Author Summary Glycans are sugar additions to proteins. Surrounding all eukaryotic cells, secreted and membrane glycans form a glycocalyx that regulates cell–cell signaling. However, the mechanisms controlling glycan-dependent intercellular communication are largely unknown. In the nervous system, glycans play important roles in the development and regulation of synapses mediating intercellular com- munication. The Drosophila neuromuscular junction serves as a genetically tractable synapse in which expression of glycan-related genes can be systematically knocked down to investigate effects on synaptic morphology and function. This study employs a transgenic RNAi screen to characterize the synaptic requirements of 130 glycan- related genes. From this screen, two functionally paired genes (hs6st and sulf1) that add or remove a sulfate at the 6-O position on heparan sulfate proteoglycans (HSPGs) were identified as being critically important for synaptic functional development. Removal of each gene produces an opposite effect on neurotransmission strength, weak- ening and strengthening communication, respectively. This mechanism controls the synaptic expression of two HSPGs, which act as co-receptors to control the abundance of anterograde WNT and retrograde BMP signals, which drive intracellular signal transduction pathways regulating gene transcription to control synaptic functional develop- ment. This screen serves as a platform for systematic investigation of glycan mechanisms regulating synaptic development. knockdown of genes in all eight categories affects synaptic morphological development, with gene-specific effects on branch- ing, bouton differentiation and synapse area. Likewise, all eight categories regulate synaptic functional development, with gene- specific effects both weakening and strengthening neurotransmis- sion. Interestingly, only a few genes affect both structure and function, suggesting separable roles for glycans in regulating these synaptogenic pathways. The results of this genomic transgenic screen are presented as a platform from which to pursue systematic investigation of glycan mechanisms in synaptic development. Two genes were selected for screen validation and mechanistic characterization; functionally-paired HS 6-O-endosulfatase (sulf1) and HS 6-O-sulfotransferase (hs6st). RNAi knockdown and mutants identically alter synaptic functional development in a bidirectional manner; loss of sulf1 elevates neurotransmission strength, whereas loss of hs6st weakens it. Heparan sulfate proteoglycan (HSPG) targets Dally-like Protein (Dlp) and Syndecan (Sdc) [19,20] are mislocalized in sulf1 and hs6st synapses. In other developmental contexts, the sulfation state of these HSPG co-receptors strongly regulates WNT and BMP intercellular signaling [20,21,22]. At Drosophila synapses, WNT (Wg) is a key anterograde [23,24] and BMP (Gbb) a key retrograde [25,26] trans-synaptic signal. Consistently, loss of sulf1 and hs6st differentially changes synaptomatrix levels of Wg and Gbb, and downstream signaling into muscle and motor neuron nuclei, respectively. Glutamate release and receptor machinery is thereby bidirectionally altered in the two nulls. Genetic restoration of Wg/ Gbb signaling to control levels restores the bidirectional changes in synaptic functional strength and pre-/post- synaptic differentiation in both sulf1 and hs6st nulls. We conclude that extracellular HSPG sulfation state in the synaptomatrix is a point of intersection between WNT/BMP trans-synaptic signaling pathways that drive functional development of the neuromuscular synapse. HSPG Regulates Trans-Synaptic Signaling Results RNAi screen of glycan-related genes identifies multiple synaptogenesis defects Synaptic glycans play important roles as ligands, modulators and co-receptors regulating cell-matrix and intercellular commu- nication [3,27,28]. Differential glycan distribution on pre- and postsynaptic surfaces, and in the cleft, of numerous protein classes, strongly suggests that glycan mechanisms mediate synaptic structural and functional development [29,30,31]. To test the genomic scope of this requirement, we used confocal imaging and electrophysiological recording at the well-characterized Drosophila glutamatergic neuromuscular junction (NMJ) [32,33,34] to screen the Vienna Drosophila RNAi Center (VDRC) library of glycan- related genes [35]. We induced UAS-RNAi knockdown using the ubiquitous UH1-GAL4 driver [15,36]. We assayed morphological defects by co-labeling for pre- and postsynaptic markers, and assayed functional defects with two-electrode voltage clamp (TEVC) recording of neurotransmission strength. A summary of the screen results is shown in Figure 1. Full numerical results of the screen are shown in Table S1. Candidate glycan-related genes were identified and classified into eight functional categories using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [37] (Figure 1). Additional genes were added to the screen based on ortholog identification using the Information Hyperlinked over Proteins (iHOP) database [38]. The candidate gene list was expanded and verified using Flybase [39]. From this list, genes were cross-referenced with available VDRC UAS-RNAi transgenic lines to generate a final candidate list containing 130 genes within eight functionally- defined categories (Figure 1): N-glycan, O-glycan and glycosami- noglycan (GAG) biosynthesis; glycan core proteins (HSPG core proteins/glycoproteins); sugar transporters; glycosyltranferases; glycan modification genes (modification and degradation of glycans); and glycan-binding lectins. On genetic knockdown, 103 lines were viable until the wandering 3rd instar, whereas 27 lines showed developmental lethality at embryonic and early larval stages of development. From the 103 genetic lines characterized by confocal microscopy and TEVC electrophysiology in the 3rd instar (Figure 1), 21 exhibited pupal stage developmental lethality. Interestingly, .50% of pupal lethal lines displayed statistically significant defects in NMJ synaptic morphology and function. For all 103 larval-viable lines, synapse morphology and function was quantified at the wandering 3rd instar NMJ (Figure 1; Table S1). Each UAS-RNAi line driven by UH1-GAL4 in the w1118 background was compared to the genetic control of w1118 crossed to UH1-GAL4 (UH1-GAL46w1118) [35]. All morphological and functional assays were done blind to genotype, with values reported as fold-change compared to genetic control, as well as statistical significance calculated using one-way ANOVA analyses (see color scheme; P,0.05 (*), P,0.01 (**); Figure 1). The data represents $6 NMJs from $3 animals from every genotype. Synapse morphology was imaged by co-labeling with presynaptic marker anti-horse radish peroxidase (HRP) and postsynaptic marker anti-Discs Large (DLG). A synaptic bouton was defined as a varicosity of $2 mm in minimum diameter labeled by both HRP and DLG, and a synaptic branch was defined as a process containing at least two boutons [40]. NMJ branch number was the least affected morphological parameter, with only 2 of 103 genes showing a statistically significant change (Figure 1). Many more genes were involved in bouton development. All 27 genes showing a statistically significant change compared to genetic control exhibited elevated bouton numbers (Figure 1), suggesting that glycan mechanisms primarily limit morphological growth. Synapse PLOS Genetics | www.plosgenetics.org 2 November 2012 | Volume 8 | Issue 11 | e1003031