mouse α-olig2  (Millipore)

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, Ang1 ISH in transverse sections from E10.5 to E13.5 mouse embryos at brachial level (top). Combined Ang1 ISH (pseudo-colored in red) with Olig2 immunofluorescence (bottom). Arrowheads point to Ang1+ co-localization with Olig2+ pMN-NpCs. Scale bar, 50 μm (bottom) and 100 μm (top). b, plot showing that Ang1 expression (% of the area of Ang1+ signal in ventral NpC domains; left y axis) sequentially overlaps with OpC specification (OpC counts; right y axis) at the pMN between E9.5 and E13.5 (see Methods for more details). Graph shows mean ± s.e.m. (Olig2 counts: E9.5: n = 5 images from one embryo, E10.5: n = 20 images from three independent embryos, E11.5: n = 16 images from two independent embryos, E12.5: n = 24 images from four independent embryos, E13.5: n = 15 images from four independent embryos; Ang1 expression: E9.5: n = 6, E10.5: n = 8, E11.5: n = 7, E12.5: n = 14, E13.5: n = 7 images from two independent embryos). c, d, Relative Ptch (c) and Ang1 (d) expression in NSps derived from E11.5 SC progenitors stimulated with recombinant Shh (500 ng ml−1) and/or Shh inhibitor Sant1 (150 nM). Graph shows mean ± s.e.m. (n = 5 independent experiments; one-way ANOVA. Ptch: Control versus Shh *P = 0.0391; Shh versus Shh+Sant1 *P = 0.0142. Ang1: Control versus Shh *P = 0.0375; Shh versus Shh+Sant1 *P = 0.0242). e, Whole-mount SC explants immunostained for Olig2 and Sox10 to detect OpCs (arrowheads) in the VZ and MZ after culturing in control (vehicle) or recombinant Tie2–Fc (2 μg ml−1) in the y–x plane of Z-stack images. On the right side of each image, an orthogonal projection is shown for the z–y plane of a Z-stack. Scale bar, 100 μm. Open arrowheads indicate OpCs in the pMN (VZ) and filled arrowheads in the MZ. f, Scheme summarizing the phenotype of explants treated with recombinant Tie2–Fc or in control conditions. The z–y–x planes are also depicted to illustrate the positioning of the cells. A scheme of explant preparation is also provided in Extended Data Fig. 2e. g, Quantification of the number of OpCs in the MZ (Olig2+) of Tie2–Fc-treated explants, normalized to vehicle-treated explants. Graph shows mean ± s.e.m. (control n = 13, Tie2–Fc n = 17; 5 independent litters). Unpaired two-sided t-test, *P < 0.0248.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Immunofluorescence, Expressing, Derivative Assay, Recombinant

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, Scheme showing the steps to obtain neural progenitor-derived neurospheres (NSPs) from a single-cell suspension collected from E11.5 SCs via ‘open book preparation’. After the second passage NSPs were stimulated for RNA extraction. b, Image of Secondary NSPs (NSP-2) used for all experiments. Data is representative from at least 3 independent experiments. Scale bar 100μm. c,d, NSPs (in first (NSP-1) or second passage (NSP-2)) express characteristic neural stem cell markers such as Sox2 and Nestin (c). NSPs express components of the Shh pathway (Shh, Ptch and Gli1), Ang1, but not its receptor Tie2, its co-receptor Tie1 nor the endothelial cell marker Cd31 (d). Data is representative from 3 independent experiments. e, Scheme illustrating the steps for preparation of flat-mount SC explants. The SC of E11.5 embryos was isolated by ‘open book preparation’. SC ‘open books’ were cultured for 24 h. The dashed line indicates the limit of the ventricular zone (VZ) and the mantle zone (MZ). V: ventral; D: dorsal; A: anterior; P: posterior. FP: floor plate. pMN: motor neuron progenitor domain. f, Example of 3D rendering of explants images (images are shown in 2D in main Fig. 1e). Scale bar 50μm. g, Transverse sections of wild-type SCs explants immunostained for Olig2 and Sox10 to detect OPCs in the VZ (open arrowheads) and MZ (filled arrowheads) after culturing with vehicle (control DMSO), Shh inhibitor Sant1 300 nM, and/or Ang1 300 ng/mL for 24 hours. Scale bar 100 μm. h,i, Relative expression of Ptch (h) and Ang1 (i) mRNA of control (vehicle) and Sant1 treated wild-type SC explants. Graphs show mean±SEM (Control n = 16, Sant1 n = 15 of 4 independent litters). Unpaired two-sided T-Test, Ptch ****p<0.0001, Ang1**p=0.0062. j, Quantification of the number of OPCs in the MZ of the different conditions shown in (g), normalized to control. Graph show mean±SEM (Control n = 12, Sant1 n = 11, Sant1+Ang1 n = 12, of 5 independent litters). One-way ANOVA, Control vs +Sant1 **p=0.0027; +Sant1 vs +Sant1+Ang1 *p=0.0155.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Derivative Assay, RNA Extraction, Marker, Isolation, Cell Culture, Expressing

