HSPG Regulates Trans-Synaptic Signaling Figure 8. Bi-directional effects of sulf1 and hs6st nulls on synaptic assembly. (A) Representative NMJ boutons from control (w1118), sulf1 and hs6st genotypes, labeled for postsynaptic Bad Reception (Brec) glutamate receptor IID subunit (GluRIID, green) and presynaptic active zone Bruchpilot (anti-nc82, red). Quantification of GluRIID mean fluorescent intensity ($18 animals per indicated genotype) (B), GluRIID field area ($40 boutons from $9 animals per indicated genotype) (C), and GluRIID punctae number per synaptic bouton ($40 boutons from $9 animals per indicated genotype) (D), all normalized to genetic control. (E) Representative mEJC traces from control (w1118), sulf1D1 and hs6std770 alleles. Quantified mean mEJC amplitude (nA) (F), mean mEJC frequency (Hz) (G) and mean quantal content (H), with genetic control levels indicated as a dotted red line in each case. Sample sizes $15 recordings per indicated genotype. Statistically significant differences calculated using student’s t-test or Mann-Whitney test for non-parametric data and indicated as, * p,0.05, ** p,0.01, *** p,0.001. Error bars indicate S.E.M. doi:10.1371/journal.pgen.1003031.g008 pathways controlling NMJ synaptic structural and functional development [16,41,72,73] are modulated by glycan mechanisms. Our screen showed CG6657 RNAi knockdown affects functional differentiation, consistent with reports that this gene regulates peripheral nervous system development [74]. The corroboration of our screen results with published reports underscores the utility of RNAi-mediated screening to identify glycan mechanisms, and supports use of our screen results for bioinformatic/meta-analysis to link observed phenotypes to neurophysiological/pathological disease states and to direct future glycan mechanism studies at the synapse. From our screen, the two functionally-paired genes sulf1 and hs6st were selected for further characterization. As in the RNAi screen, alleles of these two genes had opposite effects on synaptic functional differentiation but similar effects on synapse morphogenesis, validating the corresponding screen results. The two gene products have functionally-paired roles; Hs6st is a heparan sulfate (HS) 6-O-sulfotransferase [43], and Sulf1 is a HS 6-O-endosulfatase [75]. These activities control sulfation of the same C6 on the repeated glucosamine moiety in HS GAG chains found on heparan sulfate proteoglycans (HSPGs). At the Drosophila NMJ, two HSPGs are known to regulate synapse assembly; the GPI-anchored glypican Dally-like protein (Dlp), and the trans- membrane Syndecan (Sdc) [45]. In contrast, the secreted HSPG Perlecan (Trol) is not detectably enriched at the NMJ [76], and indeed appears to be selectively excluded from the perisynaptic domain. In other developmental contexts, the membrane HSPGs Dlp and Sdc are known to act as co-receptors for WNT and BMP ligands, regulating ligand abundance, presentation to cognate receptors and therefore signaling [20,48]. Importantly, the regulation of HSPG co-receptor abundance has been shown to be dependent on sulfation state mediated by extracellular sulfatases [77]. Consistently, we observed upregulation of Dlp and Sdc in sulf1 synapses, whereas Dlp was reduced in hs6st synapses. In the developing Drosophila wing disc, HSPG co- receptors increase levels of the Wg ligand due to extracellular stabilization [78], and the primary function of Dlp in this developmental context is to retain Wg at the cell surface [21]. Likewise, in developing Drosophila embryos, a significant fraction of Wg ligand is retained on the cell surfaces in a HSPG-dependent manner [79], with the HSPG acting as an extracellular co- receptor. Syndecan also modulates ligand-dependent activation of cell-surface receptors by acting as a co-receptor [19,20]. At the NMJ, regulation of both these HSPG co-receptors occurs in the closely juxtaposed region between presynaptic bouton and muscle subsynaptic reticulum, in the exact same extracellular space traversed by the secreted trans-synaptic Wg and Gbb