Pigment International

: 2019  |  Volume : 6  |  Issue : 1  |  Page : 9--17

Animal models in disorders of skin color − utilizing evolution and demystifying mysteries

Kiruthika Subburaj, Seema Manjunath, Muthu Sendhil Kumaran 
 Department of Dermatology, Venereology and Leprology Post graduate institute of medical education and research Chandigarh, India

Correspondence Address:
Dr. Muthu Sendhil Kumaran
Department of Dermatology Venereology and Leprology PGIMER, Chandigarh, 160012


Disorders of pigmentation are commonly encountered in dermatology outpatient clinics. Among hypopigmentary conditions, vitiligo is the most common disorder whereas hyperpigmentation can be attributed to multiple etiologies. The pathogenesis of these disorders in humans are still poorly understood. Animal models have been extensively studied and analyzed to pave way for understanding pathology as well as employ targeted treatment. In this study, we will discuss about the animal models that provide insight about pigmentation disorders.

How to cite this article:
Subburaj K, Manjunath S, Sendhil Kumaran M. Animal models in disorders of skin color − utilizing evolution and demystifying mysteries.Pigment Int 2019;6:9-17

How to cite this URL:
Subburaj K, Manjunath S, Sendhil Kumaran M. Animal models in disorders of skin color − utilizing evolution and demystifying mysteries. Pigment Int [serial online] 2019 [cited 2019 Jul 19 ];6:9-17
Available from: http://www.pigmentinternational.com/text.asp?2019/6/1/9/262046

Full Text


Animal models have been used since times immemorial to study basic pathogenesis and role of treatment in a given, thus paving, way for multitudes of research. Various models have been studied to analyze pigmentary disorders. This article briefs about animal models used to study both hypopigmentary and hyperpigmentary disorders.

 Animal models in disorders of hypopigmentation

Vitiligo is the most common hypopigmenting disorder leading to major psychosocial distress, with multiple hypothesis on pathomechanisms; it will be discussed in detail in this review. Vitiligo presents as an acquired chronic disease with unpredictable course wherein there is loss of melanocytes leading to hypopigmented or depigmented maculo-patch lesions over mucocutaneous areas. The paucity of animal studies and complicated pathogenesis of vitiligo have led to incomplete understanding of its etiology and thereby still remains a hurdle for development of newer therapies. An individual animal model might not reflect all factors comprising disease commencement and development. Thus, multiple animal models are often required to delineate different pathomechanisms.

 Use of animal models in vitiligo


Chicken models are one of the most used animal models in defining various mechanisms behind the evolution of vitiligo. The Smyth line (SL) chicken [[Figure 1]], a mutant form, first developed by Dr. J. Robert Smyth, exhibits the complete spectrum of clinical and biological manifestations of vitiligo in humans. It demonstrates a spontaneous posthatch loss of melanocytes like vitiligo in feather and choroidal tissue. The earliest abnormality detected prior to lesions are melanosomes with abnormal morphology with extensions of pigment membranes, elevated melanin synthesis, and selective autophagocytosis. These events are followed by melanocyte degeneration that without an active immune system is insufficient to produce vitiligo as proven by immunohistochemical studies demonstrating that cell-mediated immunity induces apoptosis and interferon gamma (IFN-γ) release, altered antioxidant capacity, on exposure to heightened oxidative stress. An environmental component also plays a role in expression of SL vitiligo (e.g., Turkey herpes virus that translocates to feather), a phenomenon seen in autoimmune disorder. Controls used are two MHC-matched chicken lines − parental brown line (BL) chicken with <2% incidence of vitiligo and vitiligo-resistant light brown Leghorn (LBL) chickens. Thus, the SL and control lines of chickens provide opportunity to study different phenotypes and explore treatment options though in vivo and in vitro studies.[1]{Figure 1}

Another chicken line developed by Boissy et al.[2] was mildly affected by erratic delayed amelanotic SL chicken (DAM) (Gallus gallus), identified by postnatal elimination of melanocytes and partial depigmetation as well as lack of impairment in vision that simulate vitiligo in humans. As seen in SL chicken, an abnormal melanization because of elevated production of abnormal melanosomes with increased Dihydroxyphenylalanine (DOPA)-positive cytoplasmic components and tyrosinase levels was the basic defect in preamelanotic feathers. This was attributed to increased rate of transportion of tyrosinase via tubules and cisternae and golgi apparatus. Cumulatively, this overactive melanization would lead to accumulation of toxic melanin precursors phenols, hydroquinones, etc. Excessive autophagocytosis of melanosomes results in cell necrosis with release of melanocyte antigens and hence secondary immune response. Toxic intermediates of melanin synthesis causes death of both keratinocytes and melanocytes.[2]

Immunosuppressed autoimmune “DAM” line chickens, developed by surgical bursectomy on the day of hatching, constantly demonstrated abnormal melanocytes supporting the previous studies. These chickens developed less amelanosis of feathers and maintained their plumage pigmentation without infiltration of inflammatory cells. Hence, in unbursectomized DAM birds, the depigmentation is because of the immune response to abnormal melanocytes that is insufficient by itself to cause depigmentation.[3]

