Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet: ( A, E , J ) Schematics depicting the cellular composition of the ventral telencephalic midline at embryonic day (E)12, E15 and E17. ( B, F, K ) Dcc mRNA (green), Glast-positive glia (red) and pan-Laminin (LAM)-positive leptomeninges and basement membrane (magenta) in E12, E15 and E17 wildtype mice reveal Dcc -positive/Glast-positive glial fibres (yellow arrowheads) and absence of Dcc within the interhemispheric fissure (IHF) (open yellow arrowheads). ( C, G ) DCC protein (green) and Glast protein (red) at E12 and E15 in wildtype mice reveal DCC-positive/Glast-positive glial fibres (yellow arrowheads) and absence of DCC within the IHF (open yellow arrowheads). ( D, H ) Ntn1 mRNA (green), Glast (red) and pan-LAM (magenta) in E12 and E15 wildtype mice show Ntn1 -positive/Glast-positive glial fibres (yellow arrowheads) and absence of Ntn1 within the IHF (open yellow arrowheads). ( E , inset) Schema of DCC and NTN1 expression at the E15 IHF surface, based on the results from F–I and . ( I ) NTN1 (green) and Glast (red) or β-galactosidase (β-GAL; red) immunolabelling in E15 control and Ntn1-lacZ mice identify regions of NTN1 staining present in control heterozygotes and absent in homozygous Ntn1-lacZ mice (white arrowheads) and NTN1-/β-GAL-positive puncta located in Glast-positive glia (yellow arrowheads), with insets. ( L ) DCC protein (green), glial-specific nuclear marker SOX9 (magenta) and mature astroglial marker (GFAP) in E17 wildtype mice identify DCC-positive/GFAP-positive/SOX9-positive glia (yellow arrowheads). 3V: third ventricle; Hi: telencephalic hinge. See related .

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , (1:500).

Techniques: Expressing, Staining, Marker

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet: ( A, D, F ) Schemas of key cellular components within the telencephalic midline. ( B, C ) Dcc mRNA (green), Glast-positive glia (red) and pan-Laminin (LAM)-positive leptomeninges and basement membrane (magenta) across the cortical plate (Cp) within the neocortex (NCx) or septum (Se) in horizontal sections of wildtype mice reveal Dcc -positive/Glast-positive radial glial (RG) fibres (yellow arrowheads). LV: lateral ventricle. ( E ) DCC protein (green), Gap43-positive axons (blue) and Glast-positive MZG (red) in horizontal sections of embryonic day (E)15 wildtype mice (right panels) indicate DCC-positive/Glast-positive cells (yellow arrowheads) and DCC-positive/Gap43-positive axons (blue arrowheads) that are approaching the midline and are adjacent to MZG. ( G ) Gap43-positive axons (blue), netrin 1 (NTN1) protein (green) and pan-LAM-positive leptomeninges and basement membrane (red) in horizontal sections of E15 wildtype mice reveal NTN1-positive/Gap43-positive axons (blue arrowheads) approaching the midline and NTN1-positive/LAM-positive basement membrane (BM; red arrowheads) of the interhemispheric fissure (IHF). ( H–J ) Mid-horizontal tissue sections encompassing the entire telencephalon (yellow outlines) with in situ hybridization for Dcc mRNA or Ntn1 mRNA or immunohistochemistry for DCC protein (all white or green), counterstained with DAPI (blue). Insets of the telencephalic midline are shown on the right. ( K ) DCC immunohistochemistry in horizontal sections of E17 wildtype and Dcc knockout mice with schema of key cellular components within the telencephalic midline.

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , (1:500).

Techniques: In Situ Hybridization, Immunohistochemistry, Knock-Out

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet: Nestin-positive radial glia (white; A, C, E ) and Glast-positive glia (white; B, D, F, K ) in embryonic day (E)14–E16 Dcc kanga mice ( A–F ) and E15 Ntn1-LacZ mice ( K ) demonstrate the distribution of glial processes along the interhemispheric fissure (IHF) surface (yellow brackets) and lateral to the IHF (white arrowheads) with insets ( C’, B’, D’, F’ ). Radial fibres of the glial wedge (GW) are indicated with magenta arrowheads. The mean fluorescence intensity of Glast staining between wildtype and Dcc kanga mice at E14 ( G ), E15 ( H ) and E16 ( I ) based on the results from ( B ), ( D ) and ( F ), respectively. ( J ) The ratio of glial distribution over total midline length, with schema, based on the results from ( A ) to ( F ). All graphs represent mean ± SEM. Statistics by Mann–Whitney test . n.s: not significant; *p<0.05, **p<0.01. See related and . Figure 3—source data 1. Fluorescence intensity of GLAST and ratio of glial distribution/total midline length in Dcc mouse mutants.

