Melanocytes are pigment-producing cells that colour our hair, skin and eyes. Melanocytes are evolutionary conserved in vertebrates, and in addition to contributing to pigmentation and pattern formation, can contribute to background adaptation (zebrafish) and protection against harmful UV irradiation (humans). Many of the processes involved in melanocyte development – such as migration, proliferation and differentiation - are misregulated in melanoma. Here, I use chemical biology in zebrafish to identify targetable pathways in melanocyte development and regeneration, with a view to how these processes may be misregulated in melanoma and other pigmentation syndromes. We first wanted to address the potential for small molecules to regulate multiple stages of melanocyte development and differentiation. In Chapter 3, I describe my work involved in a small molecule screen for clinically active compounds that alter melanocyte biology (Colanesi et al., 2012). In this work we have identified small-molecules that affect melanocyte migration, differentiation, survival, morphology and number. This is important as it highlights new pathways essential for normal melanocyte development and consequently provides further tools in which to study melanocytes. Identifying the target of small molecules in vivo is a challenge in chemical biology. In Chapter 4, I describe my contributions to understanding how 5-nitrofuran compounds act in zebrafish (Zhou et al., 2012). My work has contributed to understanding the activity of 5-nitrofurans is dependent upon its nitrofuran ring structure. I have also helped confirm a conserved interaction between 5-nitrofurans and ALDH2, which may contribute to the off-target effects observed in the clinic. These results are important as they aid further understand of the 5-nitrofuran class of drugs and give evidence to support combination therapy of 5-nitrofurans with ALDH2 inhibitors as a way to overcome clinical side effects. Additionally I show that NFN1 treatment limits ensuing melanocyte regeneration thereby suggesting a role at the Melanocyte Stem Cell (MSC), which provides me with a key tool to study melanocyte regeneration in zebrafish. How tissue specific cell numbers are specified and maintained is a key question in developmental biology. In Chapter 5, I describe the identification of the MITF gene in the maintenance of cell cycle arrest in differentiated melanocytes (Taylor et al., 2011). We show that the human melanoma mutation MITF4TΔ2B promotes melanocyte division, thereby suggesting a role for melanocyte division in the pathogenesis of melanoma. This work is valuable because it highlights Mitf as a molecular rheostat that controls melanocyte proliferation and differentiation in living vertebrates, and helps us to understand the role of MITF in melanoma progression. Little is known about the pathways that control melanocyte stem cells in animals. To identify new melanocyte stem cell pathways, I used NFN1 as the basis for a small molecule screen for enhancers of melanocyte regeneration (Chapter 6). I find that chemical inhibition of Phosphatase of Regenerating Liver-3 (Prl-3) in zebrafish can enhance melanocyte regeneration. Importantly, I have found that there are an increased number of melanocyte progenitor cells in PRL3-inhibitor treated zebrafish. I propose that PRL-3 may control progenitor cell number in melanocyte regeneration. This is significant because it identifies PRL-3 as a novel molecular target controlling melanocyte progenitor cells, and identifies a new chemical tool with which to study melanocyte differentiation from a progenitor population. In the final chapter, I discuss how this work relates to the larger field of melanocyte developmental biology, and the new insight it provides into the fundamental processes of how organisms control cell number and pattern formation. In addition, I discuss how this work may have implications for understanding and treating melanocyte diseases, such as vitiligo (loss of melanocytes) and melanoma (cancer of the melanocyte).

