(Figure 2). Representative TEVC records are shown as an average of 10 consecutive nerve stimulus responses in 1.0 mM extracellular Ca2+ for each transgenic genotype in Figure 2A; sulf1 knockdown (UH1-GAL46sulf1-RNAi), hs6st knockdown (UH1-GAL46hs6st- RNAi) and genetic control (UH1-GAL46w1118). There was a striking ,80% difference in EJC amplitude between sulf1 and hs6st knockdown conditions, with sulf1 elevated by ,30% and hs6st reduced by ,30% compared to control. Quantification of EJC amplitudes showed both knockdown conditions to be highly significantly different from control and each other (control, 286.2268.56 nA; sulf1-RNAi, 365.0169.502 nA, p,0.001; hs6st-RNAi, 199.19611.84 nA, p,0.001; sulf1-RNAi vs. hs6st- RNAi, p,0.001; Figure 2B). These opposite effects on neuro- transmission strength were confirmed in characterized alleles for both genes [42,43]. Representative traces from sulf1D1 and hs6std770 mutants compared to w1118 control are shown in Figure 2C. Quantification of EJC amplitudes showed mutants to be highly significantly different from control and each other (w1118, 256.1467.38 nA; sulf1D1, 372.86618.49 nA, n = 11, p,0.001; hs6st, 209.66613.44 nA, n = 14, p,0.01; sulf1D1 vs. hs6st, p,0.001; Figure 2D). These results were confirmed in an independent sulf1 allele (sulf1DP1), which shows comparable elevation compared to control (w1118, 244.9169.04 nA; sulf1DP1, 282.28613.59, p,0.05, n = 22), as well as the hs6st (hs6std770) over deficiency (Df(3R)ED6027), which shows comparable de- pression compared to control (w1118, 256.1467.38 nA; hs6st/ Df(3R)ED6027, 224.0667.65 nA, p,0.05, n = 18). These results reveal a critical role for sulf1 and hs6st genes in synaptic functional development. Given the functionally-paired nature of sulf1 and hs6st activities on 6-O-S modification, and the epistatic function of hs6st to sulf1, we predicted that knocking both genes down would produce a phenotype similar to knockdown of hs6st alone. Consistently, hs6st and sulf1 double knockdown produced EJC amplitudes significantly lower than control (w11186hs6st-RNAi; sulf1-RNAi (control), 225.1766.28 nA, n = 12; hs6st-RNAi, sulf1-RNAi6UH1-GAL4, 198.2269.77 nA, n = 15, p,0.05; Figure S2). Cell-specific knock- down in neural (elav-GAL4), muscle (24B-GAL4) and glia (repo- GAL4) also support the observed opposite effects in neurotransmis- sion strength. With sulf1 knockdown in muscle, EJC amplitude was significantly elevated compared to control (w11186sulf1-RNAi (control), 199.97621.86 nA; 24B-GAL46sulf1-RNAi (knockdown), 222.88625.78 nA, p,0.01, n = 10), but no change occurred with neural knockdown (elav-GAL46sulf1-RNAi, 196.09625.08 nA, p = 0.72, n = 10) or glial knockdown (repo-GAL46sulf1-RNAi, 208.40632.45 nA, p = 0.53, n = 7). Moreover, only neural knock- down of hs6st caused a decrease in EJC amplitude (w11186hs6st- RNAi (control), 211.496622.142 nA, elav-GAL46hs6st-RNAi (knockdown), 184.68628.97 nA, p,0.05, n = 16), while no change occurred with muscle knockdown (24B-GAL46hs6st-RNAi, 209.92624.74 nA, p = 0.88, n = 9) or glial knockdown (repo- GAL46hs6st-RNAi, 216.38637.80 nA, p = 0.32, n = 7). We con- clude that HSPG sulfation state strongly modulates NMJ functional development, with contributions from both motor neuron and muscle, but not glia. The clear next step was to test for differences in the localization and abundance of synaptic HSPG targets known to regulate NMJ synaptogenesis. HSPG abundance at the synaptic interface is dependent on sulf1 and hs6st Both GPI-anchored HSPG glypican Dally-like (Dlp) and transmembrane HSPG Syndecan (Sdc) are clearly expressed at the Drosophila NMJ (Figure S3), where they are known to regulate synaptogenesis [45]. We detect no enrichment of the secreted HSPG Regulates Trans-Synaptic Signaling HSPG perelcan (Trol) at the NMJ, although it is