Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling is required for developmental myelination. a Diagram of activin receptor signaling: Acvr2a and Acvr2b are ligand-binding receptors that require Acvr1b to induce intracellular signallng. In PDGFRa-Cre; Acvr1b fl/fl mice, the knockout of Acvr1b eliminates all activin receptor signaling from both ligand binding receptors. b Western blots of brain lysates from P16 Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice (cerebellum) labeled for CNP (46 kDa) or MBP (14–21 kDa) with β-Actin as a loading control. c Images of corpus callosum, cerebellum [counterstained with Hoechst (blue)] and dorsal spinal cord in P16 Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice immunostained for myelin protein MAG (green). Scale bar 25 μm. d Mean MAG intensity normalized to background ± s.e.m. in the corpus callosum of P16 Acvr1b fl/fl ( n = 4 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 6 mice). Two-tailed Student’s t test, * P = 0.0211, **0.0018. e Mean number of myelinated fibers ± s.e.m. per field of toluidine-blue stained semi-thin resin sections of corpus callosum at P16 in Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 3 mice per group). Two-tailed Student’s t test, ** P = 0.0034. f Toluidine-blue stained semi-thin resin sections of corpus callosum in Acvr1b fl/fl (left) and PDGFRa-Cre; Acvr1b fl/fl mice (right), with expanded field of view ( f (a) and f (b), respectively). Scale bar 20 μm. g Analysis of distribution of myelinated axons in relation to axon diameter, represented as proportion of myelinated axons only (from all diameters), in Acvr1b fl/fl (magenta) and PDGFRa-Cre; Acvr1b fl/fl mice (green) mice ( n = 3 mice per genotype) overlaid with polynomial best-fit regression curves ( R 2 = 0.8897, 0.8344, respectively). Kolmogorov–Smirnov test, ** P = 0.002

Article Snippet: In a subset of experiments, OPCs were co-treated with activin-A and neutralizing antibodies against Acvr2a or Acvr2b (30 μg ml −1 , R&D Systems; AF340, AF339) or goat IgG isotype control (30 μg ml −1 , Santa Cruz Biotechnology).

Techniques: Ligand Binding Assay, Knock-Out, Western Blot, Labeling, Control, Two Tailed Test, Staining

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor Acvr2a regulates oligodendrocyte lineage cell behavior. a Acvr2a (top row; green) and Acvr2b (bottom row; green) expression by oligodendrocyte lineage (Olig2+; red) throughout development (P1–P14; double positive cells indicated by arrows), counterstained with Hoechst (blue). Inset; isotype control for Acvrs. Scale bar 50 μm. b Expression of Acvr2a (red) by NG2+ cells (green) (top row), and by CC1+ cells (green) (bottom row). c Data-mining of oligodendrocyte lineage cell transcriptomes from P21–30 for Acvr2a expression, represented as t distributed stochastic neighbor embedding projection. d OPCs co-treated with activin-A and neutralizing antibodies for Acvr2a or isotype IgG. Mean percentage of oligodendrocytes (MBP+) normalized to isotype control ± s.e.m. n = 3 biological replicates, two-tailed Student’s t test, P = 0.0087. e Images of MBP+ cells (red) in cultures treated with activin-A (10 ng ml −1 ) and isotype IgG or Acvr2a-neutralizing IgG. Scale bar 75 μm. f Phalloidin intensity signal (arbitrary units; AU) per oligodendrocyte plotted against oligodendrocyte size (px 2 ) in cultures co-treated with activin-A (10 ng ml −1 ) and IgG control or neutralizing antibody against Acvr2a. n = 3 biological replicates. g Images of MBP+ oligodendrocytes (green) and filamentous actin (Phalloidin+; red) in cultures treated with activin-A and IgG or Acvr2a neutralizing antibody. h Log2-transformed phosphorylation signal of TGFβ superfamily pathways following treatment with activin-A, normalized to respective total protein signal then to vehicle control. Heat map: compared to vehicle, magenta indicates increased signal, black no change, and green reduced signal

Article Snippet: In a subset of experiments, OPCs were co-treated with activin-A and neutralizing antibodies against Acvr2a or Acvr2b (30 μg ml −1 , R&D Systems; AF340, AF339) or goat IgG isotype control (30 μg ml −1 , Santa Cruz Biotechnology).

Techniques: Expressing, Control, Two Tailed Test, Transformation Assay, Phospho-proteomics

