RnDSy-lu-2945 Guidance

Slits

LRR EGF Repeats Sema PSI ALPS Ig-like Repeat EGF Repeats Basic Domain C-rich

Netrins GPI-linked -like or Transmembrane EGF Repeats Heparin Binding

ROBO1, 2 ROBO3 DCC Complement Ig-like Ig-like Repeats Binding Sema Coagulation Eph PSI Factors V/VIII FN FN Type III UNC5 Meprin Type III Repeats C-rich IPT Ig-like FN Type III Thrombospondin-like Domain GTPase Split GAP Tyrosine Binding Domain Conserved ZO-5 IC Domains Kinase Conserved DCC Binding SAM Cytoplasmic Death Domain PDZ Motifs

CONTEXT-DEPENDENT ATTRACTION OR REPULSION

Filopodia

Growth Cone Body Axon

rndsystems.com , RGMs, and their Receptors Netrins are a small family of laminin-related molecules that includes signaling. DSCAM has also been reported as a , both secreted (Netrin-1, -3, and -4) and membrane-associated potentially acting alone or in combination with DCC. Neogenin is (Netrin-G1, -G2). Netrins have been shown to bind to a complex another putative Netrin receptor that shares structural similarity to combination of receptors that affect the elicited response. Netrins bind DCC and interacts with UNC5. This receptor combination has also to UNC5 and DCC family receptors to mediate context-dependent been shown to bind to members of the repulsive guidance molecule repulsive (UNC5) or attractive (DCC) effects on . UNC5 family (RGM-A, RGM-B, RGM-C) and specifically, mediate the growth and DCC receptors form homodimers and heterodimers to regulate cone collapsing activity of RGM-A.

A. B. Netrins RGM (+/-)

Neogenin/ UNC5 DCC/UNC5 Heterodimer DSCAM Heterodimer DCC UNC5 Homodimer Homodimer

C. D.

COLLAPSE REPULSION ATTRACTION

DOMAIN KEY

DCC/UNC5/Neogenin/DSCAM Netrins RGMs Ig-like Repeats Laminin-like GPI Anchor RGM-A-Induced Collapse Requires UNC5H2/UNC5b. A: An embryonic rat (E19- FN Type III Repeats EGF Repeats vWF Domain 20) cortical growth cone in culture treated with control siRNA. B: Treatment with Recombinant Mouse RGM-A (Catalog # 2458-RG) induces growth cone collapse. Thrombospondin-like Domains Heparin Binding RGD Domain C: A neuronal growth cone treated with UNC5H2/UNC5b siRNA alone has no effect on Conserved IC Domains morphology. D: UNC5H2/UNC5b knockdown with siRNA prevents RGM-A-induced growth cone collapse. Figure adapted with permission from Hata, K. et al. (2008) J. Cell Biol. ZO-5 184:737. DCC Binding Death Domain Selected Products Molecule Proteins Antibodies ELISAs DCC H* M H M DSCAM H H Neogenin H M M Netrin-1 H M Ch H M R Ch Netrin-2 Ch Ch Netrin-4 H M H M Netrin-G1a M M Netrin-G2a M M NGL-1/LRRC4C H H

Netrin-4 Expression in Mouse Cerebellum. Netrin-4 was detected in perfusion-fixed frozen Nope M M M sections of mouse brain (cerebellum) using a Goat Anti-Mouse Netrin-4 Affinity-Purified RGM-A H M H M Ch H M R Polyclonal Antibody (Catalog # AF1132). The tissue was stained using the Anti-Goat HRP- RGM-B H M H M H DAB Cell and Tissue Staining Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). RGM-C/Hemojuvelin H M H M H M R UNC5H1 H R R UNC5H2/UNC5B H* R R UNC5H3 H H UNC5H4 H H Species Key: H M Mouse R Rat Ch Chicken * Coming Soon

2 Ephrins and Eph Receptors Ephrins and their receptors, Ephs, are divided into two signal transduction pathways in both - and receptor-expressing classes, the -A and Ephrin-B ligand families. Ephrin-A ligands are cells. Ephrins/Ephs may have a number of context-dependent activities anchored to the membrane via GPI linkage and preferentially bind including mediating attraction, repulsion, cell adhesion, or migration. EphA receptors, while Ephrin-B ligands are transmembrane proteins Among their many roles, Ephrins/Ephs regulate topographic mapping that preferentially interact with EphB receptors. Most Eph receptors along the anterior-posterior axis of the and guidance are not ligand specific. Ephrins and their Eph receptors have the at the midline. unusual capacity of bidirectional signaling, involving the activation of