abundantly expressed in the motor nerve leading up to the synaptic terminal and present in lower levels throughout the muscle (Figure S4). We therefore hypothesized that membrane-associated Dlp and Sdc HSPGs are targeted by sulf1 and hs6st activity to regulate their synaptic distribution and/or function. To test this hypothesis, we assayed both Dlp and Sdc under non-permeabilized, detergent- free conditions to examine their cell surface expression at the NMJ synaptic interface of sulf1 and hs6st mutants compared to control. These data are summarized in Figure 3. In the genetic background control (w1118), Dlp shows a punctate expression pattern strongly concentrated in a halo-like array around the anti-HRP labeled presynaptic membrane (Figure 3A, top; Figure S3). In sulf1 mutants there was a clear and consistent increase in Dlp abundance, with more numerous and intense punctae at the synaptic interface surrounding NMJ boutons, while at hs6st mutant synapses there was an opposing decrease in Dlp abundance (Figure 3A). This bidirectional and differential effect on Dlp abundance was quantified as fluorescence intensity normalized to the internal HRP labeling control. There was a significant Dlp increase in sulf1 compared to control (,40% elevated over control; p,0.05; n = 11), and a significant Dlp decrease in the hs6st synapse (,15% reduced compared to control; p,0.05; n = 11; Figure 3B). Importantly, the difference between sulf1 and hs6st nulls was very highly significant (p,0.001). In comparison, cell surface Sdc labeling also showed a dense halo- like localization around NMJ synaptic boutons labeled with cell adhesion marker Fasciclin II (FasII; Figure 3C; Figure S3). Synaptic Sdc labeling intensity was consistently greater in both sulf1 and hs6st nulls compared to control (Figure 3C). Quantifi- cation of fluorescence intensity normalized to HRP revealed that Sdc abundance was greatly increased in sulf1 synapses compared to control (,35% elevated over control; p,0.01; n = 17) and, to a greater degree, also in hs6st nulls (,50% elevated over control; p,0.001; n = 12; Figure 3D). Thus, both Dlp and Sdc HSPGs are strongly altered in sulf1 and hs6st NMJ synapses, with Dlp bidirectionally misregulated and Sdc differen- tially elevated in the two mutant conditions. HSPGs act as co-receptors for WNT and BMP intercellular signaling ligands in many developmental contexts, acting to modulate extracellular ligand abundance and downstream signal- ing [46,47]. Drosophila WNT Wingless (Wg) distribution and signaling is known to be modulated by Dlp, which retains Wg at the cell surface in a mechanism that is enhanced by HS GAG chains [48]. Specifically, Wg ligand abundance and signaling activity along the dorso-ventral axis of the developing Drosophila wing disc is elevated in sulf1 mutants [22]. Likewise, BMP ligands in other cellular contexts are closely regulated by HSPG co- receptors [20]. Specifically, Dlp has been suggested to similarly regulate Drosophila BMP Glass Bottom Boat (Gbb) [20]. We therefore hypothesized that altered HSPG co-receptors Dlp and/ or Sdc in sulf1 and hs6st synapses regulate Wg and Gbb abundance to drive differentially altered trans-synaptic signaling across the synaptic cleft. HSPG sulfation regulates abundance of WNT/BMP trans- synaptic ligands Classical WNT and BMP morphogens act locally at synapses to fine tune synaptogenesis [49,50]. At the Drosophila NMJ, the WNT Wg is well-characterized as an anterograde trans-synaptic signal modulating synaptogenesis [23,24,51]. Similarly, the BMP Gbb is well-characterized as a retrograde signal driving synaptic devel- opment [25,26,52]. A third trans-synaptic signaling pathway, presynaptically-secreted Jelly Belly (Jeb) to postsynaptic Alk PLOS Genetics | www.plosgenetics.org 5 November 2012 | Volume 8 | Issue 11 | e1003031