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Abstract
Recent data have uncovered that spindle size asymmetry (SSA) is a key component of asymmetric cell division (ACD) in the mouse cerebral cortex (Delaunay et al., 2014). In the present study we show that SSA is independent of spindle orientation and also occurs during cortical progenitor divisions in the ventricular zone (VZ) of the macaque cerebral cortex, pointing to a conserved mechanism in the mammalian lineage. Because SSA magnitude is smaller in cortical precursors than in invertebrate neuroblasts, the unambiguous demonstration of volume differences between the two half spindles is considered to require 3D reconstruction of the mitotic spindle (Delaunay et al., 2014). Although straightforward, the 3D analysis of SSA is time consuming, which is likely to hinder SSA identification and prevent further explorations of SSA related mechanisms in generating ACD. We therefore set out to develop an alternative method for accurately measuring spindle asymmetry. Based on the mathematically demonstrated linear relationship between 2D and 3D analysis, we show that 2D assessment of spindle size in metaphase cells is as accurate and reliable as 3D reconstruction provided a specific procedure is applied. We have examined the experimental accuracy of the two methods by applying them to different sets of in vivo and in vitro biological data, including mouse and primate cortical precursors. Linear regression analysis demonstrates that the results from 2D and 3D reconstructions are equally powerful. We therefore provide a reliable and efficient technique to measure SSA in mammalian cells.
Highlights
Asymmetric cell division (ACD)—unequal division producing two daughter cells with distinct fates—generates cell diversity in prokaryotes and eukaryotes
We have shown that spindle shape asymmetry (SSA) is a highly conserved mechanism that operates in the mouse developing mammalian cerebral cortex (Delaunay et al, 2014), where it plays a major role in the tight spatiotemporal control of selfrenewal and differentiation during corticogenesis
Pappus’centroid theorem, we demonstrated that this relationship is independent of the spindle shape
Summary
Surgical procedures and animal experimentation were in accordance with European requirements 2010/63/UE. 104 cells per 12 mm diameter poly-D-Lysine (Sigma, 40 μg/ml) coated glass cover slips. Cultured cover slips were saturated for 1 h in PBS1X/10% goat serum and incubated with the primary antibody overnight or up to 30 h at 4◦C: mouse anti-α-tubulin (sigma, 1:500), rabbit anti-pericentrin (Covance, 1:1000). Sections were washed in PBS, followed by incubation with appropriate goat fluorescence-conjugated secondary antibodies at room temperature for 2 h (Alexa 488 goat anti Mouse (Invitrogen, 1:1000), Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen, 1/1000). Primary antibodies were co-incubated 1 day and 2 nights in TBSb + 0.5% triton at 4◦C as follows: mouse anti-alphatubulin (sigma, 1:200), rabbit anti-pericentrin (Covance, 1:2000), sheep anti-EOMES (R&D 1:800). Nuclear staining was performed using Dapi (Invitrogen, D1306, 2 μg/mL in TBS) 10 min at RT. Image pixel size was 0.045 μm (x and y) and bit depth 0.998, z-step size 0.3–0.5 μm, and pinhole diameter from 100 μm
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