HSPG Regulates Trans-Synaptic Signaling crossed to the UH1-GAL4 driver line. Target genes are indicated by Drosophila genome CG annotation number and categorized by function. Confocal imaging of co-labeled pre- and postsynaptic markers was used to quantify NMJ architecture, including branch number, bouton number and synaptic area. TEVC electrophysiology was used to quantify evoked excitatory junctional current (EJC) amplitudes. The magnitude of fold changes compared to control (w11186UH1-GAL4) is shown on a color scale (see legend below the two columns). Statistical significance was calculated using one-way ANOVA analysis, and displayed as p,0.05 (*), p,0.01 (**). doi:10.1371/journal.pgen.1003031.g001 area was determined by outlining the terminal area labeled by DLG using the thresholding function in ImageJ. The majority of gene knockdown conditions showed a decrease in NMJ area compared to control (Figure 1). 7 RNAi lines exhibited a statistically significant decrease in area, whereas only 2 lines exhibited a statistically significant increase in synaptic area. All raw values of measured morphological parameters are included in Table S1. To assay functional differentiation, the motor nerve was stimulated with a suction electrode while the evoked excitatory junctional current (EJC) was recorded in the muscle (Figure 1) [41]. Nerve stimulation was applied at 4 V for 0.5 ms at a frequency of 0.2 Hz, with the muscle clamped at 260 mV. EJC amplitudes were calculated from recorded traces in the ubiqui- tously-driven RNAi lines (w1118 background) compared to the w1118; UH1-GAL4/+ control. Recordings were obtained from $3 independent trials for each RNAi knockdown condition. All electrophysiological screening was done blind to genotype, with values reported as fold-change and statistical significance calcu- lated by one-way ANOVA analyses (see color scheme; P,0.05 (*), P,0.01 (**); Figure 1). Genes from all eight glycan classes were identified to produce changes in neurotransmission strength upon genetic knockdown. For the 103 larval-viable lines tested, 26 lines showed a trend towards increased transmission strength, and 12 were statistically elevated compared to genetic control (Figure 1). 4 gene knockdowns showed a trend towards decreased transmission strength, of which only 1 line reached statistical significance. 73 of the 103 lines tested showed no change in functional strength (Figure 1). Interestingly, only 6 RNAi lines showed statistically significant effects on both NMJ morphology parameters and EJC amplitude: CG1597, CG6657, CG7480, CG4451, CG6725 and CG11874 (Figure 1). This suggests that glycan effects on synapse morphological and functional development are largely separable. All raw values of EJC measurements are included in Table S1. To validate results, a secondary screen was conducted using independent RNAi lines obtained from the VDRC and Harvard TRiP collections (Table S2). Of the 44 genes that showed morphological and functional defects in the primary screen, 33 were retested using independent RNAi lines, with the others lacking available secondary lines from any source. Using the same screen of morphological and functional characterization, we determined that ,80% of retested secondary lines showed the reported structural (bouton number) and functional (EJC) pheno- types consistent with primary screen (Table S2). These primary and secondary RNAi screen results now represent a resource for the systematic characterization of glycan mechanisms underlying synaptic structural and functional development. Screen results were further studied by comparing synaptogenesis phenotypes of RNAi knockdown with defined genetic nulls for two genes, CG6725 and CG4451, from the glycosaminoglycan biosynthesis class (Figure 1). The RNAi screen of functional strength as measured by EJC amplitudes indicated opposite effects for these two lines, with CG6725 (RNAi-sulf1) knockdown exhibiting an increase in transmission strength and CG4451 (RNAi-hs6st) knockdown producing a decrease (Figure 1). Along with our goal to identify interesting glycan-related genes involved in synapse development, we show here characterization of alleles of two genes obtained from screen results and define the associated mechanisms driving the bidirectional regulation of synaptic functional development. Synaptogenesis is bidirectionally regulated by paired sulf1 and hs6st genes The RNAi screen identified two functionally-paired genes, sulf1 (CG6725) and hs6st (CG4451), with similar effects on morpholog- ical development but opposite effects on synaptic functional differentiation (Figure 1). Our goal was to use these genes as a test case from the completed glycan screen, by assaying phenotypes in recently characterized mutants of both genes [42,43]. The gene products Sulfated (Sulf1), an HS 6-endosulfatase, and Hs6st, an HS 6-O-sulfotransferase, drive opposing changes in sulfation state of the same C6 carbon of the repeated glucosamine unit in GAG modified heparan sulfate proteoglycans [43,44]. Viable mutants are available for both genes, e.g. sulf1 (sulf1D1) and hs6st (hs6std770) [42,43], but requirements have never been assayed in the nervous system or neuromusculature. We therefore first compared phenotypes of RNAi knockdown and alleles at the NMJ synapse by confocal imaging of synaptic morphogenesis and TEVC recording of synaptic functional neurotransmission. Using double-labeling for HRP (presynaptic) and DLG (post- synaptic), NMJ structural parameters including bouton number, branch number and synaptic area were quantified in sulf1 and hs6st alleles. The mutant results closely recapitulated the RNAi knockdown findings from the screen (Table S1). To consistently compare RNAi and mutant conditions, both animal groups were simultaneously reared and processed to visualize the NMJ (Figure S1). Structural quantification showed an increased bouton number with RNAi-mediated sulf1 knockdown (sulf1-RNAi6UH1-GAL4; 36.461.6, n = 10) and hs6st knockdown (hs6st-RNAi6UH1-GAL4; 35.161.96, n = 10) compared to the transgenic control (w11186UH1-GAL4; 21.961.84, p,0.001, n = 10; Figure S1A, S1B). Consistently, increased bouton number was observed in both sulf1 (31.961.37, n = 10) and hs6st (36.2562.58, n = 8) mutants compared to genetic control (w1118, 19.361.69, p,0.001, n = 10; Figure S1C, S1D). In contrast, no significant change in branch number was exhibited with sulf1 knockdown (3.2260.28, p.0.05, n = 9) or hs6st knockdown (3.2260.22, p.0.05, n = 9) compared to control (w11186UH1-GAL4; 2.6460.06, n = 11). Similarly, no significant change was observed in the synaptic branch number in sulf1 (2.860.33, p = 0.27, n = 10,) and hs6st (3.6360.38, p = 0.115, n = 10) nulls compared to control (w1118; 3.460.46, n = 8). Further, there was no significant difference in synaptic area in sulf1 (138.1665.82, p.0.05, n = 10,) and hs6st (138.48613.38, p.0.05, n = 8,) mutants compared to the control (w1118; 118.0468,38, n = 10), however a slight increase in synaptic area was observed in sulf1 knockdown (178.68610.64, p,0.05, n = 9), while no change was observed for hs6st knockdown (16468.47, p.0.05, n = 10) as compared to control (w11186UH1-GAL4; 134.57611.95, n = 10). Based on these imaging studies, we conclude morphological differences in synaptic architecture observed in both sulf1 and hs6st allele conditions are consistent with both RNAi knockdown conditions. Functional development was next tested with electrophysiolog- ical recording to compare RNAi and mutant phenotypes PLOS Genetics | www.plosgenetics.org 4 November 2012 | Volume 8 | Issue 11 | e1003031