HSPG Regulates Trans-Synaptic Signaling measurements. Insets indicate single synaptic boutons at higher magnification. (A) Representative NMJ boutons from control (w1118), sulf1 and hs6st genotypes, labeled for presynaptic anti-horseradish peroxidase (HRP, red) and anti-wingless (Wg, green). (B) Extracellular distribution of Wg across the diameter of a synaptic bouton. The Y-axis indicates intensity and the X-axis shows distance in microns. The HRP intensity profile is indicated in red; Wg intensity is shown in green. (C) Quantification of Wg mean intensity levels normalized to the HRP co-label, and to genetic control. Sample sizes are at least 15 animals per indicated genotypes. (D) Representative synaptic boutons labeled with presynaptic anti-Fasciclin II (FasII; green) and anti-Glass Bottom Boat (Gbb; red). (E) Gbb distribution across the diameter of a synaptic bouton. Y-axis indicates intensity and the X-axis shows distance in microns. FasII intensity profile is indicated in green; Gbb intensity is shown in red. (F) Quantification of Gbb mean intensity levels normalized to genetic control. Sample sizes are at least 11 independent NMJs of at least 7 animals per indicated genotypes. Statistically significant differences calculated using student’s t-test and Mann-Whitney test for non-parametric data, ** p,0.01, *** p,0.001. Error bars indicate S.E.M. doi:10.1371/journal.pgen.1003031.g004 compared to control (w1118) (Figure S8B). Unfortunately, no antibody probes are available for Drosophila FGF ligands, so these signals have not yet been queried. Together, these results show that both WNT (Wg) and BMP (Gbb) ligand abundance is coordinately upregulated by the sulf1 and hs6st mechanism at the NMJ synapse, but that a spatially overlapping signaling ligand (Jeb) and at least FGF receptor expression are unaffected. These results strongly predict that Wg and Gbb trans-synaptic signaling controlled by sulf1 and hs6st activity regulates synaptic functional development. Trans-synaptic WNT/BMP signaling is regulated by HSPG sulfation Wg and Gbb serve as anterograde and retrograde trans-synaptic signals, respectively, activating cognate receptors to initiate downstream signaling cascades and nuclear import pathways in muscles and motor neurons, respectively [24,26,50,51]. The anterograde Wg signal drives dFrizzled-2 (dFz2) receptor inter- nalization in the postsynaptic domain followed by cleavage of the receptor C-terminus, which then enters the muscle nuclei [57]. The muscle-derived retrograde Gbb signal activates presynaptic receptors to drive phosphorylation of the Mothers Against Decapentaplegic (Mad) transcription factor, and then P-Mad enters the motor neuron nuclei to regulate transcription [25,26,58]. Given the differential change in both HSPG co- receptor and Wg/Gbb ligand abundance in sulf1 vs. hs6st mutants, we hypothesized that these signaling pathways would be differen- tially affected during synaptogenesis. We therefore quantitatively assayed the paired muscle and motor neuron nuclear import pathways to determine whether and how trans-synaptic signaling may be modulated by sulf1 and hs6st at the NMJ synapse. Characterized antibodies specifically recognizing the N- and C- termini of the Wg dFz2 receptor allow measurements of the receptor at the NMJ synapse (dFz2N; Figure S9) and the cleaved fragment (dFz2C; Figure 5) imported into muscle nuclei [57,59]. We first assayed dFz2 receptor abundance at the NMJ with the N- terminal specific antibody. The dFz2 receptor is closely associated with the synaptic cell membrane marker FasII and occupies a domain that envelopes all type Ib boutons (Figure S9A). In hs6st nulls, the dFz2 receptor domain was spatially extended as compared to controls, however sulf1 alleles showed no detectable change in the receptor. Likewise, fluorescence intensity measure- ments showed no significant difference between control and sulf1 nulls, but hs6st synapses displayed a ,25% increase in dFz2 receptor abundance, a very significant elevation (p,0.01, n = 12; Figure S9B) in synaptic dFz2 abundance. Thus, importantly (see Discussion), significantly more dFz2 receptors occur in the hs6st compared to sulf1 synapse. To assay downstream signal transduction, the cleaved Fz2C fragment imported into muscle nuclei was quantified using the established method of counting dFz2C-positive punctae in nuclei proximal to the NMJ (Figure 5) [59]. In genetic control (w1118), most muscle nuclei contained a small number (1–3) of detectable dFz2C punctae, but some nuclei contained more and others were devoid of detectable dFz2C (Figure 5A, top). More than 100 muscle nuclei were quantified in .7 different animals to determine the control level of dFz2C nuclear import. In sulf1 and hs6st mutants, there was a clear and consistent bidirectional difference in the number and size of dFz2C punctae in muscle nuclei (Figure 5A, middle and bottom). Null sulf1 nuclei showed a highly significant decrease in number of dFz2C punctae per nuclei (.50% decreased; p,0.01; n = 163; Figure 5B). In contrast, hs6st nulls had an opposing highly significant increase in dFz2C punctae per nuclei (.60% increased; p,0.01; n = 163; Figure 5B). The difference between sulf1 and hs6st mutants was very highly significant (p,0.001), with a differential change in signaling paralleling the bidirectional change in synaptic functional differ- entiation (Figure 2). A characterized antibody specifically recognizing phosphorylated Mad (P-Mad) allowed independent measurements of Gbb signaling in the presynaptic terminal and P-Mad import into the motor neuron nuclei as a transcriptional regulator (Figure 6) [25,60]. To assay this transduction pathway, P-Mad fluorescent intensity normalized to FasII was first assayed in presynaptic boutons [61,62]. In the genetic control (w1118), P-Mad labeling was bounded by the synaptic cell adhesion molecule marker FasII, with P-Mad localized in numerous punctate domains (Figure 6A, arrows). In sulf1 and hs6st nulls, both the intensity and size of P-Mad positive punctae were obviously and consistently greater than in controls (Figure 6A, middle and bottom). In fluorescence intensity quanti- fication, sulf1 synapses displayed a significant increase in synaptic P-Mad (45% increased; p,0.05; n = 10; Figure 6C). An increase in P-Mad was also observed in the hs6st boutons (42% greater than control; p,0.01; n = 15; Figure 6C). The motor neuron nuclei at the ventral nerve cord (VNC) midline accumulate P-Mad transcription factor downstream of Gbb signaling at the NMJ [25,61,62]. In genetic control (w1118), P-Mad nuclear labeling was consistently detected in these motor neuron nuclei (Figure 6B, arrows). A similar P-Mad distribution was observed in motor neuron nuclei of sulf1 and hs6st nulls, but the intensity of P-mad expression was clearly and consistently elevated in both mutants compared to control (Figure 6B, middle and bottom). In fluores- cence intensity quantification, sulf1 neuronal nuclei displayed a very significant increase in P-Mad accumulation (15% increased; p,0.01; n = 14; Figure 6D), paralleling increased P-Mad signaling at the NMJ (Figure 6C). Likewise, hs6st motoneuron nuclei exhibited a smaller but still significant elevation in P-Mad accumulation (9% elevated over control; p,0.05; n = 21; Figure 6D), again paralleling the observed P-Mad signaling change at the NMJ (Figure 6C). We conclude that both anterograde WNT (Wg) and retrograde BMP (Gbb) trans-synaptic signaling in muscle and motor neuron nuclei, respectively, is differentially regulated by the sulf1 and hs6st HSPG sulfation mechanism. Trans-synaptic WNT/BMP signals genetically interact with sulf1 and hs6st nulls In the sulf1 and hs6st nulls we identified a bi-directional change in synaptic functional differentiation, measured as evoked junction PLOS Genetics | www.plosgenetics.org 10 November 2012 | Volume 8 | Issue 11 | e1003031