Pigment patterns of zebrafish are a beautiful example in which to study key processes of vertebrate development such as neural crest cell migration and patterning of neural crest-derived cell types. This can for example be achieved by characterizing mutants like parade in which pigment cell development is abnormal. Here, we present a chemico-genetic study of the pigment pattern mutant parade; uniquely, this mutant displays ectopic pigment cells in the ventral medial pathway of the trunk but the characteristic stripe pattern of zebrafish embryos is unaffected. Using a positional cloning approach, we have identified the parade gene as the cell surface receptor ednra2. This was further confirmed in transient knock-down assays. Combined sequencing data from three different parade alleles strongly indicates that the mutation disrupts ednra2 receptor function by deleting C-terminal regulatory and structural residues. To expand the available molecular tools in pigment cell research, notably to chemically dissect the parade phenotype, we have participated in small molecule screening for inhibitors of pigment cell development. From this, we have isolated 57 compounds which robustly alter the development of melanophores and iridophores in wild-type embryos; 26 of these compounds additionally affect the parade phenotype, primarily by rescuing the ectopic pigment cells. Notably, chemical rescue has shown that the MEK pathway is important for the development of the parade phenotype. Our study therefore adds to our understanding of pigment pattern formation in zebrafish embryos and reveals novel functions for ednra2 in dorso-ventral patterning and cell type specification of neural crest derivatives.

Melanin is the major component of skin-, hair-, and eye pigmentation in mammals. Synthesis of melanin takes place in specialized organelles in melanocytes, called melanosomes. As melanosomes mature during pigment synthesis, they are transported towards the tips of dendrites in the melanocyte, and eventually transferred to neighbouring keratinocytes to distribute pigment throughout the skin. A large number of proteins regulate melanin synthesis and distribution. Over one hundred genes have been associated with coat colour mutations in mice, and many of these genes have also been identified in human pigmentation disorders. Other proteins involved in pigmentation are part of pathways that are not unique to pigmentation alone, such as the Ras/ERK pathway. In mouse B16 cells, cAMP stimulation leads to the upregulation of melanin synthesis and dendrite extension. However, cAMP also activates the Ras/ERK pathway in these cells, which, upon prolonged stimulation, leads to an inhibition of melanin synthesis and dendrite extension. Here I show that the protein CNrasGEF, which was previously identified in our lab, is responsible for cAMP-dependent Ras activation in B16 cells, and therefore a part of the negative regulatory pathway of melanogenesis. In order to find other proteins involved in pigmentation pathways, I have developed a method to detect melanosomes using Cellomics KineticScan (KSR) high-content image analysis. This system could potentially be used in a high-throughput RNA interference screen to identify proteins that affect melanosome formation or transport. However, in a pilot study it appeared that knockdown levels achieved upon transient transfection of knockdown constructs from a mouse shRNAmir library against selected targets were in many cases not sufficient to detect an effect on melanocytes, either by confocal microscopy, or by Cellomics KSR analysis. Further reduction of expression levels is necessary before this system can be scaled up to high-content/high-throughput identification of proteins involved in pigmentation.

Melanin is the major component of skin-, hair-, and eye pigmentation in mammals. Synthesis of melanin takes place in specialized organelles in melanocytes, called melanosomes. As melanosomes mature during pigment synthesis, they are transported towards the tips of dendrites in the melanocyte, and eventually transferred to neighbouring keratinocytes to distribute pigment throughout the skin. A large number of proteins regulate melanin synthesis and distribution. Over one hundred genes have been associated with coat colour mutations in mice, and many of these genes have also been identified in human pigmentation disorders. Other proteins involved in pigmentation are part of pathways that are not unique to pigmentation alone, such as the Ras/ERK pathway. In mouse B16 cells, cAMP stimulation leads to the upregulation of melanin synthesis and dendrite extension. However, cAMP also activates the Ras/ERK pathway in these cells, which, upon prolonged stimulation, leads to an inhibition of melanin synthesis and dendrite extension. Here I show that the protein CNrasGEF, which was previously identified in our lab, is responsible for cAMP-dependent Ras activation in B16 cells, and therefore a part of the negative regulatory pathway of melanogenesis. In order to find other proteins involved in pigmentation pathways, I have developed a method to detect melanosomes using Cellomics KineticScan (KSR) high-content image analysis. This system could potentially be used in a high-throughput RNA interference screen to identify proteins that affect melanosome formation or transport. However, in a pilot study it appeared that knockdown levels achieved upon transient transfection of knockdown constructs from a mouse shRNAmir library against selected targets were in many cases not sufficient to detect an effect on melanocytes, either by confocal microscopy, or by Cellomics KSR analysis. Further reduction of expression levels is necessary before this system can be scaled up to high-content/high-throughput identification of proteins involved in pigmentation.

