[25,26,87]. Mutation of Gbb ligand, receptors or regulators of this pathway have shown that Gbb-mediated retrograde signaling is required for proper synaptic differentiation and functional development [25,52,61,86,88]. Further, loss of Gbb signaling results in significantly decreased levels of P-Mad in the motor neurons [25]. We show here that accumulation of Gbb in sulf1 and hs6st synapses causes elevated P-Mad signaling at the synapse and P-Mad accumulation in motor neuron nuclei. Importantly, sulf1 synapses show a significantly higher level of P-Mad signaling compared to hs6st synapses, and this same change is proportionally found in P-Mad accumulation within the motor neuron nuclei. These findings indicate differential activation of Gbb trans-synaptic signaling dependent on the HS sulfation state is controlled by the sulf1 and hs6st mechanism, similar to the differential effect observed on Wg trans-synaptic signaling. Our genetic interaction studies show that these differential effects on trans-synaptic signaling have functional consequences, and exert a causative action on the observed bi-directional functional differ- entiation phenotypes in sulf1 and hs6st nulls. Genetic correction of Wg and Gbb defects in the sulf1 background restores elevated transmission back to control levels. Similarly, genetic correction of Wg and Gbb in hs6st nulls restores the decreased transmission strength back to control levels. These results demonstrate that the Wg and Gbb trans-synaptic signaling pathways are differentially regulated and, in combination, induce opposite effects on synaptic differentiation. Both wg and gbb pathway mutants display disorganized and mislocalized presynaptic components at the active zone (e.g. Bruchpilot; Brp) and postsynaptic components including gluta- mate receptors (e.g. Bad reception; Brec/GluRIID) [23,86,89]. Consistently, the bi-directional effects on neurotransmission strength in sulf1 and hs6st mutants are paralleled by dysregulation of these same synaptic components. Changes in presynaptic Brp and postsynaptic GluR abundance/distribution causally explain the bi-directional effects on synaptic functional strength between sulf1 and hs6st mutant states. Alterations in active zone Brp and postsynaptic GluRs also agree with assessment of spontaneous synaptic activity. Null sulf1 and hs6st synapses showed opposite effects on miniature evoked junctional current (mEJC) frequency (presynaptic component) and amplitude (postsynaptic component). Further, quantal content measurements also support the observa- tion of bidirectional synaptic function in the two functionally paired nulls. Genetic correction of Wg and Gbb defects in both sulf1 and hs6st nulls restores the molecular composition of the pre- and postsynaptic compartments back to wildtype levels. When both trans-synaptic signaling pathways are considered together, these data suggest that HSPG sulfate modification under the control of functionally-paired sulf1 and hs6st jointly regulates both WNT and BMP trans-synaptic signaling pathways in a differential manner to modulate synaptic functional development on both sides of the cleft. We present here the first systematic investigation of glycan roles in the modulation of synaptic structural and functional develop- ment. We have identified a host of glycan-related genes that are important for modulating neuromuscular synaptogenesis, and these genes are now available for future investigations, to determine mechanistic requirements at the synapse, and to explore links to neurological disorders. As proof for the utilization of these screen results, this study has identified extracellular heparan sulfate modification as a critical platform of the intersection for two secreted trans-synaptic signals, and differential control of their downstream signaling pathways that drive synaptic development. Other trans-synaptic signaling pathways are inde- pendent and unaffected by this mechanism, although it is of course HSPG Regulates Trans-Synaptic Signaling possible that a larger assortment of signals could be modulated by this or similar mechanisms. This study supports the core hypothesis that the extracellular space of the synaptic interface, the heavily-glycosylated synaptomatrix, forms a domain where glycans coordinately mediate regulation of trans-synaptic pathways to modulate synaptogenesis and subsequent functional maturation. Materials and Methods Drosophila stocks and genetics The glycan-related gene collection was generated using the KEGG glycan databases and Flybase annotation. The 163 UAS- RNAi lines tested were obtained from the Vienna Drosophila RNAi Center (VDRC) and Harvard TriP collection. Transgenic UAS- RNAi males were crossed to GAL4 driver females, with progeny raised at 25uC on standard food, controlling for density (3 R crossed to 2 =). The UH1-GAL4 driver was used for ubiquitous knockdown of target gene expression [15]. Neural specific elav- GAL4 [90], muscle specific 24B-GAL4 [91] and glia specific repo- GAL4 lines [92] from Bloomington stock center were used to assay cell-targeted knockdown. The two sulf1 alleles used were sulf1D1 [42] and sulf1DP1 [43]. The two hs6st alleles used were hs6std770 and the deficiency Df(3R)ED6027 [93]. The wg allele wgI-12 [94] and gbb alleles gbb1 and gbb2 were used [25,87]. Multiply mutant animals were made using standard genetic crosses. The trol-GFP line was obtained from Flytrap [76]. Antibody production We generated a rabbit polyclonal anti-Gbb antibody using a 1:1 combination of two Gbb-specific peptides (SHHRSKRSASHP, NDENVNLKKYRNMIVKSC) corresponding to amino acids 319–330 and 435–452 of Gbb (Young-In Frontier, Seoul, Korea). The antibody was purified by Protein A affinity chromatography, and antibody specificity demonstrated by examining immunore- activity in the wandering third instar neuromusculature with gbb mutants and by expressing UAS-gbb9.1 under the control of the muscle driver BG57-GAL4 (Figure S11). Immunoreactivity in the wandering third instar neuromusculature was severely reduced in a strong hypomorphic gbb allele (gbb1/gbb2, UAS-gbb9.9), which has leaky expression of UAS-gbb9.9 in a allelic combination [25,87,95]. In sharp contrast, the anti-Gbb signal was strongly elevated in BG57-GAL4/UAS-gbb9.1 relative to wildtype larvae. Immunocytochemistry Wandering third instars were dissected in Ca2+-free saline and then immediately fixed in either 4% paraformaldehyde for 10 minutes (all labels except anti-Dlp) or Bouin’s fixative for 30 mins (anti-Dlp). Preparations were then washed in permeabi- lizing PBST (PBS+0.1% Triton-X) or detergent-free PBS for extracellular labeling only [16]. The following primary antibodies were used: rabbit or goat anti-HRP (1:250; Jackson ImmunoR- esearch Laboratories); mouse anti-DLG (4F3; 1:250; Develop- mental Studies Hybridoma Bank (DSHB)); mouse anti–Fasciclin II (1D4; 1:5; DSHB); mouse anti-Dlp (13G8, 1:5; DSHB) and rabbit anti-Syndecan (1:200) [96]; mouse anti-Wg (4D4; 1:2 DSHB) and rabbit anti-Gbb (1:100); rabbit anti-PcanV (1:1000) [97]; guinea pig anti-Jeb (1:100) [17]; rabbit anti-dFz2-C (1:500) and rabbit anti-dFz2-N (1:100) [57]; rabbit anti-Htl (1:100) [56]; rabbit anti- P-Mad (PS1; 1:1000) [60]; rabbit anti-GluRIID (1:500) [64] and mouse anti-BRP (1:100; DSHB). Primary antibodies were incubated at 4uC overnight. Alexa-conjugated secondary antibod- ies (Jackson ImmunoResearch Laboratories) were used at 1:250 dilutions for 2 hours at room temperature. Staining with propidium iodide (Sigma Aldrich) to visualize cell nuclei was PLOS Genetics | www.plosgenetics.org 17 November 2012 | Volume 8 | Issue 11 | e1003031