Journal: International Journal of Molecular Sciences

Article Title: Alteration of Extracellular Matrix Components in the Anterior Pituitary Gland of Neonatal Rats Induced by a Maternal Bisphenol A Diet during Pregnancy

doi: 10.3390/ijms222312667

Figure Lengend Snippet: Immunohistochemistry of NG2 on postnatal Day 1 in the anterior pituitary glands of control rats (( A , C ): higher magnification view of ( A )) and BPA-treated rats (( B , D ): higher magnification view of ( B )). The number ( E ) and mean intensity ( F ) of NG2-positive cells decreased in the BPA-treated group. Note: the capillary lumens (asterisks), * p < 0.05, ** p < 0.01. Scale bars: 100 µm ( A , B ); 10 µm ( C , D ).

Article Snippet: Anti-NG2 rabbit polyclonal antibody (pericyte marker, diluted 1:600; Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), anti-S100 rabbit polyclonal antibody (FS cell marker, diluted 1:1000; Cat. No. Z0311, Agilent DAKO, Santa Clara, CA, USA), anti-aldolase C rabbit polyclonal antibody (novel FS cell marker, diluted 1:200; Cat. No. AB_2571658, Frontier Institute Co., Ltd., Hokkaido, Japan) or anti-collagen type I rabbit polyclonal antibody (diluted 1:400; Cat. No. ab34710, Abcam, Cambridge, UK) were incubated overnight at room temperature.

Techniques: Immunohistochemistry, Control

Journal: Acta neuropathologica

Article Title: Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood

doi: 10.1007/s00401-017-1747-1

Figure Lengend Snippet: Essential role of microglia for adequate induction of oligodendrocyte progenitors. a Scheme of experimental setup. Cx3cr1GFP/Wt mice were injected intraperitoneally (i.p.) with BLZ945 at P2, P4, P6, and P7. Analysis was performed at P8. b, c Representative blots and quantification of flow cytometric analysis of CD45+CD11b+ microglia (b) and PDGFRα+ oligodendrocyte progenitors (OPCs; c) pre-gated on living cells, single cells, Gr1− cells of mice treated as depicted in a (n = 5–6, samples are from four independent experiments). Each symbol represents one mouse. Means with SEM are represented. Significant differences were determined by an unpaired t test: *P < 0.05, **P < 0.01. d Quantitative RT-PCR of Csf1r of sorted CD45+CD11b+ microglia and NG2+PDGFRα+ OPCs from P8 NG2YFP/Wt mice (n = 5). Data are normalized to Gapdh and β-Actin and presented as fold change to microglia levels. e Representative image of a primary postnatal OPC culture showing overlapping Olig2+/DAPI+ cells (left). OPC cultures were treated for 3 days with vehicle or different concentrations of BLZ945 (0.5, 1, and 5 μM). The number of DAPI+OPCs/coverslip did not change after treatment compared to the vehicle controls. f Representative immunofluorescent images (left) of the corpus callosum showing CX3CR1+ (green) microglia and PDGFRα+ (red) OPCs in vehicle or BLZ945-treated mice. Right quantification of these cells in the corpus callosum (CC), cortex (Cx), and cerebellum (Cb). Each symbol represents one mouse. Means with SEM are represented. n = 4; samples from two independent experiments. Scale bar 40 μm. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01). g Representative immunofluorescent images (left) of the corpus callosum showing Sox9+ (red) astrocytes and PDGFRα+ (green) OPCs in vehicle or BLZ945-treated mice. Right quantification of Sox9+/PDGFRα− astrocytes in the corpus callosum (CC), cortex (Cx), and cerebellum (Cb). n = 4–5; samples from two independent experiments. Scale bar 40 μm. Significant differences were examined by an unpaired t test. n.s. not significant. h Quantification of PDGFRα+ OPCs and CC1+ oligodendrocytes in the corpus callosum (CC) and cortex (Cx) of P20 Csf1r+/+ and Csf1r−/− mice. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05). n.s. not significant

Article Snippet: For immunofluorescence in Sox10-iCreER T2 brains, coronal sections (30 μm) were cut and stained as described [ 71 ], with the following primary antibodies: chick-GFP (1:1000, Cat. No. GFP-1020, Aves Lab, Tigard, Oregon, USA), rabbit (rb)-NG2 (1:500, Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), rb-Iba1 (1:500, Cat. No. 01919741, Wako, Neuss, Germany), m-S100β (1:250, Cat. No. S2532, Sigma-Aldrich, Taufkirchen, Germany), and m-GFAP (1:500, Cat. No. G3893, Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Injection, Quantitative RT-PCR

