Zebrafish PRL-3 Regulates Yolk Syncytial Layer Integrity and Actomyosin Contractility During Epiboly

Zebrafish epiboly is a critical morphogenetic event driven by the precise coordination of microtubule-mediated pulling forces and actomyosin-dependent constriction. While the phosphatase PRL-3 is known to regulate cytoskeletal remodeling in cancer metastasis, its physiological function during early vertebrate embryogenesis remains undefined. Here, we identify <i>zfPRL-3</i> as an indispensable regulator of zebrafish epiboly. Morpholino-mediated depletion of <i>zfPRL-3</i> resulted in severe developmental arrest, blastoderm destabilization, and mechanical rupture of the yolk cell. Time-lapse imaging revealed that <i>zfPRL-3</i> morphants suffer from catastrophic structural failures, characterized by either blastoderm dispersion or excessive inward constriction. At the cellular level, we demonstrate that <i>zfPRL-3</i> depletion disrupts the organization of the Yolk Syncytial Layer (YSL), evidenced by the irregular scattering of YSL nuclei-a hallmark of microtubule network collapse. Furthermore, <i>zfPRL-3</i> morphants exhibit premature assembly of the contractile actomyosin ring at 60% epiboly, indicating a failure in the inhibitory mechanisms that normally restrain marginal constriction. We propose that <i>zfPRL-3</i> functions as a molecular brake that couples YSL integrity with the timing of contractility. By maintaining microtubule stability and preventing premature actomyosin ring formation, <i>zfPRL-3</i> ensures that the opposing physical forces driving epiboly are precisely balanced. Collectively, our findings define <i>zfPRL-3</i> as a critical spatiotemporal regulator that orchestrates the successful progression of epiboly.