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling regulates remyelination. a Representative images of organotypic cerebellar slice cultures at 7 days post lysolecithin-induced demyelination, treated with vehicle control or activin receptor agonist activin-A during remyelination, immunostained against myelin basic protein (MBP; green), and axonal neurofilament-H (NF; red). Scale bar 50 μm. b Mean remyelination index ± s.e.m. in activin-A-treated explants at 7, 10, and 14 days post lysolecithin (dpl) normalized to vehicle control from the respective time point. n = 3 animals, one-sample t test compared to theoretical mean of 1 (control), ** P = 0.0057. c Representative images of slice cultures at 14 dpl treated with vehicle control or an inhibitor of activin receptor signaling inhibin-A during remyelination, immunostained against myelin basic protein (MBP; green) and axonal neurofilament-H (NF; red). Scale bar 50 μm. d Mean remyelination index ± s.e.m. in inhibin-A-treated explants at 7, 10, and 14 dpl normalized to vehicle control from the respective time point. n = 3 animals, one-sample t test compared to theoretical mean of 1 (control), * P = 0.0165, *0.0374, **0.0004, respectively. e Acvr2a and Acvr2b expression (green) in demyelinated caudal cerebellar peduncles (CCP) at 5 days post-lesion (dpl; prior to remyelination) and 10 dpl (onset of remyelination), counterstained with Hoechst (blue). Scale bar 25 μm. f Colocalization of Acvr2b or Acvr2a (green) with NG2 (top 2 rows; red; arrowheads) or Olig2 (bottom 2 rows; red; arrowheads) at 5 and 10 dpl in CCP, counterstained with Hoechst (blue). g Mean number of cells double positive for Olig2 and Acvr2a or Acvr2b per field ± s.e.m. at 5 and 10 dpl. n = 3 = 4 animals per group. Two-tailed Student’s t test, P = 0.0345 (5 dpl), 0.0298 (10 dpl). h Non-lesioned CCP shows no staining of Acvr2b (green). Scale bar 10 μm

Article Snippet: In a subset of experiments, OPCs were co-treated with activin-A and neutralizing antibodies against Acvr2a or Acvr2b (30 μg ml −1 , R&D Systems; AF340, AF339) or goat IgG isotype control (30 μg ml −1 , Santa Cruz Biotechnology).

Techniques: Control, Expressing, Two Tailed Test, Staining

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor expression dysregulation in developmental and adult human myelin disorders. a Images of activin-A subunit (INHBA; red) immunostaining in non-injured and injured developing white matter in a case of perinatal brain injury, counterstained with Hoechst (turquoise). Scale bar 25 μm. b Mean densities of INHBA+ cells ± s.e.m. per mm 2 in non-injured and injured developing white matter in perinatal brain injury. n = 5 cases (Table S1); each patient block is represented by different color. Mann–Whitney test , *P = 0.0411. c Images of oligodendrocyte lineage cells (Olig2+; green) expressing Acvr2a or Acvr2b (red) in developing white matter, indicated by arrowheads. Scale bar 25 μm. d Densities of Acvr2a+ Olig2+ and Acvr2b+ Olig2+ cells per mm 2 in non-injured versus injured areas of developing white matter. n = 5 cases; each patient block is represented by different color. Mann–Whitney test, *P = 0.0238 (non-injured Acvr2a+ Olig2+ vs Acvr2b+ Olig2+), * P = 0.0317 (non-injured Acvr2a+ Olig2+ vs injured Acvr2a+ Olig2+). e Densities of PCNA+ Olig2+ proliferating oligodendrocyte lineage cells per mm 2 in non-injured vs injured areas of developing white matter. n = 5 cases; each patient block represented by different color. f Images of INHBA+ cells (red) in control and acute active multiple sclerosis (MS) lesion tissue, counterstained with Hoechst (turquoise). Scale bar 100 μm. g Mean densities of INHBA+ cells per mm 2 in post-mortem brain tissue from healthy control, or MS lesions (remyelinated, acute active, chronic active (rim), chronic inactive). n for each lesion type indicated in Table S2. Mann–Whitney test, * P = 0.0286. h Proportion of Acvr2a+ Olig2+ or Acvr2b+ Olig2+ from total Olig2+ cells in healthy control tissue or MS lesions (remyelinated, acute active, chronic active (rim), chronic inactive). n for each lesion type indicated in Table S2. Kruskal–Wallis test and Dunn’s multiple comparison test, * p < 0.05. i Acvr2a+ (top row; red) or Acvr2b+ (bottom row; red) and Olig2+ (blue) double positive cells in MS lesions. Scale bar 5 μm. j Quantification of differentiation of OPCs into mature oligodendrocytes (MBP+; per field) following transfection with lentivirus (GFP+), either control (control-LV) or Acvr2b-expressing (Acvr2b-LV), and treated with activin-A (10 ng ml −1 ) for 3 days. * P = 0.0247, two-tailed Student’s t test. k Representative images of OPC cultures treated with activin-A and Control-LV or Acvr2b-LV for 3 days immunostained for MBP (red) and GFP (green). Scale bar 50 µm. l Representative images of maturing oligodendrocytes transfected with Control-LV or Acv2b-LV for 3 days and treated with activin-A (10 ng ml −1 ) for 5 days, stained for GFP (green), MBP (false colored yellow), and Phalloidin (red). Scale bar 20 µm. m Phalloidin intensity signal (arbitrary units; AU) per MBP+ transfected (GFP+) oligodendrocyte plotted against oligodendrocyte size (px 2 ) in cultures co-treated with activin-A (10 ng ml −1 ) and Control-LV or Acvr2b-LV. n Model for role of activin receptor signaling in oligodendrocyte lineage cells. Acvr2a is expressed during developmental myelination, inducing oligodendrocyte differentiation, myelination, and myelin membrane compaction. Following injury, successful repair involves a transition in expression from Acvr2b to Acvr2a to support new myelin formation. In myelin disorders, failed repair is associated with an upregulation of Acvr2b, impairing Acvr2a-driven responses, leading to lack of myein

Article Snippet: In a subset of experiments, OPCs were co-treated with activin-A and neutralizing antibodies against Acvr2a or Acvr2b (30 μg ml −1 , R&D Systems; AF340, AF339) or goat IgG isotype control (30 μg ml −1 , Santa Cruz Biotechnology).