<p>Vertebrates like urodele and teleost have an enhanced capacity for regeneration, when compared to mammals. Recently, the teleost zebrafish (Danio rerio) has become a popular model for studying regenerative events, due to the ability to regenerate multiple organs such as the fin and the heart, and the diverse genetic approaches available for functional studies. In my thesis studies, I have used the zebrafish caudal fin as a model system to understand molecular and cellular mechanism of appendage regeneration. </p><p>Pharmacological and genetic studies have revealed that Fgf signaling is important for appendage regeneration. To dissect the mechanism of Fgfs during zebrafish fin regeneration, lab colleagues and I have generated and utilized transgenic animals in which Fgf signaling can be experimentally increased or decreased. Through these transgenic studies, I found that position-dependent Fgf signaling directs regenerative growth and blastemal proliferation. Proximally-amputated fin regenerates grow at higher rates than the distally-amputated, owing to position-dependent amounts of Fgf activity. Further studies using new transgenics have provided an understanding of mechanisms by which Fgfs influence epidermal regulation of the blastema. Loss- and gain-of-function studies of Fgfs reveal that Fgf signaling both positively and negatively regulated shh expression in the epidermis to maintain blastemal function.</p><p>During the fin regeneration process, pigmentation pattern is re-established as along with bone structures and connective tissues. While the lineage of the blastema is not precisely clear, pigment cells in the fin regenerates are thought to be derived from melanocyte stem cells. Therefore, melanocyte regeneration is an informative system to understand the mechanism underlying regulation of adult stem cells during regeneration. As part of my thesis studies, we generated transgenic animals in which ectopic Ras expression can be experimentally induced. Transgenic studies, combined with pharmacological approaches, have revealed that Ras controls self-renewal of melanocyte stem cells during fin pigment regeneration.</p> / Dissertation

The human melanocortin receptors (hMCRs) play a fundamental role in human behavior such as satiety, feeding, sexual and more. A set of SHU9119 peptide derivatives were studied for their structure-activity relationships. These peptides contained a sequential a-N-methylation amino acid scan.A second set of peptide derivatives intended to be used to create TNF-a; inhibition, via the melanocortin receptors. These peptides were shown to bind to all of the hMCR receptors, and only exhibit cAMP stimulation at hMC1R/hMC5R.The data from both of the sets of compounds illustrate that small changes in the stereochemistry of the SH9119 and TNF-a; derivatives cause drastic changes in the binding and the agonistic/antagonist properties of the compounds.This thesis determined the effect that hMC1R mutations have on the binding and cAMP response of well characterized ligands. This study ruled out 9 different residues for being the required for the cAMP response of the hMC1R.

