, 2003). It has been shown that Eiger and Wengen can interact physically with each other through their TNF homology and TNFR homology domains, respectively. This evolutionarily conserved TNF-α-like signaling pathway has now been shown to be involved in JNK-induced cell death in the fly eye ( Kanda et al., 2002, Kauppila et al., 2003 and Babcock et al., 2009). We have determined that Wengen is expressed in motoneurons, where it could function as a receptor for glial-derived Eiger signaling. Expression of Wengen cDNA was determined by RT-PCR analysis from RNA made specifically from a FACS-sorted population of GFP-positive

GSI-IX datasheet motoneurons isolated from third-instar larval brain (Figure S4). Because Wengen is a type I membrane protein, we tagged the C-terminal tail with a Venus tag to visualize Wengen localization (Kanda et al., 2002 and Kauppila et al., 2003). When expressed in motoneurons using OK371-GAL4, Wengen-Venus accumulates along motor axons within peripheral nerves ( Figure 4A). Wengen-Venus also traffics to the presynaptic nerve terminal at the NMJ, where it is distributed in a

punctate manner throughout synaptic boutons and inter-bouton regions ( Figure 4B). It is worth noting that expression Pifithrin-�� datasheet in axons is considerably stronger than within the NMJ, even when one assesses staining in isolated axons just prior to muscle innervation (data not shown). Thus, the highest levels of Wengen occur in axons where it is in a position to receive signaling from glial-derived Eiger. Finally, as a control, we determined that the axonal staining does not colocalize with other synaptic protein markers, so the accumulation of Wengen-Venus in axons cannot be attributed to impaired axonal transport ( Figure S5). In order to examine whether Wengen plays a role in neuromuscular degeneration, we used a previously established and verified wengen RNAi

(wgnRNAi) construct ( Kanda et al., 2002, Kauppila et al., 2003, Megestrol Acetate Babcock et al., 2009, Xue et al., 2007 and Igaki et al., 2002) in an attempt to suppress the degeneration phenotype observed in ank2 mutants. Animals with knockdown of wengen in motoneurons show no evidence of NMJ degeneration ( Figures 4C, 4E, and 4F). However, neuronal expression of wgnRNAi in an ank2 background significantly suppresses the severity of synaptic degeneration when compared with ank2 animals alone ( Figures 4D–4F). Again, this suppression of neuromuscular degeneration cannot be accounted for by enhanced synaptic growth because bouton numbers are normal when wgnRNAi is neuronally expressed in an otherwise wild-type background ( Figure S3). Again, we controlled for the presence of axonal blockages and defects in synaptic microtubules following wgnRNAi in the ank2 mutant background. There is no suppression of axonal blockages nor is there an improvement in the organization of the microtubule cytoskeleton ( Figure S6).