Journal: Acta neuropathologica

Article Title: Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood

doi: 10.1007/s00401-017-1747-1

Figure Lengend Snippet: Reduced postnatal myelinogenesis in microglia-depleted mice. a Scheme of experimental setup. Cx3cr1GFP/Wt mice were injected intraperitoneally (i.p.) with BLZ945 at P2, P4, P6, and P7. Analysis was done at P20. b–f Quantification of CX3CR1+ microglia (b), PDGFRα+ OPCs (c), CC1+ oligodendrocytes (d), Sox9+/PDGFRα− astrocytes (e), and NeuN+ neurons (f) in the indicated brain regions using immunohistochemically stained brain slices. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01). Each symbol represents one mouse. Bars represent mean ± SEM. g Quantitative RT-PCR of the genes proteolipid protein (Plp), myelin basic protein (Mbp), myelin oligodendrocyte glycoprotein (Mog), chondroitin sulfate proteoglycan 4 (Cspg4 = NG2), platelet derived growth factor receptor alpha (Pdgfrα), colony stimulating 1 receptor (Csf1r), and allograft inflammatory factor 1 (Aif1) in tissue isolated from the corpus callosum and cerebellum at P20 (n = 4; samples from two independent experiments). Data were normalized to Gapdh and β-actin and are presented as fold change normed to vehicle. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). h, i Western blot analysis for the myelin proteins proteolipid protein (PLP) and myelin basic protein (MBP) in the corpus callosum and cerebellum at P20. GAPDH was used as a loading control. Quantification (right) was performed by normalization of the myelin proteins to GAPDH. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Bars indicate mean ± SEM. j Representative electron microscope images from the corpus callosum treated with vehicle or BLZ945 at P20 (left, scale bar 2 μm) and quantification thereof (right). The total number of axons (myelinated and non-myelinated cut off ≥0.5 μm), the percentage of myelinated axons of all axons, and the G-ratio (axon diameter/filament diameter) were quantified (n = 5–6; samples from two independent experiments). Each symbol represents one mouse. Mean ± SEM are presented. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05). k Scheme of experimental setup. Cx3cr1GFP/Wt mice were injected i.p. with BLZ945 at P2, P4, P6, and P7. Analysis was done at P40–42. l Western blot analysis for the myelin proteins PLP and MBP in the corpus callosum and cerebellum at P40–42. GAPDH was used as a loading control. Quantification (right) was performed by normalization of the myelin proteins to GAPDH. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05). Bars indicate mean ± SEM

Article Snippet: For immunofluorescence in Sox10-iCreER T2 brains, coronal sections (30 μm) were cut and stained as described [ 71 ], with the following primary antibodies: chick-GFP (1:1000, Cat. No. GFP-1020, Aves Lab, Tigard, Oregon, USA), rabbit (rb)-NG2 (1:500, Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), rb-Iba1 (1:500, Cat. No. 01919741, Wako, Neuss, Germany), m-S100β (1:250, Cat. No. S2532, Sigma-Aldrich, Taufkirchen, Germany), and m-GFAP (1:500, Cat. No. G3893, Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Injection, Staining, Quantitative RT-PCR, Derivative Assay, Isolation, Western Blot, Microscopy

Journal: Acta neuropathologica

Article Title: Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood

doi: 10.1007/s00401-017-1747-1

Figure Lengend Snippet: Microglia are necessary for myelin homeostasis during adulthood. a Scheme of experimental setup. 6–10-week-old Cx3cr1GFP/Wt mice were treated with BLZ945 for 7 consecutive days by oral gavage. Analysis was performed 1 day after the last application. b, c Quantification of CX3CR1+ microglia, PDGFRα+ OPCs, CC-1+ oligodendrocytes, Sox9+ astrocytes (b, d) in the corpus callosum (b) and cerebellum (c) using immunohistochemically stained brain sections at day 8. n = 4–7; samples from two independent experiments. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001; n.s not significant.) Each symbol represents one mouse. Mean ± SEM are depicted. d Quantitative RT-PCR of the genes proteolipid protein (Plp), myelin basic protein (Mbp), myelin oligodendrocyte glycoprotein (Mog), chondroitin sulfate proteoglycan 4 (Cspg4), platelet derived growth factor receptor alpha (Pdgfrα), colony stimulating 1 receptor (Csf1r), and allograft inflammatory factor 1 (Aif1) in tissue isolated from the corpus callosum and cerebellum at day 8 (n = 4–7; samples from two independent experiments). Data were normalized to Gapdh and β-actin and are presented as fold change normed to vehicle. Significant differences were examined by an unpaired t test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Bars represent mean ± SEM. e Western blot analysis for proteolipid protein (PLP) and myelin basic protein (MBP) in the corpus callosum at P8, 1 day after the last BLZ945 application. GAPDH was used as a loading control. Quantification (right) was performed by normalization of the myelin proteins to GAPDH. Bars indicate mean ± SEM. Significant differences were examined by an unpaired t test (n.s not significant). f Scheme of experimental setup. 8–10-week-old Sox10-iCreERT2 × CAG-eGFP mice were treated with BLZ945 for 5 consecutive days by oral gavage. Analysis was performed 1 day after the last application. g, i Quantification of Iba-1+ microglia, Sox10-GFP+/NG2+ OPCs (g), Sox10-GFP+/NG2− oligodendrocytes and GFAP+/S100β+ astrocytes (i) in the corpus callosum using immunohistochemically stained brain sections at day 6. n = 5–6, samples from two independent experiments. Significant differences were examined by an unpaired t test and marked with asterisks (**P < 0.01, ***P < 0.001; n.s not significant.) Each symbol represents one mouse. Mean ± SEM are depicted. h Representative confocal images of a NG2-immunofluorescence in the corpus callosum of a mouse treated with vehicle (left) or BLZ945 for 5 consecutive days. Scale bar 50 μm

Article Snippet: For immunofluorescence in Sox10-iCreER T2 brains, coronal sections (30 μm) were cut and stained as described [ 71 ], with the following primary antibodies: chick-GFP (1:1000, Cat. No. GFP-1020, Aves Lab, Tigard, Oregon, USA), rabbit (rb)-NG2 (1:500, Cat. No. AB5320, Merck Millipore, Darmstadt, Germany), rb-Iba1 (1:500, Cat. No. 01919741, Wako, Neuss, Germany), m-S100β (1:250, Cat. No. S2532, Sigma-Aldrich, Taufkirchen, Germany), and m-GFAP (1:500, Cat. No. G3893, Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Staining, Quantitative RT-PCR, Derivative Assay, Isolation, Western Blot, Immunofluorescence

