Journal: iScience
Article Title: LPLAT11/MBOAT7-driven phosphatidylinositol remodeling ensures radial glial cell integrity in developing neocortex
doi: 10.1016/j.isci.2025.114248
Figure Lengend Snippet: Mboat7 KO cortex exhibits reduced PI(4,5)P 2 levels, and pharmacological inhibition of PI(4,5)P 2 synthesis induces Golgi rounding (A) Measurement of total PI in the cortices in Mboat7 +/− and Mboat7 −/− mice at E11.5–E13.5 using LC-MS/MS-based method ( n = 3 embryos [E13.5, Mboat7 −/− ], n = 4 embryos [E11.5 and E12.5 Mboat7 +/− ], and n = 5 embryos [E12.5 Mboat7 −/− and E13.5 Mboat7 +/− ] from two independent litters). Peak areas are normalized by the area of the internal standard (25:0 PI). Ara and non-Ara indicate arachidonic acid-containing and non-arachidonic-acid-containing species, respectively. (B, C) Measurement of total PI4P (B) and PI(4,5)P 2 (C) in the cortices in Mboat7 +/− and Mboat7 −/− mice at E11.5–E13.5 using SFC-MS/MS-based method ( n = 3 embryos [E11.5, E12.5], n = 6 embryos [E13.5 Mboat7 −/− ], and n = 7 embryos [E13.5 Mboat7 +/− ] from two independent litters). Peak areas are normalized by the area of internal standard (37:4 PI4P or 37:4 PI(4,5)P 2 ). (D–F) Imaging MS analysis of PI (D), PIP (E), and PIP 2 (F) at E13.5 cortices of Mboat7 +/− and Mboat7 −/− mice. Signals were normalized by total ion current. (G) Immunofluorescence staining for PI(4,5)P 2 in the cortices of Mboat7 +/− and Mboat7 −/− mice at E13.5. The right panels show zoomed-in views of the area within the dotted frame. (H) Quantitative analysis of PI(4,5)P 2 positive dots per area within 100-μm-wide bins ( n = 5 embryos [ Mboat7 +/− ] and n = 4 embryos [ Mboat7 −/− ] from two independent litters). (I) Immunofluorescence staining for GM130 in cultured E12.5 cortical hemispheres treated with DMSO or 1 μM PIPKIγ inhibitor (UNC3230). The Golgi apparatus is rounded in the hemispheres treated with UNC3230. The right panels show zoomed-in views of the area within the dotted frame. (J) Measurement of the lengths of the GM130 + Golgi apparatus in the ventricular zone within 50-μm-wide bins ( n = 4 hemispheres [DMSO] and n = 4 hemispheres [UNC3230] from two independent litters and two independent experiments) in (I). (K–O) UNC3230 was administered into the ventricle of wild-type mice at E12.5. PBS (containing 0.1% DMSO) was administered as the control group. The E13.5 cortices were immunostained for PI(4,5)P 2 (K), GM130 (L), E-cadherin (M), Sox2 (N), and p-H3 (O). (P) Quantitative analysis of PI(4,5)P 2 -positive dots per area within 100-μm-wide bins ( n = 4 embryos [PBS] and n = 6 embryos [UNC3230] from two independent litters). (Q) Graph shows the lengths of the GM130 + Golgi apparatus in the ventricular zone within 50-μm-wide bins ( n = 703 cells from 3 embryos [PBS] and n = 670 cells from 3 embryos [UNC3230] from two independent litters). (R) Ratio of apical intensity against total intensity from VZ to MZ is shown for E-cadherin ( n = 7 embryos [PBS] and n = 6 embryos [UNC3230] from two independent litters). (S) Quantitative analysis of apical and dispersed RGCs positive for p-H3 (Sox2 + p-H3 + cells) per area within 200-μm-wide bins ( n = 6 embryos [PBS] and n = 8 embryos [UNC3230] from two independent litters). (T) Immunofluorescence staining for GM130 in cultured E12.5 cortical hemispheres from Mboat7 +/− and Mboat7 −/− mice. Rounding of the Golgi apparatus was observed in Mboat7 +/− hemispheres treated with 10 μM LPLAT11 inhibitor (Sevenin-1). (U) Measurement of the lengths of the GM130 + Golgi apparatus in the ventricular zone within 50-μm-wide bins ( n = 5 hemispheres [DMSO, Mboat7 +/− ], n = 4 hemispheres [Sevenin-1, Mboat7 +/− ], n = 5 hemispheres [DMSO, Mboat7 −/− ], and n = 4 hemispheres [Sevenin-1, Mboat7 −/− ] from two independent litters and two independent experiments). Data are shown as mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; unpaired two-tailed Student’s t test (C, H, P, R, S), unpaired two-tailed Welch’s t test (J, Q), and one-way ANOVA with Tukey’s post hoc test (U). The color of the asterisks corresponds to the color of the respective groups in the graph. Scale bars, 20 μm [K, L, T, and enlarged figures in (G, I)]; 500 μm (D–F); 100 μm (others). See also and .
