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Mitotic spindle asymmetry in rodents and primates: 2D vs. 3D measurement methodologies.

Delaunay D, Robini MC, Dehay C - Front Cell Neurosci (2015)

Bottom Line: We therefore set out to develop an alternative method for accurately measuring spindle asymmetry.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.We therefore provide a reliable and efficient technique to measure SSA in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Brain Research Institute, Institut National de la Santé et de la Recherche Médicale, U846 Bron, France ; Université de Lyon I Lyon, France.

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.

No MeSH data available.


Related in: MedlinePlus

Linear relationship between 2D and 3D SSA measurements: Experimental validation. (A,B) Linear regression of 3D vs. 2D SSA measurements. The SSA has been quantified using both 2D and 3D methods and their relationship evaluated for two experimental samples: Mouse AP dividing cells in vitro and E63 to E80 Monkey VZ progenitors in vivo. (A) 2D vs. 3D SSA quantification comparison for in vitro mouse. (B) 2D vs. 3D SSA quantification comparison for Monkey in vivo. The regression line is displayed in red, the magenta curves delimit the 95% pointwise confidence band, and the green curves delimit the 95% Working-Hotelling confidence band. (C–F) 2D and 3D SSA in the Monkey VZ. (C) Optical sections of an E80 Monkey VZ stained with α-tubulin. The white dashes delimit a cell in metaphase. (D) Manually drawn area 1 and 2 on the maximal stack projection. (E) 2D SSA area determination. The difference between the Left (green) and the Right area (yellow) is expressed in percentage and is greater than 10, typical of an asymmetric cell. (F) 3D volume rendering and calculation of the same cells. Note the proximity between 2D and 3D values. Scale bars: 10 μm.
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Figure 5: Linear relationship between 2D and 3D SSA measurements: Experimental validation. (A,B) Linear regression of 3D vs. 2D SSA measurements. The SSA has been quantified using both 2D and 3D methods and their relationship evaluated for two experimental samples: Mouse AP dividing cells in vitro and E63 to E80 Monkey VZ progenitors in vivo. (A) 2D vs. 3D SSA quantification comparison for in vitro mouse. (B) 2D vs. 3D SSA quantification comparison for Monkey in vivo. The regression line is displayed in red, the magenta curves delimit the 95% pointwise confidence band, and the green curves delimit the 95% Working-Hotelling confidence band. (C–F) 2D and 3D SSA in the Monkey VZ. (C) Optical sections of an E80 Monkey VZ stained with α-tubulin. The white dashes delimit a cell in metaphase. (D) Manually drawn area 1 and 2 on the maximal stack projection. (E) 2D SSA area determination. The difference between the Left (green) and the Right area (yellow) is expressed in percentage and is greater than 10, typical of an asymmetric cell. (F) 3D volume rendering and calculation of the same cells. Note the proximity between 2D and 3D values. Scale bars: 10 μm.

Mentions: We performed linear regression analyses to validate the quasi-linear relationship (Equation 6) between 3D and 2D spindle-pole differences. Two separate samples were analyzed: (i) dissociated cortical progenitors (from E10 to E16, Figure 3) and (ii) monkey VZ precursors in situ (from E63 to E80, Figures 5A–F). The results are summarized in Figure 5. The green and magenta curves respectively delimit the 95% simultaneous and pointwise confidence bands; that is, the true regression lines lie between the green curves with a probability of 95%, and given a 2D measurement Δ*, there is a 95% probability that the corresponding 3D measurement is bounded by the magenta curves at Δ = Δ*. In accordance with the bounds given in Equation (6), the slopes of the regression lines are close to one: the regression line L1 of the in vitro mouse data has a slope of 1.009 with a standard deviation of 0.095, and the regression line L2 of the in vivo monkey data has a slope of 0.831 with a standard deviation of 0.084 (the intercepts of L1 and L2 are smaller than 4%). The 95% confidence intervals for the true slopes of L1 and L2 are (0.82, 1.20) and (0.66, 1.00), respectively. That is, we estimate with 95% confidence that if the 2D spindle-pole difference increases by 10%, then the mean 3D spindle-pole difference increases by somewhere between 8.2 and 12% in the case of the in vitro mouse data, and between 6.6 and 10% in the case of the in situ monkey data—this further confirms the proportionality between 2D and 3D measurements, and hence their equivalent discriminative power.


