Axon Guidance. Slits ROBO1, 2 ROBO3 DCC. Neuropilin Complement Binding Coagulation Factors V/VIII Meprin. Ig-like. FN Type III.

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1 ndsy-lu-2945 Axon Guidance Slits Semaphorins Sema PSI Ig-like epeat Basic Domain L ALPS C-rich Ephrins GPI-linked or Transmembrane Netrins Laminin-like eparin Binding Eph C-rich FN Type III Tyrosine Kinase SA PDZ Plexin Sema PSI IPT GTPase Binding Split GAP Domain Neuropilin Complement Binding Coagulation Factors V/VIII eprin OBO1, 2 OBO3 DCC Ig-like FN Type III Conserved Cytoplasmic otifs Ig-like epeats FN Type III epeats Conserved IC Domains Ig-like UNC5 Thrombospondin-like Domain ZO-5 DCC Binding Death Domain CONTEXT-DEPENDENT ATTACTION O EPULSION Filopodia Cell Body Axon Growth Cone rndsystems.com

2 Netrins, Gs, and their eceptors Netrins are a small family of laminin-related molecules that includes both secreted (Netrin-1, -3, and -4) and membrane-associated proteins (Netrin-G1, -G2). Netrins have been shown to bind to a complex combination of receptors that affect the elicited response. Netrins bind to UNC5 and DCC family receptors to mediate context-dependent repulsive (UNC5) or attractive (DCC) effects on axon guidance. UNC5 and DCC receptors form homodimers and heterodimers to regulate signaling. DSCA has also been reported as a Netrin receptor, potentially acting alone or in combination with DCC. Neogenin is another putative Netrin receptor that shares structural similarity to DCC and interacts with UNC5. This receptor combination has also been shown to bind to members of the repulsive guidance molecule family (G-A, G-B, G-C) and specifically, mediate the growth cone collapsing activity of G-A. Netrins (+/-) G A. B. DSCA DCC omodimer UNC5 omodimer DCC/UNC5 eterodimer Neogenin/ UNC5 eterodimer C. D. ATTACTION EPULSION COLLAPSE DOAIN KEY DCC/UNC5/Neogenin/DSCA Netrins Gs Ig-like epeats FN Type III epeats Thrombospondin-like Domains Conserved IC Domains ZO-5 DCC Binding Death Domain Laminin-like eparin Binding GPI Anchor vwf Domain GD Domain Netrin-4 Expression in ouse Cerebellum. Netrin-4 was detected in perfusion-fixed frozen sections of mouse brain (cerebellum) using a Goat Anti-ouse Netrin-4 Affinity-Purified Polyclonal Antibody (Catalog # AF1132). The tissue was stained using the Anti-Goat P- DAB Cell and Tissue Staining Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). G-A-Induced Growth Cone Collapse equires UNC52/UNC5b. A: An embryonic rat (E19-20) cortical growth cone in culture treated with control sina. B: Treatment with ecombinant ouse G-A (Catalog # 2458-G) induces growth cone collapse. C: A neuronal growth cone treated with UNC52/UNC5b sina alone has no effect on morphology. D: UNC52/UNC5b knockdown with sina prevents G-A-induced growth cone collapse. Figure adapted with permission from ata, K. et al. (2008) J. Cell Biol. 184:737. olecule Proteins Antibodies ELISAs DCC * DSCA Neogenin Netrin-1 Ch Ch Netrin-2 Ch Ch Netrin-4 Netrin-G1a Netrin-G2a NGL-1/LC4C Nope G-A Ch G-B G-C/emojuvelin UNC51 UNC52/UNC5B * UNC53 UNC54 Species Key: uman ouse at Ch Chicken * Coming Soon 2

