HSPG Regulates Trans-Synaptic Signaling homozygous sulf1D1 null, heterozygous wg/+ and gbb/+ in sulf1 background (wgI-12/gbb2; sulf1D1/sulf1D1), homozygous hs6std770 and heterozygous wg/+ and gbb/+ in hs6st background (wgI-12/gbb2; hs6std770/hs6std770). The nerve was stimulated (arrows) in 1.0 mM external Ca2+, and TEVC records (260 mV holding potential) made from muscle 6 in segment A3. Each trace was averaged from 10 consecutive evoked EJC recordings. (B) Quantified mean EJC amplitudes (nA) for the nine genotypes shown. Sample sizes are $7 animals per indicated genotype. Statistically significant differences calculated using student’s t-test, * p,0.05, ** p,0.01. Error bars indicate S.E.M. doi:10.1371/journal.pgen.1003031.g007 area per bouton and number of GluRIID punctae normalized to field area per synaptic bouton were also bidirectionally altered in the sulf1 and hs6st nulls (Figure 8C, 8D). GluRIID receptor field area was increased in sulf1 (,30% greater; p,0.01, n = 47) but decreased in hs6st (,25% reduced; p,0.01, n = 51). Conversely, measurements of GluRIID puncta normalized to field area per synaptic bouton were decreased in sulf1 (,15% lower; p,0.05, n = 47), but increased in hs6st nulls (,40% greater; p,0.01, n = 51, Figure 8D). The bi-directional differences between sulf1 and hs6st were very highly significant (p,0.001). The active zone protein Brp also showed opposite effects (Figure 8A). Although the difference between sulf1 and control was not quite significant (p.0.05, n = 20), hs6st synapses showed a very significant decrease in Brp compared to control (,20% reduced; p,0.01, n = 21; Figure 8A). Based on these results, we next tested pre- (Brp) and postsynaptic (Brec/GluRIID) changes in sulf1 and hs6st mutants with genetic reduction of Wg and Gbb (wg/gbb;sulf1/sulf1 and wg/ gbb;hs6st/hs6st), as in Figure 7. Distribution changes of both pre- and postsynaptic components were assayed as measurements of glutamate receptor field and active zone areas (Figure S10A). To measure glutamate receptor distribution comparing wg/gbb;sulf1/ sulf1 to matched control, we counted the number of GluRIID punctae per bouton (p = 0.73, n = 48; Figure S10B) and GluRIID area (p = 0.92, n = 48; Figure S10C), and found both corrected back to control levels. Likewise, for wg/gbb;hs6st/hs6st compared to control, GluRIID puncta number (p = 0.88, n = 48) and area (p = 0.41, n = 58) were both corrected to control levels. To measure Brp-positive presynaptic active zones comparing wg/ gbb;sulf1/sulf1 to matched control, we counted the number of Brp punctae per bouton (p = 0.43, n = 48; Figure S10D) and Brp area (p = 0.39, n = 48; Figure S10D), and found both corrected back to control levels. Likewise, for wg/gbb;hs6st/hs6st compared to control, Brp number (p = 0.54, n = 58) and area (p = 0.19, n = 58) were also corrected back to control levels. These results provide strong genetic evidence that Wg and Gbb trans-synaptic signaling changes are causative for the pre- and postsynaptic molecular differenti- ation defects in the sulf1 and hs6st mutants. These bidirectional pre- and postsynaptic molecular changes parallel functional transmission changes in sulf1 and hs6st mutants (Figure 2). To assay function at the single synapse level, we finally assayed spontaneous synaptic vesicle fusion events. Representative mEJC traces for control compared to sulf1 and hs6st nulls are shown in Figure 8E. Consistent with observed bidirectional changes in evoked transmission, mEJC amplitudes in hs6st were ,25% lower than in sulf1 nulls (hs6st, 0.6060.02 nA vs. sulf1, 0.7660.05 nA; p,0.5, n = 34; Figure 8F). Moreover, hs6st nulls had a ,100% elevated mEJC frequency compared to sulf1 nulls (hs6st, 2.5660.27 vs. sulf1, 1.3060.09; p,0.001, n = 34; Figure 8G). Based on these mEJC measurements, there was a highly significant bidirectional