Wen-Rui Li,Cheng-Rang Li,Lin Lin?

Department of Dermatology,Hospital for Skin Diseases(Institute of Dermatology),Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing,Jiangsu 210042,China.

Introduction

Pigmentary disorders are a heterogeneous group of hereditary or acquired disorders characterized by varying degrees of hyperpigmentation and/or hypopigmentation.Pigmentary disorders originate from abnormalities in melanocyte development,defects in melanin synthesis,or changes in melanosome transfer.1-2Several skin pigmentation disorders and diseases are heritable, including dyschromatosis universalis hereditaria(DUH),Dowling-Degos disease(DDD),and albinism.To elucidate the pathogenesis of these heritable pigmentary disorders,a cross-species approach has been widely applied and has resulted in a detailed understanding of melanocyte physiology and pathophysiology.

Zebrafish are characterized by stripes across the body and fins,and they have become an important animal model in the study of vertebrate melanocyte development and disease.3-6The zebrafish genome has been fully sequenced and more than 26,000 protein-coding genes have been identified.7This genome sequencing shows that approximately 70%of human genes(including genes associated with disease)have at least one direct ortholog in zebrafish,highlighting a marked similarity with humans.8Furthermore,large-scale mutagenesis screening tests have identified numerous zebrafish genes that play a role in pigment biology.

Themechanisms of thespecification anddifferentiationof melanocytes (melanophores in zebrafish) share a high degree of conservation among humans,mice,and zebrafish.Melanocytes originate from multipotent neural crest cells near the dorsal aspect of the neural tube during embryonic development,and a group of Sox10+pluripotent cells from the neural crest are destined to become microphthalmiaassociated transcription factor (MITF)+melanoblasts.9MITF(mitfa in zebrafish)appears to be a highly conserved master regulator for the proper development and differentiation of melanocytes.10MITF activates genes associated with melanocyte growth,differentiation,and survival,such as dopachrome tautomerase,tyrosinase(TYR),and TYRrelated protein 1(TYRP1).11-13The melanoblasts are then differentiated into melanocytes/melanophores that produce melanin to protect the skin from UV damage.14Although the development of melanocytes from neural crest cells is similar in mice,chickens,and zebrafish,there are interspecies differences in the migration of neural crest derivatives to their final destinations;melanoblasts in zebrafish migrate along the ventral and lateral pathways to form pigment patterns,while the melanoblasts in chickens and mice travel along the lateral pathway only.15Additionally,in contrast to mammals and amphibians,the pigment cells in zebrafish do not transfer melanin packets into surrounding keratinocytes.16However,although there are some differences between zebrafish and mammals in the mechanisms of melanocyte development, most features of melanocyte specification,differentiation,and function are similar.15,17As there is a high degree of conservation in melanocyte development and cell biology between species,zebrafish are valuable as a model for the study of human hereditary pigmentation disorders.

A powerful tool for the genetic study of zebrafish:Morpholino oligos

Morpholino oligos(MOs)are synthetic P-chiral analogs of nucleic acids that can reduce gene expression by base pairing with complementary target mRNA. MOs are widely used as a powerful tool for genetic manipulation and analysis in zebrafish embryos.18In contrast to natural DNA and RNA,the ribose or deoxyribose moieties in MOs are replaced by methylenemorpholine rings,and the anionic phosphates are replaced by nonionic phosphorodiamidate linkages.Because of these structural changes,the molecule is uncharged and so it cannot be recognized by any enzymes,including DNase and RNase.19Instead of degrading RNA targets,MOs act via translation20or a splice-blocking21mechanism. MOs can prevent the translation initiation complex read-through in cytosol by targeting the 5’UTR to reduce the expression of target RNA,20or they can modify pre-mRNA splicing events in the nucleus by targeting junctions or regulatory sites involved in the splicing of pre-mRNA.21Combined with the properties of stability,efficacy,and low toxicity,MOs provide excellent specificity and do not elicit immune responses.18Hence, MOs have become the standard knockdown tools in the study of zebrafish developmental biology.22-24

Zebrafish as a model for the study of hereditary pigmentary disorders

Due to the visible melanophores in the transparent skin of zebrafish larvae,genetic similarities between zebrafish and higher vertebrates including humans, conservation of pigment cell genes between zebrafish and mammals,and genetic tractability with robust transgenic and chemical tools,zebrafish larvae are a biologically powerful model for evaluating pigment cell development,identifying novel disease genes,and understanding hereditary pigmentary disease in humans.