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a,b, ISH for Shh in transverse sections of embryo SCs (a) and relative expression of Shh mRNA (b) in the neural compartment of E12.5 Ang1 fl/fl vs Ang1 fl/flNestin:Cre embryos. Graphs show mean±SEM (Ang1 fl/fl n=17, Ang1 fl/flNestin:Cre n=15, of 4 independent litters). Unpaired two-sided T-test. Scale bar 100μm (a). c,d, ISH for Shh in transverse sections of embryo SCs (c) and relative expression of Shh mRNA (d) in the neural compartment of E12.5 Ang1 fl/fl v/s Ang1 fl/flOlig2:Cre. Graphs show mean±SEM (Ang1 fl/fl n=19, Ang1 fl/flOlig2:Cre n=15, of 4 independent litters). Unpaired two-sided T-test. Scale bar 100μm (c). e, Representative images of neural-progenitors derived NSPs derived from E11.5 wild-type embryo SCs and cultured for 72 h in differentiation conditions and treated with vehicle (control), Ang1 (100 mg/mL) and/or Tie2/FC (2 μg/mL). In vitro cultures were immunostained for Olig2 (labelling neural progenitors and OL lineage cells) and PDFGRα (labelling committed OPCs, arrowheads). Scale bar 50μm. f, Quantification of Olig2+ cells per field of view (FOV) of NSP in vitro cultures treated as in (e), normalized to control conditions. g, Quantification of PDGFRα+ cells per field of view (FOV) of NSP in vitro cultures treated as in (e), normalized to control conditions. Graphs show mean±SEM (n= 6 (independent experiments) isolated and differentiated NSPs cell cultures), one-way ANOVA.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Derivative Assay, Expressing, Cell Culture, In Vitro, Isolation