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , (1:500).

Techniques: Fluorescence, Staining, MANN-WHITNEY

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet: ( A, C, E ) Nuclear glial marker SOX9 (green) and MZG marker Glast (red) in embryonic day (E)14–E16 Dcc kanga mice reveal SOX9-positive/Glast-positive MZG at the pial IHF surface (boxed region and insets) above the base of the IHF (magenta arrowhead). ( B, D, F, G ) Quantification of SOX9-positive/Glast-positive MZG at the IHF pial surface based on the results from ( A), (C ) and ( E ). All graphs represent mean ± SEM. Statistics by Mann–Whitney test or Two-way unpaired Student's T test ( G ) or two-way ANOVA with post Sidak’s multiple comparison test ( B, D, F ): *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n.s: not significant. See related and . Figure 4—source data 1. Number and distribution of SOX9-positive midline zipper glia (MZG) in Dcc mouse mutants.

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , (1:500).

Techniques: Marker, MANN-WHITNEY

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet: ( A ) Gfap-positive mature astroglia (green or white in inset), Glast-positive glia (red) and pan-Laminin (LAM)-positive IHF and basement membrane (magenta) in embryonic day (E)17 wildtype Dcc kanga , Dcc knockout and Ntn1-LacZ mice. Yellow arrowheads indicate presence (filled) or absence (open) of midline glial populations, the MZG, indusium griseum glia (IGG) and glial wedge (GW). Fluorescence intensity of Gfap staining from insets or bins in insets (red dotted line) was quantified in ( B ) and ( C ). ( D ) Schema of MZG development, IHF remodelling and corpus callosum (CC) formation in wildtype mice and mice deficient for NTN1 or DCC. Red dotted lines indicate rostral and caudal bins that were used to calculate the ratio of GFAP fluorescence in ( C ). All graphs represent mean ± SEM. Statistics by Kruskal–Wallis test with post-hoc Dunn’s multiple comparison test. ***p<0.001; ns: not significant. See related , and . Figure 5—source data 1. Normalized fluorescence intensity of GFAP adjacent to the telencephalic midline in E17 Dcc and Ntn1 mutant mice.

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , (1:500).

Techniques: Knock-Out, Fluorescence, Staining, Mutagenesis

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet: ( A ) Axonal marker TUBB3 (green), ( B ) TDT (white) or ( C ) DCC (white) in P0 Dcc cKO demonstrate a spectrum of callosal and interhemispheric fissure (IHF) remodelling phenotypes and a reduction in DCC expression within mice expressing Emx1 iCre . The corpus callosum (CC) or CC remnant (CCR) and hippocampal commissure (HC) are indicated with white brackets or white arrowheads, and the IHF is indicated with yellow brackets. Red arrowheads indicate axon bundles that have not crossed the midline. ( D ) Schema of measurements taken for quantification shown in ( C – E ). ( E ) Quantification of the ratio of IHF length normalized to total telencephalic midline length measured for P0 Dcc cKO mice. ( F, G ) Quantification of CC length ( F ) and depth ( G ) normalized to the total telencephalic midline length in P0 Dcc cKO mice. ( H ) Quantification of HC length normalized to the total telencephalic midline length in P0 Dcc cKO mice. ( I ) Quantification of DCC expression measured from the cingulate cortex (CCx) and intermediate zone (IZ) of Dcc cKO mice. ( J, K ) Scatterplots of the relationship between CC length ( J ) or HC length ( K ) normalized to total telencephalic midline length and IHF length normalized to total telencephalic midline length for middle horizontal sections of P0 Dcc cKO mice. Pearson r correlations are shown. ( L ) DCC (white), ( M, N ) axonal marker GAP43 (green or white, insets) and TDT (magenta) in embryonic day (E)15 Dcc cKO mice, with quantification of mean DCC fluorescence in ( O ) and quantification of mean GAP43 fluorescence within 50 µm from the IHF (dotted red lines) in ( P ). ( Q ) TDT (white or magenta) and glial marker GLAST (green) in E15 Dcc cKO with insets and yellow arrowheads indicating GLAST-positive/TDT-positive MZG, and quantified in ( R ). All graphs represent mean ± SEM. Statistics by Mann–Whitney test or unpaired t-test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; n.s: not significant. See related and . Figure 6—source data 1. Measurements of interhemispheric fissure (IHF), corpus callosum (CC) and hippocampal commissure (HC) length and depth, deleted in colorectal carcinoma (DCC) fluorescence and GLAST-positive/TDTOMATO-positive midline zipper glia (MZG) cell bodies in Dcc cKO mice.

