Tug-of-war between cortical and cytoplasmic forces shaping planar of 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 to generate the characteristic planar, square 4-cell stage. We show that following the first mitotic division, CCs in the two blastomeres form at variable orientations relative to the mother spindle and progressively achieve parallel and coplanar alignment during interphase of the 2-cell stage. Final spindle orientation is established prior to nuclear envelope breakdown, contrasting with dynamic spindle reorientation reported in other systems. Our analyses reveal that CC rotation is driven by forces acting on long astral microtubules and guided by an anisotropic, ER-rich cytoplasmic domain that surrounds the CCs. This ER domain correlates with a dense astral microtubule network, enabling length-dependent microtubule pulling that orients the CCs independently of cell shape. In parallel, dynein-mediated cortical pulling refines spindle tilt and maintains coplanarity. Together, these findings uncover a cell-cycle-regulated tug-of-war between ER-mediated cytoplasmic forces and anisotropic cortical forces that ensures robust planar cleavage in ascidian early embryos.