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Lgl1 is expressed in oligodendrocyte progenitor cells (OPC) and its expression increases with differentiation into oligodendrocytes. a Scheme of OPC treatment before qRT-PCR. b Immunofluorescence for OPC marker NG2 and oligodendrocyte differentiation marker O4 of corpus callosum-derived OPC under proliferating or differentiating conditions. Differentiation increases O4-positive cell frequency. Scale bar = 100 µm. c Graph representing mRNA expression level changes with differentiation. qRT-PCR results are displayed on a log 2 scale and normalized to proliferative conditions. Data represented as mean ± s.e.m. of five independent experiments ( n = 5, * p < 0.05, ** p < 0.01, Mann–Whitney test). d , e Co-immunostaining of P35 wildtype mouse corpus callosum for Lgl1 and OPC marker NG2 (top panels) or differentiation marker CC1 (bottom panels). Insets are high magnification panels of Lgl1 positive cells (yellow arrows). Scale bar: 100 μm. f Schematic of coronal section of a P60 mouse showing the corpus callosum where cells were counted. g Quantification of PDGFRα-positive, NG2-positive, and CC1-positive cells per total Lgl1-positive cells. 14% were PDGFRα-positive, 22% were NG2-positive, and 59% were CC1-positive. Data are represented as mean ± s.e.m. of four independent experiments

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Marker, Derivative Assay, MANN-WHITNEY, Immunostaining

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Lgl1 knockout blocks OPC to OL differentiation. a Schematic of approach for clonal labeling and NG2 Cre-conditional deletion of Lgl1 in OPC. b Representative triple immunostaining of corpus callosum of Lgl1 WT and Lgl1 cKO from mice treated as in ( a ) for red fluorescent protein (Tom), OPC marker NG2 and differentiation marker CC1. Green arrows point to Tom+CC1+ oligodendrocytes. Orange arrows in the Lgl1 cKO panel point to Tom+NG2+CC1+ cells. Scale bar: 50 µm. c Quantification of NG2+, CC1+, and NG2+CC1+ cells ratios amongst Tom+ cells. Data are represented as mean ± s.e.m. from the analyses of 4 Lgl1 WT vs. 4 Lgl1 cKO treated mice (** p < 0.01, Mann–Whitney test). d Schematic of broad deletion of Lgl1 in P30 mice followed by FACS at P60. e Plot showing quantification of OPC (white) and OL (gray) in mice treated following scheme in ( d ). Data are represented as box & whiskers ± s.e.m. from the analyses of 6 Lgl1 WT vs. 5 Lgl1 cKO treated mice (** p < 0.01, Mann–Whitney test). f Pie charts of quantification for NG2+, O4+, astrocyte marker GLAST+ and neuroblast marker CD24+ cells amongst Tom+ cells, after Lgl1 knockout at P60 (see also Supplementary Fig. ). Data are represented as percentages from the analyses of 6 Lgl1 WT vs. 5 Lgl1 cKO mice (OPC p < 0.01; OL p < 0.01). g Representative immunostaining for NG2 and O4 of OPC and OL isolated from GFAP-Cre, Tom , Lgl1 wt/wt and, GFAP-Cre ERT2 , Tom , Lgl1 fl/fl mice. Scale bars: 100 µm. h Quantification of NG2 and O4 cells amongst all red fluorescent protein (Tom) positive cells show that Lgl1 knockout strongly blocks the differentiation of OPC into O4+OL. Data are represented as mean ± s.e.m. from the independent analyses of 4 Lgl1 WT vs. 4 Lgl1 cKO mice (** p < 0.01, Mann–Whitney test)

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: Knock-Out, Labeling, Triple Immunostaining, Marker, MANN-WHITNEY, Immunostaining, Isolation

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Lgl1 regulates asymmetric cell divisions of OPC. a Treatment schematic for in vivo pair assay in P30 NG2-Cre ERT2 Tom Lgl1 WT vs. Lgl1 cKO mice, which were given EdU for 14 days in drinking water. b Left panels: Representative cell pairs in the corpus callosum of treated mice and co-immunostained for red fluorescent protein (Tom) to label NG2+ cells and progeny, proliferation marker EdU and CC1. Symmetric, self-renewing divisions = two Tom+EdU+CC1− progeny, asymmetric cell division = one Tom+EdU+CC1− and one Tom+EdU+CC1+ progeny and symmetric, differentiating division = two Tom+EdU+CC1+ progeny. Scale bar: 10 µm. c Graph depicting the frequency of each cell division mode and showing a significant decrease of asymmetric divisions (yellow bars) and an increase of symmetric, self-renewing divisions (red bars) in Lgl1 knockout (cKO) mice when compared with wildtype (WT) mice. Data are represented as mean ± s.e.m. from the analyses of 4 Lgl1 WT vs. 4 Lgl1 cKO treated mice ( n = 4, * p < 0.05, Mann–Whitney test). d Graph depicting the percentage of three division modes during remyelination in Lgl1 WT and Lgl1 cKO corpus callosum, and showing a significant decrease of asymmetric divisions (yellow bars) and increase of symmetric, self-renewing divisions (red bars). Data are represented as mean ± s.e.m. from the analyses of 4 Lgl1 WT vs. 4 Lgl1 cKO treated mice ( n = 4, * p < 0.05, Mann–Whitney test). e Representative NG2+ OPC pairs stained for NG2 (red) and DNA (blue) showing an asymmetric division in Lgl1 WT (left panel) and symmetric, self-renewing division in Lgl1 cKO (right panel) cells. Scale bar = 10 μM. f Graph depicting percentage of cell pairs and showing that Lgl1 loss increases symmetric, self-renewing divisions at the expense of asymmetric divisions in vitro ( n = 6, * p < 0.05, Mann–Whitney test)

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: In Vivo, Marker, Knock-Out, MANN-WHITNEY, Staining, In Vitro