3 Ephrins and Eph eceptors Ephrins and their tyrosine kinase receptors, Ephs, are divided into two classes, the Ephrin-A and Ephrin-B ligand families. Ephrin-A ligands are anchored to the membrane via GPI linkage and preferentially bind EphA receptors, while Ephrin-B ligands are transmembrane proteins that preferentially interact with EphB receptors. ost Eph receptors are not ligand specific. Ephrins and their Eph receptors have the unusual capacity of bidirectional signaling, involving the activation of Eph eceptors Ephrin Ligands EphA1 Ephrin-A1 (low); Ephrin-B1 EphA2 Ephrin-A1, -A3, -A4, -A5 EphA3 Ephrin-A2, -A3, -A4, -A5 EphA4 Ephrin-A1, -A4, -A5; -B2; Ephrin-A2, -A3 (low) EphA5 Ephrin-A1, -A2, -A3, -A4, -A5 EphA6 Ephrin-A1 EphA7 Ephrin-A1, -A2, -A3, -A4, -A5 EphA8 Ephrin-A1, -A2, -A3, -A4, -A5 EphA10 Unknown EphB1 Ephrin-B1, -B2, -B3 EphB2 Ephrin-A5 EphB3 Ephrin-B1, -B2 EphB4 Ephrin-B2 EphB5 Unknown EphB6 Ephrin-B2 Ephrin-A2 Expression in at Embryonic Neurons. Ephrin-A2 was detected in rat embryonic hippocampal neurons using a Goat Anti-ouse Ephrin-A2 Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF603) followed by a FITC-conjugated anti-goat secondary antibody (green). Astrocytes were stained using an anti-gfap antibody (red). Ligand Binding Specificities for Eph Family eceptors. The ligand binding specificities for different Eph receptors are shown. The information in the table was adapted from Surawska,. et al. (2004) Cytokine Growth Factor ev. 15: Phospho-EphB4 Total EphB4 EphB4 Phosphorylation (O.D. 450) Untreated signal transduction pathways in both ligand- and receptor-expressing cells. Ephrins/Ephs may have a number of context-dependent activities including mediating attraction, repulsion, cell adhesion, or migration. Among their many roles, Ephrins/Ephs regulate topographic mapping along the anterior-posterior axis of the superior colliculus and guidance at the spinal cord midline. Ephrin-B2 + IgG1 EphrinB2-Induced EphB4 Phosphorylation Assessed using the DuoSet IC ELISA Development System. T47D human breast ductal carcinoma cells were left untreated or treated with ecombinant ouse Ephrin-B2 (Catalog # 496-EB) and ecombinant uman IgG1 (Catalog # 110-G) to induce clustering. EphB4 tyrosine phosphorylation was assessed using the uman Phospho-EphB4 DuoSet IC ELISA Development System (Catalog # DYC4057; bar graph). For comparison, the same lysates were also assessed for phosphorylated and total EphB4 using Western blot. olecule Proteins Antibodies ELISAs EphA1 EphA2 EphA3 EphA4 EphA5 EphA6 EphA7 EphA8 EphA10 EphB1 EphB2 EphB3 EphB4 EphB6 Ephrin-A1 Ephrin-A2 Ephrin-A3 Ephrin-A4 Ephrin-A5 Ephrin-B Ch X Ephrin-B1 Ephrin-B2 Z Z Ephrin-B3 Species Key: uman ouse at Ch Chicken X Xenopus Z Zebrafish 3

4 Semaphorins, Neuropilins, and Plexins The Semaphorins constitute a large family of secreted and membranetethered molecules that can be divided into eight classes according to their structure and species of origin. Classes 1 and 2 are found in invertebrates, classes 3 through 7 are found in vertebrates, and class 8 is viral. Of the vertebrate Semaphorins, class 3 Semaphorins are secreted, classes 4, 5, and 6 are transmembrane proteins, and class 7 molecules are GPI-anchored. Two distinct transmembrane receptor families, Neuropilins and Plexins, have been identifi ed as Semaphorin receptors. Neuropilins (NP-1 and NP-2) provide binding specifi city for class 3 Semaphorins, while Plexins serve as functional receptors for membrane-associated Semaphorins, and as signaling mediators for class 3 Semaphorins. Semaphorin-Plexin signaling regulates cytoskeletal reorganization and cell adhesion, and is involved in processes such as axon guidance, angiogenesis, hematopoiesis, organogenesis, and immune cell regulation. Neuropilins and Plexins are highly expressed on neurons. Classically described as collapsing factors and mediators of axon repulsion in vitro, Semaphorins regulate axon branching and prevent axons from entering certain regions of the nervous system during