Carrier of Wingless (Cow) Regulation of Drosophila Neuromuscular Junction Development.

Abstract The first Wnt signaling ligand discovered, Drosophila Wingless [Wg (Wnt1 in mammals)], plays critical roles in neuromuscular junction (NMJ) development, regulating synaptic architecture, and function. Heparan sulfate proteoglycans (HSPGs), consisting of a core protein with heparan sulfate (HS) glycosaminoglycan (GAG) chains, bind to Wg ligands to control both extracellular distribution and intercellular signaling function. Drosophila HSPGs previously shown to regulate Wg trans-synaptic signaling at the NMJ include the glypican Dally-like protein (Dlp) and perlecan Terribly Reduced Optic Lobes (Trol). Here, we investigate synaptogenic functions of the most recently described Drosophila HSPG, secreted Carrier of Wingless (Cow), which directly binds Wg in the extracellular space. At the glutamatergic NMJ, we find that Cow secreted from the presynaptic motor neuron acts to limit synaptic architecture and neurotransmission strength. In cow null mutants, we find increased synaptic bouton number and elevated excitatory current amplitudes, phenocopying presynaptic Wg overexpression. We show cow null mutants exhibit an increased number of glutamatergic synapses and increased synaptic vesicle fusion frequency based both on GCaMP imaging and electrophysiology recording. We find that membrane-tethered Wg prevents cow null defects in NMJ development, indicating that Cow mediates secreted Wg signaling. It was shown previously that the secreted Wg deacylase Notum restricts Wg signaling at the NMJ, and we show here that Cow and Notum work through the same pathway to limit synaptic development. We conclude Cow acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix.


Introduction
The developing nervous system requires the coordinated action of many signaling molecules to ensure proper synapse formation and function. One key class of signals is the Wnt ligands. The first discovered Wnt, Drosophila Wingless (Wg), is secreted from presynaptic neurons (Packard et al., 2002) and glia (Kerr et al., 2014) at the developing glutamatergic neuromuscular junction (NMJ) to bind to the Frizzled-2 (Fz2) receptor (Bhanot et al., 1996) in both anterograde and autocrine signaling. In the postsynaptic muscle, Wg binding to Fz2 activates the noncanonical Frizzled Nuclear Import (FNI) pathway, which leads to Fz2 endocytosis and cleavage of the Fz2 C terminus (Fz2-C; Mathew et al., 2005). The Fz2-C fragment is trafficked to the nucleus to control translation of synaptic mRNAs and glutamate receptors (GluRs; Speese et al., 2012). In presynaptic neurons, Wg binding to Fz2 activates a divergent canonical pathway inhibiting glycogen synthase kinase 3b (GSK3b ) homolog Shaggy (Sgg) to control microtubule cytoskeletal dynamics via the microtubuleassociated protein 1B (MAP1B) homolog Futsch (Miech et al., 2008), resulting in synaptic bouton growth (Franco et al., 2004;Ataman et al., 2008). The Wg signaling ligand must be tightly regulated in the synaptic extracellular space (synaptomatrix) to ensure proper NMJ development.
One critical category of proteins regulating Wg ligand in the synaptomatrix is heparan sulfate proteoglycans (HSPGs; Kamimura and Maeda, 2017). HSPGs consist of a core protein to which heparan sulfate (HS) glycosylphosphatidylinositol (GAG) chains are covalently attached. HS GAG chains are composed of repeating disaccharide subunits expressing variable sulfation patterns (the "sulfation code"; Masu, 2016). These GAG chains bind secreted extracellular ligands to regulate intercellular signaling. There are three HSPG families: transmembrane; glycerophosphatidylinositol (GPI) anchored; and secreted. The Drosophila genome encodes only five HSPGs, with the following three known to affect NMJ development: transmembrane syndecan (Johnson et al., 2006); GPI-anchored Dally-like protein (Dlp; Johnson et al., 2006;; and secreted perlecan (Kamimura et al., 2013). A second secreted HSPG recently characterized in Drosophila was named Carrier of Wingless (Cow; Chang and Sun, 2014). In the developing wing disk, Cow directly binds secreted Wg and promotes its extracellular transport in an HS-dependent manner. Cow shows a biphasic effect on Wg target genes. Removing Cow results in a Wg overexpression (OE) phenotype for shortrange targets, and a loss-of-function phenotype for longrange targets (Chang and Sun, 2014).
The mammalian homolog of Cow, Testican-2, is highly expressed within the developing mouse brain (Vannahme et al., 1999), and inhibits neurite extension in cultured neurons (Schnepp et al., 2005), although the mechanism of action is not known. We therefore set out to characterize Cow functions at the developing Drosophila NMJ. We use the larval NMJ model because it is large, accessible and particularly well characterized for HSPG-dependent Wg transsynaptic signaling (Sears and Broadie, 2018). Each NMJ terminal consists of a relatively stereotypical innervation pattern, with consistent axonal branching and synaptic bouton formation (Menon et al., 2013). Boutons are the functional unit of the NMJ, containing presynaptic components required for neurotransmission including glutamatecontaining synaptic vesicle (SV) pools and specialized active zone (AZ) sites for SV fusion. AZs contain Bruchpilot (Brp) scaffolds, which both cluster Ca 21 channels  and tether SVs (Hallermann et al., 2010). AZs are directly apposed to GluR clusters in the postsynaptic muscle membrane (Schuster et al., 1991). This spatially precise juxtaposition is critical for high-speed and efficient synaptic communication between neuron and muscle.
In this study, we sought to test Cow functions at the NMJ, with the hypothesis that Cow should facilitate extracellular Wg transport across the synapse. Structurally, cow null mutants display overelaborated NMJs with more boutons and more synapses, phenocopying Wg overexpression. This phenotype is replicated with targeted neuronal Cow knockdown, but not muscle Cow knockdown, which is consistent with Cow secretion from the presynaptic terminal. Functionally, cow null mutants display increased synaptic transmission strength. Both electrophysiology recording and postsynaptically targeted GCaMP imaging show increased SV fusion, indicating elevated presynaptic function. Replacing native Wg with a membrane-tethered Wg blocks secretion (Alexandre et al., 2014). Tethered Wg has little effect on NMJ development, but when combined with the cow null suppresses the synaptic bouton increase, indicating that Cow mediates only secreted Wg signaling. It was recently shown that Notum, a secreted Wg deacylase, also restricts Wg signaling at the NMJ (Kopke et al., 2017). We show here that combining null cow and notum heterozygous mutants causes a synergistic increase in NMJ development, indicating nonallelic noncomplementation. Moreover, combining null cow and notum homozygous mutants did not cause an increase in NMJ development compared with the single nulls, indicating an interaction within the same pathway. We conclude that Cow functions via negative regulation of Wg trans-synaptic signaling.