Techniques: Expressing, Immunostaining, Blocking Assay, MANN-WHITNEY, Control, Comparison, Transfection, Two Tailed Test, Staining, Membrane

Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A Analysis of the Bioplex 3.0 interactome identified 42 potential DLK1 binding partners. The pie chart shows the fractions of candidate proteins containing extracellular domains (magenta) and intracellular proteins (teal). ACVR2B (bold) is the only known cell surface receptor among extracellular domain-containing proteins. B Confocal microscopy images depicting wild type U2OS cells or DLK1-expressing U2OS cells stained with recombinant ACVR2B-Fc protein. ACVR2B-Fc binding was detected with an anti-Fc Alexa Fluor 488 antibody, the contours of the cells were visualized by actin staining (magenta) using phalloidin 647, and nuclei were counterstained using DAPI (blue). The images are represented as maximum projections of 5 z-slices taken 0.8 µm apart. Scale, 20 µm. The experiment was independently repeated three times. C SPR was used to determine the steady-state binding affinity between DLK1(N-EGF6) and ACVR2B-Fc. The DLK1 protein was injected over a sensor chip containing immobilized ACVR2B-Fc and the data was fitted to a 1:1 binding model. RU = resonance units. The associated SPR sensograms for this data are shown in Supplementary Fig. . D Confocal microscopy images depicting the staining of U2OS cells overexpressing ACVR2B coupled to a GFP SPARK (green) tag, with DLK1-Fc protein (magenta). DLK1 binding was detected using an anti-Fc Alexa Fluor 647 antibody. Nuclei counterstained with DAPI (blue). Scale, 20 µm. The experiment was independently repeated two times. E Flow cytometry histograms depicting the binding of DLK1-Fc protein to yeast expressing ACVR2B and eleven other TGF-β superfamily receptors. The experiment was independently repeated two times. Source data are provided as a Source Data file.

Article Snippet: For C2C12 differentiation assays, cells were plated in 24 well plates (Cellvis) and at ~70% confluency the experiment was started (Day 0) by replacing growth media with DMEM + 2% horse serum (differentiation media) and blocked for 30 min with recombinant soluble DLK1, DLK1 R193D -mutant or ACVR2B blocking antibody (Bimagrumab, MedChemExpress, Cat# HY-P99355) (100 nM) before addition of 4 μg/ml recombinant active myostatin (R&D systems).

Techniques: Binding Assay, Cell Surface Receptor Assay, Confocal Microscopy, Expressing, Staining, Recombinant, Injection, Flow Cytometry

Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A Crystal structure of DLK1 domains EGF5-6 (magenta) in complex with the extracellular domain of ACVR2B (teal). B A surface model of ACVR2B (teal) with DLK1(magenta) overlaid with a cartoon representation of myostatin (yellow). C Zoom in panel showing Trp 78 and Phe 101 of ACVR2B forming hydrophobic interactions with Arg 193 of DLK1. D Zoom in panel showing Phe 82 of ACVR2B packing against Val 209 and Val 229 of DLK1, and Arg 56 of ACVR2B forming a hydrogen bond with Gln 228 . E SPR isotherms comparing the binding between DLK1(EGF5-6) or DLK1(EGF5-6) R193D -mutant to ACVR2B-Fc. The DLK1 proteins were injected over a sensor chip containing immobilized ACVR2B-Fc and the data was fitted to a 1:1 binding model. RU = resonance units. F SPR isotherms comparing the binding between DLK1 and three ACVR2B-Fc interface mutants. The R56A mutation in ACVR2B was associated with a ~ 6-fold decrease in DLK1-binding affinity compared to WT ACVR2B, and there was a complete loss of DLK1 binding to the W78A or F101A mutants. RU = resonance units. Source data are provided as a Source Data file.

Article Snippet: For C2C12 differentiation assays, cells were plated in 24 well plates (Cellvis) and at ~70% confluency the experiment was started (Day 0) by replacing growth media with DMEM + 2% horse serum (differentiation media) and blocked for 30 min with recombinant soluble DLK1, DLK1 R193D -mutant or ACVR2B blocking antibody (Bimagrumab, MedChemExpress, Cat# HY-P99355) (100 nM) before addition of 4 μg/ml recombinant active myostatin (R&D systems).

Techniques: Binding Assay, Mutagenesis, Injection

Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A The structure of ACVR2A (PDB ID: 5NH3 ) was superimposed onto ACVR2B in the DLK1-ACVR2B complex structure (PDB ID: 9D20 ). B A zoom window shows Phe 82 of ACVR2B inserted into the pocket formed by DLK1 residues Val 209 and Val 229 . The analogous residue of ACVR2A, Ile 83 , is not predicted to fit into this pocket. C A zoom window shows Arg 193 of DLK1 forming polar interactions with Thr 93 and Glu 94 of ACVR2B. The analogous residues in ACVR2A, Lys 94 and Lys 95 , are not predicted to form charge complementary interactions. D–F Electrostatic potential surface representation of the DLK1-ACVR2B complex. The arrow indicates the ACVR2B interface glutamate (E94) residue that is substituted for a lysine (K95) in ACVR2A. D ACVR2B alone ( E ) and ACVR2A alone ( F ). G Sequence alignment of human ACVR2B and ACVR2A. Selected conserved (teal) and non-conserved (yellow) residues forming the DLK1-ACVR2B interface are highlighted. The E94 residue of ACVR2B is indicated with a black arrow.

