Fig 2: 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

Fig 3: 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 (px2) 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 TGFß 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

Fig 4: 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 mm2 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 mm2 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 mm2 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 mm2 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 (px2) 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