PCR/RT-PCR studies
Staged Drosophila eggs were dechorionated using bleach for 30 s, washed with distilled H 2 O (dH 2 O) three times, and embryos were genotyped using a GFP marker with an epifluorescent microscope. Five embryos per genotype were homogenized in 10 ml of Gloor and Engels DNA extraction buffer (10 mM Tris HCL, pH 8.2; 1 mM EDTA, pH 8.0; 25 mM NaCl; and 200 mg/ml Proteinase K) with a glass rod in an Eppendorf tube, and the homogenate was incubated at 37°C for 30 min, and then at 95°C for 2 min. For each PCR, ;10 ng of DNA was used with the following primers: forward 59-GCAACATTCTGGCTTCGTG-TCATGC-39 and reverse 59-CTCTCGACTTGCAAATAGC-AGACGATGATC-39 for the cow gene (product size, 1927); and forward 59-GTGGAAAAGCGGTTGAAATAGGG-39 and reverse 59-GTCCACATCCACAAAGATGCC-39 for the dfmr1 gene control (product size, 3850). For the RT-PCR studies, one embryo per genotype was used with the RNeasy Micro Kit (catalog #74004, Qiagen) to extract RNA. The OneStep RT-PCR Kit (catalog #210212, Qiagen) was used. For each reaction, ;7 ng of RNA was used with the following primers: forward 59-AGAACAGCAACTTGAAT-GCCTATC-39 and reverse 59-CGAAGCATCTGCACCA-TTCC-39 for the cow gene (product size, 348); and forward 59-TAAACTGCGAGAGGTTTTCC-39 and reverse 5' AT-TCGATGAGTGTACGCTG-39 for the dmgalectin gene control (product size, 321). Products were loaded on a 0.8% agarose gel in TAE buffer with purple gel loading dye (catalog #B7025S, New England Biolabs) and SYBR safe DNA gel stain (catalog #S33102, Thermo Fisher Scientific), and run at 100 V for 30 min.

Cow antibodies
We used a well characterized, published anti-Cow antibody (Chang and Sun, 2014). New rabbit anti-Cow antibodies were also made by ABclonal against amino acids 36-236. Three antiserums were recovered and affinity purified (29,30,31). Cow antibody 31 was preabsorbed against cow nulls (cow GDP ) for imaging studies. Cow antibody 31 was used for Figures 1, 2 and 4).
Research Article: New Research made with HRP signal-delineated z-stack areas of maximum projection using ImageJ threshold and wand-tracing tools.

Two-electrode voltage-clamp electrophysiology
Wandering third instars were dissected longitudinally along the dorsal midline, internal organs were removed, and body walls were glued down (Vetbond, 3M). Peripheral motor nerves were cut at the base of the ventral nerve cord (VNC). Dissections and two-electrode voltage-clamp (TEVC) recordings were both conducted at 18°C in physiological saline as follows (in mM): 128 NaCl, 2 KCl, 4 MgCl2, 1.5 CaCl2, 70 sucrose, and 5 HEPES, pH 7.2. Preparations were imaged using a Zeiss Axioskop microscope with a Zeiss 40Â water-immersion objective. Muscle 6 in abdominal segments 3-4 was impaled with two intracellular electrodes (1 mm outer diameter borosilicate capillaries; catalog #1B100F-4, World Precision Instruments) of ;15 MV resistance filled with 3 M KCl. The muscles were clamped at À60 mV using an Axoclamp-2B amplifier (Axon Instruments). Spontaneous miniature excitatory junction current (mEJC) recordings were made in continuous 2 min sessions and low-pass filtered. For EJC Figure 2. Cow expression in embryos, larval NMJ synaptic terminal, and wing disk. A, Confocal images of stage 16 embryos colabeled with anti-HRP (red) to mark neuronal membranes and anti-Cow (green) in genetic background control (w 1118 , left) and cow null (cow GDP /cow GDP , right). The ventral nerve cord (VNC) is labeled. B, Confocal images of third instar NMJ colabeled with anti-HRP (red) and anti-Cow (green) in control (w 1118 , left) and cow null (cow GDP /cow GDP , right). From nonpermeabilized labeling, Cow appears secreted from a dynamic subset of synaptic boutons (arrows) and also present in the nerve bundle (arrowhead). Cow is shown without HRP in below images. White line marks the NMJ terminal HRP domain. C, Higher-magnification images of w 1118 NMJ synaptic boutons colabeled with anti-HRP (blue), anti-Wg (green), and anti-Cow (red), with merged image on right. White line marks the NMJ terminal HRP domain. D, Cow-GAL4 driving UAS-Cow::eGFP in wandering third instar wing imaginal disk (left) and NMJ colabeled with anti-HRP (red) and anti-GFP (green, right). For the NMJ, a single confocal section (0.5 mm) shows Cow punctae (arrow) within and surrounding synaptic boutons. records, the motor nerve was stimulated with a fire-polished suction electrode using 0.5 ms suprathreshold voltage stimuli at 0.2 Hz from a Grass S88 stimulator. Nerve stimulation-evoked EJC recordings were filtered at 2 kHz. To quantify EJC amplitude, 10 consecutive traces were averaged, and the average peak value was recorded. Clampex 9.0 was used for data acquisition, and Clampfit 9 was used for data analysis (Axon Instruments).

SynapGCaMP imaging
For SynapGCaMP quantal imaging experiments, wandering third instars were dissected and type 1b NMJs were imaged in physiological saline as follows (in mM): 70 NaCl, 5 KCl, 1.5 CaCl 2 , 25 MgCl 2 , 10 NaHCO 3 , 5 trehalose, 115 sucrose, and 5 HEPES, pH 7.2. Fluorescence images were acquired with a Vivo Spinning Disk Confocal microscope (3i Intelligent Imaging Innovations), with a 63Â 1.0 numerical aperture (NA) water-immersion objective (Zeiss), LaserStack 488 nm (50 mW) laser, Yokogawa CSU-X1 A1 spinning disk, and EMCCD camera (Photometrics Evolve). Image capture and analysis were performed as reported previously (Newman et al., 2017). Briefly, spontaneous events were imaged at 20 Hz (50 ms exposures, in streaming capture mode) for 30 s. Movies 1, 2 were then filtered, registered, and bleach corrected prior to DF conversion. Using the d DF data, an XYT local maxima algorithm was applied to the thresholded DF data to identify where and when quantal release events occur (Newman et al., 2017). Quantal coordinates were used to calculate DF/F amplitudes and frequencies (normalized to the baseline SynapGCaMP6f 2D area).