Evidence of altered immune system response was further substantiated in SL vitiligo (SLV), wherein the leukocyte infiltration and immune function-related cytokine levels began to elevate in early SLV, attained peak in active stage of vitiligo, and fell closer to previtiligo levels following complete loss of pigment-producing cells. Notable rise was found in T cells (CD8+ > CD4+), B cells, and MHC II-expressing cells along with elevated levels of cytokines like IFN-γ, interleukin (IL)-10, and IL-21. Thus, postnatal loss of melanocytes can be attributed to cell-mediated immunity.[4]

An insight in the role of an apoptotic mechanism as an implicated pathomechanism of depigmentation in SLV was proposed by Wang and Erf.[5] He used terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) to identify apoptotic cells in cryostat sections of regenerating feathers from 2-week-old SL chickens as well as from normally pigmented control chickens at age of 6, 8, 10, and 12 weeks. The count of TUNEL+ cell count was higher in chickens with vitiligo compared to nonvitiliginous SL, BL, or LBL chickens and were present next to melanocyte cell bodies. The degree of apoptosis was highest during active depigmentation; 2 to 4 weeks prior to the visible onset of SLV, count of CD8+ and MHC class II+ cells in the feather pulp and the barb ridge was elevated and were localized next to the TUNEL+ cell. The count of TUNEL+ cells and CD8+ cells were directly proportional to each other throughout depigmentation. Hence, enhanced apoptosis is a mechanism involved in the melanocyte cell death and is induced by cytotoxic T cells (CD8+).[5]

Austin and Boissy[6] observed that there was high levels of circulating autoantibodies against melanocyte proteins (molecular masses 65–80 kDa) in SL chickens. Three proteins in melanocytes of mammals with isoforms in this molecular mass range are tyrosinase, TRP-1 and TRP-2. Chicken melanocytes express only tyrosinase and SL autoantibodies recognize TRP-1 as the primary antigen as the chicken melanocytes expressed messages for TRP-1. Hence, it was concluded that the SL autoantibodies recognize TRP-1 as the primary antigen in melanocytes and chicken melanocytes express a homolog of TRP-1 (the human gp75 and the murine brown/b locus protein).[6]

The genetic analysis laid a further milestone in understanding the genotype in vitiligo. The transcriptomic microarray analysis of originating autoimmune lesions in SL chicken with BL chickens as parental control was studied by Shi et al.[7] Increase in expression of genes related to immune system was observed in AV (during active loss of pigmentation-active vitiligo) samples. There was reduced gene expression for melanocyte-related proteins in AV and CV (after complete melanocyte loss-complete vitiligo) samples compared to NV (never-developed vitiligo), and EV (SLV chickens before vitiligo onset-earlier to vitiligo activity) samples. Varied expression of genes in relation to altered redox status along with apoptosis have been associated to the development of vitiligo. Hence, along with innate immune activity and impaired redox mechanisms, the adaptive immune activity has a major role in loss of pigment-producing cells.[7]


The swine model has a high incidence (54%) of spontaneous cutaneous malignant melanomas in newborn of parents having melanoma; tumors continue to develop after birth with an incidence of 85% by 1 year of age followed by spontaneous regression of primary and metastatic lesions within the first year of life demonstrated by reduction in tumor volume and subsequent alterations in tumor color from black to white. There was localized depigmentation of adjacent hair and skin as well as generalized depigmentation including iris of the eye. Cell-mediated melanocyte destruction may be attributed to the widespread depigmentation.[8] Sinclair swine, like SL chicken, demonstrated antibodies against vitiligo antigens with molecular mass ranging from 45 to 75 kDa.[9] Hence, swine models could be used in future development of treatment strategies targeted at the molecular level.


Current research on zebrafish pigmentation demonstrated its similarity to pigmentation in humans. The gene SLC24A5 (NCKX5) associated with pigmentation homologous to that in zebrafish golden mutants have an ortholog very identical to depigmentation in modern Europeans as it has results on decreased melanosome size, number, and density during melanin synthesis.[10] SLC45A2 gene encodes a membrane-associated transporter protein that regulates pH of melanosomes and activity of enzymes during melanogenesis as seen in the zebrafish model.[11] Melanin synthesis in zebra fish is comparable to that in humans. The role of endoplasmic reticulum (ER), calcium sensor protein STIM1 (stromal-interaction-molecule-1) domain in regulation of melanin synthesis via interaction with cell membrane-localized adenylyl cyclase 6 (ADCY6) has been identified by studying zebra fish models.[12] Adenylyl cyclase is coupled to melanocortin (MC) receptors where α-melanocyte-stimulating hormone (MSH) binds. Five MC receptors (MC1R–MC5R) have been demonstrated in mammals along with few melanocyte-concentrating hormone (MCH) receptors whereas zebrafish has six MC receptors − two MC5R orthologs and three MCH receptors.[13],[14],[15],[16] Cyclic adenosine monophosphate acts as a mediator for translocation of melanin and to bring intact microtubules that are vital for pigment dispersion and aggregation.

