Combinatorial regulation of cellular rotation by CUL-3-actomyosin-dependent oriented division, eggshell geometry, and Ras–MAPK signaling during dorsal–ventral axis establishment in Caenorhabditis elegans

Cellular rotation is an understudied mechanism that regulates animal morphogenesis. In C. elegans, the dorsal-ventral axis is established when the two-cell-stage AB cell rotates within the eggshell as it divides, generating the diamond-shaped blastomere arrangement at the four-cell stage that enables distinct cell fate specification. Multiple mechanisms, including actomyosin-dependent oriented division, chiral cortical flow, and eggshell shape, have been proposed to regulate this arrangement, but whether these represent conflicting hypotheses or co-acting mechanisms remains unclear. Here, we show that CUL-3-actomyosin-dependent oriented division, eggshell geometry, and the Ras-MAPK signaling pathway regulate distinct steps of cellular rotation. AB cell rotation occurred in two distinct phases: Phase I during AB cytokinesis and Phase II during cytokinesis of the neighboring P1 cell. Quantitative analysis revealed that CUL-3-actomyosin-dependent oriented division is the only one of these three pathways that regulates the AB division axis before anaphase. Actomyosin-dependent oriented division and eggshell geometry were both required for Phase I rotation, whereas Phase II rotation was independent of eggshell geometry. We further identified the Ras-MAPK signaling pathway as a regulator of AB cell rotation that acts independently of eggshell geometry. Strikingly, the CUL-3-actomyosin-dependent pathway may have two distinct roles: first, specifying the AB division axis, and second, correcting the division axis in all cell types during cytokinesis. Together, these functions contribute significantly to cellular rotation and dorsal-ventral axis establishment.