Structured illumination microscopy
Dissected wandering third instar preparations were imaged using a Nikon N-SIM in 3D SIM (structured illumination microscopy) mode, configured with a 100Â EX V-R diffraction grating, automated TiE inverted fluorescence microscope stand, 100Â SR Apo 1.49 NA objective, Andor DU-897 EM-CCD, and 488/561 nm lasers. Image acquisition was managed through NIS-Elements (Nikon Instruments), and stacks were acquired with a 0.12 mm step size. Stack reconstruction of the raw data were used prior to rendering and analysis. To acquire larger fields of view and capture whole NMJs, SIM images were stitched together using the automated tiling method within NIS-Elements software.

Laser-scanning confocal imaging analysis
We used Imaris Version 9.3.0 to quantify LSM (laserscanning confocal imaging) images using the "surfaces" function to identify the number and volume of Brp punctae, as follows: 1. Open image file and click "add new surfaces" to start the wizard. 2. Algorithm settings click "segment only a region of interest" (ROI). 3. Select ROI in X, Y, and Z. 4. Select "source channel" and thresholding conditions. 5. Adjust threshold until all spots are selected. 6. Enable "split touching objects" with seed points diameter (0.4 mm). 7. Use "quality filter" to adjust selections with minimal background. 8. Click "finish" to execute all creation steps and exit the wizard. 9. Click "edit" tab and delete extraneous spots by hand. 10. Click "statistics" tab and export values to Microsoft Excel.

SIM image analysis
We used Imaris Version 9.3.0 to quantify SIM images using the "spots" function to identify the number of Brp punctae and GluR clusters, as follows: 1. Open image file and click "add new spots" to start the wizard. 2. Algorithm settings click "segment only a region of interest" with "different spot sizes (region growing)." 3. Select ROI in X, Y, and Z. 4. Select "source channel" and click "background subtraction." 5. Classify spots with a "quality" filter type and adjust by eye.

[View online]
Movie 2. SynapGCaMP imaging of spontaneous quantal events in cow KD NMJ. Example of muscle 4 type 1b NMJ imaged following motor neuron-targeted cow RNAi (vglut-Gal4.UAScow-RNAi; SynapGCaMP6f/1) with quantified data shown in Figure 4. [View online] Research Article: New Research 6. Spot regions click "local contrast." 7. Region threshold with diameter from "region volume." 8. Click "finish" to execute all creation steps and exit the wizard. 9. Click "edit" tab and delete extraneous spots by hand. 10. Click "statistics" tab and export values to Microsoft Excel.

Statistical analyses
All statistical measurements were performed within GraphPad Prism (version 7.04 for Windows). The D'Agostino-Pearson K-squared normality test was performed on all datasets to check for normality. For comparisons of two genotypes, a t test (normally distributed) or Mann-Whitney test (not normally distributed) was performed. For all other comparisons of more than two genotypes, an ordinary one-way ANOVA (normally distributed) or Kruskal-Wallis test (not normally distributed) was performed. All graphs were made in Prism, and the data are represented in scatter plots with the mean 6 SEM.

Carrier of wingless (cow) genetic locus, mutants and expression profiles
The cow gene encodes three transcripts (cow-RC, cow-RD, cow-RE), with cow-RD containing a long 39-UTR (Fig. 1A). We acquired a reported cow null mutant (cow 5D ; Chang and Sun, 2014), two mutations from the Gene Disruption Project (cow GDP 03259 and 12802; Bellen et al., 2004;Nagarkar-Jaiswal et al., 2015), and two cow deficiencies from the Bloomington Drosophila Stock Center (Df[619] and Df[6193]). The cow 5D mutant has a 9119 bp deletion starting in the 39-UTR that does not remove cow coding sequence, but is published as a well characterized protein null (Chang and Sun, 2014). The cow GDP lines are minos-mediated integration cassette (Mi{MIC}) insertions; 03259 in cow intron 1, and 12802 in cow intron 4. Df[619] completely removes cow and 31 other genes, while cow Df[6193] removes cow and 41 other genes. PCR tests were performed using primers in the cow 5D deletion region ( Fig. 1A). As expected, there are no PCR products from cow 5D or either cow Df (Fig. 1B). Next, RT-PCR tests were performed using primers spanning an exon-exon junction to ensure mRNA amplification (Fig. 1A). The RNA extraction was confirmed using primers for a control gene (dfmr1; Fig. 1C). The cow transcript in the genetic background control w 1118 is present at similar levels in the cow 5D line (Fig. 1D). There is no detectable cow transcript in either of the cow Dfs, or in one of the cow gdp lines (03259), and only a very faint product in the other cow gdp line (12802; Fig. 1D). Thus, cow gdp 03259 is an RNA null allele.
The published cow 5D mutation has been reported to have transcript levels similar to those of wild type, but to have no detectable Cow protein expression (Chang and Sun, 2014). We therefore next examined protein levels via Western blotting using the published, well characterized Cow antibody (Chang and Sun, 2014), as well as three new antibodies made for this study (see Materials and Methods). Cow protein has a predicted molecular weight of ;75 kDa (without HS chains) and ;100 kDA (with HS chains). The two Cow protein bands are clearly present in the w 1118 controls and absent in both cow deficiency lines (Fig. 1E). Cow protein is also undetectable in the cow gdp lines, even at heightened levels of protein loading (Fig.  1E). In stark contrast to previously published work (Chang and Sun, 2014), both Cow protein bands are present at normal levels in cow 5D mutants (Fig. 1E, arrows). In our studies, cow 5D mutants typically die as early-stage larvae, and the few escapers can be raised to the third instar only with constant care. In contrast, both cow gdp protein nulls are fully adult viable, both as homozygotes and as heterozygotes over Df [619]. Thus, our evidence indicates that cow 5D does not affect Cow expression, but has a second site larval lethal mutation. Further, the Cow protein is not required for full adult viability. For the remainder of experiments, cow gdp 03259 and cow Df[619] were used, as both show complete removal of Cow RNA and protein.
To assess Cow protein expression in controls and null mutants, we performed anti-Cow labeling and Cow-Gal4 to drive UAS-Cow::eGFP (Fig. 2). In control embryos, Cow is widely expressed, including localization in the VNC ( Fig. 2A). In cow null mutants (cow GDP /cow GDP ), antibody labeling is undetectable ( Fig. 2A, right). Since Cow has a signal peptide, and has been previously established to be secreted (Chang and Sun, 2014), we tested Cow expression at the NMJ using antibody labeling with nonpermeabilizing conditions. In the w 1118 control wandering third instar NMJ, Cow appears secreted from a dynamic subset of type 1b synaptic boutons (Fig. 2B, arrows). Cow is also present in a punctate pattern along the peripheral nerve bundle (arrowhead). In cow nulls, neuronal and synaptic antibody labeling is lost (Fig. 2B, right). Within NMJ synaptic boutons colabeled for both Cow and Wg antibody, the two secreted proteins have overlapping expression patterns, colocalizing in the extracellular synaptomatrix surrounding the same boutons ( Fig. 2C). Using Cow-Gal4 to drive a UAS-Cow::eGFP, GFP is present throughout the wandering third instar wing imaginal disk, including punctae surrounding the wing pouch ( Fig. 2D, left). Cow::eGFP is also present at the NMJ in punctae within and surrounding the synaptic boutons within a single confocal slice (Fig. 2D, right). Overall, Cow is expressed in both neuronal and non-neuronal tissue in embryos, larvae, and imaginal discs, and colocalizes with Wg at the NMJ.