signals [45]. We therefore proposed that altered Dlp and Sdc HSPG co- receptors in sulf1 and hs6st mutants differentially trap/stabilize Wg and Gbb trans-synaptic signals at the interface between motor neuron and muscle, to modulate the extent and efficacy of intercellular signaling driving synaptic development. HS sulfation modification is linked to modulating the intercel- lular signaling driving neuronal differentiation [80]. In particular, WNT and BMP ligands are both regulated via HS sulfation of their extracellular co-receptors, and both signals have multiple functions directing neuronal differentiation, including synaptogen- esis [49,50,51]. In the Drosophila wing disc, extracellular WNT (Wg) ligand abundance and distribution was recently shown to be strongly elevated in sulf1 mutants [22]. Moreover, sulf1 has also recently been shown to modulate BMP signaling in other cellular contexts [81]. Consistently, we have shown here increased WNT Wg and the BMP Gbb abundance and distribution in sulf1 NMJ synapses. The hs6st also exhibits elevated Wg and Gbb at the synaptic interface, albeit the increase is lower and results in differential signaling consequences. In support of this contrasting effect, extracellular signaling ligands are known to bind HSPG HS chains differentially dependent on specific sulfation patterns [82,83,84]. It is important to note that the sulf1 and hs6st modulation of trans-synaptic signals is not universal, as Jelly Belly (Jeb) ligand abundance and distribution was not altered in the sulf1 and hs6st conditions [17]. This indicates that discrete classes of secreted trans-synaptic molecules are modulated by distinct glycan mechanisms to control NMJ structure and function. At the Drosophila NMJ, Wg is very well characterized as an anterograde trans-synaptic signal [23,24,85] and Gbb is very well characterized as a retrograde trans-synaptic signal [25,26,50,86]. In Wg signaling, the dFz2 receptor is internalized upon Wg binding and then cleaved so that the dFz2-C fragment is imported into muscle nuclei [57,59,85]. In hs6st nulls, increased Wg ligand abundance at the synaptic terminal corresponds to an increase in dFz2C punctae in muscle nuclei as expected. In contrast, the increase in Wg at the sulf1 synapse did not correspond to an increase in the dFz2C-terminus nuclear internalization, but rather a significant decrease. One explanation for this apparent discrepancy is the ‘exchange factor’ model based on the biphasic ability of the HSPG co-receptor Dlp to modulate Wg signaling [48]. In the Drosophila wing disc, this model suggests that the transition of Dlp co-receptor from an activator to repressor of signaling depends on Wg cognate receptor dFz2 levels, such that a low ratio of Dlp:dFz2 potentiates Wg-dFz2 interaction, whereas a high ratio of Dlp:dFz2 prevents dFz2 from capturing Wg [48]. In sulf1 synapses, we observe a very great increase in Dlp abundance (,40% elevated) with no significant change in the dFz2 receptor. In contrast, at hs6st synapses there is a decrease in Dlp abundance (15% decreased) together with a significant increase in dFz2 receptor abundance (,25% elevated). Thus, the higher Dlp:dFz2 ratio in sulf1 nulls could explain the decrease in Wg signal activation, evidenced by decreased dFz2-C terminus import into the muscle nucleus. In contrast, the Dlp:Fz2 ratio in hs6st is much lower, supporting activation of the dFz2-C terminus nuclear internalization pathway. This previously proposed com- petitive binding mechanism dependent on Dlp co-receptor and dFz2 receptor ratios predicts the observed synaptic Wg signaling pathway modulation in sulf1 and hs6st dependent manner [48]. At the Drosophila NMJ, Gbb is very well characterized as a retrograde trans-synaptic signal, with muscle-derived Gbb causing the receptor complex Wishful thinking (Wit), Thickveins (Tkv) and Saxaphone (Sax) to induce phosphorylation of the transcription factor mothers against Mothers against decapentaplegic (P-Mad) PLOS Genetics | www.plosgenetics.org 16 November 2012 | Volume 8 | Issue 11 | e1003031