Journal: PLoS ONE

Article Title: Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

doi: 10.1371/journal.pone.0055828

Figure Lengend Snippet: In situ hybridization on coronal mouse brain sections at E16.5 (A–C′) and P5 (L–N′). Panels A′–C′ and L′–N′ show higher magnifications of boxed areas in A–C and L–N, respectively. Immunohistochemistry (D–F, H–J) and RGMa-AP section binding (G, K) on E16.5 coronal mouse brain sections. Sections in D–F and H–J are counterstained in blue with fluorescent Nissl. (A–C′) In situ hybridization shows differential expression patterns of RGMa , RGMb and Neogenin in the olfactory bulb and olfactory epithelium (OE). In line with this, immunohistochemistry reveals that axons of olfactory sensory neurons in the OE stain strongly for RGMb and weakly for RGMa and Neogenin. Furthermore, RGMa, RGMb and Neogenin are expressed on olfactory bulb axon projections such as the lateral olfactory tract (LOT). a, apical; ACa, anterior commissure pars anterior; AOB, accessory olfactory bulb; b, basal; CP, cortical plate; CRP, cribriform plate; EPL, external plexiform layer; GL, glomerular layer; GR, granule cell layer; IPL, internal plexiform layer; IZ, intermediate zone; LV, lateral ventricle; MCL, mitral cell layer; ONL, olfactory nerve layer; OVZ, olfactory ventricular zone; S, septum; STR, striatum; VN, vomeronasal nerve. Scale bar A–C 200 µm, A′–C′ 100 µm, D–F 300 µm, G 500 µm, H–J 400 µm, K 500 µm, L–N 400 µm and L′–N′ 200 µm.

Article Snippet: In brief, sections were washed in PBS (pH 7.4) and incubated in normal blocking buffer (PBS, 4% BSA and 0.1% Triton) for 1 hr at RT and incubated with goat anti-RGMa antibody (AF2458; R&D systems) 1∶200, sheep anti-RGMb antibody (AF3597; R&D systems) 1∶200 or goat anti-Neogenin antibody (AF1079; R&D systems) 1∶200 overnight in normal blocking buffer at 4°C.

Techniques: In Situ Hybridization, Immunohistochemistry, Binding Assay, Quantitative Proteomics, Staining

Journal: PLoS ONE

Article Title: Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

doi: 10.1371/journal.pone.0055828

Figure Lengend Snippet: In situ hybridization (A–C′) and immunohistochemistry (D–L) on coronal mouse brain sections at E16.5. Panels A′–C′ and D′–F′ show higher magnifications of the boxed areas in A–C and D–F, respectively. Sections in D–L are counterstained in blue with fluorescent Nissl. (A–C′) In situ hybridization reveals strong expression of RGMa and Neogenin , and moderate expression of RGMb , in the embryonic mouse cortex. Arrow in A indicates neurons of the lateral migratory stream. (D–I) RGMa and Neogenin protein are strongly expressed in the cortex and on various cortical axon projections. Strong staining for RGMb (E), and Neogenin (F), is detected in the corpus callosum (CC). The internal capsule (IC) stains strongly for RGMa (G). (J–L) Immunostaining with isotype-matched control antibodies did show significant staining. CP, cortical plate; FIM, fimbria; IZ, intermediate zone; LV, lateral ventricle; MZ, marginal zone; SP, subplate; STR, striatum; SVZ, subventricular zone; VZ, ventricular zone. Scale bar A–C 400 µm, A′–C′ 200 µm, D–F 300 µm, D′–F′ 150 µm, G-I 300 µm and J-M 300 µm.

Article Snippet: In brief, sections were washed in PBS (pH 7.4) and incubated in normal blocking buffer (PBS, 4% BSA and 0.1% Triton) for 1 hr at RT and incubated with goat anti-RGMa antibody (AF2458; R&D systems) 1∶200, sheep anti-RGMb antibody (AF3597; R&D systems) 1∶200 or goat anti-Neogenin antibody (AF1079; R&D systems) 1∶200 overnight in normal blocking buffer at 4°C.

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing, Staining, Immunostaining, Control

Journal: PLoS ONE

Article Title: Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

doi: 10.1371/journal.pone.0055828

Figure Lengend Snippet: In situ hybridization (A–C′) and immunohistochemistry (D–I) on coronal mouse brain sections at P5. Panels A′–C′ show higher magnifications of boxed areas in A–C. Sections in D-I are counterstained in blue with fluorescent Nissl. (A–C′) In situ hybridization detects strong expression of RGMa in cortical layers 1–3, 5 and 6. RGMb is mainly expressed in layer 5 in a medial to lateral gradient and Neogenin is expressed in layers 1–5. (D–F) RGMa protein is expressed in layers 1–3 and 5. Very weak staining is detected for RGMb and Neogenin is strongly expressed in cortical layers 1–3. (G–I) High levels of RGMa and Neogenin are detected in the corticospinal tract. AC, anterior commissure; CC, corpus callosum; LV, lateral ventricle; PD, pyramidal decussation; S, septum; STR, striatum. Scale bar A–C 600 µm, A′–C′ 200 µm, D–F 200 µm and G–I 250 µm.