Article Snippet: For C2C12 differentiation assays, cells were plated in 24 well plates (Cellvis) and at ~70% confluency the experiment was started (Day 0) by replacing growth media with DMEM + 2% horse serum (differentiation media) and blocked for 30 min with recombinant soluble DLK1, DLK1 R193D -mutant or ACVR2B blocking antibody (Bimagrumab, MedChemExpress, Cat# HY-P99355) (100 nM) before addition of 4 μg/ml recombinant active myostatin (R&D systems).

Techniques: Residue, Sequencing

Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A Illustration of Myostatin-ACVR2B signaling in the presence or absence of DLK1. Binding of the canonical ligand Myostatin to ACVR2B and ACVR1B leads to the formation of a 2:2:2 complex and subsequent activation of SMAD2/3 (left). The EGF5 domain of DLK1 binds to ACVR2B to inhibit ligand signaling (right), and the DLK1(EGF5-6) region used for co-crystallization is indicated with a dashed circle. Created in BioRender. Antfolk, D. (2025) https://BioRender.com/p73c456 B DLK1 inhibits Myostatin-ACVR2B signaling in a HEK293-(CAGA) 12 reporter assay. Myostatin treatment at 2 nM is represented as 100% activation, and a decrease in activation was observed upon treatment with increasing concentrations (125 nM-16 µM) of soluble DLK1. Data is represented as normalized relative luciferase units (RLU) represented as the mean of triplicate wells from one representative experiment. The experiment was independently repeated three times. C DLK1 transfected into HEK293-(CAGA) 12 reporter cells inhibit Myostatin signaling. Data is represented as RLU based on quadruplicate wells from one representative experiment. The experiment was independently repeated two times. D Representative microscopy images showing C2C12 myoblast differentiation in the presence of Myostatin, Myostatin + DLK1, or Myostatin + DLK1 R193D (loss-of-ACVR2B-binding mutant). Control cells were allowed to differentiate for 72 h. Myostatin treatment (4 ug/ml) inhibits C2C12 myoblast differentiation into myotubes as determined by MyoHC staining. C2C12 cells were fixed with 4% PFA, immunostained with an anti-MyoHC antibody and an anti-mouse IgG Alexa Fluor 488 secondary antibody. Nuclei were counterstained with Hoechst 33342. Nuclei represented with pseudo color (magenta) in zoom in panels. Scale bar, 100 μm. The experiment was independently repeated four times. Source data are provided as a Source Data file.

Article Snippet: For C2C12 differentiation assays, cells were plated in 24 well plates (Cellvis) and at ~70% confluency the experiment was started (Day 0) by replacing growth media with DMEM + 2% horse serum (differentiation media) and blocked for 30 min with recombinant soluble DLK1, DLK1 R193D -mutant or ACVR2B blocking antibody (Bimagrumab, MedChemExpress, Cat# HY-P99355) (100 nM) before addition of 4 μg/ml recombinant active myostatin (R&D systems).

Techniques: Binding Assay, Activation Assay, Crystallization Assay, Reporter Assay, Luciferase, Transfection, Microscopy, Mutagenesis, Control, Staining

Journal: Drug Testing and Analysis

Article Title: Detection of erythropoiesis stimulating agent Luspatercept after administration to healthy volunteers for antidoping purposes

doi: 10.1002/dta.3341

Figure Lengend Snippet: Confirmation of luspatercept presence in serum samples. (a) Analysis by SDS‐PAGE and double‐blot (anti‐ActRIIB Sino Biological Inc. 10229‐T20). (b) Analysis by SAR‐PAGE and double‐blot (anti‐ActRIIB Sino Biological Inc. 10229‐T16). (c) Analysis by IEF‐PAGE (2‐10) and single‐blot (biotinylated anti‐ActRIIB R&D systems BAF339). A signal corresponding to the reference luspatercept was present in all samples evaluated post‐administration (D49 to D70) and absent before administration (C1). Positive QC (serum spiked with luspatercept) and negative QC (blank serum) were prepared with the samples and included in the analysis

Article Snippet: Luspatercept detection (confirmation procedure) : Following blotting, the membrane was blocked with 5% nonfat milk and directly incubated 1 h at room temperature with the biotinylated anti‐ActRIIB antibody solution (BAF339, R&D systems) after IEF‐PAGE or polyclonal anti‐ActRIIB antibody solution (10229‐T20 or 10229‐T16 from SinoBiological Inc.) after SDS‐/SAR‐PAGE.