Presynaptic cow restricts NMJ growth and synaptic bouton formation
Wg trans-synaptic signaling regulates NMJ growth and synaptic bouton formation (Packard et al., 2002), thus we hypothesized that if Cow regulates Wg at the NMJ, Cow loss should affect the NMJ architecture. Each NMJ terminal consists of a relatively stereotypical muscle innervation pattern, with a consistent number of axon branches and large synaptic boutons (Menon et al., 2013). Wg signaling bidirectionally regulates synaptic development, with Wg knockdown decreasing NMJ synaptic bouton number and Wg OE increasing boutons (Packard et al., 2002;Kopke et al., 2017), including an increase in satellite boutons [small boutons connected to the mature (parent) bouton or adjacent axon; Torroja et al., 1999;Gatto and Broadie, 2008]. To test Cow requirements in synaptic architectural development, we labeled the wandering third instar NMJ. Anti-HRP was used to label the NMJ terminal by binding to extracellular fucosylated N-glycans associated with the presynaptic neural membrane (Jan and Jan, 1982;Parkinson et al., 2013). Anti-DLG was used to label the postsynaptic scaffold in the subsynaptic reticulum (SSR; Lahey et al., 1994;Parnas et al., 2001). We used cow GDP /Df (referred to as cow null) to eliminate cow globally, and characterized cow RNAi lines (Chang and Sun, 2014) for both motor neuron (vglut-Gal4) and muscle (24B-Gal4) cell-targeted knock-down studies. Sample images and the summary of results are shown in Figure 3.

Cow restricts presynaptic vesicle fusion and neurotransmission strength
We used the following two methods to assay NMJ synaptic functional differentiation and neurotransmission strength: (1) TEVC electrophysiology Parkinson et al., 2013;Kopke et al., 2017); and (2) imaging genetically encoded calcium reporter SynapGCaMP6f (Newman et al., 2017). For assaying evoked transmission, muscle 6 was clamped (À60 mV), while the motor nerve was stimulated with a suction electrode (1.5 mM [Ca 21 ]). EJC traces were recorded (0.2 Hz, 10 consecutive stimuli) to measure the average amplitude. For assaying mEJC events, spontaneous synaptic vesicle fusions were recorded, measuring frequency and amplitude. The mEJC frequency indicates presynaptic vesicular release (number of active synapses, fusion probability), and mEJC amplitude indicates number of activated postsynaptic receptors. For quantal imaging, the SynapGCaMP reporter (MHC-CD8-GCaMP6f-Sh) contains a myosin heavy chain (MHC) promoter for muscle targeting, CD8 transmembrane domain for membrane targeting, and Shaker (Sh) K 1 channel C-terminal tail for postsynaptic targeting (Newman et al., 2017). By imaging transmission, we are able to specifically determine the changes in quantal activity at the convergent motor neuron inputs separately. Live-imaging recordings were made of the SynapGCaMP reporter at muscle 4, with spontaneous event frequency divided by the NMJ synaptic area, and event amplitude measured as the change in the fluorescence signal over the baseline NMJ fluorescence (DF/F 0 ). Representative recordings and summarized data are shown in Figure 5.
Cow restricts presynaptic active zone and glutamatergic synapse formation We next used imaging to assay presynaptic and postsynaptic molecular components of the synapse to test the hypothesis of increased NMJ synapse number in cow mutants. The presynaptic AZ is the specialized site of SV  (Hallermann et al., 2010). Each AZ directly apposes a postsynaptic GluR cluster to mediate fast neurotransmission (Schuster et al., 1991). We used colabeling with both anti-Brp (Wagh et al., 2006) and anti-GluRIIC (aka GluRIII; Marrus et al., 2004) to compare cow null mutants to w 1118 genetic background controls (Fig. 6). Brp AZ punctae occur much more often in cow null NMJs (Fig. 6A), but are consistently smaller in volume (Fig. 6B). In quantified measurements, the number of Brp AZ punctae per NMJ is significantly increased in the cow null mutants compared with matched controls (w 1118 , 193.1 6 10.55 vs cow GDP , 284.8 6 10.54; p , 0.0001; Fig. 6A, right), but the average volume of the Brp AZ synaptic punctae is significantly decreased in the mutants (w 1118 , 0.86 6 0.033 mm 3 vs cow GDP , 0.72 6 0.025; p = 0.0019; Fig. 6B, right). This is consistent with a previous report also showing a reciprocal relationship between Brp AZ punctae number and volume (Graf et al., 2009). Brp AZ punctae are precisely juxtaposed to GluR clusters in a functional synapse (Menon et al., 2013). For better resolution to image postsynaptic GluR clusters and quantify the synaptic apposition, SIM was used (Gustafsson, 2000). To compare with previous LSM, Brp AZs were first measured to find a consistent increase in the cow null mutants, but with larger punctae numbers, presumably due to increased resolution (w 111 , 298.66 17.2 vs cow GDP , 387.9 6 17.86; p = 0.0019; Fig. 6C). There is also a similar increase in GluR clusters (w 1118 , 382 6 23.21 vs cow GDP , 542.86 29.41; p = 0.0004; Fig.  6D). Brp punctae and GluR clusters almost always partner, with rare exceptions seen at a similar frequency in controls and mutants (Fig. 6D). There are more GluR clusters than Brp punctae in both genotypes. The GluR/Brp ratio was measured to test for defects in synaptic apposition. If there is a larger ratio in the mutants compared with controls, this would indicate more GluR clusters without a Brp AZ. Conversely, a smaller ratio would indicate more GluR clusters paired with a presynaptic partner. Quantified measurements show no difference in the GluR/Brp ratio between controls and the cow null mutants (w 1118 , 1.296 0.04 vs cow GDP , 1.366 0.05; p = 0.272). Together, these results demonstrate that Cow limits NMJ synapse formation, which is consistent with strengthened neurotransmission.

