Characterizing the effect of alcohol and wnt/β-catenin interactions on melanocyte development: insights from zebrafish (Danio rerio)

Abstract

Melanocytes, which are derived from the multipotent neural crest, are found in the skin, eyes, and other diverse anatomical locations of the vertebrates. The main aspects of melanocyte lineage are genetically determined and are influenced by a variety of extrinsic and intrinsic factors. Fetal alcohol spectrum disorder (FASD) is described as birth defects associated with prenatal alcohol exposure (PAE). PAE is likely one of the leading causes of severe congenital malformations but the pathophysiology of FASD-associated body pigmentation disorders is needed to be further investigated. Zebrafish (Danio rerio) (ZF) is identified as a well-established model organism to study FASD and melanocyte biology with remarkably high similarity to humans. The Wnt signaling pathway is a conserved signal transduction pathway that is important in embryonic development and it regulates many aspects of melanocyte lineage. The current study was designed to investigate the effect of ethanol and Wnt activity on ZF melanocyte formation, patterning, and melanogenesis. Stereo-microscopic examinations were used for screening melanocyte counts, phenotypic differences and distribution of melanocytes in zebrafish meanwhile microscopic software tools were used for analyzing the morphometric differences of melanocytes against ethanol and Wnt regulatory chemical exposures. The chemical effect on melanosome biogenesis and melanogenesis was detected using toluidine blue stained histological sections, transmission electron microscopic (TEM) observations and spectrophotometric methods respectively. Whole-mount in situ hybridization (WMISH) was used to identify the gene expressions in melanocyte precursor cells. Ethanol and W-C59 exposed samples showed low melanocyte densities, and defects in melanocyte phenotype and patterning whereas LiCl demonstrated stimulatory action in most aspects of melanocyte development. Increased activity was observed in melanogenesis, melanoblast development, dct+ and Wnt3a+ gene expressions in melanocyte precursor cells and melanosome biogenesis in the cells of the RPE layer of the LiCl-treated fish. In contrast EtOH and Wnt inhibitors down-regulated the above-mentioned factors. Combined chemical-treated fish of EtOH and Wnt modulators displayed significant adverse effects on melanocyte development. Our data confirmed that Wnt signaling plays a crucial role in melanocyte development and a balanced Wnt signaling level is important for proper melanocyte development. Further, prenatal ethanol exposure adversely affects melanocyte differentiation, patterning and melanogenesis.