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, Tgfβ1 ISH in E12.5 wild-type embryo and stained for blood vessels (IsoB4+) (arrowheads show localization of Tgfβ1 mRNA in the vasculature). Scale bar, 100 μm. b,c, Relative expression of Tgfβ1 transcript of HBMECs (b) and isolated E12.5 wild-type SC ECs (c) upon control or recombinant Ang1 stimulation. Graphs show mean ± s.e.m. (b) n = 5 independent experiments, unpaired two-sided t-test *P = 0.0146; (c) n = 6 independent experiments, unpaired two-sided t-test *P = 0.0479). d, Relative expression of Tgfβ1 transcript in HBMECs treated with vehicle (DMSO), recombinant Ang1 and/or Akt/PI3K inhibitor Ly294002. Graph shows mean ± s.e.m. (n = 5 independent experiments). One-way ANOVA, Control versus +Ang1 **P = 0.0077; +Ang1 versus +Ly294002 **P = 0.0012; +Ang1 versus +Ang1+Ly294002 ****P < 0.0001. e,f, Relative expression of Tgfβ1 mRNA in CD31+ ECs sorted from Ang1 fl/fl and Ang1 fl/flNestin:Cre E12.5 SCs (e) and Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 SCs (f). Graphs show mean ± s.e.m. (Ang1 fl/fl n = 17, Ang1 fl/flNestin:Cre n = 15, from four independent litters, unpaired two-sided t-test, *P = 0.0495; Tie2 fl/fl n = 11, Tie2 fl/fl Pdgfb: CreERT2 n = 11, from three independent litters, unpaired two-sided t-test, **P = 0.0033). g, Co-immunostaining for phospho-SMAD3 (p-SMAD3) and Olig2 at the pMN level of E12.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryo transverse section. Arrowheads show Olig2+pSMAD3+ double-positive cells. Scale bar, 25 μm. h, Quantification of p-SMAD3 fluorescence intensity per Olig2+ cell counts in the pMN of Ang1 fl/fl and Ang1 fl/flNestin:Cre normalized to control littermates. Graph shows mean ± s.e.m. (Ang1 fl/fl n = 10, Ang1 fl/flNestin:Cre n = 10, from three independent litters). Unpaired two-sided t-test, *P = 0.040. AU, arbitrary units. i, Quantification of the number of OPCs in the MZ (Olig2+) from wild-type E11.5 explants treated with α-TGFβ1 blocking antibody (α-TGFβ1 Ab) or vehicle (IgY control) (5 μg ml−1) for 24 h; values are normalized to control-treated explants. Graph shows mean ± s.e.m. (IgY control n = 12, α-TGFβ1 Ab n = 12, from three independent litters). Unpaired two-sided t-test, **P = 0.0088. j, Quantification of OPC number in the MZ (Olig2+) of Ang1 fl/fl and Ang1 fl/flNestin:Cre SC explants after culturing with control (vehicle) or recombinant TGFβ1 50 ng ml−1, normalized to control (vehicle of Ang1 fl/fl). Graph shows mean ± s.e.m. (Ang1 fl/fl n = 10 vehicle and +TGFβ1 n = 11; Ang1 fl/flNestin:Cre n = 18 vehicle and +TGFβ1 n = 10, from seven independent litters). Two-way ANOVA; Control Ang1 fl/fl versus Control Ang1 fl/flNestin:Cre *P = 0.0449; Control Ang1 fl/flNestin:Cre versus +TGFβ1 Ang1 fl/flNestin:Cre **P = 0.0029. k, Quantification of the number of OPCs in the MZ (Olig2+) of Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 SC explants after culturing with control (vehicle) or recombinant TGFβ1 50 ng ml−1, normalized to control (vehicle of Tie2 fl/fl). Graph shows mean ± s.e.m. (Tie2 fl/fl n = 15 vehicle and +TGFβ1 n = 5; Tie2 fl/fl Pdgfb:CreERT2 n = 11 vehicle and +TGFβ1 n = 10, from five independent litters). Two-way ANOVA; Control Tie2 fl/fl versus Control Tie2 fl/fl Pdgfb:CreERT2 *P = 0.0278; Control Tie2 fl/fl Pdgfb:CreERT2 versus +TGFβ1 Tie2 fl/fl Pdgfb:CreERT2 ***P = 0.0002.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Staining, Expressing, Isolation, Recombinant, Immunostaining, Fluorescence, Blocking Assay

