title: The roles of Frizzled-3 and Wnt3a on melanocyte development: in vitro studies on neural crest cells and melanocyte precursor cell lines.
abstract: 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.
authors.affiliation: Department of Dermatology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. Electronic address: miriamchangch@gmail.com.
authors.forename: Chung-Hsing
authors.initials: CH
authors.lastname: Chang
authors.affiliation: Institute of Eye Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan; Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan. Electronic address: rktsai@tzuchi.com.tw.
authors.forename: Rong-Kung
authors.initials: RK
authors.lastname: Tsai
authors.affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Ming-Hsien
authors.initials: MH
authors.lastname: Tsai
authors.affiliation: National Applied Research Laboratories, Instrument Technology Research Center, Hsinchu, Taiwan.
authors.forename: Yi-Hsiung
authors.initials: YH
authors.lastname: Lin
authors.affiliation: Fukushima Project Headquarters, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hirobe
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D019070:Cell Lineage; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051157:Frizzled Receptors / Q000378:metabolism*; D006801:Humans; D008543:Melanins / Q000378:metabolism; D008544:Melanocytes / Q000187:drug effects* / Q000378:metabolism; D008810:Mice, Inbred C57BL; D014442:Monophenol Monooxygenase / Q000378:metabolism; D009432:Neural Crest / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010641:Phenotype; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013234:Stem Cells / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D046509:Tissue Culture Techniques; D060509:Wnt3A Protein / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; C105574:FZD3 protein, human; D051157:Frizzled Receptors; C486339:Fzd3 protein, mouse; D008543:Melanins; D011994:Recombinant Proteins; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D004295:Dihydroxyphenylalanine; D014442:Monophenol Monooxygenase; D019009:Proto-Oncogene Proteins c-kit
keywords: Frizzled-3; Melanoblast; Melanocyte precursor; Neural crest; Wnt3a