Fly strains and genetics
All flies were raised on standard food medium and kept at 25°C. dTDP-43 RNAi lines 38377 and 38379 were obtained from the Vienna Drosophila RNAi Center (VDRC). The dTDP-43Q 367Xmutant allele was identified from the Seattle Drosophila TILLING Project (Fly-TILL, Fred Hutchinson Cancer Research Center) using specific tilling primers (Additional file 1). dTDP-43Q 367X/CyO, GFP flies were crossed with Pin/CyO, GFP; Gal4221, UAS-mCD8-GFP to establish the stock dTDP-43Q 367X/CyO, GFP; Gal4221, UAS-mCD8-GFP/+. The Gal4221 driver was used to label ddaE and ddaF neurons with mCD8-GFP and drive the expression of transgenes . To visualize dendritic phenotypes of ddaE and ddaF neurons in third instar larvae, we crossed dTDP-43Q 367X/CyO, GFP; Gal4221, UAS-mCD8-GFP/+ flies with dTDP-43Q 367X/CyO, GFP or w1118 flies to generate dTDP-43Q 367X/dTDP-43Q 367X; Gal4221, UAS-mCD8-GFP/+ or dTDP-43Q 367X/+; Gal4221, UAS-mCD8-GFP/+ third instar larvae. For RNAi expression, dTDP-43Q 367X/CyO, GFP; Gal4221, UAS-mCD8-GFP/+ flies were crossed with UAS-dTDP-43 RNAi lines (VDRC 38377 and 38379) to generate dTDP-43Q 367X/+; Gal4221, UAS-mCD8-GFP/38377, and dTDP-43Q 367X/38379; Gal4221, UAS-mCD8-GFP/+ third instar larvae for phenotypic analysis. For transgene overexpression, Gal4221, UAS-mCD8-GFP flies were crossed with UAS-dTDP-43,, UAS-hTDP-43, UAS-hTDP-43-M337V, or UAS-hTDP-43-Q331K transgenic lines. In the above experiments, Gal4221, UAS-mCD8-GFP/+ third larvae served as the control.
Generation of transgenic Drosophila lines
Full-length hTDP-43 cDNA was cloned from HEK293 cells (provided by Dr. J.-A. Lee). To generate UAS-hTDP-43, UAS-hTDP-43-M337V, UAS-hTDP-43-Q331K, and UAS-hTDP-43- C-terminal fragment (amino acids 209-414) constructs, the corresponding primers (Additional file 1) were used to amplify DNA fragments, which were then cloned into the pUAST vector. To generate UAS-dTDP-43 constructs, the full-length dTDP-43 coding sequence was amplified from the cDNA plasmid GH09868 (Drosophila Genomics Resource Center) and cloned into the pUAST vector. These constructs were confirmed by sequencing and microinjected into wild-type (w1118) embryos to generate transgenic lines.
Antibody production and western blot
Anti-dTDP-43 polyclonal antibody was generated by immunizing rabbits with a peptide fragment spanning amino acids 179-192 (Thermo Fisher Scientific). For protein expression analysis, adult flies were frozen in ethanol with dry ice and vortexed to remove heads. Approximately 30 heads from each genotype were homogenized in 50 μl of lysis buffer (0.137 M NaCl, 20 mM Tris, pH 8.0, 10% glycerol, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM DTT, Pierce protease inhibitors and phosphatase inhibitors). Homogenate was heated at 65°C for 10 min and centrifuged at 4°C for 10 min at 13,000 rpm. Protein concentrations were determined using Bradford Assay (Bio-Rad).
Supernatant containing 10 μg of protein was separated on a 10% acrylamide SDS gel and transferred to a PVDF membrane (Bio-Rad) in a wet transfer system at 4°C for 60 min at 100 V. The membrane was incubated in blocking solution containing 5% milk in TBST (25 mM Tris-HCl, 137 mM NaCl, 3 mM KCl, pH 7.4, and 0.1% Tween-20) at 4°C overnight, with dTDP-43 antibody (1:1000 in blocking solution) at room temperature for 3 h, and finally with anti-rabbit HRP-conjugated secondary antibody (Jackson Immunoresearch; 1:10,000) for 1 h. The signal was visualized with chemiluminescent substrate (Supersignal West Pico, Pierce). For other western blot analyses, the primary antibodies were hTDP-43 antibody (1:1000; 10782-2-AP, Proteintech), and β-actin antibody (1:1500; Cell Signaling).
Quantitative RT-PCR (qRT-PCR) analysis
Total RNAs were extracted from adult heads with Trizol (Invitrogen) and used as templates to generate cDNAs with TaqMan reverse transcription reagent (Applied Biosystems). cDNAs were used as templates for qRT-PCR in a final volume of 25 μl. A standard curve was run in each PCR reaction. Individual values were normalized to the value of the gene encoding the ribosomal protein RP-49. Two pairs of primers were designed to detect dTDP-43 transcripts (Additional file 2). All reactions were performed three times. Relative mRNA expression was calculated using the standard curve method and the delta-delta Ct method.
Mosaic analysis with a repressible cell marker (MARCM)
MARCM analysis of sensory neurons in the Drosophila PNS was performed as described . First, the dTDP-43Q 367Xallele was recombined onto the chromosome containing FRTG 13. FRTG 13, dTDP-43Q 367X/CyO or FRTG 13/CyO male flies were crossed with Gal4C 155, UAS-mCD8-GFP, hs-FLP1; FRTG 13, Gal80/CyO virgin females to generate Gal4C 155, UAS-mCD8-GFP, hs-FLP1/+; FRTG 13, Gal80/FRTG 13, dTDP-43Q 367Xand Gal4C 155, UAS-mCD8-GFP, hs-FLP1/+; FRTG 13, Gal80/FRTG 13embryos, respectively. Embryos from these crosses were collected on grape agar plates for 3 h in a 25°C incubator. The embryos were aged for 3 h and heat-shocked in a 37°C water bath for 40 min to induce mitotic recombination. The embryos were then kept in a moisture chamber at 25°C for 3-4 days. Third instar larvae were collected, and larvae that contained a single mCD8::GFP-labeled dorsal cluster PNS neuron were selected under a Nikon fluorescence dissection microscope. Images of the dendritic morphology of single DA neurons were recorded with a confocal microscope (Nikon, D-Eclipse C1). The significance of differences in dendritic branching complexity was determined with Student's t test.
Quantitative Analysis of dendritic ends of sensory neurons
The dendritic morphology of GFP-labeled dorsal sensory neurons was recorded with a confocal microscope (Nikon, D-Eclipse C1), and dendritic branches of ddaE or ddaF neurons in the A3 dorsal cluster were counted as described . Briefly, dendritic ends of DA neuron images were identified visually and highlighted with dots, which were counted with Adobe Photoshop software. The data were analyzed by Student's t test.