Techniques: SDS Page

Journal: Drug Testing and Analysis

Article Title: Detection of erythropoiesis stimulating agent Luspatercept after administration to healthy volunteers for antidoping purposes

doi: 10.1002/dta.3341

Figure Lengend Snippet: Confirmation of luspatercept in urine samples. (a) Analysis by SDS‐PAGE and double‐blot (anti‐ActRIIB Sino Biological Inc. 10229‐T20). (b) Analysis by SAR‐PAGE and single‐blot (anti‐ActRIIB Sino Biological Inc. 10229‐T20). (c) Analysis by IEF‐PAGE (2‐10) and single‐blot (biotinylated anti‐ActRIIB R&D systems BAF339). A signal corresponding to the reference luspatercept was present in all samples evaluated post‐administration (D56 to D70) and absent before administration (C1). Positive QC (urine spiked with luspatercept) and negative QC (blank urine) were prepared with the samples and included in the analysis

Article Snippet: Luspatercept detection (confirmation procedure) : Following blotting, the membrane was blocked with 5% nonfat milk and directly incubated 1 h at room temperature with the biotinylated anti‐ActRIIB antibody solution (BAF339, R&D systems) after IEF‐PAGE or polyclonal anti‐ActRIIB antibody solution (10229‐T20 or 10229‐T16 from SinoBiological Inc.) after SDS‐/SAR‐PAGE.

Techniques: SDS Page

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling is required for developmental myelination. a Diagram of activin receptor signaling: Acvr2a and Acvr2b are ligand-binding receptors that require Acvr1b to induce intracellular signallng. In PDGFRa-Cre; Acvr1b fl/fl mice, the knockout of Acvr1b eliminates all activin receptor signaling from both ligand binding receptors. b Western blots of brain lysates from P16 Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice (cerebellum) labeled for CNP (46 kDa) or MBP (14–21 kDa) with β-Actin as a loading control. c Images of corpus callosum, cerebellum [counterstained with Hoechst (blue)] and dorsal spinal cord in P16 Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice immunostained for myelin protein MAG (green). Scale bar 25 μm. d Mean MAG intensity normalized to background ± s.e.m. in the corpus callosum of P16 Acvr1b fl/fl ( n = 4 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 6 mice). Two-tailed Student’s t test, * P = 0.0211, **0.0018. e Mean number of myelinated fibers ± s.e.m. per field of toluidine-blue stained semi-thin resin sections of corpus callosum at P16 in Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 3 mice per group). Two-tailed Student’s t test, ** P = 0.0034. f Toluidine-blue stained semi-thin resin sections of corpus callosum in Acvr1b fl/fl (left) and PDGFRa-Cre; Acvr1b fl/fl mice (right), with expanded field of view ( f (a) and f (b), respectively). Scale bar 20 μm. g Analysis of distribution of myelinated axons in relation to axon diameter, represented as proportion of myelinated axons only (from all diameters), in Acvr1b fl/fl (magenta) and PDGFRa-Cre; Acvr1b fl/fl mice (green) mice ( n = 3 mice per genotype) overlaid with polynomial best-fit regression curves ( R 2 = 0.8897, 0.8344, respectively). Kolmogorov–Smirnov test, ** P = 0.002

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Ligand Binding Assay, Knock-Out, Western Blot, Labeling, Two Tailed Test, Staining

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor Acvr2a regulates oligodendrocyte lineage cell behavior. a Acvr2a (top row; green) and Acvr2b (bottom row; green) expression by oligodendrocyte lineage (Olig2+; red) throughout development (P1–P14; double positive cells indicated by arrows), counterstained with Hoechst (blue). Inset; isotype control for Acvrs. Scale bar 50 μm. b Expression of Acvr2a (red) by NG2+ cells (green) (top row), and by CC1+ cells (green) (bottom row). c Data-mining of oligodendrocyte lineage cell transcriptomes from P21–30 for Acvr2a expression, represented as t distributed stochastic neighbor embedding projection. d OPCs co-treated with activin-A and neutralizing antibodies for Acvr2a or isotype IgG. Mean percentage of oligodendrocytes (MBP+) normalized to isotype control ± s.e.m. n = 3 biological replicates, two-tailed Student’s t test, P = 0.0087. e Images of MBP+ cells (red) in cultures treated with activin-A (10 ng ml −1 ) and isotype IgG or Acvr2a-neutralizing IgG. Scale bar 75 μm. f Phalloidin intensity signal (arbitrary units; AU) per oligodendrocyte plotted against oligodendrocyte size (px 2 ) in cultures co-treated with activin-A (10 ng ml −1 ) and IgG control or neutralizing antibody against Acvr2a. n = 3 biological replicates. g Images of MBP+ oligodendrocytes (green) and filamentous actin (Phalloidin+; red) in cultures treated with activin-A and IgG or Acvr2a neutralizing antibody. h Log2-transformed phosphorylation signal of TGFβ superfamily pathways following treatment with activin-A, normalized to respective total protein signal then to vehicle control. Heat map: compared to vehicle, magenta indicates increased signal, black no change, and green reduced signal

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Expressing, Two Tailed Test, Transformation Assay

