Figure 7.
EB1 function is required for nuclear dispersion and the ncMT shift towards apical regions. (A) Cross-section of MTs (green) and nuclei (magenta) showing enhanced and persistent perinuclear MTs in ΕΒ1 shRNA (ΕΒ1) embryos compared to control. (B) Perinuclear MT intensity measurements showing enriched MT pools in perinuclear regions in ΕΒ1 embryos; n = 60 and 48 for control and EB1, respectively, at 0 min, and n = 61 and 52 perinuclear regions in control and EB1, respectively, at 20 min from k = 3 embryos for each background. (C) Orthogonal views of MT and nuclei showing perinuclear MT bundles fail to shift apically in ΕΒ1 embryos. (D) Measurement of apical MT intensities at 0 and 20 min suggesting a failure of apical MT enrichment in EB1 embryos; n = 796 and 614 apical regions for 0 and 20 min, respectively, from k = 3 embryos. (E) Centrosomal MT intensities are enhanced when ΕΒ1 function is compromised; n = 200 and 150 centrosomal regions for control and EB1, respectively, from k = 4 embryos for control and k = 3 embryos for EB1. (F) Enhanced perinuclear Patronin:GFP in EB1 embryos (arrow) as compared to control embryos. (G) Perinuclear Patronin:GFP intensities are enhanced in ΕΒ1 embryos as compared to control embryos; n = 75 perinuclear regions from k = 3 embryos for each background. (H) Fraction of nuclei in the apical 10 μm of the cell in control and ΕΒ1 embryos. Error bars indicate the standard error of mean; n = 263 and 288 nuclei for control and EB1, respectively, from k = 3 embryos for each background. (I) Fraction of nuclei invading the apical exclusion zone in control and EB1 embryos; n = 455 and 434 nuclei in control and EB1, respectively, at 0 min and n = 483 and 469 nuclei in control and EB1, respectively, at 20 min from k = 3 embryos for each background. (J) Maximum-intensity projections of nuclei color coded for distance from cell apices in control and ΕΒ1 embryos. (K) Percent of active nuclei as detected by MSD. (L) Mean nuclear speeds in control and EB1 embryos. (K and L), n = 546 and 383 nuclei in control and EB1, respectively, from k = 3 embryos for each background. (M) Model of MT network transitions during GBE, showing that a centrosomal MT network transitions to a ncMT network during GBE with the aid of EB1, CLASP, and Patronin function while nuclei are driven into deeper cell regions. Scale bar = 10 μm for (J) and 5 μm for (A, C, and F). Fig. 2 C; Fig. 3 C; Fig. 5, A and H; Fig. 7, C and F; and Fig. 4 E control images/plots reproduced for comparison purposes. All scatter plots show the mean ± SD. Statistical significance was calculated using the Mann–Whitney U-test. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

EB1 function is required for nuclear dispersion and the ncMT shift towards apical regions. (A) Cross-section of MTs (green) and nuclei (magenta) showing enhanced and persistent perinuclear MTs in ΕΒ1 shRNA (ΕΒ1) embryos compared to control. (B) Perinuclear MT intensity measurements showing enriched MT pools in perinuclear regions in ΕΒ1 embryos; n = 60 and 48 for control and EB1, respectively, at 0 min, and n = 61 and 52 perinuclear regions in control and EB1, respectively, at 20 min from k = 3 embryos for each background. (C) Orthogonal views of MT and nuclei showing perinuclear MT bundles fail to shift apically in ΕΒ1 embryos. (D) Measurement of apical MT intensities at 0 and 20 min suggesting a failure of apical MT enrichment in EB1 embryos; n = 796 and 614 apical regions for 0 and 20 min, respectively, from k = 3 embryos. (E) Centrosomal MT intensities are enhanced when ΕΒ1 function is compromised; n = 200 and 150 centrosomal regions for control and EB1, respectively, from k = 4 embryos for control and k = 3 embryos for EB1. (F) Enhanced perinuclear Patronin:GFP in EB1 embryos (arrow) as compared to control embryos. (G) Perinuclear Patronin:GFP intensities are enhanced in ΕΒ1 embryos as compared to control embryos; n = 75 perinuclear regions from k = 3 embryos for each background. (H) Fraction of nuclei in the apical 10 μm of the cell in control and ΕΒ1 embryos. Error bars indicate the standard error of mean; n = 263 and 288 nuclei for control and EB1, respectively, from k = 3 embryos for each background. (I) Fraction of nuclei invading the apical exclusion zone in control and EB1 embryos; n = 455 and 434 nuclei in control and EB1, respectively, at 0 min and n = 483 and 469 nuclei in control and EB1, respectively, at 20 min from k = 3 embryos for each background. (J) Maximum-intensity projections of nuclei color coded for distance from cell apices in control and ΕΒ1 embryos. (K) Percent of active nuclei as detected by MSD. (L) Mean nuclear speeds in control and EB1 embryos. (K and L), n = 546 and 383 nuclei in control and EB1, respectively, from k = 3 embryos for each background. (M) Model of MT network transitions during GBE, showing that a centrosomal MT network transitions to a ncMT network during GBE with the aid of EB1, CLASP, and Patronin function while nuclei are driven into deeper cell regions. Scale bar = 10 μm for (J) and 5 μm for (A, C, and F). Fig. 2 C; Fig. 3 C; Fig. 5, A and H; Fig. 7, C and F; and Fig. 4 E control images/plots reproduced for comparison purposes. All scatter plots show the mean ± SD. Statistical significance was calculated using the Mann–Whitney U-test. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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