Article Snippet: In brief, sections were washed in PBS (pH 7.4) and incubated in normal blocking buffer (PBS, 4% BSA and 0.1% Triton) for 1 hr at RT and incubated with goat anti-RGMa antibody (AF2458; R&D systems) 1∶200, sheep anti-RGMb antibody (AF3597; R&D systems) 1∶200 or goat anti-Neogenin antibody (AF1079; R&D systems) 1∶200 overnight in normal blocking buffer at 4°C.

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing, Staining

Journal: PLoS ONE

Article Title: Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

doi: 10.1371/journal.pone.0055828

Figure Lengend Snippet: In situ hybridization (A–C, J–L) and immunohistochemistry (D–I, M–O) on coronal mouse brain sections at E16.5 (A–I) and P5 (J–O). Sections in D–I and M–O are counterstained in blue with fluorescent Nissl. (A–F) RGMa mRNA and protein are expressed in the ventricular zone (VZ), dentate gyrus (DG) and cornu ammonis (CA) region. Strong expression of RGMb mRNA and protein is detected in the pial surface lining the hippocampal fissure (HF). Neogenin transcripts and protein are widely expressed in the developing hippocampus (Hip). (G–I) Immunostaining with isotype matched controls. (J–L) In situ hybridization at P5 shows strong but differential expression patterns of RGMa , RGMb and Neogenin in the CA pyramidal cell layers (Pyr). In addition, strong expression of Neogenin is detected in the granular layer (GC) of the DG. (M–O) Immunohistochemistry reveals expression of RGMa and weak expression of RGMb in the stratum lacunosum moleculare (SLM) and fimbria (FIM). Neogenin strongly labels different hippocampal layers. CX, cortex; Hb, habenula; HC, hippocampal commissure; PO, polymorph layer; SO, stratum oriens; SR, stratum radiatum; Th, thalamus. Scale bar A–C: 400 µm, D–F: 300 µm, G–I: 300 µm, J–L: 500 µm and M–O: 400 µm.

Article Snippet: In brief, sections were washed in PBS (pH 7.4) and incubated in normal blocking buffer (PBS, 4% BSA and 0.1% Triton) for 1 hr at RT and incubated with goat anti-RGMa antibody (AF2458; R&D systems) 1∶200, sheep anti-RGMb antibody (AF3597; R&D systems) 1∶200 or goat anti-Neogenin antibody (AF1079; R&D systems) 1∶200 overnight in normal blocking buffer at 4°C.

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing, Immunostaining, Quantitative Proteomics

Journal: PLoS ONE

Article Title: Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

doi: 10.1371/journal.pone.0055828

Figure Lengend Snippet: In situ hybridization (A–F, M–P), immunohistochemistry (G–L) and RGMa-AP (Q) and AP (R) section binding on coronal (A–I, M–P) and sagital (J–L, Q–R) mouse brain sections at E16.5. Sections G–L are counterstained in blue with fluorescent Nissl. (A–L) In situ hybridization and immunostaining reveal strong expression of RGMa in the medial habenula (MHb) and strong expression of RGMb in the MHb and lateral habenula (LHb). Weak Neogenin expression is detected in the LHb and MHb. In line with this, strong RGMa and RGMb immunostaining is detected on the fasciculus retroflexus (FR), the major output bundle of the Hb. (M–P) In situ hybridization for tyrosine hydroxylase ( TH ) stains dopaminergic neurons in the substantia nigra (SN) and ventral tegmental area (VTA). RGMa and Neogenin expression is detected in the SN and VTA, while RGMb is predominantly expressed in the VTA. (Q) RGMa-AP section binding shows strong staining of the stria medullaris (SM) and weak staining of the FR. (R) Section binding with AP control. CX, cortex; Hip, hippocampus; MB, midbrain; PC, posterior commissure; Th thalamus. Scale bars A–L: 400 µm, M–P: 200 µm and Q–R: 400 µm.

Article Snippet: In brief, sections were washed in PBS (pH 7.4) and incubated in normal blocking buffer (PBS, 4% BSA and 0.1% Triton) for 1 hr at RT and incubated with goat anti-RGMa antibody (AF2458; R&D systems) 1∶200, sheep anti-RGMb antibody (AF3597; R&D systems) 1∶200 or goat anti-Neogenin antibody (AF1079; R&D systems) 1∶200 overnight in normal blocking buffer at 4°C.