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling regulates remyelination. a Representative images of organotypic cerebellar slice cultures at 7 days post lysolecithin-induced demyelination, treated with vehicle control or activin receptor agonist activin-A during remyelination, immunostained against myelin basic protein (MBP; green), and axonal neurofilament-H (NF; red). Scale bar 50 μm. b Mean remyelination index ± s.e.m. in activin-A-treated explants at 7, 10, and 14 days post lysolecithin (dpl) normalized to vehicle control from the respective time point. n = 3 animals, one-sample t test compared to theoretical mean of 1 (control), ** P = 0.0057. c Representative images of slice cultures at 14 dpl treated with vehicle control or an inhibitor of activin receptor signaling inhibin-A during remyelination, immunostained against myelin basic protein (MBP; green) and axonal neurofilament-H (NF; red). Scale bar 50 μm. d Mean remyelination index ± s.e.m. in inhibin-A-treated explants at 7, 10, and 14 dpl normalized to vehicle control from the respective time point. n = 3 animals, one-sample t test compared to theoretical mean of 1 (control), * P = 0.0165, *0.0374, **0.0004, respectively. e Acvr2a and Acvr2b expression (green) in demyelinated caudal cerebellar peduncles (CCP) at 5 days post-lesion (dpl; prior to remyelination) and 10 dpl (onset of remyelination), counterstained with Hoechst (blue). Scale bar 25 μm. f Colocalization of Acvr2b or Acvr2a (green) with NG2 (top 2 rows; red; arrowheads) or Olig2 (bottom 2 rows; red; arrowheads) at 5 and 10 dpl in CCP, counterstained with Hoechst (blue). g Mean number of cells double positive for Olig2 and Acvr2a or Acvr2b per field ± s.e.m. at 5 and 10 dpl. n = 3 = 4 animals per group. Two-tailed Student’s t test, P = 0.0345 (5 dpl), 0.0298 (10 dpl). h Non-lesioned CCP shows no staining of Acvr2b (green). Scale bar 10 μm

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Expressing, Two Tailed Test, Staining

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor expression dysregulation in developmental and adult human myelin disorders. a Images of activin-A subunit (INHBA; red) immunostaining in non-injured and injured developing white matter in a case of perinatal brain injury, counterstained with Hoechst (turquoise). Scale bar 25 μm. b Mean densities of INHBA+ cells ± s.e.m. per mm 2 in non-injured and injured developing white matter in perinatal brain injury. n = 5 cases (Table S1); each patient block is represented by different color. Mann–Whitney test , *P = 0.0411. c Images of oligodendrocyte lineage cells (Olig2+; green) expressing Acvr2a or Acvr2b (red) in developing white matter, indicated by arrowheads. Scale bar 25 μm. d Densities of Acvr2a+ Olig2+ and Acvr2b+ Olig2+ cells per mm 2 in non-injured versus injured areas of developing white matter. n = 5 cases; each patient block is represented by different color. Mann–Whitney test, *P = 0.0238 (non-injured Acvr2a+ Olig2+ vs Acvr2b+ Olig2+), * P = 0.0317 (non-injured Acvr2a+ Olig2+ vs injured Acvr2a+ Olig2+). e Densities of PCNA+ Olig2+ proliferating oligodendrocyte lineage cells per mm 2 in non-injured vs injured areas of developing white matter. n = 5 cases; each patient block represented by different color. f Images of INHBA+ cells (red) in control and acute active multiple sclerosis (MS) lesion tissue, counterstained with Hoechst (turquoise). Scale bar 100 μm. g Mean densities of INHBA+ cells per mm 2 in post-mortem brain tissue from healthy control, or MS lesions (remyelinated, acute active, chronic active (rim), chronic inactive). n for each lesion type indicated in Table S2. Mann–Whitney test, * P = 0.0286. h Proportion of Acvr2a+ Olig2+ or Acvr2b+ Olig2+ from total Olig2+ cells in healthy control tissue or MS lesions (remyelinated, acute active, chronic active (rim), chronic inactive). n for each lesion type indicated in Table S2. Kruskal–Wallis test and Dunn’s multiple comparison test, * p < 0.05. i Acvr2a+ (top row; red) or Acvr2b+ (bottom row; red) and Olig2+ (blue) double positive cells in MS lesions. Scale bar 5 μm. j Quantification of differentiation of OPCs into mature oligodendrocytes (MBP+; per field) following transfection with lentivirus (GFP+), either control (control-LV) or Acvr2b-expressing (Acvr2b-LV), and treated with activin-A (10 ng ml −1 ) for 3 days. * P = 0.0247, two-tailed Student’s t test. k Representative images of OPC cultures treated with activin-A and Control-LV or Acvr2b-LV for 3 days immunostained for MBP (red) and GFP (green). Scale bar 50 µm. l Representative images of maturing oligodendrocytes transfected with Control-LV or Acv2b-LV for 3 days and treated with activin-A (10 ng ml −1 ) for 5 days, stained for GFP (green), MBP (false colored yellow), and Phalloidin (red). Scale bar 20 µm. m Phalloidin intensity signal (arbitrary units; AU) per MBP+ transfected (GFP+) oligodendrocyte plotted against oligodendrocyte size (px 2 ) in cultures co-treated with activin-A (10 ng ml −1 ) and Control-LV or Acvr2b-LV. n Model for role of activin receptor signaling in oligodendrocyte lineage cells. Acvr2a is expressed during developmental myelination, inducing oligodendrocyte differentiation, myelination, and myelin membrane compaction. Following injury, successful repair involves a transition in expression from Acvr2b to Acvr2a to support new myelin formation. In myelin disorders, failed repair is associated with an upregulation of Acvr2b, impairing Acvr2a-driven responses, leading to lack of myein

