NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

An Unexpectedly Complex Architecture for Skin Pigmentation in Africans.

Approximately 15 genes have been directly associated with skin pigmentation variation in humans, leading to its characterization as a relatively simple trait. However, by assembling a global survey of quantitative skin pigmentation phenotypes, we demonstrate that pigmentation is more complex than previously assumed, with genetic architecture varying by latitude. We investigate polygenicity in the KhoeSan populations indigenous to southern Africa who have considerably lighter skin than equatorial Africans. We demonstrate that skin pigmentation is highly heritable, but known pigmentation loci explain only a small fraction of the variance. Rather, baseline skin pigmentation is a complex, polygenic trait in the KhoeSan. Despite this, we identify canonical and non-canonical skin pigmentation loci, including near SLC24A5, TYRP1, SMARCA2/VLDLR, and SNX13, using a genome-wide association approach complemented by targeted resequencing. By considering diverse, under-studied African populations, we show how the architecture of skin pigmentation can vary across humans subject to different local evolutionary pressures.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Mechanisms Underlying the Formation and Evolution of Vertebrate Color Patterns.

Vertebrates exhibit a wide range of color patterns, which play critical roles in mediating intra- and interspecific communication. Because of their diversity and visual accessibility, color patterns offer a unique and fascinating window into the processes underlying biological organization. In this review, we focus on describing many of the general principles governing the formation and evolution of color patterns in different vertebrate groups. We characterize the types of patterns, review the molecular and developmental mechanisms by which they originate, and discuss their role in constraining or facilitating evolutionary change. Lastly, we outline outstanding questions in the field and discuss different approaches that can be used to address them. Overall, we provide a unifying conceptual framework among vertebrate systems that may guide research into naturally evolved mechanisms underlying color pattern formation and evolution.

Melanocyte lineage dynamics in development, growth and disease.

Melanocytes evolved to produce the melanin that gives colour to our hair, eyes and skin. The melanocyte lineage also gives rise to melanoma, the most lethal form of skin cancer. The melanocyte lineage differentiates from neural crest cells during development, and most melanocytes reside in the skin and hair, where they are replenished by melanocyte stem cells. Because the molecular mechanisms necessary for melanocyte specification, migration, proliferation and differentiation are co-opted during melanoma initiation and progression, studying melanocyte development is directly relevant to human disease. Here, through the lens of advances in cellular omic and genomic technologies, we review the latest findings in melanocyte development and differentiation, and how these developmental pathways become dysregulated in disease.

Zebrafish Pigment Pattern Formation: Insights into the Development and Evolution of Adult Form.

Vertebrate pigment patterns are diverse and fascinating adult traits that allow animals to recognize conspecifics, attract mates, and avoid predators. Pigment patterns in fish are among the most amenable traits for studying the cellular basis of adult form, as the cells that produce diverse patterns are readily visible in the skin during development. The genetic basis of pigment pattern development has been most studied in the zebrafish, Danio rerio. Zebrafish adults have alternating dark and light horizontal stripes, resulting from the precise arrangement of three main classes of pigment cells: black melanophores, yellow xanthophores, and iridescent iridophores. The coordination of adult pigment cell lineage specification and differentiation with specific cellular interactions and morphogenetic behaviors is necessary for stripe development. Besides providing a nice example of pattern formation responsible for an adult trait of zebrafish, stripe-forming mechanisms also provide a conceptual framework for posing testable hypotheses about pattern diversification more broadly. Here, we summarize what is known about lineages and molecular interactions required for pattern formation in zebrafish, we review some of what is known about pattern diversification in Danio, and we speculate on how patterns in more distant teleosts may have evolved to produce a stunningly diverse array of patterns in nature.

A long noncoding RNA at the cortex locus controls adaptive coloration in butterflies.

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we name ivory, transcribed from the cortex locus, in modulating color patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory. In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies.

Extracellular vesicles released by keratinocytes regulate melanosome maturation, melanocyte dendricity, and pigment transfer.