Eph Receptors 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 Ephrin-A2 Expression in Rat Embryonic . Ephrin-A2 was detected in rat embryonic hippocampal neurons using a Goat Anti-Mouse Ephrin-A2 Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF603) followed by a FITC-conjugated anti-goat secondary EphB1 Ephrin-B1, -B2, -B3 antibody (green). Astrocytes were stained using an anti-GFAP antibody (red). EphB2 Ephrin-A5 EphB3 Ephrin-B1, -B2 EphB4 Ephrin-B2 EphB5 Unknown Selected Products Molecule Proteins Antibodies ELISAs EphB6 Ephrin-B2 EphA1 H M H M H Ligand Binding Specificities for Eph Family Receptors. The ligand binding specificities for EphA2 H M H M H different Eph receptors are shown. The information in the table was adapted from Surawska, H. et al. (2004) Rev. 15:419. EphA3 H M M EphA4 H M M EphA5 H M R M R H M EphA6 H M M 3.0 EphA7 H M M Phospho-EphB4 EphA8 H M M Total EphB4 2.5 EphA10 H H EphB1 R R 2.0 EphB2 H M H M EphB3 H M H M 1.5 (O.D. 450) EphB4 H M H M H

1.0 EphB6 H M H M EphB4 Phosphorylation Ephrin-A1 H M M 0.5 Ephrin-A2 H M M Ephrin-A3 H M H 0 Ephrin-A4 H M H M Untreated Ephrin-B2 + IgG1 Ephrin-A5 H M H EphrinB2-Induced EphB4 Phosphorylation Assessed using the DuoSet® IC ELISA Development System. T47D human breast ductal carcinoma cells were left untreated or Ephrin-B H M R Ch X treated with Recombinant Mouse Ephrin-B2 (Catalog # 496-EB) and Recombinant Human Ephrin-B1 H M M IgG (Catalog # 110-HG) to induce clustering. EphB4 tyrosine phosphorylation was assessed 1 Ephrin-B2 H M Z M Z using the Human Phospho-EphB4 DuoSet® IC ELISA Development System (Catalog # DYC4057; bar graph). For comparison, the same lysates were also assessed for Ephrin-B3 H M H phosphorylated and total EphB4 using Western blot. Species Key: H Human M Mouse R Rat Ch Chicken X Z

3 Semaphorins, , and The Semaphorins constitute a large family of secreted and membrane- for membrane-associated Semaphorins, and as signaling mediators for tethered molecules that can be divided into eight classes according to class 3 Semaphorins. -Plexin signaling regulates cyto- their structure and species of origin. Classes 1 and 2 are found in skeletal reorganization and cell adhesion, and is involved in processes , classes 3 through 7 are found in vertebrates, and class such as axon guidance, , hematopoiesis, organogenesis, 8 is viral. Of the vertebrate Semaphorins, class 3 Semaphorins are and immune cell regulation. secreted, classes 4, 5, and 6 are transmembrane proteins, and class 7 Neuropilins and Plexins are highly expressed on neurons. Classically molecules are GPI-anchored. Two distinct transmembrane receptor described as collapsing factors and mediators of axon repulsion in vitro, families, Neuropilins and Plexins, have been identifi ed as Semaphorin Semaphorins regulate axon branching and prevent from entering receptors. Neuropilins (NRP-1 and NRP-2) provide binding specifi city certain regions of the during development in vivo. for class 3 Semaphorins, while Plexins serve as functional receptors

SEMAPHORIN CLASSES Selected Products Molecule Proteins Antibodies ELISAs Secreted Membrane-tethered ErbB2/Her2 H H M H         a1b1 H M L1CAM H M H M Neuropilin-1 H M R H M R H Neuropilin-2 H M R H M R NrCAM H M H H Plexin A1 M H M Vertebrate Viral Plexin A2 M H M R DOMAIN KEY Plexin A3 M R Semaphorins/Plexins Plexin A4 H H M R Immunoglobulin Loop Thrombospondin Repeats Plexin B1 H PSI Domain Basic Domain GPI Anchor Plexin B2 H M H M