Pigmentary dermatosis

DDD is a rare type of autosomal-dominant genodermatosis that is characterized by acquired reticular hyperpigmentation in flexural sites such as the neck,axilla,and groin.25In DDD,pigmentation develops after puberty and commonly occurs between the ages of 30 and 40 years.26In 2013,Li et al.27identified two mutations in the protein Ofucosyltransferase 1(POFUT1)gene(MIM 607491)in two Chinese families with DDD. To provide in vivo evidence for the role of POFUT1 in melanin synthesis and transport, they knocked down POFUT1 in zebrafish embryos by injecting pofut1-MO.27The POFUT1 morphant embryos displayed hypopigmentation at 48hours postfertilization and abnormal melanin distribution at 72 hours postfertilization,27which is similar to the characteristic clinical phenotype in individuals with DDD.The melanin protein contents and TYR activities were significantlydecreasedinthePOFUT1-MOgroupcomparedwith the control group.27These findings indicate that the loss of function of POFUT1 might influence the process of melanin synthesis in melanocytes and also demonstrate that POFUT1 mutations cause generalized DDD.

In addition to POFUT1, mutations of keratin 5(NM_000424.3) and protein O-glucosyltransferase 1(NM_152305.2)have been identified in individuals with DDD.28-29Ralser et al.30reported six unrelated families presenting primarily with DDD with partial comorbid acne inversa(AI),a chronic debilitating inflammatory hair follicle disorder.After ruling out mutations in keratin 5,POFUT1,and protein O-glucosyltransferase 1,Ralser et al.30performed exome sequencing and identified six heterozygous truncating mutations in presenilin enhancer-2(PSENEN,NM_172341.3)in six patients with AI-DDD and four patients with isolated DDD.Cases of AI-DDD comanifestation have been debated for decades in the literature.In 2010,the nicastrin(NM_015331.2),presenilin-1 (NM_000021.3), and PSENEN genes were demonstrated as causative genes in familial AI.31To analyze the role of PSENEN mutations in DDD in vivo,a psenen-knockdown zebrafish model was established with MOs.30The psenen-knockdown zebrafish larvae showed a major decrease in pigment cells and apparent haphazard distribution of pigmentation compared with controls.The MO group showed hyperpigmentation of the tail fins with a significant reduction in the number of pigment cells in the dorsal head area,which resembles the clinical phenotype observed in individuals with DDD.30Furthermore,the migration of pigment cells was disordered in the morphants and organized in controls,resulting in an abnormal or typical larvae stripe pattern,respectively.The shape of the pigment cells was also evaluated to monitor their differentiation.The pigment cells in the MO group were irregularly sized,while the pigment cells in controls were regularly sized with similar flat and elongated morphology.30Thus,Ralser et al.30proposed that the deleterious PSENEN mutation influenced the migration and differentiation of melanocytes and was associated with DDD with increased susceptibility to AI.Although the relationship between AI and DDD in PSENEN mutation carriers is still controversial,32-33zebrafish studies provide a unique way to investigate the diverse mechanisms controlling melanocyte development in vivo.

Depigmented dermatosis

Oculocutaneous albinism(OCA)is a heterogeneous and autosomal recessive disorder caused by mutations in several different genes.OCA type II(OCA2),or brown OCA,accounts for 30%of OCA cases worldwide.34-35OCA2 is characterized by decreased pigmentation in the hair,skin,and eyes,with associated visual anomalies.36Mutations in the OCA2 gene(OCA2 in humans,Oca2 in mice,and oca2 in zebrafish),also known as the P-gene,are responsible for the OCA2 phenotype(MIM 203200).36P protein,encoded by OCA2,is a melanosomal membrane protein consisting of 12 transmembrane regions.36P protein plays an important role in several processes throughout human pigmentation,such as intracellular trafficking of tyr37and melanosome biogenesis.38P protein also regulates the pH of the melanosome together with the ATP-driven proton pump.39Beirl et al.40identified a zebrafish oca2 mutant that has a similar phenotype and defects in melanin synthesis to mice and human oca2 mutants,indicating conserved gene function.In addition, the correct number of melanoblasts was observed in the unpigmented gaps on the head region,implying that a defect in differentiation accounts for the reduction in melanophore numbers.40

Mutations in C10orf11,a melanocyte-differentiation gene,have been reported in individuals with autosomalrecessive albinism. MO knockdown of c10orf11 in zebrafish resulted in substantial reduction of pigmentation and pigmented melanocytes,which could be rescued by wild-type C10orf11.41Slc45a2 increases the internal melanosome pH via inhibition of V-ATPase in zebrafish,and its human orthologous gene mutations have been implicated in the disease OCA4.42As mutations in TYRP1 and TYR are related to OCA3 and OCA1,respectively,in humans,43melanophore death and continual regeneration have been observed in zebrafish with tyrp1A mutation,while a decreased amount of melanocytes was observed in zebrafish with tyr mutation.44-45Thus,zebrafish models with OCA-related mutants offer exciting opportunities to further define the mechanisms for pigment cell function in vivo.