Malignant melanoma is one of the most rapidly increasing cancers and accounts for about three-quarter of all skin cancer deaths worldwide. Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge how various wavelength spectra affect the balance between proliferation and apoptosis controlling the homeostasis of the melanocyte population is still limited. The aim of this thesis was to elucidate the regulation of UVA/B induced apoptotic signaling in human epidermal melanocytes in vitro in relation to redox alterations and antioxidant photoprotection. UVA irradiation induced changes in plasma membrane stability, decreased cell proliferation and increased apoptosis. In comparison, melanocyte plasma membrane was markedly resistant to UVB irradiation although apoptosis was triggered. Thus, UVA irradiation should not be overlooked as an etiologic factor in melanoma development. Further, after irradiation with UVA/B we found alterations in redox state manifested by a reduction of intracellular GSH levels, translocation of nuclear factor-κB from the cytosol to the nucleus, an increase of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis, and an increased apoptosis frequency. α-Tocopherol provided photoprotection through several modes of action affecting redox alterations and signaling, stabilizing the plasma membrane, and decreased proliferation and apoptosis rate, while β-carotene did not show the same protective capacity. Altogether, α-tocopherol might be a useful substance in protecting melanocytes from UV induced damage. We demonstrate UVA/B irradiation to activate the intrinsic pathway of apoptosis in melanocytes where translocation of Bcl-2 family proteins to the mitochondria modulates the apoptosis signal. Interestingly, the anti-apoptotic Bcl-2 family proteins generally thought to be attached to membranes, were localized in the cytosol before UV irradiation and translocated to the mitochondria in the surviving population, which might be a critical event in preventing apoptotic cell death. Lysosomal cathepsins were released to the cytosol acting as pro-apoptotic mediators upstream of activation and translocation of Bax to the mitochondria. When melanocytes were exposed to UVA, p53 participated in apoptosis regulation through interaction with Bcl-2 family proteins, while UVB induced p53-transcriptional activity and apoptosis involving lysosomal membrane permeabilization. Thus, depending on the UV wavelength p53 mediated apoptosis in melanocytes by transcriptional dependent or independent activity. These results emphasize p53 as an important pro-apoptotic component in the regulation of apoptosis. This thesis gives new insight in the harmful and various effects of different wavelengths within the UV spectrum on human melanocytes in vitro. Improved knowledge of the apoptosis regulatory systems in melanocytes might lead to a better understanding of the formation of pigment nevi and malignant melanoma and, in the future, provide better strategies to prevent and eliminate tumor development and progression.

Melanocytes, pigment-producing cells, derive from the neural crest (NC), a population of pluripotent cells that arise from the dorsal aspect of the neural tube during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The deletion of the transcription factor Ets1 in mice results in hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The goal of the present study was to establish the temporal requirement and role of Ets1 in murine melanocyte development. In the mouse, Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick cranial NC, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of melanocytes, enteric ganglia, and other NC derivatives. Using a combination of immunofluorescence and cell survival assays Ets1 was found to be required between embryonic days 10 and 11, when it regulates NC cell and melanocyte precursor (melanoblast) survival. Given the requirement of Ets1 for Sox10 expression in the chick cranial NC, a potential interaction between these genes was investigated. Using genetic crosses, a synergistic genetic interaction between Ets1 and Sox10 in melanocyte development was found. Since Sox10 is essential for enteric ganglia formation, the importance of Ets1 on gut innervation was also examined. In mice, Ets1 deletion led to decreased gut innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate a Sox10 enhancer critical for Sox10 expression in melanoblasts. Furthermore, mutating Ets1 at a site I characterized in the spontaneous variable spotting mouse pigmentation mutant, led to a 2-fold decrease in enhancer activation. Overexpression and knockdown of Ets1 did not affect Sox10 expression; nonetheless, Ets1 knockdown led to a 6-fold upregulation of the transcription factor Sox9, a gene required for melanocyte and chondrocyte development, but which impairs melanocyte development when its expression is prolonged. Together, these results suggest that Ets1 is required early during melanocyte development for NC cell and melanoblast survival, possibly acting upstream of Sox10. The transcription factor Ets1 may also act indirectly in melanocyte fate specification by repressing Sox9 expression, and consequently cartilage fate.

The final publication is available at http://dx.doi.org/10.1111/pcmr.12297. Natsuko Okamoto et al. A melanocyte–melanoma precursor niche in sweat glands of volar skin. Pigment Cell & Melanoma Research. Volume 27, Issue 6, pages 1039–1050, November 2014 / 京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12890号 / 論医博第2090号 / 新制||医||1007(附属図書館) / 31644 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 羽賀 博典, 教授 鈴木 茂彦 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

NO / Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H2O2 in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 μM H2O2 increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH4Cl and elevated l-tyrosine, H2O2-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H2O2-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca2+-chelator BAPTA. Thus, BAPTA reduced the level of H2O2-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca2+ and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca2+ binding capacity and, in addition, correlated inversely with H2O2-induced increases in intracellular Ca2+. Our results show that melanin may have an important role in regulating intracellular Ca2+ homeostasis and it is suggested that melanin protects against H2O2-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca2+.