Plexin B3 H H M R A. B. Plexin C1 H M H M 100 PBS Semaphorin 3A 90 Plexin D1 H H Semaphorin 3A H M H M 80 Semaphorin 3B M M 70 Semaphorin 3C H M H M 60 Semaphorin 3E H M H M H 50 Semaphorin 3F M H M 40 Semaphorin 4A H H M

% Growth Cone Collapse Cone % Growth 30 Semaphorin 4B H M

20 Semaphorin 4C H M H M R Semaphorin 4D/CD100 H M H M 10 Semaphorin 4F M M 0 20 ng/mL 60 ng/mL 180 ng/mL Semaphorin 4G H M H M Semaphorin 5A H M H M R Semaphorin 3A-Induced Growth Cone Collapse. A. E8 chick dorsal root ganglion explants, grown in the presence of Recombinant Human b-NGF (Catalog # 256-GF), were Semaphorin 5B H M incubated with PBS or with increasing concentrations of Recombinant Human Semaphorin Semaphorin 6A H M* H M 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 Semaphorin 6B H M H M more). B. A fully extended chick dorsal root ganglion growth cone grown in the presence of Semaphorin 6C H M H M Recombinant Human b-NGF (Catalog # 256-GF) was left untreated (top) or treated with Recombinant Human Semaphorin 3A (Catalog # 1250-S3; bottom). Treatment with Semaphorin 6D H M H Semaphorin 3A induced growth cone collapse. Semaphorin 7A H M H M TEM7/PLXDC1 H TIM-2 M M VEGF R2/KDR/Flk-1 H M H M H M Species Key: H Human M Mouse R Rat Ca Canine *Coming Soon Morphogens as Axon Guidance Cues A morphogen is classically defi ned as a signaling molecule that elicits structurally and functionally distinct. Now, however, evidence suggests different cellular responses depending on its concentration. More that select, early-expressed morphogens can be temporally “recycled” specifi cally, morphogens are secreted molecules that drive the and serve as axon guidance cues. Members of the hedgehog, organization of regional groups of cells into patterns. Until recently, morphogenetic (BMP), and Wnt families have all been morphogens and guidance molecules were considered to be implicated as axon guidance factors.

Selected Products for Wnts Selected Products Molecule Proteins Antibodies ELISAs Molecule Proteins Antibodies ELISAs for BMPs APC H Pygopus-1 H M Molecule Proteins Antibodies ELISAs Axin-1 H M R Pygopus-2 H BMP-2 H M R Z H Z H M R b-Catenin H M R H M ROR1 H H BMP-2/BMP-7 H H Dishevelled-1 H ROR2 H H H Heterodimer Dishevelled-2 H R-Spondin 3 H M H M H BMP-2/BMP-6 H Heterodimer Dishevelled-3 H Ryk H M H M BMP-2/BMP-4 H Z Dkk-1 H M R H M R H M sFRP-1 H H BMP-2a Z Frizzled-1 H M H M sFRP-2 H M H M BMP-3 H H Frizzled-2 H M H M sFRP-3 H M H M BMP-3b/ H H Frizzled-3 H M sFRP-4 H H GDF-10 Frizzled-4 H M H M sFRP-5 H M H BMP-4 H M Z H M Z H Frizzled-5 H H TCF7/TCF1 H M BMP-4/BMP-7 H Frizzled-6 H M Wnt-2 H Heterodimer Frizzled-7 H M H M Wnt-2b M H M BMP-5 H M H M H Frizzled-8 H M M Wnt-3a H M H M BMP-6 H M H M H Frizzled-9 M M Wnt-5a H M H M R BMP-7 H M H M H Frizzled-10 H Wnt-7a H H BMP-8 H LRP-6 H M H M Wnt-10b H M H M BMP-9 H M H M H M Norrin H M H M BMP-10 H M H M BMP-15/ H H M R GDF-9B Stress Fibers b-Catenin Merge BMPR-IA/ALK-3 H M H BMPR-IB/ALK-6 H M H M BMPR-II H M H Untreated