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Lgl1 knockout increases proliferation and promotes gliomagenesis. a Representative immunostaining of corpus callosum of Lgl1 WT mouse (top panel) and Lgl1 cKO mouse (bottom panel) for Tom (red) and co-staining for DNA (gray) after broad deletion of Lgl1 in NG2-Cre ERT2 Tom Lgl1 WT (top panel) vs. Lgl1 cKO P30 mice followed by analyses at P60 by FACS and IF. Scale bar: 200 µm. b Quantification of Tom+ cells by FACS from Lgl1 WT and Lgl1 cKO corpus callosum, showing a significant increase following Lgl1 knockout. Data are represented as mean ± s.e.m. from the analyses of 5 Lgl1 WT vs. 5 Lgl1 cKO mice (*** p < 0.001, Mann–Whitney test). c Kaplan– Meier survival curve for Lgl1 WT ( Lgl1 wt/wt ; NG2Cre; Ink4a/Arf +/- ) and NG2Cre-driven homozygous Lgl1 cKO ( Lgl1 fl/fl ; NG2Cre; Ink4a/Arf +/- ) mice. Six animals were analyzed for each genotype. d Hematoxylin and Eosin (H&E) staining of endogenous tumors in Lgl1 cKO mice, showing infiltrative growth. Scale bar: 100 μm. e , f Immunostaining of endogenous tumors in Lgl1 NG2-Cre mice for Olig2 (blue, e ) and NG2 (green, f ). Scale bar: 50 μm

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: Knock-Out, Immunostaining, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Lgl1 cKO OPC show defect in NG2 routing to the lysosome. a Time-lapse fluorescent pictures of Lgl1 WT and Lgl1 cKO OPC labeled for NG2-EC (red) and lysosome (Lys-GFP, green) under differentiation conditions. Pictures are acquired every hour for 3 hours. White arrows indicate NG2-EC that did not reach the lysosome. Time indicated as hours:minutes. Scale bar: 25 μm. b Quantitative graph representing the ratio of labeled NG2 puncta in the lysosomes in WT (black) and Lgl1 cKO cells (gray). Data are represented as mean ± s.e.m., n = 10 cells each genotype, three independent experiments (** p < 0.01, Student’s t -test). c Immunostaining of NG2dTom cells (yellow) with lysosomal protein Lamp1 (red) showing the difference in lysosome shape between Lgl1 WT OPC and cKO OPC. Scale bar: 25 μm

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: Labeling, Immunostaining

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Non-degraded NG2 in Lgl1 cKO OPC is recycled to the membrane. a Representative co-immunostaining for NG2-EC (red) and Rab11 (green) 45 min after incubation in differentiation medium. Note the higher colocalization of NG2-EC with Rab11 in Lgl1 cKO OPC (yellow arrowheads) compared to Lgl1 WT cells (red arrowhead). Scale bar: 5 μm. b – d Fluorescent immunostaining pictures illustrating colocalization of remaining NG2 (blue) with ( b ) ARF6 (red), ( c ) Rab4 (red), and ( d ) EEA1 (green) in Lgl1 cKO cells 24 hours after incubation in differentiation conditions. Scale bars: 10 μm. e , f Frames illustrating TIRF time lapse of Lgl1 cKO and WT cells 4 hours after incubation in differentiation conditions. Pictures were taken each 20 min. Examples of recycling labeled NG2 puncta (white) are highlighted in dashed circle. NG2 recycling persists in Lgl1 cKO cells up to time point 01:20 whereas it is insignificant in WT cells. Time indicated as hours:minutes. Scale bar: 10 μm. g Graph representing quantification of the corrected total cell fluorescence reflecting NG2 recycling in Lgl1 cKO cells (blue circles) and in Lgl1 WT cells (red lozenges). Data are depicted as mean ± s.e.m., n = 8 cells for each genotype, three independent experiments (*** p < 0.01, two-way ANOVA)

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: Membrane, Immunostaining, Incubation, Labeling, Fluorescence

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Lgl1 negatively regulates internalization and recycling of NG2 in OPC. a Schematic illustration of the experimental approach assessing global changes in gene expression by microarray in Lgl1 WT vs. Lgl1 cKO OPC. FACS sorted red fluorescent PDGFRα+O4− cells. b Bar graph of ingenuity pathway analyses (IPA) of Lgl1 WT vs. cKO OPC. Receptor-mediated endocytosis pathway is amongst the upregulated pathways in Lgl1 cKO cells. c Validation by q-RT-PCR for mRNA encoding receptor-mediated endocytosis regulators that were differentially expressed in Lgl1 cKO cells as determined by microarray analyses. OPC were derived from the corpus callosum of GFAP-Cre , Tom , Lgl1 wt/wt and, GFAP-Cre , Tom , Lgl1 fl/fl mice. Data represented as mean ±s.e.m. n = 6 Lgl1 WT and 6 Lgl1 cKO animals (* p < 0.05, ** p < 0.01, Mann–Whitney test). d Steady-state frames of time lapse (1 frame/ 10 min) of NG2-EC (red) and early-endosome marker Rab5a (green) in Lgl1 cKO and WT OPC. Note that NG2 stays mostly at the membrane in Lgl1 WT cells (red arrows), whereas it co-localizes with the early endosome in the cytoplasm of Lgl1 cKO OPC (yellow arrows). Scale bar: 20 µm. e , f Frames illustrating time lapse experiment under TIRF microscopy. Pictures are acquired each 30 seconds as soon as cells were exposed to differentiation conditions. Each white puncta in focus represents labeled NG2-EC docked at the membrane. Individual recycled puncta are highlighted with dashed circles of different colors. Note that in Lgl1 cKO cells, the majority of the labeled NG2 is internalized and recycled to the membrane whereas in WT cells, the majority of NG2 puncta retains a stable localization at the membrane. Scale bars: 10 μm. g Graph representing the quantification of NG2 recycling at the membrane throughout the first 5 min of differentiation conditions. Data represented as mean ± s.e.m. n = 3 cells per genotype, three independent experiments (*** p < 0.005, Student's t -test)