Membrane-tethering Wg prevents cow null defects in NMJ development
Our starting hypothesis was that Cow regulates Wg by binding the ligand in the extracellular space and carrying  . Cow limits presynaptic active zones and glutamatergic synapse number. A, Representative muscle 4 NMJ images from confocal LSM of genetic background controls (w 1118 , left) and cow null mutants (cow GDP , right) colabeled for presynaptic membrane marker (HRP, red) and the active zone scaffold Brp (green). Brp alone is shown in right panels and the quantified Brp punctae number is shown to the right. B, High-magnification synaptic bouton images with Brp punctate identified using Imaris software (asterisks, left) and volume indicated in a heatmap (scale, 0.01-3.4 mm 3 ; right). Quantified Brp punctae volume shown to the right. C, Representative NMJ images from a SIM of controls (w 1118 ) and cow nulls (cow GDP ) colabeled for both presynaptic active zones (Brp, red) and postsynaptic glutamate receptors (GluRIIC, green). The quantified Brp punctae number is shown to the right. D, Highmagnification SIM images of juxtaposed Brp punctae and GluR clusters at synapses. Arrowheads indicate Brp or GluR domains without a partner, which are observed at equal frequency in both genotypes. Quantified GluR cluster number is shown to the right. Data shown in scatter plots, with mean 6 SEM. p Values are shown for each statistical comparison. it across the synaptic cleft (from neuron to muscle). This hypothesis is based on published work demonstrating that Cow is secreted, directly binds secreted Wg and acts to mediate intercellular transport (Chang and Sun, 2014). To test this hypothesis, we obtained transgenic lines with the wg gene cut from its native locus via FRT sites and then replaced either without (FRT-wg; transgenic control) or with (NRT-wg) a membrane tether. Importantly, HA-tagged NRT-wg is not secreted from Wg-expressing cells and fails to maintain the expression of long-range Wg targets (Alexandre et al., 2014). We tested whether tethering Wg to the membrane affects NMJ development. Comparing FRT-wg to NRT-wg, there is increased expression of the Wg ligand around presynaptic boutons (data not shown). To determine whether tethered Wg can bind Fz2 receptors, the NMJ bouton number was measured to assess presynaptic Wg signaling. Next, NRT-wg was combined with the cow null mutant (NRT-wg; cow GDP ) to test the hypothesis that Cow normally acts to regulate secreted Wg function. If Wg needs to be secreted and transported dependent on Cow function, then NRTwg and NRT-wg; cow GDP would be predicted to have the same phenotype. Representative images and summarized data are shown in Figure 7.
In comparing the control FRT-wg and tethered NRTwg, there is no change in mature NMJ bouton number, but there is a clear increase in the number of immature satellite boutons when Wg is tethered (Fig. 7A). In quantified measurements, NRT-wg has the same number of NMJ synaptic boutons as the control (FRT-wg, 26.71 6 1.04 vs NRT-wg, 27.04 6 1.72; p = 0.999; Fig. 7A,B), but a fourfold increase in the percentage of satellite boutons (FRT-wg, 2.04 6 0.77% vs NRT-wg, 8.3 6 1.62; p = 0.0019; Fig. 7C). When membrane-tethered Wg is placed in the cow null background (NRT-wg; cow GDP ), both the mature synaptic bouton number and the percentage of satellite boutons are similar to the FRT-wg control levels (Fig. 7A). In quantified measurements, the mature bouton number is no longer different between the two genotypes (FRT-wg, 26.71 6 1.04 vs NRT-wg; cow GDP , 26.78 6 0.97; p = 0.999; Fig. 7B; Table 1, all other comparisons), and the satellite boutons are also restored to near-normal levels (FRT-wg, 2.04 6 0.77% vs NRT-wg; cow GDP , 3.60 6 1.1; p = 0.999; Fig. 7C). Together, these results suggest that Cow facilitates Wg-dependent satellite bouton formation, and that Wg has to be secreted for Cow to act on it. However, in contrast to the original hypothesis, Cow acts as a negative regulator of secreted Wg signaling at the NMJ, suggesting that it should interact with other Wg-negative regulators in the extracellular synaptomatrix.

Cow and Notum function together to restrict NMJ growth and bouton formation
The secreted deacylase Notum has also been recently shown to regulate NMJ synaptic bouton formation via the negative regulation of Wg trans-synaptic signaling (Kopke et al., 2017). Notum restricts Wnt signaling by cleaving the Wg palmitoyl group that binds to Fz2 receptors (Kakugawa et al., 2015). In notum null mutants, NMJ Wg signaling is elevated both presynaptically and postsynaptically, resulting in increased synaptic bouton number, synapse number, and neurotransmission strength (Kopke et al., 2017). To test the hypothesis that the increased NMJ development in cow null mutants is similarly caused by an increase in Wg trans-synaptic signaling, we performed the genetic test of combining cow and notum null heterozygotes to assay effects on NMJ synaptic bouton development. The failure of mutant alleles at two different loci to complement one another is one method to test for an in vivo interaction of the gene products in a common signaling mechanism (nonallelic noncomplementation; Yook et al., 2001;Hawley and Gilliland, 2006). In this case, the interaction tests the hypothesis that Cow and Notum have closely associated functions in the regulation of Wg synaptic signaling via direct interaction with the Wg ligand in the extracellular synaptomatrix. We compared bouton formation in genetic background control (w 1118 ); cow null (cow GDP ), and notum null (notum KO ) homozygotes and heterozygotes; cow/notum trans-heterozygotes; and cow/ notum double null mutant (cow GDP ,notum KO /cow GDP , notum KO ). Representative images and summarized data are shown in Figure 8.
The trans-heterozygote has a clearly expanded NMJ with more synaptic boutons compared with controls, as well as other wg mutant phenotypes such as the appearance of ghost boutons (Fig. 8A, inset). Ghost boutons are immature boutons that contain the HRP marker, but do not yet contain the postsynaptic DLG protein (Ataman et al., 2006). The cow (cow GDP /1) and notum (notum KO /1) heterozygotes alone are no different from w 1118 controls and lack synaptic features of impaired Wg signaling (Fig.  8A, Table 1). In quantified measurements, trans-heterozygotes have strongly increased bouton numbers (w 1118 , 28.33 6 1.46 vs cow GDP /notum KO , 46.13 6 1.08; p , 0.0001; Fig. 8A, right; Table 1, all other comparisons). Extracellular Wg labeling without cellular permeabilization in all these genotypes indicates no difference in the Wg fluorescence intensity (Fig. 8B). In quantified measurements, there is no detectable change in Wg ligand levels between controls and cow/notum trans-heterozygotes (normalized w 1118 , 1.0 6 0.09 vs cow GDP /1; notum KO /1, 0.9 6 0.09; p = 0.852; Fig. 8B (Table 1). These results indicate that Cow and Notum act in the same pathway to restrict Wg signaling in structural development, and that the level of extracellular Wg ligand alone is not predictive of signaling activity.

