channel images of (A) anti-horseradish peroxidase (HRP; red) and anti-Wingless (Wg; green), and (B) anti-Fasciclin II (FasII; green) and anti-glass bottom boat (Gbb; red), in non-permeablized labeling conditions in the absence of detergent. Note strong localization of both Wg and Gbb at the NMJ terminal. Merged and single channel images of (C) anti-HRP (red) and anti-Wg (green), and (D) anti-FasII (green) and anti-Gbb (red) in permeablized labeling conditions with 4% paraformaldehyde added to all antibody incubations. Note that most of the synaptic localization of Wg and Gbb is lost. (TIF) Figure S6 NMJ retention of Wg/Gbb altered by highly-sulfated heparin. Confocal imaging of Wg and Gbb trans-synaptic ligand abundance at the wandering third instar NMJ (muscle 4, segment A3) following acute incubation with highly-sulfated heparin. (A) Single channel and merged images of anti-horseradish peroxidase (HRP; red) and anti-Wingless (Wg; green) following control (no heparin), 0.156 mg/ml, 0.315 mg/ml and 0.625 mg/ml heparin treatments. (B) Single channel and merged images of anti-HRP (red) and anti-glass bottom boat (Gbb; green) following control, 0.156 mg/ml, 0.315 mg/ml and 0.625 mg/ml heparin treat- ments. (C) Quantification of fluorescence intensity of Wg and Gbb normalized to the internal HRP co-label for the control and indicated heparin concentrations. Individual data points are an average of $3 animals. Dotted line shows fitted linear trend lines. Statistically significant differences calculated using student’s t-test and indicated as ***p,0.001, ** p,0.01, * p,0.05. Error bars indicate S.E.M. (TIF) Figure S7 NMJ expression of Jeb ligand unchanged in sulf1/ hs6st nulls. (A) Representative NMJ images at the wandering third instar NMJ on muscle 6 in segment A3 from control (w1118), sulf1 and hs6st nulls, labeled with neural marker anti-horseradish peroxidase (HRP; red) and anti-Jelly belly (Jeb; green). Merged images show Jeb tightly localized at synaptic boutons. (B) Quantification of anti-Jeb mean fluorescence intensity levels normalized to HRP co-label and the genetic control. Sample sizes are $8 animals per indicated genotypes. Statistically significant differences calculated using student’s t-test. N.S. indicates no significant difference. Error bars indicate S.E.M. (TIF) Figure S8 NMJ expression of FGF receptor unchanged in sulf1/ hs6st nulls. (A) Representative NMJ images at the wandering third instar NMJ on muscle 6 in segment A3 from control (w1118), sulf1 and hs6st nulls, labeled with neural marker anti-horseradish peroxidase (HRP; red) and anti-Heartless (Htl; green). Merged images show the Htl FGF receptor tightly localized at synaptic boutons. (B) Quantification of Htl mean fluorescence intensity levels normalized to HRP co-label and the genetic control. Sample sizes are $7 animals per indicated genotypes. Statistically significant differences calculated using student’s t-test. N.S. indicates no significant difference. Error bars indicate S.E.M. (TIF) Figure S9 Synaptic Frizzled-2 receptor levels in sulf1 and hs6st nulls. Frizzled-2 receptor N-terminus (dFz2-N) specific antibody shows localized expression surrounding synaptic boutons at the NMJ. (A) Representative wandering third instar NMJ images from muscle 6 in segment A3 for control (w1118), sulf1 and hs6st mutants, double-labeled with presynaptic neural marker anti- Fasciclin II (FasII, red) and dFz2-N (green). Right: dFz2-N shown alone for clarity. (B) Quantification of dFz2-N mean fluorescence intensity for the indicated genotypes, normalized to the genetic HSPG Regulates Trans-Synaptic Signaling control. Sample sizes are $12 animals per genotype. Statistically significant differences calculated using student’s t-test, ** p,0.01. Error bars indicate S.E.M. (TIF) Figure S10 Wg and Gbb signals genetically interact with sulf1 and hs6st nulls. Genetic reduction of Wg and Gbb levels in sulf1 and hs6st homozygous conditions restores molecular synaptic assembly to control levels. (A) Representative NMJ boutons from control (w1118), heterozygous wg/+ and gbb/+ in sulf1 background (wgI-12/gbb2; sulf1D1/sulf1D1) and hs6st background (wgI-12/gbb2; hs6std770/hs6std770) labeled for postsynaptic Bad Reception (Brec) glutamate receptor IID subunit (GluRIID, green) and presynaptic active zone Bruchpilot (anti-nc82, red). Quantification of GluRIID punctae/bouton (B), total GluRIID area (C), Brp punctae/bouton (D) and total Brp area (E), all normalized to the genetic control. All multiply mutant conditions are restored to control levels for all parameters, with no significant differences remaining. (TIF) Figure S11 Characterization of anti-Gbb antibody specificity. Representative confocal images of wandering third instar NMJ 6/ 7 double-labeled with anti-Gbb (red) and anti-HRP (green) under detergent permeabilized (A–C) and non-permeabilized (D–F) conditions. The genotypes analyzed include control (w1118; A,D), gbb1/gbb2,UAS-gbb9.9 (B,E), and BG57-GAL4/UAS-gbb9.1 (C,F). (TIF) Table S1 Primary screen results. Raw number values of the RNAi screen indicated by human ortholog name, Drosophila gene name and CG number. Mean value and standard deviation (SD) included for NMJ morphology parameters of bouton number, branch number and synaptic area, and for NMJ functional parameter of evoked excitatory junctional current (EJC) ampli- tude. Sample sizes $6 NMJs and $3 animals for morphology and function measurements. (XLS) Table S2 Secondary screen results. Raw number values for the secondary screen results indicated by human ortholog name, Drosophila gene name and CG number. The two independent IDs for RNAi lines are shown. For all retested lines, morphological quantification for NMJ bouton number (top) and evoked excitatory junctional current (EJC) amplitude (bottom). All results are shown as fold-changes compared to genetic control. Sample sizes are $6 individual animals per genotype. Replication of primary screen result is indicated in the final column as Y, and failure to replicate indicated as N. (XLS) Acknowledgments We are particularly grateful to Hiroshi Nakato (sulf1DP1 and hs6std770), Xinhua Lin (sulf1D1), Kristy Wharton (gbb1/gbb2, UAS-gbb9.9 and UAS- gbb9.1), the Vienna Drosophila RNAi Center (VDRC), the Harvard TriP Collection, and the Bloomington Drosophila Stock Center for providing essential Drosophila stocks. We also particularly thank Joachim Schulz (Sdc), Peter ten Dijke (P-Mad), Vivian Budnik (dFz2-C and dFz2-N), Stefan Baumgartner (PcanV), and the Iowa Hybridoma Bank for essential antibodies. We thank Samuel Friedman for providing a representative image of anti-Perlecan antibody staining at the NMJ. We thank William Parkinson, Emma Rushton, Lane Coffee, and Sean Schaffer for technical assistance. Author Contributions Conceived and designed the experiments: ND KB. Performed the experiments: ND. Analyzed the data: ND. Contributed reagents/ materials/analysis tools: MN SL. Wrote the paper: ND KB. PLOS Genetics | www.plosgenetics.org 19 November 2012 | Volume 8 | Issue 11 | e1003031