We do not detect the OvHts isoform (1156 aa) that is present in nurse cells and oocytes ( Petrella et al., 2007; data not shown). We find that presynaptic expression of Hts-M using elav-GAL4 in the hts mutant background (DfBSC26/hts1103) rescues the presynaptic retraction phenotype ( Figures 4D and 4F; retraction frequency 7% compared to 54% in

mutant animals, n > 98 NMJ). Importantly, this presynaptic rescue assay allows us to visualize Hts-M protein that is present in the presynaptic nerve terminal because we only resupply the protein in the motoneuron, not in the muscle. Hts-M protein is present within the presynaptic nerve terminal where it localizes at or near the presynaptic membrane but is not present within active zones marked by Brp ( Figure 4E). In contrast, postsynaptic expression of Hts-M causes a slight, Anti-cancer Compound Library mouse though not significant, reduction in the frequency of synapse retraction. However, we Adriamycin find that ectopic expression of Hts-M in muscle severely disrupts muscle and NMJ morphology ( Figure S4). Thus, is it not possible to accurately quantify the postsynaptic contribution of Hts-M to NMJ stability when

it is expressed via UAS-GAL4. From these data, both RNAi-mediated Hts knockdown and transgenic rescue, we conclude that Hts is required presynaptically to stabilize the NMJ. We next analyzed synaptic transmission, comparing wild-type with the hts1103/DfBSC26 allelic combination and with animals expressing htsRNAi in presynaptic neurons. We find a significant increase second in the average quantal amplitude in the hts1103/DfBSC26 mutant

animals and a corresponding decrease in average quantal content ( Figures S5A–S5C). However, the increased mepsp amplitude was not observed in animals expressing htsRNAi presynaptically. This could be due to a less severe knockdown of Hts protein. Alternatively, the increased average mepsp amplitude could reflect a postsynaptic activity of Hts. This possibility is consistent with the prior demonstration that knockdown of postsynaptic spectrin causes a comparable increase in quantal size ( Pielage et al., 2006). We also find that synaptic transmission is considerably more variable in hts loss of function animals ( Figure S5D). We plotted average mepsp versus average quantal content for individual NMJ recordings. There is more variation both in mepsp amplitude and quantal content compared to wild-type. This is consistent with prior studies demonstrating highly variable recordings at NMJ undergoing retraction ( Massaro et al., 2009, Pielage et al., 2005 and Pielage et al., 2008). The observed increase in release variability is less severe than after knockdown of α-/β-spectrin, which might be accounted for by enhanced NMJ growth that is unique to hts mutant animals (see below).