Incomplete penetrance of ventral remodeling in double mutants

was also observed by imaging. In unc-55; hbl-1 double mutants, we observed patches of the ventral nerve cord that contained an approximately normal number of synapses, while other regions totally lacked synapses (data not shown). A transgene expressing hbl-1 in the VD and DD neurons of unc-55; hbl-1 double mutants (using the unc-25 promoter) decreased the ventral IPSC rate to that observed in unc-55 single mutants ( Figure 3F) but did not rescue the non-neuronal hbl-1 defects ( Figure S3A). These results suggest that HBL-1 acts in VD neurons to promote ectopic remodeling. To further document the functional integrity of the ventral VD synapses, we analyzed the locomotion behavior of unc-55; hbl-1 double mutants. A prior study showed that ectopic remodeling of VD synapses in unc-55 mutants was accompanied by a locomotion defect ( Zhou and Walthall, 1998). During backward KPT-330 movement, unc-55 mutants assume a ventrally coiled body posture, presumably due to the absence of inhibitory input to the ventral body muscles ( Figure 3I). This unc-55 coiling defect was significantly reduced (but not eliminated) in unc-55; hbl-1 double mutants ( Figure 3I). The coiling defect was restored by transgenes driving hbl-1 expression in the D neurons (using either the unc-25 GAD or the unc-30 promoter) in unc-55; hbl-1 double mutants ( Figure 3I and Figure S3E),

as would be predicted if HBL-1 acts in VD neurons to PD-0332991 in vitro promote remodeling. Thus,

the imaging, electrophysiology, and behavioral assays all support the idea that hbl-1 is a functionally important UNC-55 target whose expression promotes ectopic remodeling of VD synapses in unc-55 mutants. The partial suppression and incomplete penetrance observed in the unc-55; hbl-1 double Rolziracetam mutants indicate that the hbl-1(mg285) mutation did not completely abolish remodeling of VD synapses. The persistent VD remodeling observed in double mutants could reflect residual hbl-1 activity in hbl-1(mg285) mutants, or the activity of other UNC-55 target genes ( Lin et al., 2003). Consistent with the latter idea, transgenic expression of hbl-1 in DD and VD neurons (with the unc-25 promoter) was not sufficient to cause ectopic remodeling of VD synapses ( Figure S3F). Thus, hbl-1 is unlikely to be the only UNC-55 target involved in D neuron remodeling. Thus far, our results show that hbl-1 promotes ectopic remodeling of unc-55 mutant VD neurons but that hbl-1 expression alone is not sufficient to cause VD remodeling. We next analyzed DD remodeling, which occurs in wild-type animals ( Walthall, 1990 and White et al., 1978). Prior to hatching, DD neurons form ventral NMJs, which can be identified as ventral UNC-57::GFP puncta. During the L1 stage, these ventral DD synapses are eliminated and new dorsal synapses are formed (visualized as dorsal UNC-57 or RAB-3 puncta; Figure 4A and Figure S4A).