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, Scheme showing Nestin expression in SC NPCs to illustrate that Nestin:Cre driver mediates Ang1 deletion in all NPCs. b, Images of transverse sections at brachial level of Ang1 fl/fl (control) and Ang1 fl/flNestin:Cre E12.5 embryos. Left: entire SC immunostained with Olig2. Right: pMN domain immunostained for Olig2 and Sox10. Bracket, pMN; open arrowheads indicate OPCs in the pMN (VZ) and filled arrowheads in the MZ. Scale bars, 100 μm (left) and 50 μm (images of pMN). c, Quantification of OPCs in the VZ and MZ of Ang1 fl/fl and Ang1 fl/flNestin:Cre mice, normalized to control littermates. d, Analysis of the number of Olig2+ progenitors in the pMN. e, Ratio of VZ Sox10+ cells within the total number of Olig2+ pMN cells. For c–e, graphs show mean ± s.e.m. (Ang1 fl/fl n = 7, Ang1 fl/flNestin:Cre n = 8, from four independent litters). Unpaired two-sided t-test (VZ ****P < 0.0001, MZ **P = 0.0098, Sox10% **P = 0.0051). f, Scheme showing Olig2 expression in SC pMN to illustrate that Olig2:Cre driver mediates Ang1 deletion only in pMN and its progeny. g, Images of transverse sections at brachial level of Ang1 fl/fl and Ang1 fl/flOlig2:Cre E12.5 embryos. Left: entire SC immunostained with Olig2. Right: pMN domain immunostained for Olig2 and Sox10. Bracket, pMN; open arrowheads indicate OPCs in the pMN and filled arrowheads in the MZ. Scale bars, 100 μm (left) and 50 μm (images of pMN). h, Quantification of OPCs in the VZ and MZ of Ang1 fl/fl and Ang1 fl/flOlig2:Cre. i, Analysis of the number of Olig2+ progenitors in the pMN. j, Ratio of VZ Sox10+ cells within the total number of Olig2+ pMN progenitor cells. For h–i, graphs show mean ± s.e.m. (Ang1 fl/fl n = 10, Ang1 fl/flOlig2:Cre n = 12, from three independent litters). Unpaired two-sided t-test (VZ **P = 0.0050, MZ *P = 0.0375, Sox10 ***P = 0.0009). Dashed lines mark the perimeter of the SC. FP, floor plate; RF, roof plate.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Expressing

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, ISH for Cre recombinase in transverse sections of E11.0 and E12.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryo showing Cre expression. Data is representative from 3 independent embryos per genotype, from 2 litters. Scale bar 100μm. b, Transverse section of mTmGNestin:Cre E11.5 reporter embryo, showing Nestin-driven recombination, and consequently GFP expression (green), in NPCs and differentiated cells. Tomato (red) is excluded from Nestin expressing tissue. Data is representative from 3 independent embryos from one litter. Scale bar 100 μm. c, ISH for Ang1 in transverse sections of E11.0 and E12.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryo showing efficient deletion of Ang1 mRNA in Ang1 fl/flNestin:Cre embryos. Data is representative 3 independent embryos per genotype, from 2 litters. Scale bar 100μm. d, Relative Ang1 mRNA expression in the neural compartment (CD31− negative fraction from CD31+ MACS isolation) from Ang1 fl/fl and Ang1 fl/flNestin:Cre E12.5 SCs. Graph show mean±SEM (Ang1 fl/fl n = 17, Ang1 fl/flNestin:Cre n = 15, from 4 independent litters). Unpaired two-sided T-Test, ***p=0.0001. e, Images of Ang1 fl/fl (control) vs Ang1 fl/flNestin:Cre (CNS-Ang1 deleted) embryos from E11.5 to E13.5. Scale bar 1 mm. f, Transverse thick sections of Ang1 fl/fl and Ang1 fl/flNestin:Cre immunostained for Nestin to visualize neural progenitor projections. Scale bar 100μm. g,h, Analysis at brachial (g) and thoracic (h) levels of SC tissue total area. Graphs show mean±SEM (Ang1 fl/fl n=9, Ang1 fl/flNestin:Cre n=10). Unpaired two-sided T-Test. i, Images of transverse sections at thoracic level of Ang1 fl/fl and Ang1 fl/flNestin:Cre E12.5 embryos showing the pMN domain immunostained for Olig2 and Sox10. Bracket: pMN; Open arrowheads indicate OPCs in the pMN and filled arrowheads OPCs in the MZ. Scale bar 50 μm. j, Quantification of OPCs in the VZ and MZ of Ang1 fl/fl and Ang1 fl/flNestin:Cre, normalized to controls (Ang1fl/fl). k, Analysis of the number of Olig2+ progenitors in the pMN. l, Ratio of pMN Sox10+ cells within the total number of Olig2+ pMN progenitor cells. For j-l: Graphs show mean±SEM (Ang1 fl/fl n=7, Ang1 fl/flNestin:Cre n=8, of 3 independent litters). Unpaired two-sided T-test. j: VZ ***p=0.0006; MZ **p=0.0028; l: Sox10% * p=0.0180. m, ISH for Pdgfrα in 18 μm transverse sections of E12.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryo at brachial and thoracic levels. Data is representative from Ang1 fl/fl n=7, Ang1 fl/flNestin:Cre n=8, from 3 independent litters. Arrowheads indicate Pdgfrα+ cells. Scale bar 100μm. n, Transverse sections at brachial level of Ang1 fl/fl and Ang1 fl/flNestin:Cre E16.5 embryos immunostained for Olig2. Scale bar 100μm. o, Quantification of Olig2+ OL lineage cells in E16.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryos, normalized to control (Ang1 fl/fl) littermates. Graphs show mean±SEM (Ang1 fl/fl n=10, Ang1 fl/flNestin:Cre n=13, of 3 independent litters). Unpaired two-sided T-test.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Expressing, Isolation

