Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Time-lapse imaging reveals prolonged roundup duration in Sp2−/− NPCs. (A) Schematic for generation and imaging of organotypic slice cultures from MADM cortices. Photomicrograph of an Emx1:MADM:wt/wt forebrain section at E14.5. (B) Schematic of mitotic NPCs with the clearly identifiable stages in our preparations marked by the blue bar. (C) Representative panels from live imaging experiments in control Emx1:MADM:wt/wt cortices. tdTomato-WT (red) and EGFP-WT (green) NPCs exhibited similar roundup durations. (D) Representative panels from Emx1:MADM:F/+ live imaging experiments. Green Sp2−/− progenitors spent more time in roundup than their sibling red Sp2+/+ cells. Elapsed time is indicated in minutes above each frame. Blue bars indicate frames in roundup. (E) Comparison of roundup durations indicates a significant increase in Sp2−/− (green) cells of Emx1:MADM:F/+ cortices compared with all other cell genotypes. Emx1:MADM:wt/wt: red, n=40 cells; green, n=45 cells. Emx1:MADM:F/+: red, n=56; green, n=59 cells; four independent litters analyzed for each genotype. One-way ANOVA, F=7.733; P<0.0001, *P<0.05 after Bonnferroni's multiple comparison correction. Scale bars: 500 µm in A; 10 µm in C,D. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Imaging, Control, Comparison

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NPCs have prolonged early mitosis and decreased metaphase durations. (A) Schematic of in utero electroporation of an H2B::mCherry construct to visualize chromatin in en face time-lapse imaging of organotypic whole-mount preparations. (B) Representative images of cortical NPCs with time noted in minutes above each frame. Prophase+prometaphase (purple) and metaphase (orange) periods are marked as indicated by bars underneath the panels. (C) Quantification of time spent in each stage of mitosis. Sp2F/F (control), n=65 cells from three experiments; Emx1:F/F, n=60 cells from four experiments; prophase+prometaphase, t=2.403, d.f.=123 P=0.02. Metaphase: t=3.75, d.f.=123, P=0.0003. Data are mean±s.e.m. Scale bars: 10 µm. *P<0.05, Student's t-test.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: In Utero, Electroporation, Construct, Imaging, Control

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Population analysis of control and Sp2−/− cells in the E14.5 MADM cortices. (A) Representative images of E14.5 Emx1:MADM:wt/wt and Emx1:MADM:F/+ cortices immunoenhanced for EGFP (green), tdTomato (red) and nuclei counterstained with DAPI (blue). Scale bar: 50 µm. (B) Overall green to red ratios (G:R) for total number of cells, and by layer in the developing cortex. VZ/SVZ, ventricular/subventricular zone; IZ, intermediate zone; CP, cortical plate. Total cells, t=1.785, P=0.11; VZ/SVZ, t=3.119, P=0.01; IZ, t=1.196, P=0.27; CP, t=0.4838, P=0.64. Emx1:MADM:wt/wt, n=5; Emx1:MADM:F/+, n=5. (C) Average densities (values ×104/mm3) of MADM cells overall and by layer in the developing cortex. All layers: Emx1:MADM:wt/wt, t=1.883, P=0.10; Emx1:MADM:F/+, t=0.3231, P=0.75. VZ/SVZ: Emx1:MADM:wt/wt, t=6.866, P=0.0001; Emx1:MADM:F/+, t=1.383, P=0.20. IZ: Emx1:MADM:wt/wt, t=1.346, P=0.22; Emx1:MADM:F/+, t=0.1167, P=0.91. CP: Emx1:MADM:wt/wt, t=0.0754, P=0.94; Emx1:MADM:F/+, t=0.4735, P=0.65. Emx1:MADM:wt/wt, n=5; Emx1:MADM:F/+, n=5. Dots represent values from individual embryos. *P<0.05, Student's t-test. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Control

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NPCs generate fewer subependymal and upper layer cells in the postnatal cortex. (A) Representative images of P0 Emx1:MADM:wt/wt and Emx1:MADM:F/+ cortices immunoenhanced for EGFP (green), tdTomato (red) and co-stained for NeuN (blue) to delineate the cortical layers. Scale bar: 100 µm. (B) Green to red ratios (G:R) of the P0 cortex for all layers or by layer. SEZ, subpendymal zone; WM, white matter; DL, deep layers; UL, upper layers. All layers: t=6.458, P=0.0007; SEZ: t=3.143, P=0.02; WM: t=2.275, P=0.06; DL: t=1.41, P=0.21; UL: t=4.526, P=0.004; Student's t-test, *P<0.05; m=4 embryos per genotype. (C) Densities (values ×104/mm3) for MADM cells of the P0 cortex for all layers or by layer. All layers: Emx1:MADM:wt/wt, t=0.3883, P=0.71; Emx1:MADM:F/+, t=5.645, P=0.0013. SEZ: Emx1:MADM:wt/wt, t=0.7553, P=0.48; Emx1:MADM:F/+, t=5.753, P=0.0012. WM: Emx1:MADM:wt/wt, t=0.3416, P=0.74; Emx1:MADM:F/+, t=4.619, P=0.0036. DL: Emx1:MADM:wt/wt, t=0.3719, P=0.72; Emx1:MADM:F/+, t=0.8907, P=0.41. UL: Emx1:MADM:wt/wt, t=0.1627, P=0.88; Emx1:MADM:F/+, t=3.393, P=0.014. Student's t-test, *P<0.05. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Staining