Discussion
The function of signaling ligands in the extracellular space is tightly regulated to ensure coordinated intercellular development, often via glycan-dependent mechanisms Parkinson et al., 2013;Shilts and Broadie, 2017). The most recently discovered Drosophila HSPG, secreted Cow, was characterized with this role (Chang and Sun, 2014). In the developing wing disk, the Wnt Wg is produced in a stripe of cells at the dorsal/ventral margin boundary, and acts as an intercellular morphogen through Fz2 receptor signaling (Bhanot et al., 1996;Zecca et al., 1996;Neumann and Cohen, 1997). The glypican HSPGs Dally and Dlp, bound to outer plasma membrane leaflets via GPI anchors, bind Wg to regulate both ligand distribution and intercellular signaling (Tsuda et al., 1999;Baeg et al., 2001;Dear et al., 2017). It has been proposed that Dally/Dlp HSPGs are involved in the movement of extracellular Wg to form a morphogen gradient (Han et al., 2005). However, in dally dlp double mutant clones, extracellular Wg is detected far away from Wg-secreting cells, suggesting that another extracellular factor can transport Wg. Cow was shown to fill this role by binding extracellular Wg to increase stability and rate of movement from producing to receiving cells (Chang and Sun, 2014). Supporting this model, cow mutants manifest Wg ligand gain-of-function/ overexpression phenotypes for short-range targets, and loss-of-function phenotypes for long-range targets.
At the NMJ, such a long-range Wg morphogen transport function is not seemingly required, except perhaps as a clearance mechanism, but Wg extracellular regulation and short-range Wg transport to cross the synaptic cleft is critical for NMJ development (Packard et al., 2002;Friedman et al., 2013;Dear et al., 2016;Parkinson et al., 2016). At the forming of NMJ, Wg from neurons and glia signals both presynaptically (neuronal) and postsynaptically (muscle) via Fz2 receptors (Packard et al., 2002;Kerr et al., 2014). In the motor neuron, Wg signaling inhibits the GSK3b homolog Sgg to regulate the MAP1B homolog Futsch to modulate microtubule dynamics controlling NMJ bouton formation (Miech et al., 2008). However, Futsch distribution and microtubule dynamics do not change with elevated Wg signaling (Kopke et al., 2017), so this pathway alone does not explain the increased bouton formation with increased Wg signaling. In the postsynaptic muscle, Wg signaling drives Fz2 endocytosis and C-terminus cleavage, with transport to the nucleus regulating mRNAs involved in synaptogenesis, including postsynaptic GluR distribution (Speese et al., 2012). In wg mutants, GluRs are more diffuse; with clusters irregular in size/shape, increased receptor numbers and a larger postsynaptic volume (Packard et al., 2002;Speese et al., 2012;Kerr et al., 2014). Thus, Wg trans-synaptic signaling controls both NMJ structure and function.
Based on the findings from Chang and Sun (2014), we hypothesized that Cow binds Wg to facilitate the transport across the synapse to Fz2 receptors on the muscle. If this is correct, we would expect a presynaptic Wg OE phenotype in the absence of Cow (Wg buildup at the source), and a postsynaptic Wg decrease/loss phenotype (failure of Wg transport). Presynaptically, we find increased synaptic bouton number in cow null mutants phenocopying the Wg OE condition (Kopke et al., 2017), consistent with this hypothesis. These results indicate that Cow normally inhibits NMJ bouton formation, consistent with the effects of inhibiting presynaptic Wg Figure 8. Cow and Notum act in the same Wg pathway to limit NMJ bouton number. A, Confocal images of the muscle 4 NMJ colabeled with presynaptic HRP marker (green) and postsynaptic DLG marker (red) in the genetic background control (w 1118 ), cow null heterozygote (cow GDP /1), notum null heterozygote (notum KO /1), and cow/notum transheterozygote (cow GDP /notum KO ). Quantified bouton number is shown to the right. B, High-magnification NMJ confocal images of anti-Wg labeling at synaptic boutons of the same indicated genotypes. The presynaptic HRP marker boundary is outlined in white. Quantified Wg fluorescence intensity is shown to the right, normalized to the background control (w 1118 ). C, Confocal images of the muscle 4 NMJ colabeled with presynaptic HRP marker (green) and postsynaptic DLG marker (red) in the genetic background control (w 1118 ), cow null (cow GDP /cow GDP ), notum null (notum KO /notum KO ), and cow/notum double null (cow GDP ,notum KO /cow GDP ,notum KO ). Quantified bouton number is shown to the right. Data shown in scatter plots, with mean 6 SEM. p Values are shown for each statistical comparison. signaling (Packard et al., 2002). Postsynaptically, we find an increased number of GluR clusters due to elevated synapse formation in cow null mutants, but no evidence of diffuse GluR clusters of irregular size/shape and larger volume, as has been reported in wg mutants (Packard et al., 2002;Speese et al., 2012;Kerr et al., 2014). Therefore, we do not find strong support for the second prediction of the hypothesis. GluR changes within single postsynaptic domains are challenging to see even with enhanced resolution microscopy (e.g., the SIM used here; Gustafsson, 2000), but future studies could focus more on GluRIIA cluster size/shape/intensity in cow mutants. If GluR defects are detected in cow nulls, it would be interesting to test the FNI pathway (Mathew et al., 2005).
Wg signaling regulates multiple steps of NMJ development including branching, satellite bouton budding, and synaptic bouton maturation (Koles and Budnik, 2012). None of the cow manipulations cause changes in branching, indicating that Cow does not regulate this Wg signaling, likely working in concert with other Wg regulators. Wg loss (wg ts ) decreases bouton formation (Packard et al., 2002), while neural Wg OE increases branching, satellite, and total bouton numbers (Packard et al., 2002;Miech et al., 2008;Kopke et al., 2017). Satellite boutons represent an immature stage of development, with small boutons connected to the mature (parent) bouton or adjacent axon (Torroja et al., 1999;Gatto and Broadie, 2008). Neuronal Cow OE does not change mature bouton number, but increases satellite bouton budding. Neuronal Cow RNAi also increases satellite boutons. Thus, changing neural Cow levels in either direction elevates satellite bouton numbers, suggesting different consequences on budding versus developmental arrest. It also appears that the cellular source of secreted Cow, or the balance between sources, may be important for proper Wg regulation. Importantly, glia-secreted Wg regulates distinct aspects of synaptic development (Kerr et al., 2014), with loss of glial-derived Wg accounting for some, but not all, of wg mutant phenotypes. Similarly, cell-targeted cow manipulations cause different NMJ phenotypes. There is no evidence for normal Cow function in postsynaptic muscle, but it remains possible that Cow secreted from glia could regulate Wg trans-synaptic signaling.
