HSPG Regulates Trans-Synaptic Signaling done at 1:100 dilution of 1 mg/ml propidium iodide incubated for 30 minutes at room temperature. using student’s t-test or the Mann-Whitney test for non-parametric data. Imaging quantification Images were taken with on an upright Zeiss LSM 510 META laser-scanning confocal using a Plan Apo 636 oil objective. For structural quantification, including NMJ synapse branch number, bouton number and area, preparations were double-labeled with anti-HRP and anti-DLG, with counts made at muscle 4 in segment A3. For nuclear import studies, nuclei were identified by propidium iodide staining with fluorescent punctae counted and intensity quantified [59]. For synaptic functional protein quanti- tation, glutamate receptor and Brp punctae were quantified for muscle 4, segment 3. Glutamate receptor number and field area was quantified in consecutive boutons of .3 mm diameter. All preparations were fixed, stained and processed simultaneously to allow for intensity comparisons. All analyses were done with ImageJ software (National Institutes of Health) using the threshold function to outline areas and Z-stacks made using the maximum projection function. Statistics were done either with one-way ANOVA analysis followed by Dunnett’s post-test, student’s t-test or Mann-Whitney test for non-parametric data. All analyses were done blind to genotypes during all stages of experimentation and analysis. All figure images were projected in LSM Image Examiner (Zeiss) and exported to Adobe photoshop. Heparin treatment Stock solution of heparin (Sigma, H3393) in 16PBS was prepared and serially diluted to obtain concentrations (e.g. 0.625, 0.315 and 0.156 mg/ml). Dissected wandering third instar larvae were incubated with these heparin concentrations for 5 minutes at RT, followed by a 1 minute wash with 16PBS and then 10 minute fix with 4% paraformaldehyde in 16PBS. After fixation, anti-Wg or anti-Gbb antibodies were used as above with appropriate secondary antibodies. Processed animals were ana- lyzed for changes in intensity measurements as above in the image quantification section. All fluorescence intensity measurements were compared to preparations treated identically with only 16PBS and no heparin, and the processed simultaneously for immunolabeling, microscopy and quantification. Electrophysiology Two-electrode voltage-clamp (TEVC) records were made from the wandering third instar NMJ as previously described [41]. In brief, staged control, mutant and transgenic RNAi animals were secured on sylgard-coated coverslips with surgical glue (liquid suture), dissected longitudinally along the dorsal midline, and glued flat. The segmental nerves were cut near the base of the ventral nerve cord. Recording was performed in 128 mM NaCl, 2 mM KCl, 4 mM MgCl2, 1.0 mM CaCl2, 70 mM sucrose, and 5 mM Hepes. Recording electrodes (1-mm outer diameter capillaries; World Precision Instruments) were filled with 3 M KCl and had resistances of .15 MV. Spontaneous mEJCs were collected using continuous (gap-free) recording and evoked EJC recordings were made from the voltage-clamped (Vhold = 260 mV) muscle 6 in segment A3 with a TEVC amplifier (Axoclamp 200B; MDS Analytical Technologies). The cut segmental nerve was stimulated with a glass suction electrode at a suprathreshold voltage level (50% above baseline threshold value) for a duration of 0.5 ms. Records were made with 0.2 Hz nerve stimulation in episodic acquisition setting and analyzed with Clampex software (version 7.0; Axon Instruments). Each n = 1 represents a recording from a different animal. Statistical comparisons were performed Supporting Information Figure S1 NMJ synaptic bouton number in sulf1 and hs6st mutants. (A) Representative NMJ images from muscle 4 in segment A3 showing anti-horseradish peroxidase (HRP; red) and anti-Discs Large (DLG; green) in control (w11186UH1-GAL4), sulf1 RNAi (UH1-GAL46UAS-CG6725) and hs6st RNAi (UH1- GAL46UAS-CG4451). (B) Quantification of synaptic bouton number in RNAi-knockdown conditions for sulf1 and hs6st, normalized to genetic control (w11186UH1-GAL4). Sample sizes are $10 animals per indicated genotypes. (C) Representative NMJ images of anti-HRP (red) and anti-DLG (green) in w1118 control, sulf1 and hs6st mutants. (D) Quantification of synaptic bouton number in mutant conditions normalized to genetic control. Sample sizes are $8 animals per indicated genotype. Statistically significant differences were calculated using student’s t-test and indicated as ***p,0.001. Error bars indicate S.E.M. (TIF) Figure S2 Double knockdown of sulf1 and hs6st measure of EJC amplitude. (A) Representative evoked excitatory junctional current (EJC) traces from control (w11186UH1-GAL-4) and double knockdown with both sulf1 and hs6st RNAi transgenic lines (UH1-GAL46UAS-sulf1-RNAi; UAS-hs6st-RNAi). (B) Quantified mean EJC amplitudes (nA) for the two genotypes shown in panel A normalized to control. Sample sizes are $12 animals per indicated genotype. Statistically significant differences calculated using student’s t-test, * p,0.05, Error bars indicate S.E.M. (TIF) Figure S3 NMJ synaptic localization of Dally-like and Syndecan HSPGs. Representative confocal images showing HSPG synaptic localization at the larval NMJ. (A) Single channel images of presynaptic anti-horseradish peroxidase (anti-HRP, blue), Dally- like Protein (anti-Dlp, green) and postsynaptic glutamate receptor subunit IID (anti-GluRIID, red). (B) Single channel images showing presynaptic anti-horseradish peroxidase (anti-HRP, blue), syndecan (anti-Sdc, red) and postsynaptic Discs Large (anti-DLG, green). (C) Merged image showing Dlp localization with respect to presynaptic HRP, postsynaptic GluRIID and the triple-labeled terminal. (D) Merged image showing Sdc localization with respect to presynaptic HRP, postsynaptic DLG and the triple-labeled terminal. (TIF) Figure S4 HSPG Perlecan (Trol) is absent from the NMJ synaptic terminal. (A) Representative confocal image showing Perlecan expression at the wandering third instar larval NMJ using the Trol-GFP Flytrap line ZCL1700 from the Flytrap GFP Resource. Single channel and merged images show presynaptic anti-horseradish peroxidase (anti-HRP, red) and Trol-GFP (green). (B) Representative confocal image showing Perlecan (anti-PcanV) antibody staining, shown at a much higher confocal gain than in A to emphasize muscle expression. Perlecan is strongly expressed in the motor nerve, and clearly present on the muscle surface, but is never detectably enriched at the NMJ terminal. In many cases, as in the example shown, Perlecan appears at lower levels in the perisynaptic region surrounding the NMJ than elsewhere on the muscle. (TIF) Figure S5 Permeabilized versus non-permeabilized Wg and Gbb labeling. Representative NMJ images of muscle 6/7 in segment A3 from the wandering third instar. Merged and single PLOS Genetics | www.plosgenetics.org 18 November 2012 | Volume 8 | Issue 11 | e1003031