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: Gene Expression, Microarray, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, MANN-WHITNEY, Marker, Membrane, Microscopy, Labeling

Journal: Nature Communications

Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

doi: 10.1038/s41467-018-05099-3

Figure Lengend Snippet: Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p < 0.01, Student's t -test)

Article Snippet: The following antibodies were used at the given concentration: α-Rb NG2 (Millipore 1:200), αMs-IgM O4 (R&D, 1:400), α-Ms CC1 (Calbiochem, 1:100), α-Gt Dcx (Santa Cruz Biotechnology, 1:300), α-Gt Hugl-1 (Lgl1, Santa Cruz biotechnology, 1:200), α-Rb Hugl-1 (Lgl1, Santa Cruz Biotechnology, 1:200), α-Ms Nestin (Chemicon international, 1:400), α-Rb PlP (abcam105784, 1:1000), α-Rat MBP (abcam7349, 1:400), α-Rb-beta galactosidase (ab616, 1:200), gt-α-Olig2 (EMD Millipore; 1:200), α-Rb-Rab11 (Life Technologies Corporation, 1:200); α-Rb-p19Arf (Santa Cruz; 1:100); α-Gt-Lamp1 (R and D systems AF4320, 1:100); α-ARF 6 (Santa Cruz sc-7971; 1:200), α-Ms EEA1(Santa Cruz sc-365652, 1:150); α-Rb-Rab4 (Millipore 07-655; 1:200); α-Ms-Rab10 (Life Technologies Corporation, 1:100).

Techniques: Membrane, Labeling, Expressing

Journal: bioRxiv

Article Title: Activity-Dependent Modulation of Synapse-Regulating Genes in Astrocytes

doi: 10.1101/2020.12.30.424365

Figure Lengend Snippet: See also Fig S1, Table S1. A. Schematic of time points of VC development analyzed, corresponding to synapse development. B. Diagram of VC depicting neuronal (blue) laminar arrangement and connectivity (arrows). Astrocytes (green) are present in all layers. C-E. Astrocyte number increases in the VC across development. C . Example images of the VC from Aldh1l1-GFP mice at time points analyzed. GFP marks astrocytes (green), DAPI (magenta) labels nuclei. Layers labeled by numbers on the right in each panel. D. Quantification of C, astrocytes as a percentage of total cells within each cortical layer. E. Quantification of C, number of astrocytes per mm 2 of VC within each layer. F-I. Developmental expression pattern of the presynaptic proteins VGlut1 and VGlut2 in each cortical layer. F, H. Example images of VGlut1 or VGlut2 protein levels (white puncta). G, I. Quantification of F, H, density of VGlut1 or VGlut2 signal as threshold area per μm 2 . J-M. Developmental expression pattern of the postsynaptic AMPAR subunits GluA1 and GluA2 within each cortical layer. J, L. Example images of GluA1 or GluA2 protein levels (white puncta), DAPI (blue) labels nuclei. K, M. Quantification of J, L number of GluA1 or GluA2 positive puncta per cortical volume (μm 3 ). Scale bars in C: 50μm; In F, H, J, L: 10μm. In C-E: N=4 mice for P1; N=3 mice for P4-P28. In F-M: N= 3 mice/ age. Graphs show mean ±s.e.m., red squares average of individual mouse. *P ≤ 0.05 **P<0.01, ***P<0.001, ns (not significant) by one-way ANOVA comparing expression between time points within each layer.

Article Snippet: The following antibodies were used: Chk anti GFP (Millipore #06-896, 1:500), Rb anti Sox9 (Abcam #ab185966, 1:2000), Rb anti Aldh1l1 (Abcam #ab-87117, 1:500), Rb anti HA (CST #3724), Rb anti S100β (Abcam #ab52642, 1:100), Ms anti Neun (Millipore #MAB377), Rb anti NG2 (Millipore # Ab5320), Rb anti MOG (Proteintech # 12690-1-ap), Rb anti Iba1 (Wako #016-20001) Gp anti VGlut1 (Millipore #AB5905, 1:2000), Gp anti VGlut2 (Millipore #AB2251 1:3000, 1:5000), Rb anti GluA1 (Millipore #AB1504, 1:400), Rb anti GluA2 (Millipore #AB1768-I 1:400), Ms anti Bassoon (Enzo #VAMP500, 1:500).

Techniques: Labeling, Expressing

Journal: bioRxiv

Article Title: Activity-Dependent Modulation of Synapse-Regulating Genes in Astrocytes