development in vivo. Invertebrate DOAIN KEY Semaphorins/Plexins Sema Domain PSI Domain 20 ng/ml Secreted SEAPOIN CLASSES embrane-tethered 60 ng/ml Vertebrate Immunoglobulin Loop Basic Domain A. B. 100 PBS 90 Semaphorin 3A % Growth Cone Collapse ng/ml Viral Thrombospondin epeats GPI Anchor Semaphorin 3A-Induced Growth Cone Collapse. A. E8 chick dorsal root ganglion explants, grown in the presence of ecombinant uman b-ngf (Catalog # 256-GF), were incubated with PBS or with increasing concentrations of ecombinant uman Semaphorin 3A (Catalog # 1250-S3). The percent of growth cone collapse was assessed following a thirty minute incubation (Collapsed = Less than 3 fi lopodia; Non-collapsed = 3 fi lopodia or more). B. A fully extended chick dorsal root ganglion growth cone grown in the presence of ecombinant uman b-ngf (Catalog # 256-GF) was left untreated (top) or treated with ecombinant uman Semaphorin 3A (Catalog # 1250-S3; bottom). Treatment with Semaphorin 3A induced growth cone collapse. olecule Proteins Antibodies ELISAs ErbB2/er2 Integrin a1b1 L1CA Neuropilin-1 Neuropilin-2 NrCA Plexin A1 Plexin A2 Plexin A3 Plexin A4 Plexin B1 Plexin B2 Plexin B3 Plexin C1 Plexin D1 Semaphorin 3A Semaphorin 3B Semaphorin 3C Semaphorin 3E Semaphorin 3F Semaphorin 4A Semaphorin 4B Semaphorin 4C Semaphorin 4D/CD100 Semaphorin 4F Semaphorin 4G Semaphorin 5A Semaphorin 5B Semaphorin 6A * Semaphorin 6B Semaphorin 6C Semaphorin 6D Semaphorin 7A TE7/PLXDC1 TI-2 VEGF 2/KD/Flk-1 Species Key: uman ouse at Ca Canine * Coming Soon

5 orphogens as Axon Guidance Cues A morphogen is classically defi ned as a signaling molecule that elicits different cellular responses depending on its concentration. ore specifi cally, morphogens are secreted molecules that drive the organization of regional groups of cells into patterns. Until recently, morphogens and guidance molecules were considered to be structurally and functionally distinct. Now, however, evidence suggests that select, early-expressed morphogens can be temporally recycled and serve as axon guidance cues. embers of the hedgehog, bone morphogenetic protein (BP), and Wnt families have all been implicated as axon guidance factors. for Wnts olecule Proteins Antibodies ELISAs olecule Proteins Antibodies ELISAs APC Pygopus-1 Axin-1 Pygopus-2 b-catenin O1 Dishevelled-1 O2 Dishevelled-2 -Spondin 3 Dishevelled-3 yk Dkk-1 sfp-1 Frizzled-1 sfp-2 Frizzled-2 sfp-3 Frizzled-3 sfp-4 Frizzled-4 sfp-5 Frizzled-5 TCF7/TCF1 Frizzled-6 Wnt-2 Frizzled-7 Wnt-2b Frizzled-8 Wnt-3a Frizzled-9 Wnt-5a Frizzled-10 Wnt-7a LP-6 Wnt-10b Norrin Stress Fibers b-catenin erge Untreated for BPs olecule Proteins Antibodies ELISAs BP-2 Z Z BP-2/BP-7 eterodimer BP-2/BP-6 eterodimer BP-2/BP-4 BP-2a BP-3 BP-3b/ GDF-10 Z Z BP-4 Z Z BP-4/BP-7 eterodimer BP-5 BP-6 BP-7 BP-8 BP-9 BP-10 BP-15/ GDF-9B BP-IA/ALK-3 BP-IB/ALK-6 BP-II Wnt-3a Wnt-5a Wnt-Induced Stress Fiber Formation and Nuclear b-catenin Accumulation. &D Systems ecombinant ouse Wnt-3a (Catalog # 1324-WN) and ecombinant uman/ouse Wnt-5a (Catalog # 645-WN) promote stress fi ber formation in NI- 3T3 mouse embryonic fi broblast cells, while only Wnt-3a promotes nuclear b-catenin accumulation. Images Courtesy of Dr. aymond abas, obert Wood Johnson School of edicine, Piscataway, NJ. for edgehog olecule Proteins Antibodies ELISAs Patched 1/PTC1 Patched 2/ PTC2 Desert edgehog/ Dhh Indian edgehog/ Ihh Sonic edgehog/ Shh ip GLI-1 GLI-2 GLI-3 BOC CDO DISP1 Species Key: uman ouse at Z Zebrafi sh