Multiple pigment-specific genetic components and proteins like SOX9 and SOX10 are present in zebrafish, determining the fate of neural crest and regulating melanin synthesis. SOX10 controls the transcription of MITF and transactivates the transcription of TRP-2 genes needed for melanin synthesis where MITF is not able to activate on its own.[17] Pigment cell-specific premelanosomal (PMEL) protein is essential in premelanosomal fibril formation and shape of the melanosomes and its defect leads to loss of melanocyte viability in PMEL-mutant zebrafish.[17] Zebrafish embryos (aged 48 h postfertilization) demonstrated a high melanosomal dispersion on illumination with visible light as the pigmentation in the body expands on a bright background. The light-induced melanosomal dispersion may serve as a protective mechanism from the impact of ultraviolet (UV) irradiation.[18],[19]

As pigmentation in zebrafish simulates human pigmentation in many aspects, it paves way for multitudes of research in exploring the genotypic as well as the phenotypic expressions of human pigmentary disorders.


Several techniques have been developed to model vitiligo in mice and most of them induced immune activity against melanoma as a therapeutic approach with vitiligo as an unexpected side effect.

The Jackson Laboratory developed the mivit/mivit mouse which is the first inbred strain which had spontaneous loss of pigment because of loss of function of the melnocytes without a functional immune system. A point mutation was induced in MITF gene that influences melanocyte growth and regulates genes vital for melanin synthesis. Because of monogenic nature and absence of autoimmune mechanisms, the mivit/mivit mouse does not mimic vitiligo pathomechanism in humans.[20],[21] It was identified that a single arginine deletion in the basic domain of MITF in humans, comparable to the mutation in mice, does not lead to vitiligo but partial albinism and loss of hearing.[22] Thus, MITF mutations are not genetically linked to human.[23]

The AAD+ (alpha-1 and alpha-2 domains of human HLA-A2 and alpha-3 transmembrane and cytoplasmic domains of the mouse H-2D gene) transgenic mouse expresses an MHC I molecule with peptide-binding region of human HLA-A*021 and presents tyrosinase epitope Tyr369. They were bred with albino mice that could not express tyrosinase. A T-cell clone against Tyr369 was obtained. TCR genes from the clone mice were utilized in generating TCR transgenic mice with CD8+ T cells against Tyr369 (i.e., FH mice). Spontaneous loss of pigmentation occurred in the hair and less prominently in skin of tail on crossing FH mice with AAD+ mice. Immunohistochemistry (IHC) of the depigmented regions showed CD8+ and CD4+ T cells infiltration. Depigmentation was mediated by CD8+ T cells, whereas CD4+ T cells are not essential also causing negative regulation of the disease. IFN-γ as well as CXCR3 and CCR5, which bind the IFN-γ-induced chemokines CXCL9, CXCL10, and CCL5, have been identified to have a role in depigmentation.[24]

Muranski et al.[25] studied Bw or “white-based brown mutation mice” that represent an immunologic knockout of TRP-1. On backcrossing with C57BL/6n background for eight generations along with multiple vaccination rounds, T cells were fused and a hybridoma against MHC class II-overexpressing B16 melanoma cell line was separated. TCR was identified and cloned and expressed in transgenic C57BL/6 mice. A founder with TRP-1 TCR transgene was crossed onto a recombination activating gene (RAG) background to prevent rearrangement of endogenous TCR. CD4-selected splenocytes were adoptively transferred from TCR transgenic mice into RAG-1 recipients followed by autoimmune response thereby vitiligo and disruption of retinal architecture. Instead of Th1 cells that are vital for tumor rejection, Th17-cell led to destruction of B16 melanoma that was interferon (IFN) dependent whereas depletion of IL–17A and IL-23 had less effect. Thus, polarization of effector CD4+ T cells is vital for melanoma eradication.[25]

Cote et al.[26] induced melanocyte antigen-specific CD8+ T cells by depleting the regulatory T cells (Treg) in B16 tumor-bearing mice by treating with anti-CD4. Lack of CD4 T cells led to notable priming of IFN-γ-producing CD8 T-cell activity against TRP-2 and gp100. Use of anti-CD25 in depletion of Treg cells, leaving help unaffected, did not result in priming CD8 T cells. Following excision of primary tumors, the mice lacking CD4 T-cell help developed vitiligo and maintained antigen-specific memory CD8 T-cell responses, cytokine production, thereby protection against B16 melanoma. Thus, CD4 T-cell help is dispensable for protective memory T-cell responses against melanoma and depletion of the same can induce long-term immunity to cancer.[26]

Mehrotra et al.[27] produced TCR transgenic mouse using human tyrosinase reactive, CD8-independent, high-affinity TCR obtained from MHC class I-restricted CD4+ T cells from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient. The HLA-A2–restricted TCR was positively selected on CD4+ and CD8+ single-positive cells. CD3+ CD4− CD8− double-negative T cells primarily expressed the transgenic TCR.TIL 1383I TCR transgenic CD4+, CD8+, and double-negative T cells retained the capacity to restrict tumor expansion without vaccination or cytokines. Spontaneous hair depigmentation and visual defects were found in HLA-A2+/human tyrosinase TCR double-transgenic mice. Thus, the expression of only TIL 1383I TCR in CD3+ T cells is adequate in restraining the growth of murine and human melanoma, without depending on CD4 and CD8, which hence stands for the depigmentation associated with melanoma.[27] The controversial findings from the above studies regarding the role of CD4 T cells in depigmentation require further evaluation in future.