Techniques: In Situ Hybridization, Immunohistochemistry, Binding Assay, Immunostaining, Expressing, Staining, Control

Journal: PLoS ONE

Article Title: Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

doi: 10.1371/journal.pone.0055828

Figure Lengend Snippet: In situ hybridization (A–C, G–I) and immunohistochemistry (D–F) on coronal mouse brain sections at P5 (A–F) and in the adult (G–I). Sections D–F are counterstained in blue with fluorescent Nissl. (A–C, G–I) At P5 and in the adult, in situ hybridization reveals strong expression of RGMa and RGMb in the medial habenula (MHb). The lateral habenula (LHb) strongly expresses RGMb , while only weak expression of Neogenin is detected in Hb. (D–F) Immunohistochemistry detects strong expression of RGMa and RGMb and weak expression of Neogenin in the fasciculus retroflexus (FR). 3V, third ventricle; CX, cortex; SM, stria medullaris; PVT, paraventricular thalamic nucleus. Scale bars A–I: 200 µm.

Article Snippet: In brief, sections were washed in PBS (pH 7.4) and incubated in normal blocking buffer (PBS, 4% BSA and 0.1% Triton) for 1 hr at RT and incubated with goat anti-RGMa antibody (AF2458; R&D systems) 1∶200, sheep anti-RGMb antibody (AF3597; R&D systems) 1∶200 or goat anti-Neogenin antibody (AF1079; R&D systems) 1∶200 overnight in normal blocking buffer at 4°C.

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

doi: 10.1371/journal.pone.0055828

Figure Lengend Snippet: In situ hybridization (A–C, G–I, M–O) and immunohistochemistry (D–F, J–L, P–R) on coronal mouse brain sections at E16.5 (A–F), P5 (G–L) and in the adult (M–R). (A–I, M–O) In situ hybridization and immunohistochemistry reveals strong and broad expression of RGMa, RGMb and Neogenin in the cerebellum (CB). (J–L) Immunostaining shows expression of RGMa and Neogenin in all cerebellar layers at P5. RGMb is expressed in the internal granular layer (IGL), Purkinje cell layer (PCL) and external granular layer (EGL). (P–R) In the adult, RGMa, RGMb and Neogenin protein are expressed in Purkinje cells (PCs) and axons in the granular cell layer (GCL), PCL and molecular layer (ML). Neogenin strongly labels PC dendrites in the ML. DCN, deep cerebellar nuclei; WM, white matter; VZ, ventricular zone. Scale bar A–C: 300 µm, D–F: 250 µm, G–I: 150 µm, J–L: 150 µm, M–O: 100 µm and P–R: 50 µm.

Article Snippet: In brief, sections were washed in PBS (pH 7.4) and incubated in normal blocking buffer (PBS, 4% BSA and 0.1% Triton) for 1 hr at RT and incubated with goat anti-RGMa antibody (AF2458; R&D systems) 1∶200, sheep anti-RGMb antibody (AF3597; R&D systems) 1∶200 or goat anti-Neogenin antibody (AF1079; R&D systems) 1∶200 overnight in normal blocking buffer at 4°C.

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing, Immunostaining

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet: Identification of the minimal NEO1-NET1 interaction region, related to Figure 1 (A, B) SPR equilibrium binding experiments of different NET1 and NEO1 constructs. Graphs show a plot of the equilibrium binding response against used NEO1 construct concentrations (left panels: full-length NEO1 ectodomain (eNEO1), right panels: NEO1 FN type III domains 4 to 6 (NEO1 FN456 ). Ligands immobilized on SPR sensor chip: A , full-length NET1; B , NET1 ΔNTR . (C) Immunofluorescence staining of FLAG-tagged full-length human DCC (DCC FL ) and mouse NEO1 (NEO1 FL ) overexpressed in COS-7 cells (green). Left panel: bound NET1 ΔNTR is stained via a Rho ID4 tag (red); right panel: transfected cells were incubated with buffer only as a negative control and stained as in the left panel. (D) Western blot of COS-7 cells transfected with the indicated plasmids used in C . α-tubulin serves as a loading control. (E, F) Proximity ligation assay (PLA) to test for simultaneous binding of NET1 and RGMB to NEO1. (E) COS-7 cells were transfected with a NEO1-mVenus fusion protein or the corresponding empty vector, and with full-length RGMB (wild type or RGMB-A186R). Transfected cells were incubated with NET1 ΔNTR before performing the PLA assay. PLA signals are shown in red and NEO1-mVenus transfected cells in green with nuclei in blue. (F) PLA signals were quantified and values from 3 individual experiments were plotted. A two-tailed, unpaired t test showed the statistical significance as p = 0.0107.

