As an additional control, PCR was also performed for Vps35 genomic sequence. 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. ( C) Western blot from adult heads probed with anti-Vps29 antibody, confirming Vps29 1 is a protein null allele. ( 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/+). Quantitation based on n = 200–235 per group. ( 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. Flies were raised using an alternating 12 hr light/dark cycle (12h-LD) or in complete darkness. Loss of either Vps29 or Vps35 disrupted light-induced depolarization (arrow) in 10-day-old flies. On- and off- transient ERG potentials (top and bottom arrowheads, respectively) were also lost. Raising flies in the dark restored ERG depolarization, but not the transients, indicating persistent defects in synaptic transmission. ( F–G) Quantification (n = 6–8) of ERG depolarization and on-transient potentials. ‘X’ denotes undetectable on-transients in 20-day-old animals. ( H) Compared with controls ( FRT40A clones), ERG transient potentials are extinguished by rapid stimulation in Vps29 1 clones. ( 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).Ĭonsistent results were obtained using either ey-FLP or ey 3.5-FLP, which targets presynaptic neurons only.
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. Statistical analysis was based on log-rank test with Bonferroni's correction ( D) or one-way ANOVA ( F, G, I).
( A) Adult flies lacking Vps29 ( Vps29 1 homozygotes and Vps29 1/Df) are recovered at ratios below Mendelian expectations (~25% vs. For statistical analysis, a chi-square test was performed. ( B) Loss of Vps29 causes light- and age-dependent retinal degeneration. 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. GMR-w-RNAi was used to remove eye pigment. Flies were raised in 12 hr light/12 hr dark cycle (L) or in the dark ( D). ( C) Compared with controls ( FRT40A clones), ERG transient potentials are partially extinguished by rapid stimulation in Vps35 MH20 clones. Flies were raised in complete darkness and examined at 1 day.
( 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).
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