Extracellular vesicles (EVs) facilitate the transfer of proteins, lipids, and genetic material between cells and are recognized as an additional mechanism for sustaining intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to warrant skin pigmentation. Melanocytes synthesize the melanin pigment in melanosomes that are transported along the dendrites prior to the transfer of melanin pigment to keratinocytes. EVs secreted by keratinocytes modulate pigmentation in melanocytes [(A. Lo Cicero et al., Nat. Commun. 6, 7506 (2015)]. However, whether EVs secreted by keratinocytes contribute to additional processes essential for melanocyte functions remains elusive. Here, we show that keratinocyte EVs enhance the ability of melanocytes to generate dendrites and mature melanosomes and promote their efficient transfer. Further, keratinocyte EVs carrying Rac1 induce important morphological changes, promote dendrite outgrowth, and potentiate melanin transfer to keratinocytes. Hence, in addition to modulating pigmentation, keratinocytes exploit EVs to control melanocyte plasticity and transfer capacity. These data demonstrate that keratinocyte-derived EVs, by regulating melanocyte functions, are major contributors to cutaneous pigmentation and expand our understanding of the mechanism underlying skin pigmentation via a paracrine EV-mediated communication.

The influence of transposable elements on animal colouration.

Transposable elements (TEs) are mobile genetic sequences present within host genomes. TEs can contribute to the evolution of host traits, since transposition is mutagenic and TEs often contain host regulatory and protein coding sequences. We review cases where TEs influence animal colouration, reporting major patterns and outstanding questions. TE-induced colouration phenotypes typically arise via introduction of novel regulatory sequences and splice sites, affecting pigment cell development or pigment synthesis. We discuss if particular TE types may be more frequently involved in the evolution of colour variation in animals, given that examples involving long terminal repeat (LTR) elements appear to dominate. Currently, examples of TE-induced colouration phenotypes in animals mainly concern model and domesticated insect and mammal species. However, several influential recent examples, coupled with increases in genome sequencing, suggest cases reported from wild species will increase considerably.

Haplotype-based analysis resolves missing heritability in oculocutaneous albinism type 1B.

Oculocutaneous albinism (OCA) is a rare disorder of pigment production. Affected individuals have variably decreased global pigmentation and visual-developmental changes that lead to low vision. OCA is notable for significant missing heritability, particularly among individuals with residual pigmentation. Tyrosinase (TYR) is the rate-limiting enzyme in melanin pigment biosynthesis and mutations that decrease enzyme function are one of the most common causes of OCA. We present the analysis of high-depth short-read TYR sequencing data for a cohort of 352 OCA probands, ∼50% of whom were previously sequenced without yielding a definitive diagnostic result. Our analysis identified 66 TYR single-nucleotide variants (SNVs) and small insertion/deletions (indels), 3 structural variants, and a rare haplotype comprised of two common frequency variants (p.Ser192Tyr and p.Arg402Gln) in cis-orientation, present in 149/352 OCA probands. We further describe a detailed analysis of the disease-causing haplotype, p.[Ser192Tyr; Arg402Gln] ("cis-YQ"). Haplotype analysis suggests that the cis-YQ allele arose by recombination and that multiple cis-YQ haplotypes are segregating in OCA-affected individuals and control populations. The cis-YQ allele is the most common disease-causing allele in our cohort, representing 19.1% (57/298) of TYR pathogenic alleles in individuals with type 1 (TYR-associated) OCA. Finally, among the 66 TYR variants, we found several additional alleles defined by a cis-oriented combination of minor, potentially hypomorph-producing alleles at common variant sites plus a second, rare pathogenic variant. Together, these results suggest that identification of phased variants for the full TYR locus are required for an exhaustive assessment for potentially disease-causing alleles.

A gene regulatory network combining Pax3/7, Sox10 and Mitf generates diverse pigment cell types in medaka and zebrafish.

Neural crest cells generate numerous derivatives, including pigment cells, and are a model for studying how fate specification from multipotent progenitors is controlled. In mammals, the core gene regulatory network for melanocytes (their only pigment cell type) contains three transcription factors, Sox10, Pax3 and Mitf, with the latter considered a master regulator of melanocyte development. In teleosts, which have three to four pigment cell types (melanophores, iridophores and xanthophores, plus leucophores e.g. in medaka), gene regulatory networks governing fate specification are poorly understood, although Mitf function is considered conserved. Here, we show that the regulatory relationships between Sox10, Pax3 and Mitf are conserved in zebrafish, but the role for Mitf is more complex than previously emphasized, affecting xanthophore development too. Similarly, medaka Mitf is necessary for melanophore, xanthophore and leucophore formation. Furthermore, expression patterns and mutant phenotypes of pax3 and pax7 suggest that Pax3 and Pax7 act sequentially, activating mitf expression. Pax7 modulates Mitf function, driving co-expressing cells to differentiate as xanthophores and leucophores rather than melanophores. We propose that pigment cell fate specification should be considered to result from the combinatorial activity of Mitf with other transcription factors.

UV-Protection Timer Controls Linkage between Stress and Pigmentation Skin Protection Systems.

