Myosin 10 is involved in murine pigmentation.

Myosins are molecular motors that are well known for their role in cell movement and contractile functions. Although extensively studied in muscle physiology, little is known about the function of myosins in mammalian skin. As part of the Sanger Institute Mouse Genetics Project, we have identified a role for Myo10 in pigmentation, with a phenotype unlike those of Myo5a or Myo7a. Adult mice homozygous for a disrupted Myo10 allele on a C57BL/6N background displayed a high degree of penetrance for white patches on their abdomen and dorsal surface. Forepaw syndactyly and hind paw syndactyly were also observed in these mice. Tail epidermal wholemounts showed a complete lack of melanocytes in the hair follicles and interfollicular epidermis. Myo10 has previously been implicated in human pigmentation. Our current study reveals involvement of Myo10 in murine skin pigmentation.

adenosine triphosphate

Hair Gene Expression Library

Myosin 10

Myosin 5a

Myosin 7a

tyrosinase‐related protein 1

ultraviolet

wild type

BACKGROUND

Skin pigmentation is a highly variable and conserved trait among living organisms. It plays a critical role in social communication, camouflage, mimicry and protection against the harmful effect of UV radiation.1 Melanin pigments mediate the pigmentation process in skin. Melanogenesis is a process whereby melanin pigments are produced and deposited in melanocytes under complex regulatory control by multiple cellular events. Upon maturation, melanosomes—the melanin‐containing granules—are transported from melanocytes to neighbouring keratinocytes in the epidermis.2, 3

Myosins are among the motor proteins involved in melanosome cargo transportation. Functionally, myosins are a large superfamily of ATP‐dependent motor proteins that translocate actin filaments. Other functions include cell adhesion, motility, endocytosis, exocytosis and cytokinesis.4 There are 35 classes of myosins that have been classified based on evolutionary conservation of their motor domains.5, 6 Myosin 5a (Myo5a), Myosin 7a (Myo7a) and Myosin 10 (Myo10) are unconventional myosins and are known to have functions in melanocytes and neurons.7, 8 The roles of Myo5a and Myo7a are known in skin pigmentation, whereas the in vivo involvement of Myo10, while previously studied in primary human cell cultures9 and being advertised as a key target by at least one commercial cosmetic product, is not fully understood.10

QUESTION ADDRESSED

Does Myosin 10 play a role in murine skin pigmentation?

EXPERIMENTAL DESIGN

To investigate the role of Myo10 in mouse skin pigmentation, we analysed Myo10 C57BL/6N mice from the Sanger Institute Mouse Genetics Project,11 which carry an inserted cassette 3′ to exon 18 and an in‐frame deletion of exon 19 in Myo10 (Figure 1A). See Data S1.

Figure 1

Myo10 disruption leads to digit and pigmentation defects. (A) Schematic diagram of the deletion (tm2) construct for Myo10. Exon numbers indicated (with exon 19 deleted) and corresponding protein domains are illustrated. (B) Coat of adult C57BL/6N Myo10 mice. (C) Pale tail phenotype in Myo10 mice (assessed in 3 Myo10 mice). (D) Fused digits in forepaws of Myo10 mice. Digit fusion prevalence: Forepaw: 10/14 Myo10 abnormal vs 0/34 local WTs and 0/1688 baseline WTs. Hind paw: 8/14 Myo10 vs 0/34 local WTs and 0/1688 baseline WTs. (E) Stacked bar graphs show coat colour abnormalities. Legend—Control, WT mice phenotyped each week alongside each batch of Myo10 mice to ensure standard testing conditions; Baseline, cumulative C57BL/6N controls over lifetime of phenotyping pipeline

RESULTS

To assess the efficiency of knockout of the Myo10 allele, we performed real time‐qPCR analysis, which showed that Myo10 mice have ~15% of wild‐type Myo10 transcript levels (Figure S1A). Similar to the reported Myo10 allele10 (http://www.informatics.jax.org/allele/MGI:5578506), Myo10 mice typically displayed abnormal dorsoventral coat patterning with white belly spots and extensive depigmented areas in the tail. Depigmentation was limited to the tail tip in control mice (Figure 1B‐C). Myo10 mice showed ocular abnormalities and also typically presented with interdigital webbing—a condition in which the skin between paw digits is not lost during development (Figure 1D). X‐ray analysis confirmed that this abnormality is not due to fusion of bones (http://www.mousephenotype.org/data/genes/MGI:107716). Unlike Xenopus Myo10 mutants, Myo10 mice did not show any cranial or skeletal abnormalities.12 Abnormal coat colour pattern was observed in all male mutants, whereas females showed slightly reduced penetrance of the phenotype (Figure 1E). Skin histopathology revealed no obvious structural abnormalities in the epidermis or dermis (Figure S1B).

The apparent lack of pigment in the tail of Myo10 mice prompted us to investigate whether the skin had any melanocyte abnormalities. Confocal imaging of tail epidermal wholemounts revealed a complete lack of pigmented melanocytes in the hair follicles and interfollicular epidermis (Figure 2A). Similar results were observed by imaging the wholemounts under bright field combined with fluorescent detectors (Figure 2B). We confirmed the lack of melanocyte stem cells and differentiated melanocytes by labelling with antibodies to c‐Kit and tyrosinase‐related protein 1 (Trp1), respectively. Immunostaining for c‐Kit and Trp1 showed no staining in mutant hair follicles and interfollicular epidermis, whereas distinct accumulation of melanocytes with characteristic dendritic projections was observed in WT epidermis (Figure 2C; Figure S1C). This indicates that melanocyte distribution in the epidermis requires Myo10.

