Other synaptic vesicle proteins, SNB-1/synaptobrevin and SNG-1/synaptogyrin, also showed a similar phenotype to RAB-3 in cyy-1 cdk-5 double mutants ( Figure S2). These results suggest that CYY-1 and CDK-5 are not required for patterning of synapses in the L1 stage, but they are essential for DD synaptic remodeling. Because other synaptic vesicle proteins, SNB-1/synaptobrevin and SNG-1/synaptogyrin, displayed a similar phenotype to RAB-3 in the double

mutants (Figure S2) and GFP::RAB-3 reliably visualized DD synaptic remodeling process (Figure S1), we used GFP::RAB-3 (wyIs202) for further experiments to label synaptic vesicles during the DD remodeling process. To ensure that the GFP::RAB-3 phenotype in the mutants indeed represents synaptic remodeling defects, we colabeled synaptic vesicles Doxorubicin and active zones with RAB-3 and SYD-2/Liprin-α, respectively. In the cyy-1 cdk-5 double mutants, SYD-2 exhibited a similar phenotype as synaptic vesicle proteins—the majority of SYD-2 signals were found in the ventral process, which colocalize with

the RAB-3 puncta in the ventral process at the L4 stage ( Figure 1B). Furthermore, we performed serial electron microscopy (EM) reconstruction to definitively examine the synaptic structural defects in the double mutants. We reconstructed dorsal nerve cord and analyzed the Selleckchem VE822 appearance of synaptic vesicles and active zones of DD neurons in wild-type and cyy-1 cdk-5 double-mutant worms. Consistent with the results from the

fluorophore-tagged synaptic markers ( Figures 1A, and1B; Figure S2), the number of synaptic vesicles and active zones in the DD dorsal process is significantly reduced in the adult double-mutant worms ( Figures 1C–1E). Taken together, our findings suggest that CYY-1 and CDK-5 combined are necessary for eliminating presynapses from the ventral process and forming new synapses in the dorsal process of DD neurons. Because both CYY-1 and CDK-5 are necessary for the completion of synaptic remodeling, we next tested if these two proteins are sufficient to initiate DD synaptic remodeling. In adult cyy-1 cdk-5 double mutants, the majority of the GFP::RAB-3 remains in the ventral processes, suggesting that the DD synaptic remodeling is almost completely blocked in Montelukast Sodium the absence of these two molecules ( Figure S3B, B1; quantified in Figure S3C). To ask whether CYY-1 and CDK-5 play instructive roles in the remodeling process, we tested if expression of CYY-1 and CDK-5 at the mid-L3 stage, a time point long after the normal remodeling process has been completed in wild-type worms, can rescue the remodeling defect in cyy-1 cdk-5 double-mutant animals ( Figure S3A). To induce CYY-1 and CDK-5 expression, we generated transgenic worms expressing CYY-1 and CDK-5 under the control of the heat-shocked promoter (Phs).