Bowne et al.[28] studied that DNA plasmids encoding human TRP-2 led to depigmentation of regrowing hairs mediated by CD8+ T cells and perforin along with anti-TRP-2 antibodies when injected into the skin of mice. Human TRP-2 and human HSP70i encoding plasmids led to depigmentation whereas without HSP70i plasmid did not, thereby suggesting that HSP70i produced by melanocytes under oxidative stress leads to functional immune response and depigmentation. Mutated HSP70i did not lead to vitiligo and hence plasmids coding specific proteins could be used for vaccination and action of adjuvants like HSP70i in pathogenesis can be studied.[28],[29]

IFN-γ expression of CXCL10 and CXCR3 on T cells have been demonstrated in mouse model. Injection of CXCR3(−/−) T cells as well as depletion of CXCL10 led to minimal depigmentation of CXCL9 that increases autoreactive T-cell recruitment to skin but not effector function, whereas CXCL10 was essential for effector function and skin localization. There was reversal of disease in mice with established depigmentation following CXCL10 neutralization as evidenced by repigmentation. Hence, CXCL10 plays a crucial role in vitiligo and inhibition of CXCL10 can be used as a targeted treatment strategy.[30]

The following studies in mice have also led to demystification of vitiligo pathogenesis:Immunization of B6 mice with recombinant vaccinia virus expressing human TRP-1 that resulted in depigmentation mediated by CD4+ T cells.[31]Loss of contact immune response in mouse associated with loss of pigment cells like humans in association with decreased Langerhans cell count in the depigmenting sites both before and after depigmentation.[32]40% monobenzone-induced depigmentation on exposed and nonexposed sites in mice with associated loss of melanocytes and perilesional CD8+ T cells.[33]

Difficulties arise when the vitiligo lesion are nonresponsive to treatment and this urges the invention of new drugs or other modalities of therapy. Butin,[34] a flavonoid in the seeds of Vernonia anthelmintica, was used in Uyghur medicine for treating vitiligo. Malondialdehyde (MDA), an end product of lipid peroxidation, is an indicator of oxidative stress. Increase in cholinesterase (CHE) activity leads to autonomic dysfunction in vitiligo patients. Abnormal rise in MDA and CHE may decrease melanogenesis. The mechanism of action of butin may be related to a rise in the count of melanin-containing hair follicles, increased expression of TYP and TPR-1 via MITF, and a reduction in the MDA content and the activity of CHE in B16 cells of mice. Hence, butin can be used in the treatment of vitiligo and needs further in-depth study in human skin models.

Programmed cell death 1 (PD-1)[35] is a member of the extended CTLA-4 family and a receptor acting as an immune checkpoint that decreases immune responses. Upon binding by corresponding ligands, PD-L1 (B7-H1 or CD274) and PD-L2 (B7-DC or CD273), it suppresses the T-cell activation and function. The expression of PD-L1 is induced upon activation of T cell. Pembrolizumab and lambrolizumab, humanized monoclonal IgG4 PD-1 antibody, have been FDA approved for the treatment of advanced or unresected melanoma, as the lack of PD-1 made the mice resistant to viral infection and controlled tumor growth and metastasis by decreasing the antigen-recognition threshold and enhancing the cytotoxicity of CD8+ T cells.

PMEL-1 vitiligo mice (The Jackson Laboratory melanoma model mice, Bar Harbor, ME, USA) when injected with a PD-L1 fusion protein that acts on PD-1 receptor, PD-1 had significant reversal of depigmentation. Tregs, specified by expression of the forkhead box P3 (FoxP3), are vital in the maintenance of peripheral tolerance and in preventing T-cell autoimmune activity. There was increase in Tregs in the skin and a fall in TCR+ cells indicating suppression of effector T-cell chemotaxis to prevent loss of pigmentation in PMEL-1 mice, indicating it could be the mechanism of action of PD-L1 fusion protein. PD-L1 fusion protein treatment repressed the abundance of melanocyte-reactive T cells in vitiligo in vivo, providing a new potential therapeutic strategy for patients with vitiligo.

 Other animal models of vitiligo

Horses like Arabians, Andalusians, and Lipizzaners with gray allele encoding STX17 (vesicle transport) develop depigmentation on their own.[36] Vitiligo in these horses has an association to increased incidence of melanoma and early graying of the hair similar to Sinclair swine. The mechanism of vitiligo in horses is not fully understood, but sera from affected horses demonstrated antibodies targeted against melanocyte-specific antigens. Hence, horses might be useful in assessing antibody formation in vitiligo.

