Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A) Control pCAG-GFP plasmids (control-IUE) or pCAG-ShhN-ires-GFP plasmids (ShhN-IUE) were electroporated into the cortical VZ on E13.5. The E18.5 brains were analyzed. The distribution patterns of electroporated cells (GFP + ) in the cortex are shown. Note that the mRNA levels of Gli1 , Ptch1 , Gad1 , Tshz1 , and Prokr2 were dramatically increased in the ShhN-IUE cortex. (B) The expressions of GSX2, ASCL1, DLX2, SP8, SP9, and OLIG2 were greatly increased in the ShhN- IUE cortex. (C) RNA-seq analysis revealed increased expression levels for SHH pathway target genes, OB interneuron lineage and oligodendrocyte lineage genes in the ShhN-IUE cortices at P0. Data are presented as means ± SEM; n = 3. ***p < 0.001, *p < 0.05; n.s., non-significant; Student’s t test in (C). Scale bars, 200 mm in (A) and (B).

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Control, RNA Sequencing, Expressing

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A and B) Scatterplot of cells after principal-component analysis and t-SNE visualization, colored according to Seurat clustering and annotated by major cell types for all the cells in the wild-type sample (A) and the ShhN -IUE sample (B). (C and D) t-SNE of cells colored by mean expression of Gsx2 and Olig2 in wild-type (C) and ShhN -IUE (D) samples. (E) The eight Gsx2 + cells in the E16.5 wild-type sample consisted of four tri-IPCs and four OB-IPCs, based on the expressions of specific genes.

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Expressing

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A) Seurat clustering was performed on all the progenitor cells in the ShhN-IUE sample. Seven clusters were identified and annotated to six cell types based on gene expression features. (B) Heatmap showing marker gene expressions in the seven cell clusters. Each column represents expressions in one cell, and each row represents expressions of one gene. (C) The t-SNE plots of cells colored by mean expression of specific marker genes. (D) Monocle analysis of all the progenitors in the ShhN-IUE samples revealed differentiation trajectories and pseudo-timelines along the cell differentiation axis. Each point represents a cell, colored by cluster identity (top) or pseudo-timeline (bottom). (E) Seurat clusters shown along the predicted pseudo-timeline differentiation trajectory.

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Gene Expression, Marker, Expressing, Cell Differentiation

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A and B) The expression of GSX2, OLIG2, and DLX2 in the cortical VZ/SVZ of wild-type (A) and ShhN-IUE (B) mice at E17. Note that very few GSX2 + cells (green) were present in the cortical SVZ. Arrows indicate GSX2 + OLIG2 + DLX2 tri-IPCs, and arrowheads indicate GSX2 + DLX2 + OB-IPCs. (C and D) The expression of SP9 and SP8 in the cortical VZ/SVZ of wild-type (C) and ShhN-IUE (D) mice at E17. (E and F) More OB interneuron lineage cells (E) and more tri-IPCs and OB-IPCs (F) were observed in the ShhN-IUE cortices than in the controls. (G) The sequential expression of GSX2/DLX2/SP9/SP8 is linked to lineage differentiation from tri-IPCs/OB-IPCs/OB neuroblasts, indicating the core transcriptional network for OB interneuron generation. Data are presented as means ± SEM; n = 3 mice for each condition. ***p < 0.001, **p < 0.01, *p < 0.05; Student’s t test in (E) and (F). Scale bars, 50 mm in (A)–(D) and (G).

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Expressing

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet:

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques:

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Recombinant, Imaging, Software

Journal: Developmental biology

Article Title: PKA-mediated Gli2 and Gli3 phosphorylation is inhibited by Hedgehog signaling in cilia and reduced in Talpid3 mutant

doi: 10.1016/j.ydbio.2017.06.035

Figure Lengend Snippet: Hedgehog signaling inhibits PKA-mediated Gli2 and Gli3 phosphorylation in cilia. (A) Immunoblots showing the levels of Gli2FL, Gli3FL, and phosphorylated Gli2 and Gli3 (pGli) in C3H10T1/2 cells. Graphs show the relative pGli levels either with or without being normalized to those of Gli2FL and Gli3FL. Two-tailed Student t-test p-value = 0.034 < 0.5. (B) Immunoblots showing the time course of the levels of Gli2FL, Gli3FL, and pGli in response to SAG. Graphs to the right show the relative pGli/(Gli2+Gli3) or pGli values. P-values ≤ 0.0067 for bar graphs marked with *. (C) Representative images showing Gli2, Gli3, and pGli staining in cilia before and after treatment with ShhN or SAG. (D) Fifteen randomly chosen cilia that show positive staining for Gli2, Gli3, and pGli. The graphs show the arbitrary intensity of pGli staining per cilia by either with (upper graph) or without (lower graph) being normalized against Gli2FL and Gli3FL levels. Two-tailed Student t-test p-value ≤ 0.00057 (upper graph) or 0.036 (lower graph), respectively.

Article Snippet: ShhN conditioned medium (1:10 dilution) and SAG (300 nM) (Cayman Chemical) were used to stimulate C3H10T1/2 for overnight or times as indicated.

Techniques: Western Blot, Two Tailed Test, Staining