Migration and establishment of progenitor pool of melanocytes is governed by SEMA3E-PLXND1 signaling.

Transitory signaling events often hinder the identification of pathways governing cell migration and stem cell establishment during development. Using zebrafish and cultured mammalian melanocytes, we identify a previously unrecognized role for PlexinD1 signaling in neural crest-derived melanocyte lineage cells. In zebrafish, loss of PlexinD1 disrupts melanocyte migration and reduces midline melanophores derived from the regeneration-competent melanocyte stem cell (McSC) pool. In mammalian melanocytes, Semaphorin 3E-mediated PlexinD1 activation induces F-actin remodeling, altering cell polarity, protrusive dynamics, migration velocity, and directional persistence, thereby biasing migration towards attractive cues such as SCF. Beyond these acute effects, PlexinD1 activation elicits EGFR signaling and transcriptionally induces GNAS, a key effector of the MC1R pathway associated with melanocyte maturation. In zebrafish, melanocyte lineage-specific Plxnd1 ablation followed by single-cell RNA sequencing revealed altered melanocyte progenitor populations and loss of PlexinD1 activation signatures. Collectively, our findings establish PlexinD1 as a central integrator of cytoskeletal dynamics and orchestrator of signaling defining a negative chemotactic axis that is also essential for melanocyte stem cell establishment and pigment pattern formation.