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , (1:500).

Techniques: Marker, Expressing, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation

doi: 10.7554/eLife.61769

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , (1:500).

Techniques: Droplet Countercurrent Chromatography, Western Blot, Immunofluorescence, Recombinant, shRNA, CRISPR, In Situ Hybridization, Sequencing, Mutagenesis, Generated, Imaging, Software

Journal: STAR Protocols

Article Title: Protocol for enhancing RNA yield and quality from single nuclei isolated from mouse brain tissue

doi: 10.1016/j.xpro.2024.103495

Figure Lengend Snippet: Single nuclei sorting and RNA quality assessment (A) Schematic overview of the FACS sorting process. (B) Representative FACS plot showing the separation of nuclei populations based on the expression of EAAT1 and NeuN markers, with distinct clusters identified for each marker. (C) Bioanalyzer gel depicting RNA extracted from EAAT1 + and NeuN + nuclei fixed in glyoxal or PFA solutions. (D) Electropherogram displaying 18S and 28S rRNA peaks from EAAT1 + and NeuN + nuclei fixed in glyoxal or PFA solutions. (E) Analysis of RNA concentration, RIN, and DV200 scores from EAAT1 + and NeuN + nuclei fixed in glyoxal or PFA solutions (N = 3–4 per group). Statistical analysis was performed using an unpaired, two-tailed Student’s t-test.

Article Snippet: Mouse monoclonal anti-EAAT1 (1:100 dilution) , Santa Cruz Biotechnology , Cat# SC-515839, not in RRID.

Techniques: Expressing, Marker, Concentration Assay, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol for enhancing RNA yield and quality from single nuclei isolated from mouse brain tissue

doi: 10.1016/j.xpro.2024.103495

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-EAAT1 (1:100 dilution) , Santa Cruz Biotechnology , Cat# SC-515839, not in RRID.

Techniques: Recombinant, Electron Microscopy, Staining, Amplification, SYBR Green Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity

doi: 10.3390/ijms241612605

Figure Lengend Snippet: Expression of EAAT1 and EAAT2 in hiPSC-derived neural cells. ( A ) The significant increase in the mRNA expression levels of EAAT1 ( a1 ) and EAAT2 ( a2 ) along with culture days was confirmed by qRT-PCR ( n = 3). ** p < 0.01 vs. DIV 0 group, Tukey’s test following ANOVA. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( B ) Representative immunoblot at 14 and 63 DIV ( b1 ). The expression level of each marker was normalized to 14 DIV. The expression levels of EAAT1 ( b2 ) and EAAT2 ( b3 ) protein tended to increase with culture days. Similar results were obtained in four independent experiments. ( C ) Identification of cell types expressed EAAT1 and EAAT2 at 63 DIV. We used the following cell markers: GFAP+Nestin+ for radial glial cells, GFAP+S100β+ for astrocytes, and HuC/D+ or MAP2+ for neurons. ( c1 ) EAAT1 was localized in radial glial cells (top) and astrocytes (bottom). ( c2 ) EAAT2 was localized in radial glial cells (top), astrocytes (middle), and neurons (bottom). Scale bar, 100 µm. Similar results were obtained in three independent experiments.