Increasing Wg signaling elevates evoked transmission strength and functional synapse number (Kopke et al., 2017), which is phenocopied in cow null mutants. Block of postsynaptic Wg signaling causes increased SV fusion frequency and amplitude of miniature excitatory junctional potentials (Speese et al., 2012). With neuronal cow RNAi, there is a similar increase in event frequency and amplitude. These results suggest a decrease in postsynaptic Wg signaling when cow is lost, supporting the Wg transport hypothesis. Blocking Wg secreted from neurons or glia increases muscle GluR cluster size, albeit with differential effects on neurotransmission efficacy (Kerr et al., 2014). Reducing neuronal Wg has no effect on mEJC frequency, but reducing glial-derived Wg increases SV fusion frequency (Kerr et al., 2014). Both nerve-evoked and spontaneous neurotransmission are increased in cow null mutants, together with increased Brp active zones and postsynaptic GluR clusters forming supernumerary synapses. SynapGCaMP is an exciting new tool to test function at individual synapses (Newman et al., 2017). With targeted neuronal cow RNAi, there is an increase in both the number of SV fusion events and the postsynaptic Ca 21 signal amplitude, which is consistent with both presynaptic and postsynaptic regulation of Wg signaling (Packard et al., 2002;Speese et al., 2012;Kerr et al., 2014). These functional phenotypes, combined with coordinated changes in presynaptic and postsynaptic formation suggest Cow regulates trans-synaptic Wg transport.
There were differences between spontaneous synaptic vesicle fusion findings between TEVC electrophysiological recordings and SynapGCaMP reporter (MHC-CD8-GCaMP6f-Sh) Ca 21 imaging (Newman et al., 2017). Motor neurons that presynaptically targeted cow RNAi showed stronger impacts on SV fusion frequency with imaging in contrast to recordings, comparable to effects in the cow GDP null mutants. Moreover, SynapGCaMP imaging revealed significantly larger SV fusion event magnitudes in contrast to the lack of change found with TEVC recording. While the basis of these differences in unknown, we speculate that it is due to the differential nature or sensitivity of these two methods. The Ca 21 imaging is based on measuring the change in the fluorescence signal over the baseline NMJ fluorescence (DF/F 0 ; Newman et al., 2017), and it may be that glutamate receptor Ca 21 permeability or intracellular Ca 21 signaling dynamics is changed in a way not directly related to detectable membrane current changes in the cow mutants. TEVC recordings capture whole NMJ activity, whereas with imaging we only captured type 1b bouton activity normalized to area. In future studies, SynapGCaMP imaging can be used to map spatial changes in synapse function by assaying quantal activity separately in convergent type 1s and 1b motor neuron inputs and within discrete synaptic boutons (Newman et al., 2017). Moreover, differences between cow GDP and cow GDP /Df conditions could be influenced by second site-enhancing mutations on the Df chromosome. Overall, it should be noted that the changes in spontaneous SV fusion frequency and amplitude in cow mutants are subtle and variable, and need to be further studied in the future.
Wg is lipid modified via palmitoylation to become strongly membrane associated (Zhai et al., 2004). The hydrophobic moiety is located at the interface of Wg and Fz2 binding, shielded from the aqueous environment by multiple extracellular transporters until signaling interaction with the receptor (Takada et al., 2017). There have been many modes of extracellular Wg transport demonstrated, primarily from work in the wing disk, including microvesicles, lipoproteins, exosomes, and cytoneme membrane extensions (Greco et al., 2001;Panáková et al., 2005;Gross et al., 2012;Huang and Kornberg, 2015). These multiple mechanisms of transport are much less studied at the synapse; however, exosome-like vesicles containing the Wg-binding protein Evenness Interrupted (Evi) have been demonstrated at the Drosophila NMJ (Korkut et al., 2009). Cow could be considered an alternative extracellular Wg transport method (Chang and Sun, 2014), acting to shield Wg while facilitating transport through the extracellular synaptomatrix Dear et al., 2016). In addition, HSPGs have been shown to regulate ligands by stabilizing, degrading, or sequestering the ligand, or as bifunctional coreceptors, or as facilitators of transcytosis (Lin, 2004;Dear et al., 2017). Results presented here are consistent with the hypothesis that Cow is mediating Wg transport across the NMJ synapse (Chang and Sun, 2014), but also that Cow has an additional role in the negative regulation of Wg synaptic signaling.
The need for secreted Wg has been recently challenged, with Wg tethering to the membrane (NRT-wg) showing Wg secretion to be largely dispensable for development (Alexandre et al., 2014). In contrast, other recent studies suggest that Wg release and spreading is necessary (Beaven and Denholm, 2018;Pani and Goldstein, 2018;Stewart et al., 2019). We find tethering Wg at the NMJ synapse increases extracellular Wg ligand levels, with no change in mature bouton numbers. This Wg accumulation shows that NRT-wg is more stable at the synaptic signaling interface, consistent with other studies (Morata and Struhl, 2014;Chaudhary et al., 2019). However, although Wg levels increase, Wg signaling is less effective. With NRT-wg, only the budding of new satellite bouton is increased, with no increase in mature bouton formation. Reducing Wg function causes Fz2 upregulation (Cadigan et al., 1998;Chaudhary et al., 2019), so we hypothesize that Wg signaling could be maintained by increased presynaptic Fz2 receptors. When Wg is tethered, Cow cannot mediate intercellular transport, so the hypothesis predicts a similar phenotype with Cow (NRT-wg) or without Cow (NRT-wg; cow GDP ). Indeed, Cow removal in the NRT-wg condition does not impact synaptic bouton number, although it does block the increase in satellite boutons, consistent with a Cow role in greater Wg stability (Chang and Sun, 2014). These results show that Wg secretion is required for the elevated NMJ development characterizing cow mutant animals.