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Expressing, Immunostaining, Blocking Assay, MANN-WHITNEY, Transfection, Two Tailed Test, Staining

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling regulates oligodendrocyte differentiation. a Mean number of oligodendrocyte lineage cells (Olig2+) per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 3 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). b Mean proportion of oligodendrocyte lineage cells (Olig2+) which are mature oligodendrocytes (CC1+) versus immature cells (CC1−), per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). Multiple t tests with false discovery rate of 1%, *** P = 0.000026. c Images of differentiating oligodendrocytes (cytoplasmic Olig1+ and nuclear Olig2+) in corpus callosum of P16 Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice. Scale bar 25 μm. d Mean number of cytoplasmic Olig1 and Olig2 double positive cells per field ± s.e.m. in corpus callosum of P16 Acvr1b fl/fl ( n = 3 mice), PDGFRa-Cre; Acvr1b fl/+ ( n = 4 mice) and PDGFRa-Cre; Acvr1b fl/fl mice ( n = 7 mice). Two-tailed Student’s t test, ** P = 0.0047, 0.0026, respectively. e Images of maturing oligodendrocytes (MAG+ MBP−) at P1 in corpus callosum of Acvr1b fl/fl and PDGFRa-Cre; Acvr1b fl/fl mice. Scale bar 25 μm. f Mean number of MAG+ cultured oligodendrocytes per field in vehicle control-treated or activin-A-treated conditions (10 ng ml −1 ) in vitro. n = 3 biological replicates. Two-tailed paired Student’s t test, * P = 0.0484. g Images of cultured OPCs treated with vehicle or 10 ng ml −1 activin-A and immunostained for MAG (green), counterstained with Hoechst (blue). Scale bar 25 μm. h Data-mining of microarray of human fetal brain at 9 and 12 gestational weeks (gw) represented as fold change in expression (normalized to 9 gw), showing paralleled expression changes between activin-A ( INHBA ) and oligodendrocyte differentiation-associated genes ( MAG , MOG ) in development

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Two Tailed Test, Cell Culture, In Vitro, Microarray, Expressing

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor Acvr2a regulates oligodendrocyte lineage cell behavior. a Acvr2a (top row; green) and Acvr2b (bottom row; green) expression by oligodendrocyte lineage (Olig2+; red) throughout development (P1–P14; double positive cells indicated by arrows), counterstained with Hoechst (blue). Inset; isotype control for Acvrs. Scale bar 50 μm. b Expression of Acvr2a (red) by NG2+ cells (green) (top row), and by CC1+ cells (green) (bottom row). c Data-mining of oligodendrocyte lineage cell transcriptomes from P21–30 for Acvr2a expression, represented as t distributed stochastic neighbor embedding projection. d OPCs co-treated with activin-A and neutralizing antibodies for Acvr2a or isotype IgG. Mean percentage of oligodendrocytes (MBP+) normalized to isotype control ± s.e.m. n = 3 biological replicates, two-tailed Student’s t test, P = 0.0087. e Images of MBP+ cells (red) in cultures treated with activin-A (10 ng ml −1 ) and isotype IgG or Acvr2a-neutralizing IgG. Scale bar 75 μm. f Phalloidin intensity signal (arbitrary units; AU) per oligodendrocyte plotted against oligodendrocyte size (px 2 ) in cultures co-treated with activin-A (10 ng ml −1 ) and IgG control or neutralizing antibody against Acvr2a. n = 3 biological replicates. g Images of MBP+ oligodendrocytes (green) and filamentous actin (Phalloidin+; red) in cultures treated with activin-A and IgG or Acvr2a neutralizing antibody. h Log2-transformed phosphorylation signal of TGFβ superfamily pathways following treatment with activin-A, normalized to respective total protein signal then to vehicle control. Heat map: compared to vehicle, magenta indicates increased signal, black no change, and green reduced signal

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Expressing, Two Tailed Test, Transformation Assay

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor signaling regulates remyelination. a Representative images of organotypic cerebellar slice cultures at 7 days post lysolecithin-induced demyelination, treated with vehicle control or activin receptor agonist activin-A during remyelination, immunostained against myelin basic protein (MBP; green), and axonal neurofilament-H (NF; red). Scale bar 50 μm. b Mean remyelination index ± s.e.m. in activin-A-treated explants at 7, 10, and 14 days post lysolecithin (dpl) normalized to vehicle control from the respective time point. n = 3 animals, one-sample t test compared to theoretical mean of 1 (control), ** P = 0.0057. c Representative images of slice cultures at 14 dpl treated with vehicle control or an inhibitor of activin receptor signaling inhibin-A during remyelination, immunostained against myelin basic protein (MBP; green) and axonal neurofilament-H (NF; red). Scale bar 50 μm. d Mean remyelination index ± s.e.m. in inhibin-A-treated explants at 7, 10, and 14 dpl normalized to vehicle control from the respective time point. n = 3 animals, one-sample t test compared to theoretical mean of 1 (control), * P = 0.0165, *0.0374, **0.0004, respectively. e Acvr2a and Acvr2b expression (green) in demyelinated caudal cerebellar peduncles (CCP) at 5 days post-lesion (dpl; prior to remyelination) and 10 dpl (onset of remyelination), counterstained with Hoechst (blue). Scale bar 25 μm. f Colocalization of Acvr2b or Acvr2a (green) with NG2 (top 2 rows; red; arrowheads) or Olig2 (bottom 2 rows; red; arrowheads) at 5 and 10 dpl in CCP, counterstained with Hoechst (blue). g Mean number of cells double positive for Olig2 and Acvr2a or Acvr2b per field ± s.e.m. at 5 and 10 dpl. n = 3 = 4 animals per group. Two-tailed Student’s t test, P = 0.0345 (5 dpl), 0.0298 (10 dpl). h Non-lesioned CCP shows no staining of Acvr2b (green). Scale bar 10 μm