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Clonal analysis of cortical NPCs in the presence and absence of Sp2. (A) Schematic of experimental paradigm to induce individual cortical clones for analysis using the Nestin-creERT2 transgenic mouse that expresses tamoxifen-inducible cre under the control of the Nestin promoter (NcreER) in combination with MADM and floxed Sp2 alleles. Tamoxifen administration at E11.5 permits cre entry into the nucleus to induce highly sparse MADM recombination events in mitotic NPCs. Embryos were sacrificed at E18.5 for fixation, serial sectioning and immunohistochemistry to visualize and quantify clones. (B) Images of serial sections containing individual clones induced at E11.5 in NcreER:MADM:wt/wt and NcreER:MADM:F/+ embryos and harvested at E18.5. Scale bar: 50 µm. (C) Representative mapped clones reconstructed from NcreER:MADM:wt/wt and NcreER:MADM:F/+ embryos. (D) G minus R (G-R) values for total clone composition and by layer in the E18.5 cortex. All layers: NcreER:MADM:wt/wt, t=0.7405, P=0.46; NcreER:MADM:F/+, t=1.894, P=0.067. SEZ/WM: NcreER:MADM:wt/wt, t=0.4133, P=0.68; NcreER:MADM:F/+, t=1.738, P=0.091. DL: NcreER:MADM:wt/wt, t=0.415, P=0.68; NcreER:MADM:F/+, t=0.6746, P=0.50. UL: NcreER:MADM:wt/wt, t=0.8134, P=0.42; NcreER:MADM:F/+, t=2.865, P=0.0071. NcreER:MADM:wt/wt, n=37; NcreER:MADM:F/+, n=35 clones. One-sample t-test. *P<0.05. (E) Laminar distribution of red and green MADM cells in individual clones (dots) by genotype. SEZ/WM: NcreER:MADM:wt/wt, t=0.9489, P=0.35; NcreER:MADM:F/+, t=0.6531, P=0.43. DL: NcreER:MADM:wt/wt, t=0.1238, P=0.9; NcreER:MADM:F/+, t=0.25, P=0.80. UL: NcreER:MADM:wt/wt, t=1.157, P=0.251; NcreER:MADM:F/+, t=1.851, P=0.067. NcreER:MADM:wt/wt, n=37; NcreER:MADM:F/+, n=35 clones. Student's t-test. *P<0.05. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Clone Assay, Transgenic Assay, Control, Immunohistochemistry

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2 is dispensable for proliferation and expansion of NSCs. (A) Representative time-lapse en face images of FoxG1:MADM:wt/wt and FoxG1:MADM:F/+ cortical wholemounts (time indicated in minutes above each panel). Arrows indicate cells tracked for each genotype and blue bars indicate frames in roundup. Scale bar: 10 µm. (B) Quantification of the time spent in roundup. FoxG1:MADM:wt/wt: red, n=30 cells; green, n=30 cells. FoxG1:MADM:F/+: red, n=34 cells; green, n=37 cells. One-way ANOVA, F=1.828, P=0.15. (C) Representative images of E10.5 FoxG1:MADM:wt/wt and FoxG1:MADM:F/+ cortices immunoenhanced for EGFP (green) and tdTomato (red). Nuclei are counterstained with DAPI (blue). Scale bar: 25 µm. (D) G:R for cells in E10.5 cortices, t=1.113, P=0.33, n=3 embryos per genotype. (E) Red and green cell density; density values are ×104/mm3. FoxG1:MADM:wt/wt, t=2.082, P=0.11; FoxG1:MADM:F/+, t=1.519, P=0.20. n=3 animals per genotype from three independent litters. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NSCs proliferate and differentiate in the E10.5 cortex. (A) Sample western blot of E10.5 cortical lysates blotted for Sp2 and actin shows depletion of Sp2 in FoxG1:F/+ and FoxG1:F/F cortices compared with Sp2F/F controls. Weight markers are in kilodaltons (kDa). (B) E10.5 cortices counterstained with DAPI for comparison of average width in FoxG1:wt/wt (n=5) and FoxG1:F/F (n=6) mice; t=0.5565, P=0.59. Scale bar: 100 µm. (C) High-power confocal images of E10.5 cortices immunostained for PH3 and counterstained with DAPI. Quantification of PH3 densities in FoxG1:wt/wt (n=5) and FoxG1:F/F (n=5) cortices; t=1.147, P=0.28. (D) Spindle angle distribution of PH3+ cells in late anaphase or early telophase, as indicated by bins (percent per segment). Angles were measured for the cleavage planes relative to the apical surface. FoxG1:wt/wt, n=33; FoxG1:F/F, n=42; t=0.9107, P=0.37. (E) High-power images of E10.5 cortices immunostained for Tbr2 and counterstained with DAPI. Quantification of Tbr2 densities in FoxG1:wt/wt (n=3) and FoxG1:F/F (n=3) cortices; t=0.8435, P=0.45. (F) High-power images of E10.5 cortices immunostained for Tuj1 and counterstained with DAPI. Quantification of Tuj1 densities in FoxG1:wt/wt (n=4) and FoxG1:F/F (n=5) cortices; t=0.8903, P=0.40. Densities are values ×105 cells/mm3. Dots in charts represent values for individual embryos. Scale bars: 25 µm in C,E,F. Embryos were from at least three independent litters for each genotype. Student's t-test. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Western Blot, Comparison