To further test how Cow is working through the Wg pathway to negatively regulate NMJ development, we turned to genetic interaction tests with the Wg-negative regulator Notum (Gerlitz and Basler, 2002;Giráldez et al., 2002;Kakugawa et al., 2015). At the NMJ, Wg trans-synaptic signaling is elevated in the absence of Notum, and null notum mutants display larger NMJs with more synaptic boutons, increased synapse number and elevated neurotransmission (Kopke et al., 2017). All these defects are phenocopied by neuronal Wg OE, showing that the positive synaptogenic phenotypes arise from lack of Wg signaling inhibition. Consistently, genetically correcting Wg levels at the synapse in notum nulls alleviates synaptogenic phenotypes (Kopke et al., 2017). We show here that cow null mutants have the same phenotypes of expanded NMJs, supernumerary synaptic boutons, greater synapse number/function, and strengthened transmission, suggesting that Cow acts like Notum in regulating Wg signaling. We performed a genetic test to ask whether Cow and Notum work in this same pathway. While cow and notum null heterozygotes do not exhibit NMJ defects, cow/ notum trans-heterozygotes display grossly expanded NMJs with excess boutons. This combined haplo-insufficiency (type 3 SSNC) of nonallelic noncomplementation suggests that Cow and Notum share related roles (Yook et al., 2001;Hawley and Gilliland, 2006). When we tested full double mutants, there is no additive effect, showing that Cow and Notum restrict Wg signaling in the same pathway. However, this pathway convergence appears restricted only to the control of structural synaptogenesis but not of functional neurotransmission, although the control neurotransmission amplitude was elevated in these studies.
Cow now joins the list of synaptic HSPGs with key roles in NMJ development. HSPGs have been implicated in vertebrate NMJ synapse formation for .3 decades (Kamimura and Maeda, 2017;Condomitti and de Wit, 2018). The Agrin HSPG is secreted from presynaptic terminals to maintain postsynaptic acetylcholine receptor clustering (Godfrey et al., 1984;Hubbard and Gnanasambandan, 2013). Another secreted HSPG, perlecan, regulates acetylcholinesterase localization (Peng et al., 1999;Arikawa-Hirasawa et al., 2002). Drosophila NMJ analyses have begun to more systematically elucidate HSPG roles in NMJ formation and function (Ren et al., 2009;Kamimura and Maeda, 2017). In particular, the glypican HSPG Dlp regulates Wg signaling to modulate both NMJ structure and function, including the regulation of active zone formation and SV release (Johnson et al., 2006;Friedman et al., 2013;Dear et al., 2017). Wg binds the core Dlp, with HS chains enhancing this binding, to retain Wg on the cell surface, where it can both compete with Fz2 receptors and facilitate Wg-Fz2 binding (Yan et al., 2009). This biphasic activity depends on the ratio of Wg, Fz2, and Dlp HSPG as expounded in the "exchange factor model" (Yan et al., 2009;Dear et al., 2016). Cow may impact this exchange factor mechanism as a fourth player, acting with Dlp to modulate Wg transport and Wg-Fz2 binding at the synaptic interface. It will be important to test Dlp levels and distribution in cow nulls to see how Cow fits into this model.
In addition to Cow, perlecan (Trol) is another secreted HSPG reported to regulate bidirectional Wg signaling at the Drosophila NMJ (Kamimura et al., 2013). Trol has been localized near the muscle membrane, where it promotes postsynaptic Wg accumulation. In the absence of Trol, Wg builds up presynaptically, causing excess satellite bouton formation (Kamimura et al., 2013). It is interesting to note that cow mutants enhance Wg signaling without increasing satellite boutons. In trol mutants, ghost boutons increase due to decreased postsynaptic Wg signaling (Kamimura et al., 2013). Note that cow mutants do not exhibit ghost boutons, which fails to support decreased postsynaptic Wg signaling. Other postsynaptic defects in trol mutants (e.g., reduced SSR, increased postsynaptic pockets; Kamimura et al., 2013) are NMJ ultrastructural features that could be a future focus using electron microscopy studies. Similar to cow mutants, extracellular Wg levels are decreased in the absence of Trol, speculated due to increased Wg proteolysis, since HS protects HS-binding proteins from degradation (Saksela et al., 1988). In cow mutants, it is not yet known whether Wg is decreased due to elevated signaling (ligand/receptor endocytosis) or to increased degradation due to Cow no longer protecting/stabilizing the ligand. Given that synaptic Fz2 is internalized with Wg binding (Mathew et al., 2005), future experiments could test internalized Fz2 levels in cow mutants as a proxy of Wg signaling.
In summary, we have confirmed here new tools to study Cow HSPG function, and have discovered that Cow from presynaptic motor neurons restricts NMJ bouton formation, glutamatergic synapse number, and NMJ functional differentiation. Cow acts within the same Wg trans-synaptic signaling pathway as Notum by regulating the Wg ligand in the extracellular synaptomatrix. Secreted Cow modulates extracellular Wg ligand levels, with additional functions controlling Wg signaling efficacy, which may be independent of or dependent on Wg transport. It will be interesting to determine whether Cow core protein and/or its HS chains are important for the synaptic structural and functional phenotypes. Wg must be secreted for Cow to act on it, as shown by the membrane-tethered interaction studies, showing that secreted Cow must work on the freely diffusible Wg ligand. Perhaps most informative for our future studies will be dissection of the interactions, coordination or redundancy of the multiple synaptic HSPGs at the NMJ, to further the understanding of extracellular Wg trans-synaptic signaling regulation during synaptic development. Drosophila is a particularly well suited model to study HSPGs because of the relatively reduced complexity in this system (17 HSPGs in mammals vs 5 HSPGs in Drosophila; Sarrazin et al., 2011). We look forward to expanding future studies to examine multiple synaptic HSPGs in parallel, with the goal of elucidating the surprisingly complex control of trans-synaptic signaling occurring within the extracellular synaptomatrix.