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: Z a, Schematic of Pdgfb expression in ECs of blood vessels to illustrate that Pdgfb:CreERT2 driver mediates Tie2 deletion in ECs. b, Protocol for tamoxifen administration to Tie2 fl/flPdgfb:CreERT2 pregnant females and embryo harvest for analysis. c, Tie2 ISH in E12.5 Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 embryos and combined with IsoB4 staining. Scale bar, 100 μm. d, Relative expression of Tie2 mRNA in isolated ECs from E12.5 Tie2 fl/fl (control) and Tie2 fl/flPdgfb:CreERT2 SCs. Graph shows mean ± s.e.m. (Tie2 fl/fl n = 10, Tie2 fl/flPdgfb:CreERT2 n = 11, from three independent litters). Unpaired two-sided t-test (****P < 0.0001). e, Images of transverse sections at brachial level of Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 embryos. Left: entire SC immunostained with Olig2. Right: pMN domain immunostained for Olig2 and Sox10. Bracket, pMN; open arrowheads indicate OPCs in the pMN and filled arrowheads in the MZ. Scale bars, 100 μm (left) and 50 μm (images of pMN). f, Quantification of OPCs in the VZ (Tie2 fl/fl n = 8, Tie2 fl/flPdgfb:CreERT2 n = 4, from two independent litters) and MZ (Tie2 fl/fl n = 11, Tie2 fl/flPdgfb:CreERT2 n = 6, from two independent litters) of Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2, normalized to controls. g, Ratio of VZ Sox10+ cells within the total number of Olig2+ pMN progenitor cells (Tie2 fl/fl n = 8, Tie2 fl/flPdgfb:CreERT2 n = 4, from two independent litters). h, Analysis of the number of Olig2+ progenitors in the pMN (Tie2 fl/fl n = 8, Tie2 fl/flPdgfb: CreERT2 n = 4, from two independent litters). Graph shows mean ± s.e.m. Unpaired two-sided t-test (VZ **P = 0.0086, MZ *P = 0.0264, Sox10 **P = 0.0068). FP, floor plate; RF, roof plate.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Expressing, Staining, Isolation