doi: 10.1101/2020.12.30.424365

Figure Lengend Snippet: See also Fig S4, Table S4. A. Schematic of the experiment: VGlut2 is removed from presynaptic terminals of neurons in the lateral geniculate nucleus of the thalamus (LGN), that project to the visual cortex (VC), by crossing VGlut2 f/f mouse (WT) with RORαcre mouse line (VGlut2 cKO). Bottom: image of tdTomato reporter expression in the LGN and the VC, when RORαcre mouse is crossed with cre-dependent tdTomato reporter mouse. B. VGlut2 expression in the VC is significantly reduced in VGlut2 cKO mice. Example images of VGlut2 immunostaining in each genotype and quantification of the thresholded signal within each cortical layer. C. VGlut1 level is unaltered in VGlut2 cKO mice. Example images of VGlut1 immunostaining and quantification. In B, C. plots show mean signal ± s.e.m. Squares and circles are the average of signal in each mouse. N=5 mice/genotype. Scale bar = 50 μm. Statistical analysis by t-test within each layer. P-value on each plot. D-I. mRNA expression of astrocyte synapse-regulating genes is altered in VGlut2 cKO at P14. D, F, H. Example images of in situ hybridization of Gpc4, Gpc6 and Chrdl1 mRNA (white) as labeled; astrocyte marker Slc1a3 (Glast, green). Merged panel on the left, single-channel probe panel on the right. Arrowheads in single-channel panel mark astrocytes. Scale bar = 20 μm. E, G, I. Quantification of D, F, G respectively. E. Gpc4 mRNA expression is increased in L1; G. Gpc6 mRNA expression is decreased in L4-6; I. Chrdl1 mRNA expression is decreased in L1-4 in VGlut2 cKO mice. Data presented as scatter with mean + range, large circles are the average signal calculated from data per mouse. Grey or red dots are signals in individual astrocytes in WT and VGlut2 cKO respectively. N=5 mice/genotype, n=~200-450 astrocytes/ per age total (average and statistical analysis is calculated based on N=5 i.e. per mouse). Statistical analysis by paired t-test within each layer. P value on each plot. J-M. Increase in GluA1 protein levels and colocalization between GluA1 and VGlut1 in L1 of the VC in VGlut2 cKO mice at P14. J. Example images from WT (top) and cKO (bottom), VGlut1 in cyan and GluA1 in red. K, L, M. Quantification of VGlut1, GluA1 and colocalized puncta respectively, normalized to WT. N-Q. Decrease in GluA2 protein levels and colocalization between GluA2 and VGlut1 in L1 of the VC in VGlut2 cKO mice at P14. N. Example images from WT (top) and cKO (bottom), VGlut1 in cyan and GluA2 in red. O, P, Q. Quantification of VGlut1, GluA2 and colocalized puncta respectively, normalized to WT. In K-M and O-Q data presented as mean ± s.e.m, squares and circles each mouse. N=5 mice/ genotype. Arrowheads mark representative colocalized puncta in J, N. Scale bar = 5 μm. Statistical analysis by t-test, p-value on each plot.

Article Snippet: The following antibodies were used: Chk anti GFP (Millipore #06-896, 1:500), Rb anti Sox9 (Abcam #ab185966, 1:2000), Rb anti Aldh1l1 (Abcam #ab-87117, 1:500), Rb anti HA (CST #3724), Rb anti S100β (Abcam #ab52642, 1:100), Ms anti Neun (Millipore #MAB377), Rb anti NG2 (Millipore # Ab5320), Rb anti MOG (Proteintech # 12690-1-ap), Rb anti Iba1 (Wako #016-20001) Gp anti VGlut1 (Millipore #AB5905, 1:2000), Gp anti VGlut2 (Millipore #AB2251 1:3000, 1:5000), Rb anti GluA1 (Millipore #AB1504, 1:400), Rb anti GluA2 (Millipore #AB1768-I 1:400), Ms anti Bassoon (Enzo #VAMP500, 1:500).

Techniques: Expressing, Immunostaining, In Situ Hybridization, Labeling, Marker

Journal: bioRxiv

Article Title: Activity-Dependent Modulation of Synapse-Regulating Genes in Astrocytes

doi: 10.1101/2020.12.30.424365

Figure Lengend Snippet: See also Fig S5, Table S4. A. Schematic of comparison. Lack of the Ip3r2 receptor results in diminished Ca 2+ transients in astrocytes. B. Validation of Ip3r2 KO model. Western blot shows absence of Ip3r2 signal in VC of KO mice. C-H. mRNA expression of astrocyte synapse-regulating genes is altered in Ip3r2 KO mice at P14. C, E, G. Example images of in situ hybridization of Gpc4, Gpc6 and Chrdl1 mRNA (white); astrocyte marker Slc1a3 (Glast, green). Merged panel on the left, single-channel probe panel on the right. Arrowheads in single-channel panel mark astrocytes. Scale bar = 20 μm. D, F, H. Quantification of C, E, G respectively. D. Gpc4 mRNA expression is decreased in several layers of the VC in Ip3r2 KO mice. F. Gpc6 mRNA expression is unaltered in the VC in Ip3r2 KO mice. H. Chrdl1 mRNA expression is increased in several layers of the VC in Ip3r2 KO mice. Data presented as scatter with mean + range, large circles are calculated from average signal per mouse. Grey or purple dots are signal per astrocyte in WT and Ip3r2 KO respectively. N=5 mice/genotype, n=~200-450 astrocytes/ per age total (average and statistical analysis is calculated based on N=5 i.e. per mouse). Statistical analysis by paired t-test within each layer. P value on each plot. I-L. Decrease in VGlut1, GluA1 protein levels, and colocalization between GluA1 and VGlut1 in L1 of the VC in Ip3r2 KO mice at P14. I. Example images from WT (top) and KO (bottom), VGlut1 in cyan and GluA1 in red. J, K, L. Quantification of VGlut1, GluA1 and colocalized puncta respectively, normalized to WT. M-P. Decrease in VGlut1, and increase GluA2 protein levels, with no change in colocalization between GluA2 and VGlut1 in L1 of the VC in Ip3r2 KO mice at P14. M. Example images from WT (top) and KO (bottom), VGlut1 in cyan and GluA2 in red. N, O, P. Quantification of VGlut1, GluA2 and colocalized puncta respectively, normalized to WT. In J-L and N-P data presented as mean ± s.e.m, squares and circles represent mice. N=5 mice/genotype. Arrowheads mark representative colocalized puncta. Scale bar = 5 μm. Statistical analysis by t-test, p-value on each plot.

