HSPG Regulates Trans-Synaptic Signaling Figure 5. Loss of sulf1 and hs6st causes opposite effects on WNT signaling. (A) Representative images of muscle nuclei from control (w1118), sulf1 and hs6st nulls, labeled with nuclear marker propidium iodide (PI, red) and for the C-terminus of the Wingless receptor Frizzled 2 (dFz2-C, green). Arrows indicate punctate dFz2-C nuclear labeling. Nuclei shown from muscle 6 in segment A3. (B) Quantification of the number of dFz2-C punctae per nuclei, normalized to genetic control. The total number of nuclei analyzed is indicated in each column; 119 for control (w1118) and 163 nuclei each for sulf1 and hs6st mutants. Sample sizes are $9 animals per indicated genotypes. Statistically significant differences calculated using student’s t- test; ** p,0.01 *** p,0.001. Error bars indicate S.E.M. doi:10.1371/journal.pgen.1003031.g005 current amplitudes increased in sulf1 and decreased in hs6st synapses (Figure 2). We therefore hypothesized that these functional changes are driven by the differential Wg and Gbb trans-synaptic signaling defects characterized above in sulf1 and hs6st mutants (Figure 3, Figure 4, Figure 5, Figure 6). We reasoned that correcting Wg and Gbb levels in sulf1 and hs6st nulls should restore neurotransmission to control levels. To test this hypothesis, we crossed heterozygous wg/+ and gbb/+ mutants into both sulf1 and hs6st homozygous backgrounds, both singly and in combination, and compared them to both positive and negative controls. The resulting 9 genotypes were all assayed with TEVC electrophysiology to compare EJC transmission strength. A summary of these data is given in Figure 7. Representative transmission records are shown as an average of 10 consecutive EJC responses (1.0 mM extracellular Ca2+) for the genotypes in Figure 7A, with quantification of mean peak amplitudes in all genotypes shown in Figure 7B. First testing sulf1 nulls, we examined the consequences of heterozygous genetic reduction of Wg and Gbb, alone and in combination. Compared to the elevated EJC amplitude of the sulf1 condition (381.2861 62.24 nA, p,0.01, n = 9; Figure 7B), genetic reduction of Wg (wg/+; sulf1/sulf1) caused very significantly reduced transmission, similar to genetic reduction of Gbb (gbb/+; sulf1/ sulf1) with a comparable effect, restoring EJC amplitude to control levels (267.16616.33, p,0.01, n = 9; Figure 7B). Combinatorial genetic reduction of both Wg and Gbb in the sulf1 (wg/ gbb;sulf1/sulf1) similarly returned EJC amplitudes to control levels (278.78623.17, n = 7; Figure 7B). Secondly testing hs6st nulls, genetic reduction of either Wg or Gbb alone was not sufficient to significantly change the depressed synaptic function (Figure 7B). In this case, combinatorial genetic reduction of both Wg and Gbb in the hs6st (wg/gbb;hs6st/hs6st) was required to raise the depressed EJC amplitude, a very significant increase back to control levels (272.98618.58, p,0.01, n = 8; Figure 7B). There- fore, we conclude that combinatorial Wg and Gbb trans-synaptic signaling defects are causative for the observed bi-directional PLOS Genetics | www.plosgenetics.org 11 November 2012 | Volume 8 | Issue 11 | e1003031