Hermansky-Pudlak syndrome(HPS)comprises a set of genetically heterogeneous diseases that can present as OCA,bleeding diathesis,pulmonary fibrosis,and congenital nystagmus.In addition to mouse models for HPS subtypes,the zebrafish mutant snow white(snw),which harbors a mutation in the HPS gene (hps5), could contribute to our understanding of the molecular and cellular mechanisms underlying HPS.46The zebrafish snw/hps5 mutants show a significant reduction in the quantity,size,and abnormity of maturity in melanosomes,resulting in a hypopigmented appearance.46Additionally, the zebrafish fade out mutant presents with syndromic defects,including hypopigmented skin and retinal pigmented epithelium,along with defects in blood clotting and outer retinal cell function.47This mimics the processes observed in humans with HPS and indicates that fade out might be an underlying pathogenic gene for HPS.

Nutrient supply is an essential environmental variable during growth;thus,varying degrees of nutrient deprivation result in several developmental abnormalities.The ex utero development of zebrafish makes it possible to manipulate the nutrient level available to the larvae.The combination of environmental control and forward genetic screening means that the zebrafish model provides a powerful tool with which to examine the relationship between genetic alterations and nutrient availability,and the effect of their interaction on the metabolism and viability of an embryo.For example,Menkes disease(OMIM #309400), a lethal multisystemic X-linked recessive hereditary disorder,is characterized by generalized copper deficiency.48-49The clinical features of Menkes disease result from mutations in the ATP7A gene and include seizures,neurodegeneration,and hypopigmentation.The ATP7A gene encodes a transmembrane coppertransporting P-type ATPase that is responsible for transporting copper across a membrane using the energy released by the hydrolysis of ATP.49-50The mutant calamity,a viable hypomorphic allele of atp7a in zebrafish,has been identified by chemical genetic screening.Exposure to mild nutritional copper deprivation leads to a loss of melanin pigmentation in this mutant.51Similarly,punctate melanocytes have been observed in catastrophe mutants (inactivating mutation in vacuolar ATPase),underlining the influences of copper in melanin synthesis in melanocytes.51Therefore, zebrafish could provide additional resources for studying the effects of genenutrient interactions on melanocytes.

Vitiligo is a skin depigmentation disorder with multifactorial inheritance. Individuals with vitiligo develop melanocytes normally at birth,but they then undergo progressive disappearance of epidermal melanocytes.Multiple mechanisms might contribute to the cell death in vitiligo, including autoimmune responses, genetic susceptibility,metabolic abnormalities,oxidative stress,and generation of inflammatory mediators.52Clinical and experimental observations have revealed that stress can induce or aggravate skin disorders via psychosomatic mechanisms.As stress exposure induces hypopigmentation in mice with increased interleukin-17 in serum,Zhou et al.53treated zebrafish with interleukin-17 and observed autophagic cell death in pigment cells and dyspigmentation.

Others

DUHisa rare inheriteddyschromatosischaracterizedbythe appearanceofbothhypo-andhyperpigmentedmaculesthat can involve almost all parts of the body.54-55DUH usually has autosomal dominant inheritance,with a few autosomal recessive and sporadic cases reported.56-57The molecular basis of DUH remained unknown until ABCB6 was identified as a causative gene in 2013.58Liu et al.59knocked down the abcb6 in zebrafish with MOs to confirm the functional role ofABCB6 inmelanocytes andpigmentation.They observed a significantly decreased number of mature pigmented melanocytes in the abcb6 morphant embryos compared with the control embryos.Furthermore,coinjection of the full-length hABCB6 mRNA transcript was shown to rescue,at least partially,the reduction in pigment cells in the abcb6 morphants.59This indicates that the knockdown of abcb6 expression is associated with phenotype in abcb6 morphant zebrafish.ABCB6 has been confirmed as a disease gene for DUH in humans,coupled with the genome-wide linkage,exome sequencing,and functional investigation in human tissues.

Thyroid hormones mediate the melanogenesis of adult zebrafish in a reversible and sexually dimorphic way,60which indicates that sex should be considered in future studies of fish melanogenesis.In addition to the critical influence of thyroid hormones on pigment pattern,pigment cells from distinct lineages in zebrafish have distinct responses to thyroid hormones.61

Conclusion

Zebrafish have many advantages as a model for elucidating the mechanisms of human hereditary pigmentary disorders.Additional zebrafish models of human pigmentary disorders are warranted for the evaluation of the mechanism of pigment cell development and the pathogenesis of these diseases.