Article Snippet: The following antibodies were used: Chk anti GFP (Millipore #06-896, 1:500), Rb anti Sox9 (Abcam #ab185966, 1:2000), Rb anti Aldh1l1 (Abcam #ab-87117, 1:500), Rb anti HA (CST #3724), Rb anti S100β (Abcam #ab52642, 1:100), Ms anti Neun (Millipore #MAB377), Rb anti NG2 (Millipore # Ab5320), Rb anti MOG (Proteintech # 12690-1-ap), Rb anti Iba1 (Wako #016-20001) Gp anti VGlut1 (Millipore #AB5905, 1:2000), Gp anti VGlut2 (Millipore #AB2251 1:3000, 1:5000), Rb anti GluA1 (Millipore #AB1504, 1:400), Rb anti GluA2 (Millipore #AB1768-I 1:400), Ms anti Bassoon (Enzo #VAMP500, 1:500).

Techniques: Western Blot, Expressing, In Situ Hybridization, Marker

Journal: bioRxiv

Article Title: Activity-Dependent Modulation of Synapse-Regulating Genes in Astrocytes

doi: 10.1101/2020.12.30.424365

Figure Lengend Snippet: See also Fig S1, Table S1. A. Schematic of time points of VC development analyzed, corresponding to synapse development. B. Diagram of VC depicting neuronal (blue) laminar arrangement and connectivity (arrows). Astrocytes (green) are present in all layers. C-E. Astrocyte number increases in the VC across development. C . Example images of the VC from Aldh1l1-GFP mice at time points analyzed. GFP marks astrocytes (green), DAPI (magenta) labels nuclei. Layers labeled by numbers on the right in each panel. D. Quantification of C, astrocytes as a percentage of total cells within each cortical layer. E. Quantification of C, number of astrocytes per mm 2 of VC within each layer. F-I. Developmental expression pattern of the presynaptic proteins VGlut1 and VGlut2 in each cortical layer. F, H. Example images of VGlut1 or VGlut2 protein levels (white puncta). G, I. Quantification of F, H, density of VGlut1 or VGlut2 signal as threshold area per μm 2 . J-M. Developmental expression pattern of the postsynaptic AMPAR subunits GluA1 and GluA2 within each cortical layer. J, L. Example images of GluA1 or GluA2 protein levels (white puncta), DAPI (blue) labels nuclei. K, M. Quantification of J, L number of GluA1 or GluA2 positive puncta per cortical volume (μm 3 ). Scale bars in C: 50μm; In F, H, J, L: 10μm. In C-E: N=4 mice for P1; N=3 mice for P4-P28. In F-M: N= 3 mice/ age. Graphs show mean ±s.e.m., red squares average of individual mouse. *P ≤ 0.05 **P<0.01, ***P<0.001, ns (not significant) by one-way ANOVA comparing expression between time points within each layer.

Article Snippet: The following antibodies were used: Chk anti GFP (Millipore #06-896, 1:500), Rb anti Sox9 (Abcam #ab185966, 1:2000), Rb anti Aldh1l1 (Abcam #ab-87117, 1:500), Rb anti HA (CST #3724), Rb anti S100β (Abcam #ab52642, 1:100), Ms anti Neun (Millipore #MAB377), Rb anti NG2 (Millipore # Ab5320), Rb anti MOG (Proteintech # 12690-1-ap), Rb anti Iba1 (Wako #016-20001) Gp anti VGlut1 (Millipore #AB5905, 1:2000), Gp anti VGlut2 (Millipore #AB2251 1:3000, 1:5000), Rb anti GluA1 (Millipore #AB1504, 1:400), Rb anti GluA2 (Millipore #AB1768-I 1:400), Ms anti Bassoon (Enzo #VAMP500, 1:500).

Techniques: Labeling, Expressing

Journal: bioRxiv

Article Title: Activity-Dependent Modulation of Synapse-Regulating Genes in Astrocytes

doi: 10.1101/2020.12.30.424365

Figure Lengend Snippet: See also Fig S4, Table S4. A. Schematic of the experiment: VGlut2 is removed from presynaptic terminals of neurons in the lateral geniculate nucleus of the thalamus (LGN), that project to the visual cortex (VC), by crossing VGlut2 f/f mouse (WT) with RORαcre mouse line (VGlut2 cKO). Bottom: image of tdTomato reporter expression in the LGN and the VC, when RORαcre mouse is crossed with cre-dependent tdTomato reporter mouse. B. VGlut2 expression in the VC is significantly reduced in VGlut2 cKO mice. Example images of VGlut2 immunostaining in each genotype and quantification of the thresholded signal within each cortical layer. C. VGlut1 level is unaltered in VGlut2 cKO mice. Example images of VGlut1 immunostaining and quantification. In B, C. plots show mean signal ± s.e.m. Squares and circles are the average of signal in each mouse. N=5 mice/genotype. Scale bar = 50 μm. Statistical analysis by t-test within each layer. P-value on each plot. D-I. mRNA expression of astrocyte synapse-regulating genes is altered in VGlut2 cKO at P14. D, F, H. Example images of in situ hybridization of Gpc4, Gpc6 and Chrdl1 mRNA (white) as labeled; astrocyte marker Slc1a3 (Glast, green). Merged panel on the left, single-channel probe panel on the right. Arrowheads in single-channel panel mark astrocytes. Scale bar = 20 μm. E, G, I. Quantification of D, F, G respectively. E. Gpc4 mRNA expression is increased in L1; G. Gpc6 mRNA expression is decreased in L4-6; I. Chrdl1 mRNA expression is decreased in L1-4 in VGlut2 cKO mice. Data presented as scatter with mean + range, large circles are the average signal calculated from data per mouse. Grey or red dots are signals in individual astrocytes in WT and VGlut2 cKO respectively. N=5 mice/genotype, n=~200-450 astrocytes/ per age total (average and statistical analysis is calculated based on N=5 i.e. per mouse). Statistical analysis by paired t-test within each layer. P value on each plot. J-M. Increase in GluA1 protein levels and colocalization between GluA1 and VGlut1 in L1 of the VC in VGlut2 cKO mice at P14. J. Example images from WT (top) and cKO (bottom), VGlut1 in cyan and GluA1 in red. K, L, M. Quantification of VGlut1, GluA1 and colocalized puncta respectively, normalized to WT. N-Q. Decrease in GluA2 protein levels and colocalization between GluA2 and VGlut1 in L1 of the VC in VGlut2 cKO mice at P14. N. Example images from WT (top) and cKO (bottom), VGlut1 in cyan and GluA2 in red. O, P, Q. Quantification of VGlut1, GluA2 and colocalized puncta respectively, normalized to WT. In K-M and O-Q data presented as mean ± s.e.m, squares and circles each mouse. N=5 mice/ genotype. Arrowheads mark representative colocalized puncta in J, N. Scale bar = 5 μm. Statistical analysis by t-test, p-value on each plot.