Rottweilers, German Shepherds, Old English Sheepdogs, Doberman Pinschers, Dachshunds, and German Shorthaired Pointers are few dog breeds genetically predisposed to develop vitiligo. Serum autoantibodies have been detected against melanocyte antigens. Dog breeds are genetically homogeneous, enabling strong genome-wide association studies. Human genome-wide association studies are preferred; but identifying additional genes becomes cost-prohibitive because of the meagre contributions of individual genes to vitiligo pathogenesis. Thus, dogs could be used to investigate genetic contributions to vitiligo.

In summary, animal models help in mechanistic studies to define vitiligo pathogenesis [Table 1]. Each model has its strengths and weaknesses, and should be selected based on the experimental questions being addressed.{Table 1}

 Animal models in hyperpigmentation disorders


Clinically, normal human pigmentation comprises an array of color of skin and hair apart from punctate pigmentation like melanocytic nevi or freckles, whereas clinically abnormal human pigmentation includes markedly increased or decreased pigmentation, known as hyperpigmentation and hypopigmentation, respectively.[37] Various animal models have been utilized to study skin pigmentation disorders; however, typical one for hyperpigmentation disorder is yet to be found. Few models used are enlisted below.

Xenotransplant models

It refers to xenotransplantation of human skin onto an animal. Mammals like rat, rabbit, monkey, mouse, and hamster are preferred considering the convenience in skin grafting, ideally an immunodeficient animal, to prevent graft detachment. For mice models, three different types have been utilized − athymic nude mice, spontaneous AGR129 mice, and severe combined immunodeficient (SCID) mice models. Immunological potential of these mice is low when compared to normal mice. Athymic nude mice lack T cells as a result of absence of thymus. SCID mice are deficient of both T and B cells. AGR129 mice not only lack T and B cells but also have immature natural killer cells. B-17/Icr-Scid, BALBcA-nu/Scid, and F344Jca-rnu are some SCID mouse examples.[38]

In a study conducted by Hachiya and Sriwiriyanont,[37] a black person’s skin was grafted on to an immunodeficient mouse and formation of pigment spots, which includes lentigo senilis, pigmentation after inflammation, and freckles were studied. After 7 to 15 days, decolorization was seen in grafted skin that reduced the pigmentation to a level of 20% by 1 month. Later, darkly pigmented part was found to gradually increase to more than 50% at around 6 months after grafting. Thereafter, the darkly pigmented part did not increase and was maintained stably. On Fontana-Masson staining, the darkly pigmented part showed a remarkable increase in epidermis thickness, similar to lentigo senilis, and the expression of genes involved in melanin synthesis (endothelin-1, SCF, a melanocyte-stimulating hormone gene). In addition, this model could be used for assessment of a pigment spot formation, suppressant or remover, by evaluating the RNA derived from the epidermis of the pigment spot.

As pigmentation system of human skin, especially formation of pigment spots, was simulated reliably and preserved for a long time in this model, this animal can be a potential model for assessing pigment spots in human skin.[37]

Transgenic models

Use of HR-1 (hairless) mice to explore different properties of skin is widespread, which includes wrinkles and sagging. Although UV exposure is found to have no effect on melanocytes in albino HR-1 mice, hair follicle defects have been noticed in epidermis location in adult skin. Development of acute pigmentation of pigmented hairy or hairless mice because of UV exposure have been studied.[39],[40] F1 mice of HR-1(aabbccDDPPhrhr)HR/De(AABBCCDDpphrhr) are homozygous or heterozygous dominant for the main coat color genes (AaBbCcDDPphrhr) and represent a unique model demonstrating UV irradiation-induced delayed pigmented spots.[41]

Induction of delayed pigmented spots like solar lentigines experimentally has been reported. Delayed pigmentation is thought to be different from acute pigmentation, which is uniform on UV irradiated area. In this study, at the start of UV irradiation, no tanning was seen in F1 hairless mice of HR-1HR/De. After 2 weeks of the first UV irradiation, an apparently homogeneous acute pigmentation was noticed, which persisted during irradiation. Pigmentation was at its peak at fourth week and did not increase thereafter even with continued irradiation. After cessation of UV irradiation, pigmentation was found to gradually decrease and match as that of nonirradiated control skin after about 2 weeks. On further monitoring, no pigmentation was found around 10 weeks. Small pigmented spots were noticed on the dorsal skin of mice at around 25 weeks, which progressively increased in size and number. The spots were less than 2 mm in diameter and light brown in color. Skin biopsies after 5-week UV irradiation showed epidermal hyperplasia along with collection of melanin granules. DOPA staining revealed various active dendritic melanocytes. Pigmentation was noticed in melanocytes all over the epidermis, except in perifollicular region. Skin biopsies from the irradiated area taken 37 weeks after completion of UV irradiation revealed epidermal hyperplasia. Only delayed pigmented spots showed active melanocytes that were similar to those observed during the acute pigmentation period.