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Binding Assay, Construct, Immunofluorescence, Staining, Transfection, Incubation, Negative Control, Western Blot, Proximity Ligation Assay, Plasmid Preparation, Two Tailed Test

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet: NET1 and RGMB can simultaneously bind NEO1 and form a ternary complex (A) Schematics of NEO1, NET1, and RGMB. SP, signal peptide; TM, transmembrane helix; IG, immunoglobulin-like domain; FN, fibronectin type III domain; CD, cytoplasmic domain; LN, laminin domain; LE, laminin epidermal growth factor-like repeats; LC, netrin (NTR) domain; N-RGM, RGM N-terminal domain identified to bind to BMP ligands ( Healey et al., 2015 ); vWfD, von Willebrand factor D-like domain; GPI, glycosylphosphatidylinositol anchor. (B and C) Proximity ligation assays (PLA) were performed to test for simultaneous binding of NET1 and RGMB to NEO1. (B) Cos-7 cells were either transfected with a NEO1-mVenus fusion protein or empty vector. Cells were incubated with NET1 ΔNTR and RGMB ECD (wild type or RGMB ECD -A186R). NEO1-mVenus positive cells are shown in green, nuclei are stained with DAPI and PLA signals in red. (C) Number of PLA signals per NEO1-mVenus positive cells. Individual values are plotted from 4 independent experiments. Statistical significance was determined using a two-tailed, unpaired t test with p < 0.0001. (D) Ribbon representation of the NEO1-NET1-RGMB protomer observed in the 3.25 Å resolution crystal structure, with NEO1 FN456 in red, NET1 ΔNTR in blue and RGMB CORE in yellow. A schematic is shown. See also Figure S1 .

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Ligation, Binding Assay, Transfection, Plasmid Preparation, Incubation, Staining, Two Tailed Test

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet: Interface analysis of the ternary NEO1-NET1-RGMB super-complex (A) Close-up views of the observed NET1-NEO1 interfaces (right: interface 1, left: interface 2). Residues are displayed in stick representation and labelled according to domain color-coding. A Ca 2+ ion bound to NET1 LN (grey sphere) and hydrogen bonds (dashed black lines) are displayed. Mutated residues are in bold and underlined. (B) SPR equilibrium binding curves for the NET1-NEO1 interaction. A schematic of the experiment and the calculated K d values are shown. (C) AUC analysis of the NEO1 FN456 -NET1 ΔNTR -RGMB ECD complex, using NET1 ΔNTR WT and mutants. Both NET1 interface-1 and -2 mutants abolish the 3:3:3 stoichiometry of the NEO1-NET1-RGMB super-complex. (D) Overlapping expression of NET1 RNA (in situ hybridization), and NEO1 and RGMB protein (immunohistochemistry) in consecutive coronal sections of E16 mouse striatum. Boxed area is shown at higher magnification for NEO1 and RGMB. Scale bar, 100 μm. (E) RGMB immunoprecipitation (IP) from adult mouse cortex was followed by immunoblotting. Input samples (lane 1), IP using control non-specific IgGs (cntrl) (lane 2), and anti-RGMB IP (lane 3). NEO1 and NET1 co-IP with RGMB from adult mouse brain lysates. (F and G) Functional analysis of the effect of NET1 on RGMA-mediated growth cone collapse. (F) Representative examples of growth cones from mouse P0 cortical neurons. Neurons were stained with the microtubule marker Tuj1 (green) and F-actin marker phalloidin (red). Scale bar, 10 μm. (G) Quantification of growth cone collapse. Growth cones were treated with control or RGMA alone and in combination with different NET1 variants. Proportions of collapsed growth cones relative to control are displayed. n = 3 experiments, one-way ANOVA followed by Tukey’s multiple comparison test. ∗ p < 0.05. Data are shown as means ± SEM. (H–J) Comparison of binary NEO1-RGM (PDB ID 4BQ6 [ Bell et al., 2013 ]) and the ternary NEO1-NET1-RGMB complexes shown as ribbons. The ternary NEO1-NET1-RGMB protomer complex architecture (I) clashes with the NEO1-RGM dimer-of-dimers signaling conformation (H) when superimposed on NEO1 (marked with an asterisk) (J). See also Figure S3 , Figure S4 , Figure S5 .

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Binding Assay, Expressing, RNA In Situ Hybridization, Immunohistochemistry, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Functional Assay, Staining, Marker

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet:

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Produced, Recombinant, Modification, Protease Inhibitor, Immunoprecipitation, Staining, Infection, Plasmid Preparation, Sequencing, Purification, Transformation Assay, Proximity Ligation Assay, In Situ, Imaging, Software, Western Blot