Skin sun exposure induces two protection programs: stress responses and pigmentation, the former within minutes and the latter only hours afterward. Although serving the same physiological purpose, it is not known whether and how these programs are coordinated. Here, we report that UVB exposure every other day induces significantly more skin pigmentation than the higher frequency of daily exposure, without an associated increase in stress responses. Using mathematical modeling and empirical studies, we show that the melanocyte master regulator, MITF, serves to synchronize stress responses and pigmentation and, furthermore, functions as a UV-protection timer via damped oscillatory dynamics, thereby conferring a trade-off between the two programs. MITF oscillations are controlled by multiple negative regulatory loops, one at the transcriptional level involving HIF1α and another post-transcriptional loop involving microRNA-148a. These findings support trait linkage between the two skin protection programs, which, we speculate, arose during furless skin evolution to minimize skin damage.

Dangerous liaisons: Loss of keratinocyte control over melanocytes in melanomagenesis.

Melanomas arise from transformed melanocytes, positioned at the dermal-epidermal junction in the basal layer of the epidermis. Melanocytes are completely surrounded by keratinocyte neighbors, with which they communicate through direct contact and paracrine signaling to maintain normal growth control and homeostasis. UV radiation from sunlight reshapes this communication network to drive a protective tanning response. However, repeated rounds of sun exposure result in accumulation of mutations in melanocytes that have been considered as primary drivers of melanoma initiation and progression. It is now clear that mutations in melanocytes are not sufficient to drive tumor formation-the tumor environment plays a critical role. This review focuses on changes in melanocyte-keratinocyte communication that contribute to melanoma initiation and progression, with a particular focus on recent mechanistic insights that lay a foundation for developing new ways to intercept melanoma development.

Seed color patterns in domesticated common bean are regulated by MYB-bHLH-WD40 transcription factors and temperature.

Seed colors and color patterns are critical for the survival of wild plants and the consumer appeal of crops. In common bean, a major global staple, these patterns are also essential in determining market classes, yet the genetic and environmental control of many pigmentation patterns remains unresolved. In this study, we genetically mapped variation for several important seed pattern loci, including T, Bip, phbw, and Z, which co-segregated with candidate genes PvTTG1, PvMYC1, PvTT8, and PvTT2, respectively. Proteins encoded by these genes are predicted to work together in MYB-bHLH-WD40 (MBW) complexes, propagating flavonoid biosynthesis across the seed coat as observed in Arabidopsis. Whole-genome sequencing of 37 accessions identified mutations, including seven unique parallel mutations in T (PvTTG1) and non-synonymous SNPs in highly conserved residues in bipana (PvMYC1) and z (PvTT2). A 612 bp intron deletion in phbw (PvTT8) eliminated motifs conserved since the Papilionoideae origin and corresponded to a 20-fold reduction in transcript abundance. In multi-location field trials of seven varieties with partial seed coat pigmentation patterning, the pigmented seed coat area correlated positively with ambient temperature, with up to 11-fold increases in the pigmented area from the coolest to the warmest environments. In controlled growth chamber conditions, an increase of 4°C was sufficient to cause pigmentation on an average additional 21% of the seed coat area. Our results shed light on key steps of flavonoid biosynthesis in common bean. They will inform breeding efforts for seed coat color/patterning to improve consumer appeal in this nutritious staple crop.

Stabilization of ß-catenin promotes melanocyte specification at the expense of the Schwann cell lineage.

The canonical Wnt/ß-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, ß-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of ß-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of ß-catenin in cells expressing tyrosinase. Constitutive activation of ß-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating ß-catenin at various stages of development (E8.5-E11.5), we showed that the activation of ß-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/ß-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, ß-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

The inhibition of VDAC1 oligomerization promotes pigmentation through the CaMK-CRTCs/CREB-MITF pathway.

The voltage-dependent anion channel 1 (VDAC1) forms an oligomeric structure on the mitochondrial outer membrane, which plays critical roles in many physiological processes. Research studies have demonstrated that the knockout of VDAC1 increases pigment content and up-regulates the expression of melanogenic genes. Due to its involvement in various physiological processes, the depletion of VDAC1 has significant detrimental effects on cellular functions and the inhibition of VDAC1 oligomerization has recently emerged as a promising strategy for the treatment of several diseases. In this study, we found that VDAC1 oligomerization inhibitors, VBIT-12 and NSC-15364, promote melanogenesis, dendrite formation and melanosome transport in human epidermal melanocytes (HEMCs). Mechanistically, treatment of HEMCs with an oligomerization inhibitor increased the level of cytoplasmic calcium ions, which activated calcium-calmodulin dependent protein kinase (CaMK) and led to the phosphorylation of CREB and the nuclear translocation of CREB-regulated transcription coactivators (CRTCs). Subsequently, CRTCs, p-CREB and CREB-binding protein (CBP) in the nucleus cooperatively recruit the transcription machinery to initiate the transcription of MITF thus promoting pigmentation. Importantly, our study also demonstrates that VDAC1 oligomerization inhibitors increase pigmentation in zebrafish and in human skin explants, highlighting their potential as a therapeutic strategy for skin pigmentation disorders.

