Tug-of-war between cortical and cytoplasmic forces shaping planar 4-cell stage embryos

Early embryonic cleavages often follow conserved geometric rules, resulting in species-specific cleavage patterns. How these rules are mechanistically implemented, however, varies widely across species and remains poorly understood. Here, using quantitative 3D live imaging, mechanical and biochemical manipulations, we dissect the mechanisms governing centrosomal complex (CC) migration and spindle orientation in ascidian 2-cell stage embryos, which generate the characteristic planar, square 4-cell stage. We show that following the first mitotic division, the CCs in each blastomere form at variable orientations relative to the mother spindle and progressively achieve parallel and coplanar alignment during interphase of the 2-cell stage. Final CC orientation is established before nuclear envelope breakdown, contrasting with the dynamic spindle reorientation during mitosis reported in other organisms. Our analyses reveal that CC rotation is driven by forces acting on long astral microtubules and guided by an anisotropic, endoplasmic reticulum (ER)-rich domain that surrounds the CCs. This ER domain is associated with a dense astral microtubule network, enabling efficient length-dependent microtubule pulling that can orient the CCs even in the absence of cell shape cues. In parallel, dynein-mediated cortical pulling refines CCs tilt and maintains coplanarity. Together, these findings uncover a cell-cycle-regulated cooperation between ER-mediated cytoplasmic forces and anisotropic cortical forces that ensures robust planar cleavage in ascidian early embryos.