The roles of Frizzled-3 and Wnt3a on melanocyte development: In vitro studies on neural crest cells and melanocyte precursor cell lines

Background: Wnt3a and Frizzled-3 are both expressed in the dorsal neural tube that gives rise to the neural crest in Xenopus, zebrafish and mice. Melanocytes originate from the neural crest (NC) and postnatally, melanocyte stem cells reside in the hair follicle bulge and in the dermis. However, the roles of Wnt3a and Frizzled-3 in melanocyte development have not been clarified. Objective: The aim of this study was to delineate the expression of Frizzled-3 in murine melanocyte lineage and human melanocytes, and to study the effects of Wnt3a on melanocyte development at various stages. Methods: Murine NC explant cultures and three NC-derived melanocyte lineage cell lines, including NCCmelb4M5 (Kit(-) melanocyte precursors), NCCmelb4 (Kit(+) melanoblasts) and NCCmelan5 (differentiated melanocytes), and human epidermal melanocytes were treated with pure recombinant Wnt3a protein and their cell behaviors were analyzed including their proliferation, Kit expression, tyrosinase (Tyr) activity, melanin production, dendrite formation and migration. Results: Frizzled-3 was expressed in Tyr-related protein (TRP)-1(+) cells in NC explant cultures, in all 3 melanocyte precursor cell lines and in human melanocytes. Wnt3a increased the population of TRP-1(+) cells, the number of L-3,4-dihydroxyphenylalanine (DOPA)(+) cells and dendrite formation in NC explant cultures. Wnt3a stimulated the proliferation of all 3 melanocyte precursor cell lines in a dose-dependent manner and also stimulated human melanocyte proliferation. Moreover, Wnt3a increased Tyr activity and melanin content of differentiated melanocytes, but did not activate Tyr activity in melanoblasts. Wnt3a stimulated dendrite formation in differentiated melanocytes, but not in melanoblasts. Wnt3a did not affect melanoblast or melanocyte migration. Wnt3a did not induce c-Kit expression in Kit(-) NCCmelb4M5 cells and did not affect c-Kit expression in any cell line tested. Conclusions: Frizzled-3 is constitutively expressed in murine melanocyte precursors, melanocytes and human melanocytes. Wnt3a and Frizzled-3 signalings play important roles in regulating the proliferation and differentiation of murine NCCs and various developmental stages of melanocyte precursors. The effect of Wnt3a on human melanocytes is similar to its effects on murine melanocytes. Therefore Wnt3a/Frizzled-3 signaling is a promising target for human melanocyte regeneration. (C) 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.