Selected Products for Hedgehog Wnt-3a Molecule Proteins Antibodies ELISAs Patched 1/PTCH1 H M Patched 2/ PTCH2 H M Desert Hedgehog/ H M M Dhh Indian Hedgehog/ H M H M Ihh Wnt-5a / H M H M H M Shh Hip M M M GLI-1 H M Wnt-Induced Stress Fiber Formation and Nuclear b-Catenin Accumulation. R&D Systems Recombinant Mouse Wnt-3a GLI-2 H M (Catalog # 1324-WN) and Recombinant Human/Mouse Wnt-5a (Catalog # 645-WN) promote stress fi ber formation in NIH- 3T3 mouse embryonic fi broblast cells, while only Wnt-3a promotes nuclear b-Catenin accumulation. Images Courtesy of Dr. GLI-3 H M Raymond Habas, Robert Wood Johnson School of Medicine, Piscataway, NJ. BOC H H M CDO H H M DISP1 H Species Key: H Human M Mouse R Rat Z Zebrafi sh Cell Adhesion Molecules Growing axons experience spatiotemporal changes in adhesion that affect their ability to reach a specific target. These interactions can be A. between adjacent cells (cell-cell adhesion), or between cells and the . Cell adhesion can affect axon guidance by enhancing or inhibiting outgrowth, fasciculation, and/or regeneration. Cell adhesion molecules (CAMs) of the Ig superfamily, extracellular matrix-associated proteins, , cadherins, and are among the adhesion-related factors reported to affect axonal outgrowth and guidance.

Selected Products Molecule Proteins Antibodies ELISAs AMIGO H H B. AMIGO2 H H M AMIGO3 M ApoE R2 H CD44 H M R P H M R Ca E P CHL1/L1CAM-2 H M H M Contactin-1 H H M R Contactin-2/TAG1 H M H M R H Contactin-3 H H M R

Contactin-4 H M H M (RGC) Axon Fasciculation Requires Homophilic Contactin-2/TAG1 +/– –/– Contactin-5 H H Interactions. Retinal explants were isolated from E14.5 TAG1 or TAG1 . In vitro assays of RGC axonal growth were performed on glass coverslips coated with alternating Contactin-6 H M M stripes of Laminin and Recombinant Human Contactin-2/TAG1 (Catalog # 1714-CN). DSCAM H H 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. RGC axons were DSCAM-L1 H H immunolabeled with anti-neurofilament antibodies (red). Data Courtesy of Dr. Jean-Léon H B H H Thomas, Université Pierre et Marie Curie, Paris. F-spondin/SPON1 H M H Kilon/NEGR1 H H M Please see our website for a large selection Integrins of Integrin-related research tools L1CAM H M H M Laminin S H R Ch A. B. Mindin M H N-Cadherin H M H M R NCAM-1/CD56 H M H M R H H M H M R Neurofascin H R H M R Neuroglycan C/CSPG5 H M H M R Neuroplastin M H M Neurotrimin H NrCAM H M H M R H Reelin H M M R-Spondin-1 H M H M H

R-Spondin-2 H M H Neurofascin-Induced Outgrowth. A: Recombinant Rat Neurofascin (Catalog # 3235-NF) R-Spondin-3 H M H M H immo­bilized on a microplate promotes neurite outgrowth in rat cortical neurons. B: Cortical neurons cultured under the same conditions in the absence of Neurofascin R-Spondin-4 H M M H exhibit little outgrowth. Tenascin C H H M Tenascin R H H M R VLDL R H M M Species Key: H Human M Mouse R Rat B Bovine Ca Canine Ch Chicken E Equine P Porcine *Coming Soon and Receptors 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, , , and . This family signals through a multimolecular complex that includes receptors of the GFR-a family and RET. Other neurotrophic factors include the : NGF, BDNF, NT-3, and NT-4. Pro-neurotrophins preferentially bind the receptor NGF R/p75NTR in combination with the co-receptor Sortilin to promote , while Trk tyrosine kinase receptors (TrkA, TrkB, TrkC) preferentially bind mature neurotrophins and promote survival. There is also an intriguing relationship between NGF R/p75NTR and myelin-associated factors shown to inhibit neurite outgrowth and regeneration. NGF R/p75NTR, Nogo R, Lingo-1, and/or the TNF receptor superfamily member, TROY, may act in a receptor complex mediating GFR a-1 and GDNF Expression in Rat Dorsal Root Ganglion. GFR a-1 and GDNF were activities of the outgrowth-inhibiting proteins Nogo-A, MAG, and OMgp. detected in perfusion-fixed frozen sections of rat dorsal root ganglion using a Biotinylated Goat Anti-Rat GFR a-1 Affinity-Purified Polyclonal Antibody (Catalog # BAF560; red) and a Goat Anti-Human/Rat GDNF Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF-212- NA; green). The tissue was stained with streptavidin-conjugated Cy™3 and a FITC-conjugated Selected Products anti-goat secondary antibody. Molecule Proteins Antibodies ELISAs Artemin H M H M H M