Mitotic spindle asymmetry in rodents and primates: 2D vs. 3D measurement methodologies.

Delaunay D, Robini MC, Dehay C - Front Cell Neurosci (2015)

Linear relationship between 2D and 3D SSA measurements: Experimental validation. (A,B) Linear regression of 3D vs. 2D SSA measurements. The SSA has been quantified using both 2D and 3D methods and their relationship evaluated for two experimental samples: Mouse AP dividing cells in vitro and E63 to E80 Monkey VZ progenitors in vivo. (A) 2D vs. 3D SSA quantification comparison for in vitro mouse. (B) 2D vs. 3D SSA quantification comparison for Monkey in vivo. The regression line is displayed in red, the magenta curves delimit the 95% pointwise confidence band, and the green curves delimit the 95% Working-Hotelling confidence band. (C–F) 2D and 3D SSA in the Monkey VZ. (C) Optical sections of an E80 Monkey VZ stained with α-tubulin. The white dashes delimit a cell in metaphase. (D) Manually drawn area 1 and 2 on the maximal stack projection. (E) 2D SSA area determination. The difference between the Left (green) and the Right area (yellow) is expressed in percentage and is greater than 10, typical of an asymmetric cell. (F) 3D volume rendering and calculation of the same cells. Note the proximity between 2D and 3D values. Scale bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Linear relationship between 2D and 3D SSA measurements: Experimental validation. (A,B) Linear regression of 3D vs. 2D SSA measurements. The SSA has been quantified using both 2D and 3D methods and their relationship evaluated for two experimental samples: Mouse AP dividing cells in vitro and E63 to E80 Monkey VZ progenitors in vivo. (A) 2D vs. 3D SSA quantification comparison for in vitro mouse. (B) 2D vs. 3D SSA quantification comparison for Monkey in vivo. The regression line is displayed in red, the magenta curves delimit the 95% pointwise confidence band, and the green curves delimit the 95% Working-Hotelling confidence band. (C–F) 2D and 3D SSA in the Monkey VZ. (C) Optical sections of an E80 Monkey VZ stained with α-tubulin. The white dashes delimit a cell in metaphase. (D) Manually drawn area 1 and 2 on the maximal stack projection. (E) 2D SSA area determination. The difference between the Left (green) and the Right area (yellow) is expressed in percentage and is greater than 10, typical of an asymmetric cell. (F) 3D volume rendering and calculation of the same cells. Note the proximity between 2D and 3D values. Scale bars: 10 μm.
Mentions: We performed linear regression analyses to validate the quasi-linear relationship (Equation 6) between 3D and 2D spindle-pole differences. Two separate samples were analyzed: (i) dissociated cortical progenitors (from E10 to E16, Figure 3) and (ii) monkey VZ precursors in situ (from E63 to E80, Figures 5A–F). The results are summarized in Figure 5. The green and magenta curves respectively delimit the 95% simultaneous and pointwise confidence bands; that is, the true regression lines lie between the green curves with a probability of 95%, and given a 2D measurement Δ*, there is a 95% probability that the corresponding 3D measurement is bounded by the magenta curves at Δ = Δ*. In accordance with the bounds given in Equation (6), the slopes of the regression lines are close to one: the regression line L1 of the in vitro mouse data has a slope of 1.009 with a standard deviation of 0.095, and the regression line L2 of the in vivo monkey data has a slope of 0.831 with a standard deviation of 0.084 (the intercepts of L1 and L2 are smaller than 4%). The 95% confidence intervals for the true slopes of L1 and L2 are (0.82, 1.20) and (0.66, 1.00), respectively. That is, we estimate with 95% confidence that if the 2D spindle-pole difference increases by 10%, then the mean 3D spindle-pole difference increases by somewhere between 8.2 and 12% in the case of the in vitro mouse data, and between 6.6 and 10% in the case of the in situ monkey data—this further confirms the proportionality between 2D and 3D measurements, and hence their equivalent discriminative power.

Bottom Line: We therefore set out to develop an alternative method for accurately measuring spindle asymmetry.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.We therefore provide a reliable and efficient technique to measure SSA in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Brain Research Institute, Institut National de la Santé et de la Recherche Médicale, U846 Bron, France ; Université de Lyon I Lyon, France.

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.

No MeSH data available.


Related in: MedlinePlus