Cooperation between cortical and cytoplasmic forces shapes planar 4-cell stage embryos

Introduction

Early embryonic cleavages follow remarkably conserved geometric principles across species despite variation in embryo shape and size. Two classical rules describing these divisions are: Hertwig’s rule, which states that the mitotic spindle aligns with the longest axis of the cell, and Sachs’ rule, whereby successive divisions occur at right angles. Together, these principles explain the emergence of a planar square arrangement of the 4-cell stage. However, this planar arrangement, unlike the mechanically stable tetrahedral arrangement, is intrinsically unstable in unconfined embryos and embryos with a non-restrictive confinement.

How mitotic spindles orient to reliably generate this conserved pattern appears to depend on cell size. In large blastomeres, spindle positioning relies mainly on length-dependent cytoplasmic pulling forces acting on astral microtubules. In contrast, in smaller cells, dynein-dependent cortical pulling forces play a dominant role in spindle orientation. In this preprint, the authors examine the ascidian Phallusia mammillata, whose intermediate-sized blastomeres provide a unique system to probe how these mechanisms are integrated.

Key Findings

Observation: Centrosomal complexes (CCs) in ascidian embryos initially form at random orientations and progressively become parallel during interphase of the 2-cell stage. From the time of their formation, they remain largely coplanar.

Methods and results: To investigate the mechanisms that regulate CC migration and spindle orientation in 2-cell stage ascidian embryos, authors used chemical or mechanical perturbations and quantified the subsequent spindle orientation.

• Perturbation A: Continuous ablation experiments during live imaging showed that alignment of CCs does not depend on contact with the nucleus nor on interactions between the two centrosomes within a complex.
• Perturbation B: Chemical treatment of microtubules abolished both parallelism and coplanarity. While this shows that both rotation and tilt are corrected by a microtubule-dependent mechanism, this does not disentangle the role of cytoplasmic versus cortical forces.
  • Cytoplasmic forces: Laser ablation of astral microtubules within the plane of rotation led to defective CC orientation without affecting coplanarity. The endoplasmic reticulum (ER) formed an anisotropic domain that is more elongated than the cell itself, suggesting it provides a cytoplasmic cue guiding CC rotation. Disruption of cell shape using calcium- and magnesium-free seawater caused both the cell and ER to round up, impairing CC orientation but not CC tilt.
  • Cortical forces: Indirect biophysical measurements indicated that microtubule pulling forces at the cortex increase during interphase of the 2-cell stage. Genetic induction of an asymmetric enhancement of cortical pulling forces perturbed CC tilt without affecting CC rotation.

Conclusion: CC rotation is driven by cytoplasmic pulling forces acting on an ER domain elongated along the cell axis, whereas CC tilt and coplanarity are maintained by cortical pulling forces.

What we like about this preprint

What we particularly like about this preprint is how it explores new perspectives on the long-standing question of spindle orientation guiding cleavages. Previously, cytoplasmic and cortical pulling forces appeared exclusively in large Xenopus embryos and small C. elegans embryos, respectively. Here, authors cleverly used the intermediate-sized Phallusia mammillata embryo to demonstrate that both forces contribute to CC alignment, acting on rotation and tilt, respectively. Rather than arguing for one mechanism over the other, they show that both mechanisms can operate simultaneously and that they are finely balanced to generate a particular cleavage pattern.

The combination of the quantitative imaging, laser ablations and genetic perturbations provide convincing mechanistic evidence of how cytoplasmic forces mediated via endoplasmic reticulum drive CCs parallelisation. Similarly, the induction of asymmetric distribution of cortical forces via a genetic construct demonstrates their contribution to CCs coplanarity.

We also like how the authors study the emergence of the 4-cell square not as a consequence of extrinsic forces/mechanisms/geometric constraints, but as a feature that emerges from internal biophysical forces in the embryo – making this study relevant well beyond ascidian embryos.

 

doi: https://doi.org/10.1242/prelights.43005

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