Thus, this model proves to be useful to evaluate the dynamics of delayed pigmentation in F1 mice of HR-1HR/De by concurrent evaluation of gene expression at regular intervals during the course of formation of pigmentation.[46]

Changes in epidermal IL-10, IL-12, and IFN-γ for 5 days following irradiation of albino Skh:HR-1 mice with contact hypersensitivity-modulating doses of UVA, UVB, or UVA + UVB was evaluated in a study.[46] A raise in epidermal IL-10 expression was observed only by UVB exposure that peaked at 3 days. Whereas, only UVA irradiation was observed to cause an increase in epidermal IL-12 expression that peaked at 3 days and an increase in epidermal IFN-γ expression that peaked at 1 day. Irradiation with both UVA and UVB nullified the UVB-induced amplified expression of IL-10 and remarkable increase in IL-12 and IFN-γ at days 3 and 1, respectively, were observed. These findings propose that photoimmunoprotection by UVA is through suppression of IL-10 production and amplification of IL-12 and IFN-γ, which are also known to antagonize IL-10 in various studies, thus maintaining balance between Th1 and Th2. The early rise of IFN-γ suggests that it responds earlier to UVA and may sequentially stimulate the enhanced expression of IL-12.[43] Thus, these mice model plays an invaluable role in studying the effects of UV rays at immunological level.

Spontaneous mutation models

These models are used to study diseases with mutations comparable to that in human diseases. Very few spontaneous mutation models are available in melanocyte-associated skin diseases. In a study conducted by Millikan et al.,[44] a Sinclair swine model was utilized to study pigment tumors. It was noticed that lesions developed by those swine resembled various human tumors histologically and clinically. The flat lesions resembled human junctional nevus, elevated lesions resembled human compound nevus, raided blue tumors resembled human blue nevus, peripheral depigmentation resembled vitiligo, and ulcerative tumors resembled melanoma.[44] As the lesions corresponding to various human pigmentary disorders could be reproduced in this model, it can be used in understanding the pathogenesis as well as therapeutic implications.

A model was proposed for studying the role of UV irradiations and chemical carcinogen in the development of skin melanoma by Atillasoy et al.[45] In this model, human newborn foreskin was grafted onto RAG-1 mice. Mice were grouped into four groups. No treatment was given to the first group and this was labeled as the control group. The second group was treated with dimethyl benzanthracene (DMBA), a chemical carcinogen. The third group was treated with UVB. The fourth group was treated with both DMBA and UVB irradiations. DMBA treatment alone induced only melanocytic hyperplasia in 16% and was not conclusive. UVB treatment alone induced solar lentigo in 23% and melanocytic hyperplasia in 68% and was a little better. Whereas, with combined treatment 38% had solar lentigo, 77% had melanocytic hyperplasia, and 2.1% had melanoma after 15 months.[45] This study highlights the use of RAG-1 mice in understanding the role of UVB and DMBA in development of solar lentigo.

In a study conducted by Imokawa et al.[46] it was noticed that in moderately colored guinea pig skin, hyperpigmentation induced by UVB, psoralen ultraviolet A (PUVA), and allergic contact dermatitis was found to resemble the pigmentary changes noticed in mongoloid human skin. On UVB irradiation of varying energies for three successive days, apparent black pigmentation of the irradiated areas was found, which peaked by 1 week and resembled the pigmented human skin changes. It was also proved by an enhanced number of strongly DOPA-positive melanocytes with stout dendrites (800–1000 cells/mm2). Similar results were also observed with UVA irradiation following an intraperitoneal injection of 8-methoxypsoralen and allergic contact dermatitis produced by the application of 1-phenylazo-2-naphtol. In contrast to routinely used mice, the skin of lightly colored guinea pigs have moderate number of melanocytes and melanosomes not only around hair follicle but also in epidermis similar to human skin.[46] This similarity between guinea pig and human skin enhances the reproducibility of the same pathogenesis in human skin.

In vivo animal models with human skin substitutes

In a study on role of animal models in human skin substitutes (HSS), mice with melanocytes functioning similar to normal human skin in situ was used for preparing HSS using the new spontaneous cell-sorting technique. Human melanocytes were spontaneously sorted to the basal layer of the reconstituted epidermis without interfering with neighboring keratinocytes and fibroblasts, and produced melanin was then transferred to contiguous keratinocytes. Compared with HSS derived from Caucasian donors, those derived from donors of African descent had darker skin pigmentation corresponding to the donors’ original complexion. Increases of melanin and acanthosis in the HSS from Caucasian donors were found after UVB exposure. Hence, this technique of forming HSS with human melanocytes was proposed to be very useful for treating skin wounds or pigmentary disorders like senile fleck and vitiligo in patients of various complexions by allowing control over the color and the population of donor melanocytes.[47]

[Table 2] summarizes all the available animal models used to study hyperpigmentation disorders.{Table 2}


The difficulties in elucidation of various pathomechanisms in pigmentary disorders among humans has been eased by the use of animal models to a great advantage. As there exists a difference in genetic information in animals compared to humans, the inferences cannot be completely relied upon. Moreover, the newer in vitro pigmented skin substitutes developed by tissue engineering may act as a good alternative.