6 Cell Adhesion olecules Growing axons experience spatiotemporal changes in adhesion that affect their ability to reach a specific target. These interactions can be between adjacent cells (cell-cell adhesion), or between cells and the extracellular matrix. Cell adhesion can affect axon guidance by enhancing or inhibiting outgrowth, fasciculation, and/or regeneration. Cell adhesion molecules (CAs) of the Ig superfamily, extracellular matrix-associated proteins, integrins, cadherins, and proteoglycans are among the adhesion-related factors reported to affect axonal outgrowth and guidance. olecule Proteins Antibodies ELISAs AIGO AIGO2 AIGO3 ApoE 2 CD44 P Ca E P CL1/L1CA-2 Contactin-1 Contactin-2/TAG1 Contactin-3 Contactin-4 Contactin-5 Contactin-6 DSCA DSCA-L1 Fibronectin B F-spondin/SPON1 Kilon/NEG1 Integrins Please see our website for a large selection of Integrin-related research tools L1CA Laminin S indin Ch N-Cadherin NCA-1/CD56 Neurocan Neurofascin Neuroglycan C/CSPG5 Neuroplastin Neurotrimin NrCA eelin -Spondin-1 -Spondin-2 -Spondin-3 -Spondin-4 Tenascin C Tenascin VLDL Species Key: uman ouse at B Bovine Ca Canine Ch Chicken E Equine P Porcine *Coming Soon A. B. etinal Ganglion Cell (GC) Axon Fasciculation equires omophilic Contactin-2/TAG1 Interactions. etinal explants were isolated from E14.5 TAG1 +/ or TAG1 / embryos. In vitro assays of GC axonal growth were performed on glass coverslips coated with alternating stripes of Laminin and ecombinant uman Contactin-2/TAG1 (Catalog # 1714-CN). A: TAG1 +/ axons prefer Contactin-2/TAG1 to Laminin. B: Contactin-2/TAG1 -/- axons display no preference between Contactin-2/TAG1- and Laminin-coated stripes. GC axons were immunolabeled with anti-neurofilament antibodies (red). Data Courtesy of Dr. Jean-Léon Thomas, Université Pierre et arie Curie, Paris. A. B. Neurofascin-Induced Neurite Outgrowth. A: ecombinant at Neurofascin (Catalog # 3235-NF) immo bilized on a microplate promotes neurite outgrowth in rat cortical neurons. B: Cortical neurons cultured under the same conditions in the absence of Neurofascin exhibit little outgrowth.

7 Neurotrophic Factors and eceptors olecule Proteins Antibodies ELISAs Artemin BDNF Ca E Ca P GDNF GFa-1/GDNF a-1 GFa-2/GDNF a-2 GFa-3/GDNF a-3 GFa-4/GDNF a-4 NGF /TNFSF16 Ca LINGO-1 LINGO-2 AG/Siglec-4a BP b-ngf NELL1 NELL2 * * Ng2/Ng B OG GF a-1 and GDNF Expression in at Dorsal oot Ganglion. GF a-1 and GDNF were detected in perfusion-fixed frozen sections of rat dorsal root ganglion using a Biotinylated Goat Anti-at GF a-1 Affinity-Purified Polyclonal Antibody (Catalog # BAF560; red) and a Goat Anti-uman/at GDNF Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF-212NA; green). The tissue was stained with streptavidin-conjugated Cy 3 and a FITC-conjugated anti-goat secondary antibody. Proliferation (FU) Neurotrophic factors are involved in an array of nervous system activities including regulating neuronal survival, neurite outgrowth, and synaptic plasticity. These include members of the GDNF family: GDNF, Artemin, Persephin, and Neurturin. This family signals through a multimolecular complex that includes receptors of the GF-a family and ET. Other neurotrophic factors include the neurotrophins: NGF, BDNF, NT-3, and NT-4. Pro-neurotrophins preferentially bind the receptor NGF /p75nt in combination with the co-receptor Sortilin to promote apoptosis, while Trk tyrosine kinase receptors (TrkA, TrkB, TrkC) preferentially bind mature neurotrophins and promote survival. There is also an intriguing relationship between NGF /p75nt and myelin-associated factors shown to inhibit neurite outgrowth and regeneration. NGF /p75nt, Nogo, Lingo-1, and/or