A Targeted Glycan-Related Gene Screen Reveals Heparan Sulfate Proteoglycan Sulfation Regulates WNT and BMP Trans-Synaptic Signaling Neil Dani1, Minyeop Nahm2, Seungbok Lee2, Kendal Broadie1* 1 Department of Biological Sciences and Department of Cell and Developmental Biology, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, Tennessee, United States of America, 2 Department of Cell and Developmental Biology, Seoul National University, Seoul, Republic of Korea Abstract A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of- product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects. Citation: Dani N, Nahm M, Lee S, Broadie K (2012) A Targeted Glycan-Related Gene Screen Reveals Heparan Sulfate Proteoglycan Sulfation Regulates WNT and BMP Trans-Synaptic Signaling. PLoS Genet 8(11): e1003031. doi:10.1371/journal.pgen.1003031 Editor: Norbert Perrimon, Harvard Medical School, Howard Hughes Medical Institute, United States of America Received May 10, 2012; Accepted August 26, 2012; Published November 8, 2012 Copyright: 2012 Dani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These studies were funded by NIH RO1 grants GM54544, MH084989, and MH096832 to KB (www.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kendal.broadie@vanderbilt.edu Introduction Glycans coat cell surfaces, and glycosylation decorates secreted molecules of the pericellular space and extracellular matrix (ECM) [1,2]. It is well known that glycan modifications mediate critical functions of intercellular signaling and regulate interactions of numerous growth factors with the ECM [3,4]. The synthesis, modification and degradation of glycoconjugates, including O/N- linked glycoproteins, glycosaminoglycan (GAG) proteoglycans and glycan-binding lectins, is controlled by a dedicated cadre of genes [5,6]. In the nervous system, these glycan-related genes play key roles in development, including neuron fate specification, migra- tion, formation of axon tracts and synapse maturation [7]. At synapses, glycosylated ECM molecules, membrane receptors and outer-leaflet glycolipids together form the highly specialized synaptomatrix interface [4,8], which interacts with trans-synaptic signals to modulate synaptogenesis [9]. A prime example is the classic Agrin proteoglycan, which bears heparan sulfate (HS) chains, O/N-linked glycans and also a glycan-binding lectin domain that binds other glycoconjugates [10,11,12]. Reduction of GAG sulfation perturbs the Agrin signaling that drives postsynaptic acetylcholine receptor (AChR) cluster maintenance at the neuromuscular synapse [13]. Likewise, Galbeta1,4GlcNAc and Galbeta1,3GalNAc glycans inhibit Agrin signaling by suppressing muscle specific kinase (MuSK) autophos- phorylation, a key step during synaptogenesis [14]. Analogous glycan-dependent mechanisms at the Drosophila neuromuscular synapse involve the secreted Mind-the-Gap (Mtg) lectin, which assembles the glycosylated synaptomatrix between presynaptic active zone and postsynaptic glutamate receptor (GluR) domains [15]. This glycan mechanism induces GluR clustering, synaptic localization of integrin ECM receptors, and shapes trans-synaptic signaling by controlling ligand/receptor abundance [16,17,18]. Thus, many long-term studies in vertebrate and invertebrate genetic models suggest that glycan mechanisms are a core foundation of synapse development. In the current study, we conducted a broad transgenic RNA interference (RNAi) screen of synaptic glycan function, assaying requirements in both structural and functional development of the Drosophila neuromuscular junction (NMJ). We tested 130 genes from 8 functional categories: N-glycan, O-glycan and GAG biosynthesis; glycosyltransferases and glycan modifying/degrading enzymes; glycoprotein and proteoglycan core proteins; sugar transporters and glycan-binding lectins. We found that RNAi- PLOS Genetics | www.plosgenetics.org 1 November 2012 | Volume 8 | Issue 11 | e1003031