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Expressing, Two Tailed Test, Staining

Journal: Acta Neuropathologica

Article Title: Activin receptors regulate the oligodendrocyte lineage in health and disease

doi: 10.1007/s00401-018-1813-3

Figure Lengend Snippet: Activin receptor expression dysregulation in developmental and adult human myelin disorders. a Images of activin-A subunit (INHBA; red) immunostaining in non-injured and injured developing white matter in a case of perinatal brain injury, counterstained with Hoechst (turquoise). Scale bar 25 μm. b Mean densities of INHBA+ cells ± s.e.m. per mm 2 in non-injured and injured developing white matter in perinatal brain injury. n = 5 cases (Table S1); each patient block is represented by different color. Mann–Whitney test , *P = 0.0411. c Images of oligodendrocyte lineage cells (Olig2+; green) expressing Acvr2a or Acvr2b (red) in developing white matter, indicated by arrowheads. Scale bar 25 μm. d Densities of Acvr2a+ Olig2+ and Acvr2b+ Olig2+ cells per mm 2 in non-injured versus injured areas of developing white matter. n = 5 cases; each patient block is represented by different color. Mann–Whitney test, *P = 0.0238 (non-injured Acvr2a+ Olig2+ vs Acvr2b+ Olig2+), * P = 0.0317 (non-injured Acvr2a+ Olig2+ vs injured Acvr2a+ Olig2+). e Densities of PCNA+ Olig2+ proliferating oligodendrocyte lineage cells per mm 2 in non-injured vs injured areas of developing white matter. n = 5 cases; each patient block represented by different color. f Images of INHBA+ cells (red) in control and acute active multiple sclerosis (MS) lesion tissue, counterstained with Hoechst (turquoise). Scale bar 100 μm. g Mean densities of INHBA+ cells per mm 2 in post-mortem brain tissue from healthy control, or MS lesions (remyelinated, acute active, chronic active (rim), chronic inactive). n for each lesion type indicated in Table S2. Mann–Whitney test, * P = 0.0286. h Proportion of Acvr2a+ Olig2+ or Acvr2b+ Olig2+ from total Olig2+ cells in healthy control tissue or MS lesions (remyelinated, acute active, chronic active (rim), chronic inactive). n for each lesion type indicated in Table S2. Kruskal–Wallis test and Dunn’s multiple comparison test, * p < 0.05. i Acvr2a+ (top row; red) or Acvr2b+ (bottom row; red) and Olig2+ (blue) double positive cells in MS lesions. Scale bar 5 μm. j Quantification of differentiation of OPCs into mature oligodendrocytes (MBP+; per field) following transfection with lentivirus (GFP+), either control (control-LV) or Acvr2b-expressing (Acvr2b-LV), and treated with activin-A (10 ng ml −1 ) for 3 days. * P = 0.0247, two-tailed Student’s t test. k Representative images of OPC cultures treated with activin-A and Control-LV or Acvr2b-LV for 3 days immunostained for MBP (red) and GFP (green). Scale bar 50 µm. l Representative images of maturing oligodendrocytes transfected with Control-LV or Acv2b-LV for 3 days and treated with activin-A (10 ng ml −1 ) for 5 days, stained for GFP (green), MBP (false colored yellow), and Phalloidin (red). Scale bar 20 µm. m Phalloidin intensity signal (arbitrary units; AU) per MBP+ transfected (GFP+) oligodendrocyte plotted against oligodendrocyte size (px 2 ) in cultures co-treated with activin-A (10 ng ml −1 ) and Control-LV or Acvr2b-LV. n Model for role of activin receptor signaling in oligodendrocyte lineage cells. Acvr2a is expressed during developmental myelination, inducing oligodendrocyte differentiation, myelination, and myelin membrane compaction. Following injury, successful repair involves a transition in expression from Acvr2b to Acvr2a to support new myelin formation. In myelin disorders, failed repair is associated with an upregulation of Acvr2b, impairing Acvr2a-driven responses, leading to lack of myein

Article Snippet: Antibodies used included mouse CD68 (1:100, DAKO; M0814), rat anti-MBP (AbD Serotec, 1:250; MCA409S), rabbit anti-INHBA (activin-A subunit; 1:100, Sigma-Aldrich; HPA020031), rabbit anti-Acvr2a (1:100, Abcam; ab135634), rabbit anti-Acvr2b (1:100, Abgent; AP7105a), rabbit anti-PCNA (1:400, Abcam; ab18197), and mouse anti-Olig2 (1:100, EMD Millipore; MABN50).

Techniques: Expressing, Immunostaining, Blocking Assay, MANN-WHITNEY, Transfection, Two Tailed Test, Staining