BDNF H M R Ca E H H M R Ca P 2300 GDNF H R H R H 2200 GFRa-1/GDNF Ra-1 H R H R 2100 GFRa-2/GDNF Ra-2 H M H M GFRa-3/GDNF Ra-3 H M H M 2000 a a GFR -4/GDNF R -4 H M 1900 NGF R/TNFRSF16 H M H M R Ca H M Proliferation (RFU) Proliferation 1800 LINGO-1 H LINGO-2 H 1700 MAG/Siglec-4a M R R R 1600 0.001 0.01 0.1 1 10 100 MBP H M R B Persephin (ng/mL) MOG H M H M b -NGF H M R H R H R Persephin-Induced Proliferation. Human thyroid carcinoma (TT) cells proliferate in a NELL1 H M H M re­sponse to increasing concentrations of Recombinant Mouse Persephin (Catalog # 2479-PS). Proliferation was assessed using Resazurin (Catalog # AR002) fluorescence. NELL2 H* M* M NgR2/NgRH1 H NgR3/NgRH2 H M H Nogo-A H R H R Nogo-B H M Nogo-C H Nogo Receptor/NgR H M H M NT-3 H H H NT-4 H M H H Oligodendrocyte Marker O1 H M R Ch Oligodendrocyte Marker O4 H M R Ch OMgp H M H M Neurturin H M H M NGF R/TNFRSF16 Expression in Mouse Brain. NGF R/TNFRSF16 was detected in perfusion- Persephin H M H M H fixed frozen sections of mouse brain (cortex) using a Goat Anti-Mouse NGF R/TNFRSF16 Ret H M H M H Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF1157; red). The tissue was counterstained (green). ROCK1 H H M R ROCK2 H M R Sortilin H M H M TrkA H R H R H R TrkB H M H M H TrkC H M H M H TROY/TNFRSF19 H M H M M Species Key: H Human M Mouse R Rat B Bovine Ca Canine Ch Chicken E Equine P Porcine *Coming Soon Proteins and ROBO Receptors The Slit family of proteins consists of three members (Slit1–3) that signal by binding to one of four Roundabout (ROBO1–4) receptors. A. B. Slits are large, secreted that are subject to proteolytic cleavage resulting in fragments with variable activities. ROBO1, ROBO2, and ROBO3 are predominantly expressed in the nervous system and Slit-ROBO interactions have been shown to regulate axon repulsion and neuronal outgrowth.

Slits

LRR

EGF Repeats ALPS EGF Repeats C-rich Slit2 Enhances Neurite Outgrowth. Cultured chick dorsal root ganglion neurons were grown in the presence of Recombinant Human b-NGF (Catalog # 256-GF), with (A) or without (B) Recombinant Mouse Slit2 (Catalog # 5444-SL). The presence of the Slit2 protein signifi cantly ROBO1, 2 ROBO3 enhanced neurite outgrowth.

Endothelia Specific ROBO4

DOMAIN KEY ROBOs Ig-like Repeats FN Type III Repeats Conserved Cytoplasmic Motifs

Slit Proteins Direct Cell Migration by Binding to ROBO Family Receptors. The Slit family of proteins binds to members of the ROBO family of receptors to initiate signaling pathways that affect cell motility. This binding is mediated by the leucine-rich repeat (LRR) region of the Slit proteins and the immunoglobulin-like (Ig-like) repeats of the ROBO family ROBO1 in Rat Neural Tube. Roundabout Receptor 1 (ROBO1) was detected in immersion- of receptors. fi xed frozen sections of rat using a Goat Anti-Rat ROBO1 Antigen Affi nity-Purifi ed Polyclonal Antibody (Catalog # AF1749). The tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue) Selected Products Molecule Proteins Antibodies ELISAs ROBO1 H* R H R R ROBO2 H M H LEARNrndsystems.com/ MORE ROBO3 H M H M H AxonGuidance ROBO4 H H M Slit1 H M Slit2 H M H M Slit3 H M R Species Key: H Human M Mouse R Rat *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.

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