Orientation of mitotic spindles during the 8- to 16-cell stage transition in mouse embryos

Abstract

Background: Asymmetric cell divisions are involved in the divergence of the first two lineages of the pre-implantation mouse embryo. They first take place after cell polarization (during compaction) at the 8-cell stage. It is thought that, in contrast to many species, spindle orientation is random, although there is no direct evidence for this.

Methodology/principal findings: Tubulin-GFP and live imaging with a spinning disk confocal microscope were used to directly study spindle orientation in whole embryos undergoing the 8- to 16-cell stage transition. This approach allowed us to determine that there is no predetermined cleavage pattern in 8-cell compacted mouse embryos and that mitotic spindle orientation in live embryo is only modulated by the extent of cell rounding up during mitosis.

Conclusions: These results clearly demonstrate that spindle orientation is not controlled at the 8- to 16-cell transition, but influenced by cell bulging during mitosis, thus reinforcing the idea that pre-implantation development is highly regulative and not pre-patterned.

Figure 1. Determination of spindle poles and embryo centre coordinates.

For a given spindle, the position of three points were determined, by moving through the stack of images (z1, z2, z3, …): the two poles of the spindle (P1 and P2) and the centroid of the embryo (O). The position of the centre of the spindle (C) and the value of the α angle were calculated using the coordinates of these 3 points.

Figure 2. Timing of the eight mitotic divisions in 8-cell stage mouse embryos.

Each colour corresponds to a given embryo. Timing of metaphase (Y axis) was used for each blastomere (X axis). Inset: distribution of the timing of metaphase in the population of embryos studied (the Y axis corresponds to the percentage of blastomeres dividing at a given time).

Figure 3. Dispersed distribution of the angle between the spindle and the radial axis.

Distributions of the spindle size (A-B, red), distance from the centroid (A-B, blue) and α angle value (C). In B, the same populations (spindle size and distance from the centroid) were plotted as a frequency distribution (every 4 µm). The dashed lines correspond to our data and the plain lines to the curve fits.

Figure 4. Orientation of the eight mitotic spindles in 8-cell stage mouse embryos.

Each colour corresponds to a given embryo. Spindle orientation (in degrees; Y axis) was measured for each blastomere (X axis). Blastomeres were ranked according to the timing of mitosis. No define pattern could be observed.

Figure 5. Spindle orientation in 8-cell stage embryos.

A: Schematic representation of the probability of spindle orientation distribution. Since the spindle can orient in a 3D space, and not on a 2D plane, the probability for the spindle to be in a given range of angles is proportional to a «stripe» of the surface of a sphere. Therefore, this probability is proportional to cosine (α) rather than to α itself. This is illustrated on these three colours balloons, viewed from the top and the side. If the ranges are proportional to α, then the surfaces covered by each of the three colours are different (top). When the ranges are proportional to cosine (α), each colour covers the same area of the surface (bottom). B: Distribution of α according to cosine (α): the cosine of the angles (X axis) shown corresponds to multiple of 0.166 (since cosine (α) varies between 0 and 1). The Y axis corresponds to the percentage of spindle oriented with a given angle. A non-random distribution is observed, with an increase for the two extreme ranges suggesting that spindle orientation is not completely random. The dash line corresponds to the expected percentage for each “α” angle value if the spindle orientation was random.

Figure 6. Spindle orientation and asymmetric divisions.

A: The number of inside cells was measured in a population of 78 fixed 16-cell stage embryos. The dotted line corresponds to the experimental values and the plain line to the best fit. The mean number of inside cells was 2.8, corresponding to 35% of asymmetric divisions. B: Cumulative distribution of the α angle in living blastomeres (n = 64). Blastomeres were sorted according to the value of the α angle. Each point on the graph corresponds to an increment of 2°. The X axis corresponds to the α angle. The Y axis corresponds to the percentage of the population with an α angle smaller or equal to a given value. The best fit line was plotted using the least square method. 35% of asymmetric divisions correspond to a threshold angle of about 40° (arrows).

Figure 7. Spindle orientation is not modulated by the timing of division nor the position of blastomeres.

Correlation between spindle orientation (X axis) and the timing of division (A; Y axis) or the distance from the embryo centre (B; Y axis).

Figure 8. Measurement of blastomere bulging during mitosis.

A-C: The measurements (A) required to estimate blastomere bulging were performed on image stacks (C). The surface of S₁ (corresponding to half of an ellipsoid) and S₂ (corresponding to a truncated circle) was then calculated (B). An example is shown in C where both fluorescence and transmitted light images were used. D: Distribution of the «bulging» index. The Y axis corresponds to the percentage of blastomeres with a given bulging index (X axis).

Figure 9. Blastomere bulging during mitosis influences spindle orientation.

Correlation between spindle orientation (Y axis) and the blastomere bulging index (S1/(S1+S2); X axis).

Figure 10. Spindle orientation and bulging in early dividing blastomeres.

Neither blastomere bulging during mitosis (A) nor spindle orientation (B) is influenced in early dividers. Each point corresponds to a blastomere.