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, ISH for Cre recombinase in transverse sections of E12.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryo showing Cre expression only in the pMN domain. Data is representative from at least 3 independent embryos per genotype, from 2 litters. Scale bar 100μm. b, ISH for Ang1 (pseudo-coloured in red) combined with immunostaining for Olig2 and Isl1/2. Filled arrowheads point to the pMN and open arrowheads point to MNs in transverse sections of E11.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryos. Note the efficient deletion of Ang1 in the pMN and MNs of Ang1 fl/flOlig2:Cre embryos. Data is representative from at least 3 independent embryos per genotype, from 2 litters. Scale bar 50μm (inset) 100μm (upper panel). c, Images of Ang1 fl/fl (control) vs Ang1 fl/flOlig2:Cre embryos from E11.5 to E13.5. Scale bar 1 mm. d, Nissl staining in transverse sections of Ang1 fl/fl and Ang1 fl/flOlig2:Cre E12.5 embryos. Data is representative from at least 3 embryos per genotype, from 2 independent litters. Scale bar 100μm. e,f, Analysis at brachial (e) and thoracic (f) levels of SC total area. Graphs show mean±SEM (Ang1 fl/fl n=7, Ang1 fl/flOlig2:Cre n=14, from 3 independent litters). Unpaired two-sided T-Test. g, Images of transverse sections at thoracic level of Ang1 fl/fl (control) and Ang1 fl/flOlig2:Cre E12.5 embryos immunostained for Olig2 and Sox10. Bracket: pMN; Filled arrowhead point to MZ OPCs and open arrowheads to VZ OPCs. Scale bar 50μm. h, Quantification of OPCs in the VZ and MZ of Ang1 fl/fl and Ang1 fl/flOlig2:Cre, normalized to controls (Ang1fl/fl). i, Analysis of the number of Olig2+ progenitors in the pMN. j, Ratio of pMN Sox10+ cells within the total number of Olig2+ pMN progenitor cells. For h-j: Graphs show mean±SEM (Ang1 fl/fl n=10, Ang1 fl/flOlig2:Cre n=12, of 3 independent litters). Unpaired two-sided T-test. h: VZ *p=0.0110; MZ **p=0.0076; j: Sox10% **p=0.0033. k, ISH for Pdgfrα in 40 μm transverse sections of E12.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryo at brachial and thoracic levels. Data is representative from Ang1 fl/fl n=7 and Ang1 fl/flOlig2:Cre n=9, from 2 independent litters. Arrowheads indicate Pdgfrα positive cells. Scale bar 100μm. l, Transverse sections at brachial level of Ang1 fl/fl (control) and Ang1 fl/flOlig2:Cre E16.5 embryos immunostained for Olig2. Scale bar 100μm. m, Quantification of Olig2+ cells (OL lineage) in E16.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryos, normalized to control (Ang1 fl/fl) littermates. Graphs show mean±SEM (Ang1 fl/fl n=13, Ang1 fl/flOlig2:Cre n=8, of 3 independent litters). Unpaired two-sided T-test.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Expressing, Immunostaining, Staining

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, 100-μm transverse sections of Ang1 fl/fl and Ang1 fl/flNestin:Cre SC explants immunostained for Olig2 and Sox10 to detect OPCs in the VZ (open arrowheads) and MZ (filled arrowheads) after culturing with control (vehicle) or recombinant Ang1 300 ng ml−1 for 24 h. Scale bar, 100 μm. b, Quantification of the number of OPCs in the MZ (Olig2+), normalized to control (Ang1 fl/fl) untreated explants. Mean ± s.e.m. (Ang1 fl/fl n = 15 vehicle and n = 14 +Ang1; Ang1 fl/flNestin:Cre n = 15 vehicle and n = 14 +Ang1, from six independent litters). Two-way ANOVA (Ang1 fl/fl versus Ang1 fl/flNestin:Cre (vehicle) *P = 0.0164; Ang1 fl/flNestin:Cre vehicle versus Ang1 fl/flNestin:Cre +Ang1 **P = 0.0015). c, 100-μm transverse sections of Ang1 fl/fl and Ang1 fl/flOlig2:Cre SC explants immunostained for Olig2 and Sox10 to detect OPCs in the VZ (open arrowheads) and MZ (filled arrowheads) after culturing with control (vehicle) or recombinant Ang1 300 ng ml−1 for 24 h. Scale bar, 100 μm. d, Quantification of the number of OPCs in the MZ (Olig2+), normalized to control (Ang1 fl/fl) untreated explants. Graph shows mean ± s.e.m. (Ang1 fl/fl n = 7 vehicle and n = 4 +Ang1, Ang1 fl/flOlig2:Cre n = 3 vehicle and n = 4 +Ang1, from four independent litters). Two-way ANOVA (Control Ang1 fl/fl versus Control Ang1 fl/flOlig2:Cre **P = 0.0093; Control Ang1 fl/flOlig2:Cre versus +Ang1 Ang1 fl/flOlig2:Cre **P = 0.0074).