Figure 2

Loss of pigmentation markers in Myo10 epidermis and hair follicles. (A) 3D maximal projected epidermal wholemount images show absence of melanocytes in hair follicles in mutant mice when compared to WT (arrows). (B) Bright field images combined with fluorescent labelling confirm the lack of melanocytes in hair follicles (arrows). (C) Single Z‐plane image of immunolabelled epidermal wholemounts of mutant mice with antibody to the melanocyte differentiation marker Trp1 shows the absence of melanocytes in interfollicular epidermis and hair follicles when compared to WT (arrows). Asterisks indicate nonspecific staining in sebaceous glands. Krt14, keratin 14; Krt15, keratin 15; Trp1, tyrosinase‐related protein 1; DAPI, 4′,6‐diamidino‐2‐phenylindole; Scale bars 100 μm

As Myo5a, Myo7a and Myo10 are unconventional myosins and loss of function mutations result in different patterns of pigmentation defects, we explored putative links in predicted protein‐protein interaction networks. STRING network analysis (http://www.string-db.org/) showed two distinct clusters of interactions for Myo5a and Myo10 (Figure S1D). Myo5a is predicted to interact with Myo7a. The other interaction partners are mostly exocyst complex component (Exoc) genes, which are involved in the docking of exocytic vesicles.13

Surprisingly, the interaction network of Myo10 was separate from that of Myo5a. Top scoring predicted interaction partners included Itgb5, Calm13 and Neo1, all of which are expressed in the plasma membrane. The distinct class of interactions of Myo10 and Myo5a suggests that Myo10 may play a different role from Myo5a in pigmentation. Myo10 is highly expressed in mouse skin,14 so we analysed expression of Myo10 in different skin compartments using a publicly available skin gene expression (Hair‐GEL) database.15 Interestingly, while Myo10 is predominantly expressed in melanocytes in embryonic and neonatal mice, it is also expressed in other skin compartments such as epidermis and dermal fibroblasts (Figure S1E).

CONCLUSIONS

The role of myosins in melanoblast migration is known through studies on Rac1 mouse mutants,16, 17 although direct evidence implicating the involvement of Myo10 has not been reported. An actin‐bundling protein, Fascin1 (Fscn1), which is a key interacting partner of Myo10, is known to promote migration and proliferation of melanoblasts.18 In the network analysis of Myo10 interaction partners, we found Fscn1 as a key protein that is known to physically interact with Myo10. This suggests that Myo10 may have a similar role to Fscn1 in actin bundling.

Like most cells, melanoblasts migrate with the help of cytoplasmic projections called filopodia.19, 20 HeLa cells that lack Myo10 fail to form filopodia,21 indicating that Myo10 is an essential component for filopodia formation. In addition to its role in melanoblast migration, Myo10 has been shown to function in melanosome transportation. Exposure to UV light increases the number of filopodia and Myo10 expression in differentiated melanocytes. Knockdown of Myo10 leads to decreased numbers of filopodia and reduced melanosome transfer.9 The digit webbing phenotype may also indicate a role for Myo10 in apoptosis, for example as a dependence receptor required for apoptosis induction. Recent data suggest that Myo10 determines tumour invasiveness and cell cycle regulation (reviewed in),22 leading us to suggest that it could exert these functions both in melanocytes and in other skin cell populations.

While Myo10 mutant tail epidermis lacked melanocytes, dorsal and ventral skin had a chimeric pattern of normal and abnormal pigmentation. It is intriguing that the pigmentation process in the tail is distinct from dorsoventral skin and reduced penetrance of the phenotype among females, although the exact mechanism of this for sexual dimorphism is unclear.23 Developmental profiling of melanoblast migration using Myo10 mutants during embryogenesis would be helpful to address this phenomenon. It is also possible that Myo10 has other cellular roles in the skin, for example during wound healing or tumour formation.

CONFLICT OF INTEREST

The authors have declared no conflicting interest.

AUTHOR CONTRIBUTIONS

KL, CJL and FMW designed the study. KL, VEV and IS performed the experiments. KL, VEV, IS, CJL and FMW analysed and interpreted the data. KL and CJL wrote the manuscript with input from all authors. All authors approved the submitted manuscript.

Figure S1 Histology of Myo10 skin, interaction network comparison of Myo10 and Myo5a and expression of Myo10. (A) Myo10 expression in WT and Myo10 tail. Myo10 probe spans exons 28‐29. RQ value is relative to endogenous control gene B2m. (B) Haematoxylin and eosin staining of the tail and dorsal skin of Myo10 and WT mice. (C) Myo10 tail skin sections immunolabelled with the antibody to the melanocyte stem cell marker c‐Kit show the absence of melanocytes in hair follicles when compared to WT (arrows). Asterisks indicate nonspecific staining. (D) STRING interaction network of Myo10 and Myo5a shows two distinct classes of interacting proteins. (E) Gene expression results obtained from Hair‐GEL database show high expression of Myo10 in melanocytes at E14.5 and widespread expression in all skin subpopulations at P5. FPKM, Fragments Per Kilobase of transcript per Million mapped reads; Scale bars 100 μm.

Click here for additional data file.

Data S1 Materials and Methods

Click here for additional data file.