Figure 7. Key spindle and cell features in APs are not altered by 30 pM nocodazole.

All measurements were performed on the same cell images from which the astral microtubule data were obtained (see Figures 2 & 4). (A) Cartoon and example image (α-tubulin immunofluorescence) of the mitotic spindle in an AP, indicating the main spindle region (solid red line) where α-tubulin immunofluorescence measurements were performed to control for variations in spindle features (B–D), between proliferating and neurogenic APs, either without (no treatment) or with BRO culture of Tis21::GFP E14.5 forebrains, with either solvent (DMSO) only (Control) or with 30 pM nocodazole (Noc). The main spindle region was defined on the basis of the cell body shape and the centrosomal region (both revealed by α-tubulin immunofluorescence) and the apical- and basal-most (peri-)centromeric foci (DAPI staining) (see Figure 2A). The α-tubulin immunofluorescence used for illustration on the right side in A and E is the same as in Figure 2C. The key in A applies also for the cartoon in E. To obtain values from the central part of the spindle, the mean of the measurements from the 2 central-most 0.75 μm confocal sections was obtained. (B) Quantification of the area of the main spindle region. (C) Quantification of the amount of spindle microtubules, as obtained from the mean fluorescence intensity in the main spindle region normalized to the mean fluorescence intensity in the entire image. (D) Spatial distribution of α-tubulin in the main spindle region, as revealed by the mean standard deviation (SD) between the per-pixel fluorescence intensity values in the main spindle region, normalized to the mean fluorescence intensity of the main spindle region. (E) Cartoon and example image of the mitotic spindle showing the AP diameter measurement at the spindle plane (red dashed line). (F) AP cell body diameter at the spindle plane, indicating also spindle length. (B–D, F) Significant differences were not found with K–W with DMC post test. Error bars are SEM. Scale bars = 5 μm. See also Figure 7—source data 1 and Figure 7—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.02875.013

Figure 7—figure supplement 1. Key subcellular, cellular, and tissue features in APs are not significantly altered by 30 pM nocodazole.

E14.5 forebrains from wt mice were incubated for 2.5 hr in BRO culture either with solvent (DMSO) only (control) or with 30 pM nocodazole, and immunofluorescences of dorsolateral telencephalon sections were performed. (A, B) Maximum intensity projections of two 0.9 μm optical sections showing F-actin (phalloidin, white), DNA (DAPI, cyan), γ-tubulin (green) as centriolar marker and Arl13b (red) as ciliary marker, showing representative examples of primary cilia protruding from the apical domain (negative F-actin staining) with normal architecture in controls or with nocodazole. Scale bar = 5 μm. (C–F) Maximum intensity projections of four 0.9 μm optical sections of GAP43-GFP (green) showing representative examples of elongated APs, from basal (top) to apical (bottom), together with DNA (DAPI, cyan) and F-actin (phalloidin, red), with normal architecture in controls or with nocodazole. Scale bar = 20 μm. (D, F) Zoom-ins of the apical region (dashed grey boxes in the merges in C and E, but without DNA), with the apical end-foot of the APs embedded in the apical ventricular surface, in DMSO and nocodazole, respectively. Scale bar = 5 μm. (G, H) Maximum intensity projections of two 0.9 μm optical sections of α-tubulin (red) and DNA (cyan), showing normal cytoarchitecture of the cortical wall as revealed by the pattern of microtubules in representative coronal sections of the entire developing cortical wall of controls or with nocodazole. Scale bar = 20 μm; n = 3 brains from 2 independent litters and experiments per category.

DOI: http://dx.doi.org/10.7554/eLife.02875.015