the TNF receptor superfamily member, TOY, may act in a receptor complex mediating activities of the outgrowth-inhibiting proteins Nogo-A, AG, and Ogp Persephin (ng/ml) Persephin-Induced Proliferation. uman thyroid carcinoma (TT) cells proliferate in a re sponse to increasing concentrations of ecombinant ouse Persephin (Catalog # 2479-PS). Proliferation was assessed using esazurin (Catalog # A002) fluorescence. Ng3/Ng2 Nogo-A Nogo-B Nogo-C Nogo eceptor/ng NT-3 NT-4 Oligodendrocyte arker O1 Ch Oligodendrocyte arker O4 Ch Ogp Neurturin Persephin et OCK1 OCK2 Sortilin TrkA TrkB TrkC TOY/TNFSF19 Species Key: uman ouse at B Bovine Ca Canine Ch Chicken E Equine P Porcine *Coming Soon NGF /TNFSF16 Expression in ouse Brain. NGF /TNFSF16 was detected in perfusionfixed frozen sections of mouse brain (cortex) using a Goat Anti-ouse NGF /TNFSF16 Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF1157; red). The tissue was counterstained (green).

8 Slit Proteins and OBO eceptors The Slit family of proteins consists of three members (Slit1 3) that signal by binding to one of four oundabout (OBO1 4) receptors. Slits are large, secreted glycoproteins that are subject to proteolytic cleavage resulting in fragments with variable activities. OBO1, OBO2, and OBO3 are predominantly expressed in the nervous system and Slit-OBO interactions have been shown to regulate axon repulsion and neuronal outgrowth. A. B. Slits L ALPS C-rich Slit2 Enhances Neurite Outgrowth. Cultured chick dorsal root ganglion neurons were grown in the presence of ecombinant uman b-ngf (Catalog # 256-GF), with (A) or without (B) ecombinant ouse Slit2 (Catalog # 5444-SL). The presence of the Slit2 protein significantly enhanced neurite outgrowth. OBO3 OBO1, 2 Endothelia Specific OBO4 DOAIN KEY OBOs Ig-like epeats FN Type III epeats Conserved Cytoplasmic otifs Slit Proteins Direct Cell igration by Binding to OBO Family eceptors. The Slit family of proteins binds to members of the OBO family of receptors to initiate signaling pathways that affect cell motility. This binding is mediated by the leucine-rich repeat (L) region of the Slit proteins and the immunoglobulin-like (Ig-like) repeats of the OBO family of receptors. olecule Proteins Antibodies ELISAs OBO1 * OBO2 OBO3 OBO4 Slit1 Slit2 OBO1 in at Neural Tube. oundabout eceptor 1 (OBO1) was detected in immersionfixed frozen sections of rat embryo using a Goat Anti-at OBO1 Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF1749). The tissue was stained with the Anti-Goat P-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue) LEAN OE rndsystems.com/ AxonGuidance Slit3 Species Key: uman ouse at *Coming Soon On the cover: The growth cone at the tip of an extending axon is exquisitely sensitive to repulsive and attractive guidance cues in its environment. These molecules may be diffusible and work from a distance, or be bound to a membrane or substrate and work at close range. It is the complex integration of these repulsive and attractive signals that guide the axon to its appropriate target. Axon guidance molecules play critical roles during nervous system development and may regulate the regenerative capacity of neurons during nervous system disease. B_axonguidance_1691_ud Trademarks and registered trademarks are the property of their respective owners. For research use or manufacturing purposes only. Global info@bio-techne.com techsupport@bio-techne.com North America TEL Europe iddle East Africa TEL +44 (0) China info.cn@bio-techne.com TEL +86 (21) est of World bio-techne.com/find-us/distributors TEL bio-techne.com