( A) Vps29 loss of function causes an increased number of synaptic terminal boutons at the larval neuromuscular junction (NMJ). NMJ preparations from Vps29 1 homozygotes or control larvae ( GR = Vps29 1 GR/+) were stained with an antibody against horseradish peroxidase, and Type IIb boutons at abdominal segments A2 and A3 were quantified (n = 5 animals per group). Flies were raised in complete darkness and examined at 1 day. ( C) Compared with controls ( FRT40A clones), ERG transient potentials are partially extinguished by rapid stimulation in Vps35 MH20 clones. Flies were raised in 12 hr light/12 hr dark cycle (L) or in the dark ( D). GMR-w-RNAi was used to remove eye pigment. Frontal sections through the retina are shown, stained with hematoxylin and eosin, revealing vacuolar changes, which was suppressed by either introducing the Vps29 genomic rescue construct (GR) or raising animals in dark conditions. ( B) Loss of Vps29 causes light- and age-dependent retinal degeneration. For statistical analysis, a chi-square test was performed. ( A) Adult flies lacking Vps29 ( Vps29 1 homozygotes and Vps29 1/Df) are recovered at ratios below Mendelian expectations (~25% vs. Statistical analysis was based on log-rank test with Bonferroni's correction ( D) or one-way ANOVA ( F, G, I). Quantification of ERG on-transients in 15-day-old flies (n = 9–12 per group) from the following genotypes: (1) Vps29 1 nSyb-Gal4/+ (2) Vps29 1 nSyb-Gal4/UAS-dVps29 (3-5) Vps29 1 nSyb-Gal4/UAS-hVps29 and (6) Vps29 1 nSyb-Gal4/UAS-dVps35. ( I) Rescue of Vps29 1 synaptic transmission defects by pan-neuronal expression ( nSyb-GAL4 driver) of either Drosophila Vps29 or Vps35 ( dVps29 and dVps35) or human Vps29 ( hVps29-1,−2, or −3, representing three alternate isoforms).
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Consistent results were obtained using either ey-FLP or ey 3.5-FLP, which targets presynaptic neurons only. ( H) Compared with controls ( FRT40A clones), ERG transient potentials are extinguished by rapid stimulation in Vps29 1 clones. ‘X’ denotes undetectable on-transients in 20-day-old animals. ( F–G) Quantification (n = 6–8) of ERG depolarization and on-transient potentials. Raising flies in the dark restored ERG depolarization, but not the transients, indicating persistent defects in synaptic transmission. On- and off- transient ERG potentials (top and bottom arrowheads, respectively) were also lost. Loss of either Vps29 or Vps35 disrupted light-induced depolarization (arrow) in 10-day-old flies. Flies were raised using an alternating 12 hr light/dark cycle (12h-LD) or in complete darkness. ( E) Representative ERG traces at 1- and 10 days after generation of ey-FLP clones from (i) control ( FRT40A), (ii) Vps29 1, or (iii) Vps35 MH20. Quantitation based on n = 200–235 per group. ( D) Vps29 1 homozygotes and Vps29 1/Df transheterozygotes ( Vps29 1/Df(2L)Exel6004) show reduced survival that is rescued by the Vps29 genomic rescue strain ( Vps29 1/Df GR/+). ( C) Western blot from adult heads probed with anti-Vps29 antibody, confirming Vps29 1 is a protein null allele. We also performed PCRs using primer pairs that span both sides of the breakpoint junctions abutting the inserted ywing 2+ marker gene cassette (not shown), and these products were Sanger sequenced to confirm the depicted molecular lesion. As an additional control, PCR was also performed for Vps35 genomic sequence. P1, P2, and P3 denote expected PCR products from primer pairs targeting Vps29 genomic sequence. ( B) Genomic polymerase chain reaction (PCR) showing loss of Vps29 coding sequence in Vps29 1 homozygotes versus control (w) flies. A bacterial artificial chromosome (BAC) (yellow) was used for transgenic genomic rescue ( GR).
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A chromosomal deficiency Df(2L)Exel6004 is shown, with the deleted regions indicated by dashed lines. Vps29 WT.GFP and Vps29 L152E.GFP are identical N-terminal tagged-Vps29 alleles, except for the L152E variant. ( A) The Vps29 genomic locus is shown, highlighting reagents used in this study. In the null allele, Vps29 1, the gene coding sequence (blue, 2L: 1150852–1151420) is replaced by a ywing 2+ marker gene.