons and quantified axon regeneration separately for each group. NICD-GFP(+) axons had significantly decreased regeneration compared to control wild-type animals ( Figure 4B), similar to gain-of-function Notch/lin-12 mutant axons ( Figure 1C). By contrast, NICD-GFP(−) axons from the same animals had normal regeneration ( Figure 4B). Third, we observed a similar overall inhibition of regeneration when we overexpressed full-length Notch/lin-12 cDNA only in the GABA neurons (

Figure 4C). Fourth, we found that NICD-GFP is able to cell autonomously learn more inhibit regeneration in animals that otherwise lack Notch/lin-12. We expressed NICD-GFP only in the GABA neurons of null Notch/lin-12 mutant animals. The gross phenotype of this strain was identical to nontransgenic Notch/lin-12 null mutants: animals had protruding vulvas and were completely sterile. However, these animals had decreased regeneration in their GABA neurons ( Figure 4D), compared to the increased regeneration normally found in Notch/lin-12 null mutants ( Figure 1C).

selleck compound Together, these results suggest that cell-autonomous Notch signaling is sufficient to inhibit axon regeneration. To determine whether intrinsic Notch signaling is necessary to inhibit regeneration, we performed tissue-specific rescue of ADAM10/sup-17. Regenerating GABA neurons contact only two tissues: body-wall muscles and skin. ADAM10/sup-17 null mutants have increased regeneration ( Figure 3B).

We found that expression of wild-type ADAM10/sup-17 in muscles or skin did not affect this phenotype. Only when wild-type ADAM10/sup-17 was expressed in GABA neurons was regeneration inhibited back to wild-type levels ( Figure 4E). Additionally, we found that overexpression in wild-type animals of ADAM10/sup-17 in the GABA neurons inhibits regeneration ( Figure 4F). ADP ribosylation factor Consistent with Notch/lin-12 being the relevant target of ADAM10/sup-17, overexpression of ADAM10/sup-17 in Notch/lin-12 null mutants does not inhibit regeneration ( Figure 4G). Taken together, these data demonstrate that Notch acts cell autonomously to inhibit regeneration and establish that Notch signaling is an intrinsic inhibitor of axon regeneration. In C. elegans, Notch itself and the ADAM metalloprotease that mediates Notch activation are encoded by two genes, with overlapping but different functions ( Figure 3A) ( Jarriault and Greenwald, 2005). However, only one Notch gene (Notch/lin-12) and one ADAM (ADAM/sup-17) inhibit regeneration in GABA neurons ( Figure 1 and Figure 3). Because Notch inhibition of regeneration is cell autonomous, we tested whether the remaining Notch components could also limit regeneration when overexpressed in GABA neurons. We found that GABA-specific overexpression of Notch/glp-1 NICD-mCh inhibited regeneration ( Figure 4H), similar to overexpression of Notch/lin-12 NICD-GFP ( Figure 3H).