Article Snippet: The following antibodies were used: Chk anti GFP (Millipore #06-896, 1:500), Rb anti Sox9 (Abcam #ab185966, 1:2000), Rb anti Aldh1l1 (Abcam #ab-87117, 1:500), Rb anti HA (CST #3724), Rb anti S100β (Abcam #ab52642, 1:100), Ms anti Neun (Millipore #MAB377), Rb anti NG2 (Millipore # Ab5320), Rb anti MOG (Proteintech # 12690-1-ap), Rb anti Iba1 (Wako #016-20001) Gp anti VGlut1 (Millipore #AB5905, 1:2000), Gp anti VGlut2 (Millipore #AB2251 1:3000, 1:5000), Rb anti GluA1 (Millipore #AB1504, 1:400), Rb anti GluA2 (Millipore #AB1768-I 1:400), Ms anti Bassoon (Enzo #VAMP500, 1:500).

Techniques: Expressing, Immunostaining, In Situ Hybridization, Labeling, Marker

Journal: bioRxiv

Article Title: Activity-Dependent Modulation of Synapse-Regulating Genes in Astrocytes

doi: 10.1101/2020.12.30.424365

Figure Lengend Snippet: See also Fig S5, Table S4. A. Schematic of comparison. Lack of the Ip3r2 receptor results in diminished Ca 2+ transients in astrocytes. B. Validation of Ip3r2 KO model. Western blot shows absence of Ip3r2 signal in VC of KO mice. C-H. mRNA expression of astrocyte synapse-regulating genes is altered in Ip3r2 KO mice at P14. C, E, G. Example images of in situ hybridization of Gpc4, Gpc6 and Chrdl1 mRNA (white); astrocyte marker Slc1a3 (Glast, green). Merged panel on the left, single-channel probe panel on the right. Arrowheads in single-channel panel mark astrocytes. Scale bar = 20 μm. D, F, H. Quantification of C, E, G respectively. D. Gpc4 mRNA expression is decreased in several layers of the VC in Ip3r2 KO mice. F. Gpc6 mRNA expression is unaltered in the VC in Ip3r2 KO mice. H. Chrdl1 mRNA expression is increased in several layers of the VC in Ip3r2 KO mice. Data presented as scatter with mean + range, large circles are calculated from average signal per mouse. Grey or purple dots are signal per astrocyte in WT and Ip3r2 KO respectively. N=5 mice/genotype, n=~200-450 astrocytes/ per age total (average and statistical analysis is calculated based on N=5 i.e. per mouse). Statistical analysis by paired t-test within each layer. P value on each plot. I-L. Decrease in VGlut1, GluA1 protein levels, and colocalization between GluA1 and VGlut1 in L1 of the VC in Ip3r2 KO mice at P14. I. Example images from WT (top) and KO (bottom), VGlut1 in cyan and GluA1 in red. J, K, L. Quantification of VGlut1, GluA1 and colocalized puncta respectively, normalized to WT. M-P. Decrease in VGlut1, and increase GluA2 protein levels, with no change in colocalization between GluA2 and VGlut1 in L1 of the VC in Ip3r2 KO mice at P14. M. Example images from WT (top) and KO (bottom), VGlut1 in cyan and GluA2 in red. N, O, P. Quantification of VGlut1, GluA2 and colocalized puncta respectively, normalized to WT. In J-L and N-P data presented as mean ± s.e.m, squares and circles represent mice. N=5 mice/genotype. Arrowheads mark representative colocalized puncta. Scale bar = 5 μm. Statistical analysis by t-test, p-value on each plot.

Article Snippet: The following antibodies were used: Chk anti GFP (Millipore #06-896, 1:500), Rb anti Sox9 (Abcam #ab185966, 1:2000), Rb anti Aldh1l1 (Abcam #ab-87117, 1:500), Rb anti HA (CST #3724), Rb anti S100β (Abcam #ab52642, 1:100), Ms anti Neun (Millipore #MAB377), Rb anti NG2 (Millipore # Ab5320), Rb anti MOG (Proteintech # 12690-1-ap), Rb anti Iba1 (Wako #016-20001) Gp anti VGlut1 (Millipore #AB5905, 1:2000), Gp anti VGlut2 (Millipore #AB2251 1:3000, 1:5000), Rb anti GluA1 (Millipore #AB1504, 1:400), Rb anti GluA2 (Millipore #AB1768-I 1:400), Ms anti Bassoon (Enzo #VAMP500, 1:500).

Techniques: Western Blot, Expressing, In Situ Hybridization, Marker