The authors would like to thank Dr. Ganesan, Director CPDO, Chandigarh, for giving photographs of SL chicken.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Wick G, Andersson L, Hala K, Gershwin ME, Selmi C, Erf GF et al. Avian models with spontaneous autoimmune diseases. Adv Immunol 2006;92:71-117.
2Boissy RE, Smyth JR Jr, Fite KV. Progressive cytologic changes during the development of delayed feather amelanosis and associated choroidal defects in the DAM chicken line. A vitiligo model. Am J Pathol 1983;111:197-212.
3Boissy RE, Lamont SJ, Smyth JR Jr, Persistence of abnormal melanocytes in immunosuppressed chickens of the autoimmune “DAM” line. Cell Tissue Res 1984;235:663-8.
4Shi F, Erf GF. IFN-gamma, IL-21, and IL-10 co-expression in evolving autoimmune vitiligo lesions of Smyth line chickens. J Invest Dermatol 2012;132(3 Pt 1):642-9.
5Wang X, Erf GF. Apoptosis in feathers of Smyth line chickens with autoimmune vitiligo. J Autoimmun 2004;22:21-30.
6Austin LM, Boissy RE. Mammalian tyrosinase-related protein-1 is recognized by autoantibodies from vitiliginous Smyth chickens. An avian model for human vitiligo. Am J Pathol 1995;146:1529-41.
7Shi F, Kong BW, Song JJ, Lee JY, Dienglewicz RL, Erf GF. Understanding mechanisms of vitiligo development in Smyth line of chickens by transcriptomic microarray analysis of evolving autoimmune lesions. BMC Immunol 2012;13:18.
8Hook RR Jr, Berkelhammer J, Oxenhandler RW. Melanoma: Sinclair swine melanoma. Am J Pathol 1982;108:130-3.
9Misfeldt ML, Grimm DR. Sinclair miniature swine: an animal model of human melanoma. Vet Immunol Immunopathol 1994;43(1-3):167-75.
10Lamason RL, Mohideen MA, Mest JR, Wong AC, Norton HL, Aros MC et al. SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science 2005;310:1782-6.
11Bin BH, Bhin J, Yang SH, Shin M, Nam YJ, Choi DH et al. Membrane-associated transporter protein (MATP) regulates melanosomal pH and influences tyrosinase activity. PLoS One 2015;10:e0129273.
12Motiani RK, Tanwar J, Raja DA, Vashisht A, Khanna S, Sharma S et al. STIM1 activation of adenylyl cyclase 6 connects Ca(2+) and cAMP signaling during melanogenesis. EMBO J 2018;37:e97597.
13Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004;84:1155-228.
14Singh AP, Nusslein-Volhard C. Zebrafish stripes as a model for vertebrate colour pattern formation. Curr Biol 2015;25:R81-92.
15Berman JR, Skariah G, Maro GS, Mignot E, Mourrain P. Characterization of two melanin-concentrating hormone genes in zebrafish reveals evolutionary and physiological links with the mammalian MCH system. J Comp Neurol 2009;517:695-710.
16Logan DW, Bryson-Richardson RJ, Pagan KE, Taylor MS, Currie PD, Jackson IJ. The structure and evolution of the melanocortin and MCH receptors in fish and mammals. Genomics 2003;81:184-91.
17Singh AP, Dinwiddie A, Mahalwar P, Schach U, Linker C, Irion U et al. Pigment cell progenitors in zebrafish remain multipotent through metamorphosis. Dev Cell 2016;38:316-30.
18Mueller KP, Neuhauss SC. Sunscreen for fish: co-option of UV light protection for camouflage. PLoS One 2014;9:e87372.
19Shiraki T, Kojima D, Fukada Y. Light-induced body color change in developing zebrafish. Photochem Photobiol Sci 2010;9:1498-504.
20Lerner AB, Shiohara T, Boissy RE, Jacobson KA, Lamoreux ML, Moellmann GE. A mouse model for vitiligo. J Invest Dermatol 1986;87:299-304.
21Lamoreux ML, Boissy RE, Womack JE, Nordlund JJ. The vit gene maps to the mi (microphthalmia) locus of the laboratory mouse. J Hered 1992;83:435-9.
22Tassabehji M, Newton VE, Liu XZ, Brady A, Donnai D, Krajewska-Walasek M et al. The mutational spectrum in Waardenburg syndrome. Hum Mol Genet. 1995;4:2131-7.
23Tripathi RK, Flanders DJ, Young TL, Oetting WS, Ramaiah A, King RA et al. Microphthalmia-associated transcription factor (MITF) locus lacks linkage to humanvitiligo or osteopetrosis: an evaluation. Pigment Cell Res 1999;12:187-92.
24Gregg RK, Nichols L, Chen Y, Lu B, Engelhard VH. Mechanisms of spatial and temporal development of autoimmune vitiligo in tyrosinase-specific TCR transgenic mice. J Immunol 2010;184:1909-17.
