Journal: Science advances
Article Title: Perturbed cell fate decision by schizophrenia-associated AS3MT d2d3 isoform during corticogenesis.
doi: 10.1126/sciadv.adp8271
Figure Lengend Snippet: Fig. 4. AS3MTd2d3 expression perturbs NSC fate and spindle orientation with evidence of centrosome localization. (A) Overview of the experimental approach. Cortical cells were isolated from E15.5 littermate control or AS3MTd2d3-Tg mice across two biological replicates. (B) UMAP embedding of integrated scRNA-seq cell type annotation (n = 30,246). (C and D) Trajectory inference and pseudo-time analysis between control and AS3MTd2d3-Tg. (E) E15.5 brain sections stained as indicated. in utero electroporation at E13.5 and BrdU injection at E14.5. (F and G) Quantification of the percentage of RFP+BrdU+Sox2+ or RFP+BrdU+Tuj1+ cells among the RFP+BrdU+ cells (Control, n = 5; AS3MTd2d3, n = 7). (H) Subcellular localization of Flag-AS3MTfull or Flag-AS3MTd2d3 in E14.5 brain sections, stained as indicated. (I) Quantification of the centrosome integrated intensity marked by γ-tubulin (AS3MTfull, n = 7; AS3MTd2d3, n = 18). (J) E14.5 brain sections stained as indicated. in utero electroporation at E13.5. Dotted line indicates orientation of the sister chromatids (α). (K) Quantification of the percentage of cortical progenitors within each 30° interval (Control, n = 6; AS3MTd2d3, n = 6). Scale bars, (E) 20 μm and [(H) and (J)] 10 μm. Bar graphs show the means ± SEM. Statistical significance is defined by unpaired two-tailed t tests for Sox2 and Tuj1 staining analysis, Welch’s t test for centrosome staining analysis, Kolmogorov-Smirnov test for trajectory analysis, and two-way ANOVA with Bonferroni’s post hoc test for comparisons among multiple groups; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; *****P < 0.00001. NSC, neural stem cell; NSC_M, NSC in mitosis; IPC, intermedi- ate progenitor cell; IPC_M, IPC in mitosis; PN, projection neuron; MG, microglia; CR, Cajal-Retzius; VZ, ventricular zone.
Article Snippet: Anti- FLAG rabbit polyclonal and mouse monoclonal (catalog no. F7425 and catalog no. F1804, Sigma- Aldrich), anti- FLAG rat monoclonal (catalog no. MA1- 142, Thermo Fisher Scientific), anti–α- tubulin mouse monoclonal (catalog no. 66031- 1- Ig, Proteintech Group), anti- BrdU rabbit polyclonal (catalog no. PA5- 32256, Invitrogen), anti- BrdU mouse monoclonal (catalog no. sc- 32323, Santa Cruz Biotechnology), anti–hemagglutinin (HA) rabbit polyclonal (catalog no. A190- 108A, RRID:AB_67465, Bethyl Laboratories), anti- cre mouse monoclonal (catalog no. ab24607, Abcam), anti- SOX2 (catalog no. ab5603, Merck Millipore), anti–γ- tubulin mouse monoclonal (catalog no. sc- 17787, Santa Cruz Biotechnology and catalog no. T5326, Sigma- Aldrich) and rabbit polyclonal (catalog no. ab11317, Abcam), anti- Tshz2 rabbit polyclonal (catalog no. 140189, Abcam), anti- NeuN mouse monoclonal (catalog no. MAB377), anti- NeuN guinea pig (catalog no. ABN90P, Merck Millipore), anti- Tuj1 mouse monoclonal (catalog no. MMS- 435P), anti- Cux1 rabbit polyclonal (catalog no. 11733- 1- AP, ProteinTech), anti- Ctip2 rat monoclonal (catalog no. ab18465, Abcam), anti- Satb2 mouse monoclonal (catalog no. ab51502, Abcam), anti- GABA rabbit polyclonal (catalog no. A2052, Sigma- Aldrich), anti–caspase- 3 rabbit polyclonal (catalog no. 9662, Cell Signaling Technology), anti–Tbr2 rabbit polyclonal (catalog no. AB2283, Merck Millipore), anti- Pax6 mouse monoclonal (catalog no. AB_528427, DSHB), anti- Tbr1 rabbit polyclonal (catalog no. ab31940, Abcam), anti–cleaved caspase- 3 rabbit polyclonal (catalog no. 9661, Cell Signaling Technology), anti- ASPM rabbit polyclonal (catalog no. IHC- 00058, Thermo Fisher Scientific), anti- CDK5RAP2 rabbit polyclonal (catalog no. IHC- 00063, Bethyl Laboratories), anti- WDR62 rabbit polyclonal (catalog no. A301- 560A, Thermo Fisher Scientific), and anti–p- VIM (S55) (catalog no. DO76- 3, MBL) were used for immunofluorescence and immunoblotting.
Techniques: Expressing, Isolation, Control, Staining, In Utero, Electroporation, Injection, Two Tailed Test
![Fig. 4. AS3MTd2d3 expression perturbs NSC fate and spindle orientation with evidence of centrosome localization. (A) Overview of the experimental approach. Cortical cells were isolated from E15.5 littermate control or AS3MTd2d3-Tg mice across two biological replicates. (B) UMAP embedding of integrated scRNA-seq cell type annotation (n = 30,246). (C and D) Trajectory inference and pseudo-time analysis between control and AS3MTd2d3-Tg. (E) E15.5 brain sections stained as indicated. in utero electroporation at E13.5 and BrdU injection at E14.5. (F and G) Quantification of the percentage of RFP+BrdU+Sox2+ or <t>RFP+BrdU+Tuj1+</t> cells among the RFP+BrdU+ cells (Control, n = 5; AS3MTd2d3, n = 7). (H) Subcellular localization of Flag-AS3MTfull or Flag-AS3MTd2d3 in E14.5 brain sections, stained as indicated. (I) Quantification of the centrosome integrated intensity marked by γ-tubulin (AS3MTfull, n = 7; AS3MTd2d3, n = 18). (J) E14.5 brain sections stained as indicated. in utero electroporation at E13.5. Dotted line indicates orientation of the sister chromatids (α). (K) Quantification of the percentage of cortical progenitors within each 30° interval (Control, n = 6; AS3MTd2d3, n = 6). Scale bars, (E) 20 μm and [(H) and (J)] 10 μm. Bar graphs show the means ± SEM. Statistical significance is defined by unpaired two-tailed t tests for Sox2 and <t>Tuj1</t> staining analysis, Welch’s t test for centrosome staining analysis, Kolmogorov-Smirnov test for trajectory analysis, and two-way ANOVA with Bonferroni’s post hoc test for comparisons among multiple groups; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; *****P < 0.00001. NSC, neural stem cell; NSC_M, NSC in mitosis; IPC, intermedi- ate progenitor cell; IPC_M, IPC in mitosis; PN, projection neuron; MG, microglia; CR, Cajal-Retzius; VZ, ventricular zone.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_3497/pm40153497/pm40153497__page6_image1.jpg)