Figure 7.
NSCs from NT require vinculin to progress from G2 to M phase. (A) Images of transverse sections of chicken NTs transfected at stage HH12 for 24, 36, and 48 h with shControl or shVCL, stained with anti N-cadherin (grayscale) and anti PH3 (magenta), GFP (green) indicates transfection. The transfected halves of the NTs are shown without the GFP channel at the right hand of each image for clarity. Scale bar = 50 μm. (B) Bar graph showing the percentage of mitotic cells (PH3+) relative to GFP population in NTs transfected with shControl or shVCL for 24, 36, and 48 h. Mean ± SEM. One-way ANOVA plus Tukey’s multi-comparisons test, n = 6–20 sections per condition from three experiments. (C) Bar graph showing the percentage of mitosis relative to transfected cells in ex-vivo chicken NT slices from Fig. 5; cultured in control, blebbistatin, or shVCL conditions. Mean ± SEM. One-way ANOVA plus Tukey’s multi-comparisons test, n = 29,072 cells for control, 22,087 for blebbistatin and 18,951 for shVCL, from two movies. (D) Time schedule used for the experiments shown in Fig. 7, E–H. (E) Images of transverse sections of chick NTs electroporated at stage HH12 for 24 h with shControl or shVCL, the embryos were treated in ovo with EdU for the last 4 h before fixation. The sections were stained with anti-PH3 (magenta) and anti-EdU (green). GFP (blue) shows transfection. Scale bar = 50 μm. (F) Dot plot showing the apicobasal position of EdU+/GFP+ nuclei from NTs transfected at stage HH12 for 24 h with shControl or shVCL. The black bars indicate the median. t test, n = 8 sections for control and 11 sections for shVCL, from two independent experiments using at least three embryos per condition. (G) Bar graphs showing the percentage of mitotic cells (PH3+) that had incorporated EdU among the GFP population in NTs transfected at stage HH12 for 24 h with shControl or shVCL. Mean ± SEM. t test, n = 15 sections for control and 15 for shVCL from three independent experiments using at least three embryos per condition. (H) As in G but counting all the cells that incorporated EdU among the GFP population. Mean ± SEM. t test, n = 15 sections for control and 16 for shVCL from three experiments using at least three embryos per condition. (I) Images of transverse sections of chicken NTs transfected at stage HH12 for 24 h with geminin·AG (green), H2B·RFP (magenta), and pSUPER-shControl or pSUPER-shVCL (vectors without GFP), stained with phalloidin (cyan) and DAPI (blue). Scale bar = 50 μm. (J) Dot plot showing the relative FI of Geminin·AG versus H2B·RFP in ShControl or ShVCL transfected NTs. Mean ± SEM. t test, n = 7 slices from two experiments using at least three embryos per condition. (K) Plot profiles showing the FI of Geminin·AG (green) and H2B·RFP (purple) along the apicobasal axis 24 hpe of ShControl or ShVCL. Mean ± SD. n = 7 sections for control and 7 for shVCL from two experiments using at least three embryos per condition. (L) Flow cytometry analysis profiles showing DNA content (Hoechst fluorescence intensity) and cell number of GFP+ population from chicken NTs transfected at stage HH12 with shControl or shVCL for 24 h (M) Bar graph showing the percentage of cells in each cell cycle phase calculated using the profiles shown in Fig. 7 L. Mean ± SEM. t test. Two experiments using at least three embryos per condition. A minimum of 2,000 cells were analyzed in each experiment. ns = non-significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

NSCs from NT require vinculin to progress from G2 to M phase. (A) Images of transverse sections of chicken NTs transfected at stage HH12 for 24, 36, and 48 h with shControl or shVCL, stained with anti N-cadherin (grayscale) and anti PH3 (magenta), GFP (green) indicates transfection. The transfected halves of the NTs are shown without the GFP channel at the right hand of each image for clarity. Scale bar = 50 μm. (B) Bar graph showing the percentage of mitotic cells (PH3+) relative to GFP population in NTs transfected with shControl or shVCL for 24, 36, and 48 h. Mean ± SEM. One-way ANOVA plus Tukey’s multi-comparisons test, n = 6–20 sections per condition from three experiments. (C) Bar graph showing the percentage of mitosis relative to transfected cells in ex-vivo chicken NT slices from Fig. 5; cultured in control, blebbistatin, or shVCL conditions. Mean ± SEM. One-way ANOVA plus Tukey’s multi-comparisons test, n = 29,072 cells for control, 22,087 for blebbistatin and 18,951 for shVCL, from two movies. (D) Time schedule used for the experiments shown in Fig. 7, E–H. (E) Images of transverse sections of chick NTs electroporated at stage HH12 for 24 h with shControl or shVCL, the embryos were treated in ovo with EdU for the last 4 h before fixation. The sections were stained with anti-PH3 (magenta) and anti-EdU (green). GFP (blue) shows transfection. Scale bar = 50 μm. (F) Dot plot showing the apicobasal position of EdU+/GFP+ nuclei from NTs transfected at stage HH12 for 24 h with shControl or shVCL. The black bars indicate the median. t test, n = 8 sections for control and 11 sections for shVCL, from two independent experiments using at least three embryos per condition. (G) Bar graphs showing the percentage of mitotic cells (PH3+) that had incorporated EdU among the GFP population in NTs transfected at stage HH12 for 24 h with shControl or shVCL. Mean ± SEM. t test, n = 15 sections for control and 15 for shVCL from three independent experiments using at least three embryos per condition. (H) As in G but counting all the cells that incorporated EdU among the GFP population. Mean ± SEM. t test, n = 15 sections for control and 16 for shVCL from three experiments using at least three embryos per condition. (I) Images of transverse sections of chicken NTs transfected at stage HH12 for 24 h with geminin·AG (green), H2B·RFP (magenta), and pSUPER-shControl or pSUPER-shVCL (vectors without GFP), stained with phalloidin (cyan) and DAPI (blue). Scale bar = 50 μm. (J) Dot plot showing the relative FI of Geminin·AG versus H2B·RFP in ShControl or ShVCL transfected NTs. Mean ± SEM. t test, n = 7 slices from two experiments using at least three embryos per condition. (K) Plot profiles showing the FI of Geminin·AG (green) and H2B·RFP (purple) along the apicobasal axis 24 hpe of ShControl or ShVCL. Mean ± SD. n = 7 sections for control and 7 for shVCL from two experiments using at least three embryos per condition. (L) Flow cytometry analysis profiles showing DNA content (Hoechst fluorescence intensity) and cell number of GFP+ population from chicken NTs transfected at stage HH12 with shControl or shVCL for 24 h (M) Bar graph showing the percentage of cells in each cell cycle phase calculated using the profiles shown in Fig. 7 L. Mean ± SEM. t test. Two experiments using at least three embryos per condition. A minimum of 2,000 cells were analyzed in each experiment. ns = non-significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

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