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Recombinant

Journal: Nature neuroscience

Article Title: Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

doi: 10.1038/s41593-020-00788-z

Figure Lengend Snippet: a, Images of E12.5 Tie2 fl/fl (control) and Tie2 fl/flPdgfb:CreERT2 embryos. Scale bar 1 mm. b, Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 transverse 40 μm sections stained with IsoB4 showing the SC. Scale bar 100μm. c, Blood vessel (BV) density quantification of Tie2 fl/fl (control) and Tie2 fl/flPdgfb:CreERT2 and normalized to control (Tie2 fl/fl) littermates (c). d, Analysis of SC total area in Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 embryos (d). For c,d: Graphs show mean±SEM (Tie2 fl/fl n=11, Tie2 fl/flPdgfb:CreERT2 n= 6, of 2 independent litters). Unpaired two-sided T-test. e, Images of thoracic transverse sections of Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 SCs. Left images: entire SC immunostained with Olig2. Right images: pMN domain immunostained for Olig2 and Sox10. Bracket: pMN; open arrowheads indicate OPCs in the pMN and filled arrowheads in the MZ. Scale bars 100μm (left panels) and 50 μm (right (pMN) panels). f, Quantification of OPCs in the VZ (Tie2 fl/fl n=8, Tie2 fl/flPdgfb:Cre n=4, of 2 independent litters) and MZ (Tie2 fl/fl n=11, Tie2 fl/flPdgfb:Cre n=6, of 2 independent litters) of Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2, normalized to controls (Tie2 fl/fl). Graphs show mean±SEM. Unpaired two-sided T-test (MZ *p=0.0338). g, Percentage of pMN Sox10+ cells within the total number of Olig2+ pMN progenitor cells. h, Analysis of Olig2+ progenitor number in the pMN. Graphs show mean±SEM. Unpaired two-sided T-test (Sox10% *p=0.0379). Graphs (g) and (h) show mean±SEM (Tie2 fl/fl n=8, Tie2 fl/flPdgfb:Cre n= 4, of 2 independent litters). Unpaired two-sided T-test.

Article Snippet: For immunofluorescence, the following primary antibodies were used at the indicated dilutions: mouse α-Isl1/2 (39.4D5, 1:200, DSHB), rabbit α-Olig2 (AB9610, 1:300, Millipore), mouse α-Olig2 (MABN50, 1:100, Millipore), rat α-PDGFRα (558774, BD Pharmingen), rabbit α-phospho Tie2 (AF2720, 1:100, R&D Systems), rat α-Tie2 (16-587-82, 1:50, eBioscience), rabbit α-phospho SMAD3 (ab52903, 1:100, Abcam), goat α-Sox10 (AF2864, 1:300, R&D Systems), rat α-CD31 (557355, 1:100, BD Pharmingen), chicken α-Nestin (NES, 1:1000, Aves), rabbit α-NG2 (AB5320, 1:100, Millipore), rabbit α-GFP (A11122, 1:100, Invitrogen), rat α-MBP (MCA409S, 1:500, AbD Serotec), rabbit α-Neurofilament-M (1:800, 841001, BioLegend) and mouse α-Neurofilament-M 2H3 (1:50, AB_2314897, DSHB).

Techniques: Staining

Journal: bioRxiv

Article Title: Oligodendrocyte Precursor Cells Are Co-Opted by the Immune System to Cross-Present Antigen and Mediate Cytotoxicity