An enhancer of Agouti contributes to parallel evolution of cryptically colored beach mice.

Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the oldfield mouse (Peromyscus polionotus), which occurs in the southeastern United States, where it exhibits considerable color variation. Dorsal coats range from dark brown in mainland mice to near white in mice inhabiting sandy beaches; this light pelage has evolved independently on Florida's Gulf and Atlantic coasts as camouflage from predators. To facilitate genomic analyses, we first generated a chromosome-level genome assembly of Peromyscus polionotus subgriseus. Next, in a uniquely variable mainland population (Peromyscus polionotus albifrons), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We find that pigment variation is strongly associated with an ∼2-kb region ∼5 kb upstream of the Agouti signaling protein coding region. Using a reporter-gene assay, we demonstrate that this regulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this Agouti region indicate that the light allele experienced recent and strong positive selection. Notably, this same light allele appears fixed in both Gulf and Atlantic coast beach mice, despite these populations being separated by >1,000 km. Together, our results suggest that this identified Agouti enhancer allele has been maintained in mainland populations as standing genetic variation and from there, has spread to and been selected in two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.

Genetic adaptation of skin pigmentation in highland Tibetans.

Strong ultraviolet (UV) radiation at high altitude imposes a serious selective pressure, which may induce skin pigmentation adaptation of indigenous populations. We conducted skin pigmentation phenotyping and genome-wide analysis of Tibetans in order to understand the underlying mechanism of adaptation to UV radiation. We observe that Tibetans have darker baseline skin color compared with lowland Han Chinese, as well as an improved tanning ability, suggesting a two-level adaptation to boost their melanin production. A genome-wide search for the responsible genes identifies GNPAT showing strong signals of positive selection in Tibetans. An enhancer mutation (rs75356281) located in GNPAT intron 2 is enriched in Tibetans (58%) but rare in other world populations (0 to 18%). The adaptive allele of rs75356281 is associated with darker skin in Tibetans and, under UVB treatment, it displays higher enhancer activities compared with the wild-type allele in in vitro luciferase assays. Transcriptome analyses of gene-edited cells clearly show that with UVB treatment, the adaptive variant of GNPAT promotes melanin synthesis, likely through the interactions of CAT and ACAA1 in peroxisomes with other pigmentation genes, and they act synergistically, leading to an improved tanning ability in Tibetans for UV protection.

Identifying the niche controlling melanocyte differentiation.

Melanocytes present in hair follicles are responsible for their pigmentation. Melanocyte differentiation and hair pigmentation depend on the stem cell factor (SCF)/c-Kit signaling pathway, but the niche that regulates melanocyte differentiation is not well characterized. In this issue of Genes & Development, Liao and colleagues (pp. 744-756) identify Krox20+-derived cells of the hair shaft as the niche and the essential source of SCF required for melanocyte maturation. This study delineates the niche factors regulating melanocyte differentiation and hair pigmentation and opens up new avenues to further characterize the cross-talk between the hair follicle and melanocytes that controls melanocyte maintenance and differentiation.

Identification of hair shaft progenitors that create a niche for hair pigmentation.

Hair differentiates from follicle stem cells through progenitor cells in the matrix. In contrast to stem cells in the bulge, the identities of the progenitors and the mechanisms by which they regulate hair shaft components are poorly understood. Hair is also pigmented by melanocytes in the follicle. However, the niche that regulates follicular melanocytes is not well characterized. Here, we report the identification of hair shaft progenitors in the matrix that are differentiated from follicular epithelial cells expressing transcription factor KROX20. Depletion of Krox20 lineage cells results in arrest of hair growth, confirming the critical role of KROX20+ cells as antecedents of structural cells found in hair. Expression of stem cell factor (SCF) by these cells is necessary for the maintenance of differentiated melanocytes and for hair pigmentation. Our findings reveal the identities of hair matrix progenitors that regulate hair growth and pigmentation, partly by creating an SCF-dependent niche for follicular melanocytes.