Article Snippet: Mouse anti-HuC/D monoclonal antibody (1:100, A21271, Thermo Fisher Scientific), chicken anti-GFAP polyclonal antibody (1:400, ab4674, Abcam), rabbit anti-S100β polyclonal antibody (1:500, ab52642, Abcam), goat anti-Vglut2 polyclonal antibody (1:500, Go-Af310-1, Frontier Institute, Hokkaido, Japan), rabbit anti-Syn1 polyclonal antibody (1:1000, AB1543, Chemicon), chicken anti-MAP2 polyclonal antibody (1:5000, ab5392, Abcam), mouse anti-PSD95 monoclonal antibody (1:500, 7E3-1B8, Thermo Fisher Scientific), mouse anti-EAAT1 monoclonal antibody (1:200, ab49643, Abcam), rabbit anti-Nestin polyclonal antibody (1:200, ABD69, Millipore), or guineapig anti-EAAT2 polyclonal antibody (1:1000, AB1783, Chemicon) were used.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot, Marker

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity

doi: 10.3390/ijms241612605

Figure Lengend Snippet: Inhibition of EAATs increases the extracellular L-Glu concentration and leads to excitotoxicity at 63 DIV. ( A ) The effect of L-Glu alone on cell viability at 14 and 63 DIV ( n = 6). Cell viability was assessed using the MTT reduction assay. At 14 ( a1 ) and 63 ( a2 ) DIV, the application of L-Glu at 100 μM for 24 h did not change MTT reductions compared with the application of DMSO at 0.4% (Cont), which was used as a vehicle. Unpaired t -test. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( B ) The effects of TFB-TBOA (TFB, nonspecific EAAT inhibitor, 30 nM) when coapplied with L-Glu on cell viability at 14 and 63 DIV ( n = 4–6). At 14 DIV, TFB caused no effect on MTT reductions ( b1 ). On the other hand, at 63 DIV, TFB significantly decreased MTT reductions, and AP5 (NMDAR antagonist, 100 μM) blocked the decrease in MTT reductions by TFB ( b2 ). Similar results were obtained in three independent experiments. ** p < 0.01 vs. L-Glu(+)TFB(−)AP5(−) group by Tukey’s test following ANOVA. ## p < 0.01 vs. L-Glu(+)TFB(+)AP5(−) group by Tukey’s test following ANOVA. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( C ) Identification of the contribution of specific EAATs to the decrease in exogenously applied L-Glu. ( c1 ) Change in the concentration of L-Glu in the medium ([L-Glu] out ) after L-Glu (100 μM) was applied at 63 DIV ( n = 3). [L-Glu] out was nearly zero at 60 min. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( c2 ) The effects of EAAT inhibitors on L-Glu uptake at 63 DIV ( n = 5–7). The effects of EAAT inhibitors on the decrease in [L-Glu] out were assessed at 30 min, which was the time for the 50% decrease in [L-Glu] out from C(c1). The percentage inhibition of EAAT inhibitors on L-Glu uptake activity was calculated as 100% of the decrease in extracellular L-Glu concentration in the absence of EAAT inhibitors. UCPH-101 (UCPH, EAAT1 selective EAAT1 inhibitor, 100 μM), dihydrokainic acid (DHK, a competitive EAAT2 inhibitor, 300 μM), WAY213613 (WAY, 10 μM), or TFB inhibited L-Glu uptake. Data are expressed as the means ± standard deviations. ( D ) Inverse correlation between the strength of L-Glu uptake inhibition and the cell viability caused by EAAT inhibitors. The Pearson’s correlation coefficient (PCC) = −0.7110. EAAT inhibitors are shown as follows: UCPH: blue dot; DHK: green dot, WAY: pink dot, and TFB: red dot.

Article Snippet: Mouse anti-HuC/D monoclonal antibody (1:100, A21271, Thermo Fisher Scientific), chicken anti-GFAP polyclonal antibody (1:400, ab4674, Abcam), rabbit anti-S100β polyclonal antibody (1:500, ab52642, Abcam), goat anti-Vglut2 polyclonal antibody (1:500, Go-Af310-1, Frontier Institute, Hokkaido, Japan), rabbit anti-Syn1 polyclonal antibody (1:1000, AB1543, Chemicon), chicken anti-MAP2 polyclonal antibody (1:5000, ab5392, Abcam), mouse anti-PSD95 monoclonal antibody (1:500, 7E3-1B8, Thermo Fisher Scientific), mouse anti-EAAT1 monoclonal antibody (1:200, ab49643, Abcam), rabbit anti-Nestin polyclonal antibody (1:200, ABD69, Millipore), or guineapig anti-EAAT2 polyclonal antibody (1:1000, AB1783, Chemicon) were used.

Techniques: Inhibition, Concentration Assay, MTT Reduction Assay, Activity Assay