change in quantal content between the two mutant conditions, with sulf1 quantal content ,50% greater than hs6st (sulf1, 539.98622.02 vs. hs6st, 350.6968.92; p,0.001, n = 34; Figure 8H). Taken together, these results show a bi-directional change in presynaptic glutamate release machinery and vesicle fusion probability, as well as postsynaptic glutamate receptor levels and functional responsiveness. We conclude that these changes underlie the bi-directional switch in neurotransmis- sion strength characterizing sulf1 and hs6st mutants. Discussion It is well known that synaptic interfaces harbor heavily- glycosylated membrane proteins, glycolipids and ECM molecules, but understanding of glycan-mediated mechanisms within this synaptomatrix is limited [9]. Our genomic screen aimed to systematically interrogate glycan roles in both structural and functional development in the genetically-tractable Drosophila NMJ synapse. 130 candidate genes were screened, classified into 8 functional families: N-glycan biosynthesis, O-glycan biosynthesis, GAG biosynthesis, glycoprotein/proteoglycan core proteins, glycan modifying/degrading enzymes, glycosyltransferases, sugar transporters and glycan-binding lectins. From this screen, 103 RNAi knockdown conditions were larval viable, whereas 27 others produced early developmental lethality. 35 genes had statistically significant effects on different measures of morphological devel- opment: 27 RNAi-mediated knockdowns increased synaptic bouton number, 9 affected synapse area (2 increased, 7 decreased) and 2 genes increased synaptic branch number. These data suggest that overall glycan mechanisms predominantly serve to limit synaptic morphogenesis. 13 genes had significant effects on the functional differentiation of the synapse, with 12 increasing transmission strength and only 1 decreasing function upon RNAi knockdown. Thus, glycan-mediated mechanisms also predomi- nantly limit synaptic functional development. A very small fraction of tested genes (CG1597; pgant35A, CG7480; veg, CG6657; hs6st, CG4451; sulf1, CG6725 and CG11874) had effects on both morphology and function. A large percentage of genes (,30%) showed morphological defects with no corresponding effect on function, while only 7% of genes showed functional alterations without morphological defects, and ,5% of all genes affect both. These results suggest that glycans have clearly separable roles in modulating morphological and functional development of the NMJ synapse. A growing list of neurological disorders linked to the synapse are attributed to dysfunctional glycan mechanisms, including muscular dystrophies, cognitive impairment and autism spectrum disorders [65,66,67]. Drosophila homologs of glycosylation genes implicated in neural disease states include ALG3 (CG4084), ALG6 (CG5091), DPM1 (CG10166), FUCT1 (CG9620), GCS1 (CG1597), MGAT2 (CG7921), MPDU1 (CG3792), PMI (CG33718) and PPM2 (CG12151) [65]. Two of these genes, Gfr (CG9620) and CG1597, showed synaptic morphology phenotypes in our RNAi screen. Given that connectivity defects are clearly implicated in cognitive impairment and autism spectrum disorders [68,69], it would be of interest to explore the glycan mechanism affecting synapse morphology in Drosophila models of these disease states. Glycans are well known to modulate extracellular signaling, including ligands of integrin receptors, to regulate intercellular communication [70,71]. In our genetic screen, several O- glycosyltransferases mediating this mechanism were identified to show morphological (GalNAc-T2, CG6394; pgant35A, CG7480, O- fut2, CG14789; rumi, CG31152) and functional (pgant5, CG31651; pgant35A, CG7480) synaptic defects upon RNAi knockdown. These findings suggest that known integrin-mediated signaling PLOS Genetics | www.plosgenetics.org 14 November 2012 | Volume 8 | Issue 11 | e1003031