HSPG Regulates Trans-Synaptic Signaling Figure 6. Loss of sulf1 and hs6st causes differential effects on BMP signaling. (A) Representative NMJ synaptic boutons on muscle 6 in segment A3 from control (w1118), sulf1 and hs6st nulls, labeled with neural marker anti-Fasciclin II (FasII, red) and for phosphorylated Mothers against decapentaplegic (P-Mad; green) activated downstream of Gbb signaling. Arrows indicate representative P-Mad punctae in the indicated genotypes. (B) Representative ventral nerve cord (VNC) midlines from the same 3 genotypes, labeled with anti-FasII (red) and P-Mad (green). Labeled motor neuron nuclei are indicated by arrows. Quantification of the mean fluorescent intensity level of P-Mad labeling normalized to FasII co-label at the NMJ synapse (C) and in motor neuron nuclei (D), normalized to genetic control. Sample sizes are $14 animals per indicated genotypes. Statistically significant differences calculated using the Mann-Whitney test for non-parametric data, * p,0.05, ** p,0.01. Error bars indicate S.E.M. doi:10.1371/journal.pgen.1003031.g006 effects on synaptic functional differentiation in the sulf1 and hs6st mutant conditions. The sulf1 and hs6st mechanism regulates pre- and postsynaptic differentiation The consequence of WNT (Wg) and BMP (Gbb) trans-synaptic signaling is nuclear import and transcriptional regulation in both synaptic partner cells [49,51]. We therefore hypothesized that sulf1 and hs6st mutants would show bidirectional changes in pre- and postsynaptic molecular components that would explain the bidirectional change in synaptic functional differentiation (Figure 2 and Figure 7). To test this hypothesis, we examined a key component of the presynaptic active zone (Bruchpilot; Brp) [63], and an essential subunit of the postsynaptic glutamate receptor (Bad Reception (Brec); GluRIID) [64]. In parallel, we also performed a miniature EJC (mEJC) analysis to compare functional presynaptic vesicle release probability and postsynaptic response amplitude. A summary of these data is shown in Figure 8. First, NMJ synapses were double-labeled for GluRIID recog- nized with anti-Brec (green) and Brp recognized with anti-nc82 (red) to compare genetic control (w1118) with sulf1 and hs6st nulls (Figure 8A). We found that GluRIID was very significantly elevated at sulf1 synapses compared to control (,30% increased; p,0.01, n = 20; Figure 8B). In the opposing direction, hs6st synapses showed a significant decrease in GluRIID abundance (,15% reduced; p,0.05, n = 21; Figure 8B). The GluRIID field PLOS Genetics | www.plosgenetics.org 12 November 2012 | Volume 8 | Issue 11 | e1003031