25Muranski P, Boni A, Antony PA, Cassard L, Irvine KR, Kaiser A et al. Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood 2008;112:362-73.
26Cote AL, Byrne KT, Steinberg SM, Zhang P, Turk MJ. Protective CD8 memory T cell responses to mouse melanoma are generated in the absence of CD4 T cell help. PLoS One 2011;6:e26491.
27Mehrotra S, Al-Khami AA, Klarquist J, Husain S, Naga O, Eby JM et al. A coreceptor-independent transgenic human TCR mediates anti-tumor and anti-self immunity in mice. J Immunol 2012;189:1627-38.
28Bowne WB, Srinivasan R, Wolchok JD, Hawkins WG, Blachere NE, Dyall R et al. Coupling and uncoupling of tumor immunity and autoimmunity. J Exp Med 1999;190:1717-22.
29Mosenson JA, Zloza A, Nieland JD, Garrett-Mayer E, Eby JM, Huelsmann EJ et al. Mutant HSP70 reverses autoimmune depigmentation in vitiligo. Sci Transl Med 2013;5:174ra28.
30Rashighi M, Agarwal P, Richmond JM, Harris TH, Dresser K, Su MW et al. cCXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo. Sci Transl Med 2014;6:223ra23.
31Overwijk WW, Lee DS, Surman DR, Irvine KR, Touloukian CE, Chan CC et al. Vaccination with a recombinant vaccinia virus encoding a "self" antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4(+) T lymphocytes. Proc Natl Acad Sci U S A 1999;96:2982-7.
32Palkowski MR, Nordlund ML, Rheins LA, Nordlund JJ. Langerhans’ cells in hair follicles of the depigmenting C57Bl/Ler-vit.vit mouse. A model for human vitiligo. Arch Dermatol 1987;123:1022-8.
33Zhu Y, Wang S, Xu A. A mouse model of vitiligo induced by monobenzone. Exp Dermatol 2013;22:499-501.
34Huo SX, Wang Q, Liu XM, Ge CH, Gao L, Peng XM et al. The effect of butin on the vitiligo mouse model induced by hydroquinone. Phytother Res 2017;31:740-6.
35Miao X, Xu R, Fan B, Chen J, Li X, Mao W et al. PD-L1 reverses depigmentation in Pmel-1 vitiligo mice by increasing the abundance of Tregs in the skin. Sci Rep 2018;8:1605.
36Essien KI, Harris JE. Animal models of vitiligo: matching the model to the question. Dermatol Sinica 2014;32:240-7.
37Hachiya A, Sriwiriyanont P. (Inventors). Animal model for pigment spots (75). Cincinnati, OH, USA.
38Gendreau I, Angers L, Jean J, Pouliot R. Pigmented skin models: understand the mechanisms of melanocytes, regenerative medicine and tissue engineering. Jose A. Amdrades, IntechOpen.
39Sato T, Kawada A. Mitotic activity of hairless mouse epidermal melanocytes: its role in the increase of melanocytes during ultraviolet radiation. J Invest Dermatol 1972;58:392-5.
40Uesugi T. Electron microscopic characterization in the processes of activation and differentiation of dormant melanocytes in epidermis of C57 black mouse skin after UV-irradiation (UV-A). Jpn J Dermatol 1978;88:685-700.
41Naganuma M, Yagi E, Fukuda M. Delayed induction of pigmented spots on UVB irradiated hairless mice. J Dermatol Sci 2001;25:29-35.
42Aoki H, Moro O. Upregulation of the IFN-c-stimulated genes in the development of delayed pigmented spots on the dorsal skin of F1 mice of HR-1HR/De. J Invest Dermatol 2005;124:1053-61.
43Shen J, Bao S, Reeve VE. Modulation of IL-10, IL-12, and IFN-gamma in the epidermis of hairless mice by UVA (320-400 nm) and UVB (280-320 nm) radiation. J Invest Dermatol 1999;113:1059-64.
44Millikan LE, Boylon JL, Hook RR, Manning PJ. Melanoma in sinclair swine-new animal model. J Investig Dermatol 1974;62:20-30.
45Atillasoy ES, Seykora JT, Soballe PW, Elenitsas R, Nesbit M, Elder DE et al. UVB induces atypical melanocytic lesions and melanoma in human skin. Am J Pathol 1998;152:1179-86.
46Imokawa G, Kawai M, Mishima Y, Motegi I. Differential analysis of experimental hypermelanosis induced by UVB, PUVA, and allergic contact dermatitis. Using a brownish guinea pig model. Arch Dermatol Res 1986;278:352-62.
47Hachiya A, Sriwiriyanont P, Kaiho E, Kitahara T, Takema Y, Tsuboi R. An in vivo mouse model of human skin substitute containing spontaneously sorted melanocytes demonstrates physiological changes after UVB irradition. J Invest Dermatol 2005;125:364-72.