HSPG Regulates Trans-Synaptic Signaling Figure 3. Synaptic HSPG co-receptor abundance is modified by 6-O-S sulfation. (A) Representative NMJ synaptic boutons imaged from control (w1118), sulf1 and hs6st nulls, probed with presynaptic neural marker anti-HRP (green) and Dally-like (Dlp; red). Right: Dlp distribution without the HRP signal is shown for clarity. (B) Quantification of mean fluorescent intensity levels of anti-Dlp labeling normalized to the HRP co-label at the muscle 6 NMJ, normalized to genetic control. (C) Boutons labeled with neural marker anti-Fasciclin II (FasII, green) and anti-Syndecan (Sdc, red). Right: Sdc distribution is shown alone for clarity. (D) Quantification of the mean fluorescent intensity levels of anti-Sdc labeling at the muscle 6 NMJ, normalized to genetic control. Sample sizes are at least 12 independent NMJs of at least 7 animals per indicated genotypes. Statistically significant differences calculated using student’s t-test, * p,0.01, ** p,0.01, ***p,0.001. Error bars indicate S.E.M. doi:10.1371/journal.pgen.1003031.g003 receptor [17], has no known interaction with HSPGs and therefore would not be expected to be affected in sulf1 and hs6st nulls, providing a comparison for specificity. To test the hypothesis that the observed alterations of HSPG co-receptor abundance will drive specific changes in WNT and BMP intercellular pathways, we labeled NMJ synapses with antibodies under non-permeablized conditions to reveal extracellular trans-synaptic signaling ligands (Figure S5), and compared protein abundance and distribution in controls, sulf1 and hs6st mutants. The data are summarized in Figure 4. NMJ synapses were first labeled with Wg antibody (green) together with anti-HRP (red) to label the presynaptic membrane (Figure 4A). In control animals (w1118), external Wg localized at large type Ib synaptic boutons in a dynamic pattern of punctuate distribution at the synaptic interface between motor neuron and muscle (Figure 4A, top; Figure S5). In sulf1 and hs6st mutants, Wg was consistently elevated and concentrated uniformly in the extracellular domain adjacent to, and overlapping with, the anti- HRP-labeled presynaptic membrane (Figure 4A, middle and bottom). The elevated Wg levels in mutants were clearly observed at the level of individual synaptic boutons, as shown in the magnified insets in Figure 4A. To examine changes in Wg spatial distribution, cross-sectional planes were examined in single confocal line scans through the diameter of individual synaptic boutons (Figure 4A, white lines). Representative distribution plots for membrane-marker HRP (red) and external Wg (green) are shown in Figure 4B. In all genotypes, extracellular Wg was closely associated with the HRP-labeled presynaptic membrane, but both sulf1 and hs6st nulls displayed a consistent increase in Wg label intensity and broadening of the spatial domain occupied by the secreted Wg ligand (Figure 4B, middle and bottom). To quantify changes in extracellular Wg abundance, the mean fluorescent signal intensity was normalized to the internal HRP co-label, and then normalized to analogous control intensity ratios. In sulf1D1 nulls, there was very highly significant elevation of Wg compared to control (,90% increased; p,0.001; n = 16; Figure 4C). A similar increase was observed in the independent sulf1DP1 (p,0.001; n = 11). The hs6st displayed a smaller significant increase in Wg abundance (,40% increased; p,0.001; n = 15; Figure 4C), which was again recapitulated in hs6st over deficiency (Df(3R)ED6027) condition. Importantly, Wg abun- dance is differentially elevated in sulf1 vs. hs6st mutants (p,0.01, Figure 4C). To test whether the sulf1/hs6st mechanism might coordinately regulate multiple trans-synaptic signals, we next assayed the BMP Gbb, a muscle-derived retrograde signal [25]. A barrier to previous Gbb analyses has been the absence of an anti-Gbb antibody. We therefore generated a specific anti-Gbb antibody for this study (see Methods). As above, labeling