doi: 10.1101/461434

Figure Lengend Snippet: Isolated and PDGF (20 ng/mL) expanded postnatal rat OPCs (P4-P6) were differentiated with T3 ( ), T3 + IFNγ (10 ng/mL; ), T3 + IL17 (50 ng/mL; ), or T3 + IFNγ + IL-17 ( ) for 96 hrs prior to assessment using qPCR (a) and ICC staining for MBP and Olig2 (b) . Isolated and PDGF (20 ng/mL) expanded postnatal rat OPCs (P4-P6) were left undifferentiated (0 hrs; ) or differentiated with T3 (10 nM), IFNγ (10 ng/mL), T3 + IFNγ, for 8 hrs ( ), 24 hrs ( ), 48 hrs ( ) and 96 hrs ( ) to compare gene expression between groups while obtaining information at multiple time points. (c-e) Affymetrix gene arrays analysis was performed from three independent biological replicates. (c) Venn diagram summarizing the number and overlap of up-regulated (top) and down-regulated (bottom) genes based on time point. (d) Global heat map of all probes clustered into three gene expression patterns comparing PDGF, T3, and T3 + IFNγ at all time points. (e) Targeted heat map comparing PDGF, T3, IFNγ, and T3 + IFNγ at all time points. Displayed genes were identified from GSEA analysis (supplement 3). (f) Quantitative PCR validation of OPCs differentiated with T3 ( ), T3 + IFNγ (10 ng/mL; ) or T3 + IL17 (50 ng/mL; ) for 96 hrs prior to assessment. Error bars represent the standard deviation from 3 independent primary isolations and experiments. Significance for qPCR analysis was determined by one-way ANOVA analysis followed by Dunnett’s multiple comparison analysis where T3 + IFNγ or T3 + IL17 were compared to T3 alone control (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001).

Article Snippet: In addition, α-mouse-Olig2 (1:1000; Millipore) was used in combination with α-chicken-YFP to verify that PDGFRα driven CRE recombination within the corpus callosum was restricted to the oligodendrocyte lineage.

Techniques: Isolation, Staining, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: bioRxiv

Article Title: Oligodendrocyte Precursor Cells Are Co-Opted by the Immune System to Cross-Present Antigen and Mediate Cytotoxicity

doi: 10.1101/461434

Figure Lengend Snippet: (a) TRE/IFNγ x GFAP/tTA mice were fed CPZ for a total of 6 weeks. At the start of CPZ mice were either kept on doxycycline (DOXY) ( ; n=5) or removed from doxycycline ( ; n=5) to induce astrocyte-derived IFNγ expression. Two weeks after replacement of normal diet mice were sacrificed and the corpus callosum was dissected and prepared for qPCR analysis. Significance for qPCR analysis was determined by two-tailed, unpaired t-test between doxycycline and no doxycycline conditions (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bars represent standard deviation (b) Whole brains from TRE/IFNγ x GFAP/tTA mice under the same experimental paradigm were isolated for flow cytometry analysis. The OPC population was determined by CD11b negativity and Olig2, A2B5, and PDGFRα positivity. H2Kb expression is shown in the histogram plot in which the staining control FMO ( ; pooled) is compared to mice kept on doxycycline ( ; n=5) and mice removed from doxycycline ( ; n=5). Significance for flow cytometry analysis was determined by two-tailed, unpaired Student’s t-test between doxycycline and no doxycycline conditions (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bars represent standard deviation. (c) The same gating strategy was used to determine MHC Class II (IA/IE) expression by H2Kb. Significance for flow cytometry analysis was determined by two-tailed, unpaired t-test between doxycycline ( ; n=6) and no doxycycline ( ; n=6) conditions were compared (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bars represent standard deviation. CD4 + and CD8 + populations were also analyzed within the whole brain tissue as determined by flow cytometry. The CD4+ population remains unchanged between doxycycline ( ; n=6) and doxycycline removal ( ; n=6). However, the removal of doxycycline significantly increased the number of CD8 + cells in the brain. Significance for flow cytometry analysis was determined by twotailed, unpaired Student’s t-test between doxycycline and no doxycycline conditions were compared (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bars represent standard deviation. ( e ) Percent OPC numbers were also analyzed for PDGFRα, A2B5 and O4. PDGFRα + ( top ), PDGFRα + /A2B5 + ( middle ) and PDGFRα + /O4 + ( bottom ) OPC percentages were all significantly decreased in animals in which doxycycline was removed. Significance for flow cytometry analysis was determined by two-tailed, unpaired Student’s t-test between doxycycline and no doxycycline conditions were compared (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bars represent standard deviation.

Article Snippet: In addition, α-mouse-Olig2 (1:1000; Millipore) was used in combination with α-chicken-YFP to verify that PDGFRα driven CRE recombination within the corpus callosum was restricted to the oligodendrocyte lineage.

Techniques: Derivative Assay, Expressing, Two Tailed Test, Standard Deviation, Isolation, Flow Cytometry, Staining