Article Snippet: For PIPKIγ inhibitor treatment, UNC3230 (TargetMol Chemicals Inc.) dissolved in DMSO was added to the culture medium at a final concentration of 1 μM, which reduced the level of PI(4,5)P 2 without any apparent toxicity among the tested concentrations (0.25, 0.5, and 1 μM).
Techniques: Inhibition, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Imaging, Immunofluorescence, Staining, Cell Culture, Control, Two Tailed Test
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![Mboat7 KO cortex exhibits reduced PI(4,5)P 2 levels, and pharmacological inhibition of PI(4,5)P 2 synthesis induces Golgi rounding (A) Measurement of total PI in the cortices in Mboat7 +/− and Mboat7 −/− mice at E11.5–E13.5 using LC-MS/MS-based method ( n = 3 embryos [E13.5, Mboat7 −/− ], n = 4 embryos [E11.5 and E12.5 Mboat7 +/− ], and n = 5 embryos [E12.5 Mboat7 −/− and E13.5 Mboat7 +/− ] from two independent litters). Peak areas are normalized by the area of the internal standard (25:0 PI). Ara and non-Ara indicate arachidonic acid-containing and non-arachidonic-acid-containing species, respectively. (B, C) Measurement of total PI4P (B) and PI(4,5)P 2 (C) in the cortices in Mboat7 +/− and Mboat7 −/− mice at E11.5–E13.5 using SFC-MS/MS-based method ( n = 3 embryos [E11.5, E12.5], n = 6 embryos [E13.5 Mboat7 −/− ], and n = 7 embryos [E13.5 Mboat7 +/− ] from two independent litters). Peak areas are normalized by the area of internal standard (37:4 PI4P or 37:4 PI(4,5)P 2 ). (D–F) Imaging MS analysis of PI (D), PIP (E), and PIP 2 (F) at E13.5 cortices of Mboat7 +/− and Mboat7 −/− mice. Signals were normalized by total ion current. (G) Immunofluorescence staining for PI(4,5)P 2 in the cortices of Mboat7 +/− and Mboat7 −/− mice at E13.5. The right panels show zoomed-in views of the area within the dotted frame. (H) Quantitative analysis of PI(4,5)P 2 positive dots per area within 100-μm-wide bins ( n = 5 embryos [ Mboat7 +/− ] and n = 4 embryos [ Mboat7 −/− ] from two independent litters). (I) Immunofluorescence staining for GM130 in cultured E12.5 cortical hemispheres treated with DMSO or 1 μM <t>PIPKIγ</t> <t>inhibitor</t> (UNC3230). The Golgi apparatus is rounded in the hemispheres treated with UNC3230. The right panels show zoomed-in views of the area within the dotted frame. (J) Measurement of the lengths of the GM130 + Golgi apparatus in the ventricular zone within 50-μm-wide bins ( n = 4 hemispheres [DMSO] and n = 4 hemispheres [UNC3230] from two independent litters and two independent experiments) in (I). (K–O) UNC3230 was administered into the ventricle of wild-type mice at E12.5. PBS (containing 0.1% DMSO) was administered as the control group. The E13.5 cortices were immunostained for PI(4,5)P 2 (K), GM130 (L), E-cadherin (M), Sox2 (N), and p-H3 (O). (P) Quantitative analysis of PI(4,5)P 2 -positive dots per area within 100-μm-wide bins ( n = 4 embryos [PBS] and n = 6 embryos [UNC3230] from two independent litters). (Q) Graph shows the lengths of the GM130 + Golgi apparatus in the ventricular zone within 50-μm-wide bins ( n = 703 cells from 3 embryos [PBS] and n = 670 cells from 3 embryos [UNC3230] from two independent litters). (R) Ratio of apical intensity against total intensity from VZ to MZ is shown for E-cadherin ( n = 7 embryos [PBS] and n = 6 embryos [UNC3230] from two independent litters). (S) Quantitative analysis of apical and dispersed RGCs positive for p-H3 (Sox2 + p-H3 + cells) per area within 200-μm-wide bins ( n = 6 embryos [PBS] and n = 8 embryos [UNC3230] from two independent litters). (T) Immunofluorescence staining for GM130 in cultured E12.5 cortical hemispheres from Mboat7 +/− and Mboat7 −/− mice. Rounding of the Golgi apparatus was observed in Mboat7 +/− hemispheres treated with 10 μM LPLAT11 inhibitor (Sevenin-1). (U) Measurement of the lengths of the GM130 + Golgi apparatus in the ventricular zone within 50-μm-wide bins ( n = 5 hemispheres [DMSO, Mboat7 +/− ], n = 4 hemispheres [Sevenin-1, Mboat7 +/− ], n = 5 hemispheres [DMSO, Mboat7 −/− ], and n = 4 hemispheres [Sevenin-1, Mboat7 −/− ] from two independent litters and two independent experiments). Data are shown as mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; unpaired two-tailed Student’s t test (C, H, P, R, S), unpaired two-tailed Welch’s t test (J, Q), and one-way ANOVA with Tukey’s post hoc test (U). The color of the asterisks corresponds to the color of the respective groups in the graph. Scale bars, 20 μm [K, L, T, and enlarged figures in (G, I)]; 500 μm (D–F); 100 μm (others). See also and .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7648/pmc12757648/pmc12757648__gr7.jpg)