was done under non- permeabilized conditions to reveal only the extracellular Gbb, together with labeling for HRP or the cell adhesion molecule marker FasII to reveal the presynaptic membrane (Figure S5). In the control (w1118), extracellular Gbb concentrated in a ring of punctate domains around boutons (Figure 4D, top). Gbb was similarly punctate in sulf1 and hs6st nulls, but consistently more extensive and denser (Figure 4D, middle and bottom; see magnified insets). To examine Gbb spatial distribution, cross- sectional planes of confocal line scans were made through individual synaptic boutons (Figure 4D, white lines). Representa- tive plots for FasII (green) and Gbb (red) show extracellular Gbb closely associated with the FasII-labeled presynaptic membrane in all genotypes (Figure 4E). However, sulf1 and hs6st nulls consistently displayed increased Gbb intensity and broadened expression compared to the control. Upon quantifying signal intensity of Gbb normalized to HRP co-label, sulf1D1 exhibited a significantly higher Gbb abundance than control (65% increased; p,0.01; n = 12; Figure 4F). The independent sulf1DP1 allele showed a similar increase (p,0.001; n = 12). The hs6st also showed Gbb elevation compared to control (59% increased; p,0.01; n = 11; Figure 4E), which was confirmed in hs6st over deficiency (Df(3R)ED6027; p,0.05; n = 23). To test further whether extracellular Wg and Gbb abundance was sensitive to the sulfation state of GAGs, a biochemical approach was next used to determine effects on Wg and Gbb trans- synaptic signals (Figure S6). Specifically, NMJs were acutely exposed to heparin, the most sulfated form of GAG [53], and then synaptic Wg and Gbb abundance was measured by immunolabel- ing as above. We found that both trans-synaptic signals were rapidly altered by heparin incubation in a dose-dependent manner. Specifically, incubation with increasing concentrations of heparin caused a reciprocal decrease in Wg labeling intensity in the NMJ synaptic domain (Figure S6A, S6C), with a significant decrease first detected with 0.315 mg/ml heparin incubation (,50% less than control, p,0.01, n = 4). Interestingly, increasing heparin concentrations caused a parallel increase in Gbb abundance in the NMJ synaptic domain (Figure S6B, S6C) in a dose-dependent manner, with significant increases again first detected at 0.315 mg/ml heparin (,25% greater than control, p,0.05) and rising further at 0.625 mg/ml heparin (,40% greater than control, p,0.001). These results indicate that HSPG sulfation state does indeed affect trans-synaptic signal abundance, supporting the observed alterations in Wg and Gbb abundance in mutants of heparan sulfate modifying genes, sulf1 and hs6st. To examine effects on other trans-synaptic signaling pathways in the sulf1 and hs6st mutant synapses, we also assayed for changes in Jeb [17] and FGF [17] signaling. In both control and mutants, extracellular Jeb labeling was tightly associated with NMJ type Ib boutons and, like other trans-synaptic ligands, occupied an extracellular domain closely associated with the presynaptic membrane (Figure S7A). However, in stark contrast to Wg and Gbb ligands in the same extracellular synaptomatrix domain, no change was observed in Jeb abundance or spatial distribution in sulf1 (p = 0.99, n = 10) or hs6st (p = 0.36, n = 8) compared to control (w1118) NMJ synapses (Figure S7B). FGF signaling is also well established to be affected by HSPGs [54], and one pioneering study has investigated roles for FGF signaling at the Drosophila NMJ [55]. The probe used in the previous study was an antibody against the FGF receptor Heartless (Htl) [56]. Using this antibody, we confirmed that the Htl receptor beautifully localizes to NMJ boutons to mediate FGF signaling (Figure S8A). However, Htl receptor synaptic abundance and distribution was very similar for the sulf1 (p = 0.89, n = 9) and hs6st (p = 0.69, n = 7) mutants PLOS Genetics | www.plosgenetics.org 8 November 2012 | Volume 8 | Issue 11 | e1003031