**2. Origin and development of melanocytes**

**1. Introduction**

262 Muscle Cell and Tissue

solely.

suprabasal layers [6].

that is, melanoblasts [7].

The biology of skin pigmentation plays a critical role in assorted physiological faculties from lower to higher vertebrates, like social interaction, camouflage, mimicry, sexual display as well as distinct racial coloration as seen in human beings. The pigmented biopolymer referred to as melanin is predominant in contributing color to skin, hair, and eyes of mammals. Melanin destined for pigmentation is produced in melanosomes, which are exclusively synthesized within melanocytes and in retinal pigment epithelial (RPE) [1]. Melanocytes are the key components of the pigmentary system because of their ability to produce melanin. These cells are found at many locations throughout the body. Mammalian melanocytes can be classified as "cutaneous" (follicular and epidermal) and "extracutaneous" (e.g., choroidal, cochlear) [2]. Though all of them have derived from pluripotent, NCC and have the capacity to synthesize melanin, their distinct task in all target places are much broader than the melanin synthesis

The existence of melanocytes is not only restricted to the epidermis but they are also found in other locations of the human body such as hair, iris, part of heart, nervous system, inner ear, etc. Here it is important to notice that ability of melanin synthesis is not confined to melanocytes only, but also other cells such as pigmented epithelium of retina, epithelia of iris and ciliary body of the eye, some neurons and adipocytes, which can also generate melanin [3]. Two types of melanocytes have been found: first are the differentiated melanocytes of neural crest origin which are also present at various locations within the body and the other are the RPE cells, originated from the outer layer of the optic cup of brain [4]. The RPE cells perform a critical role in the phagocytosis, that is, turnover and renewal of shed photoreceptor membrane as

Melanocytes and their production of melanin pigment have important roles in determining the physiology of mammal skin. They synthesize melanin inside a special membrane bound organelle, termed as melanosomes. Melanosomes are transferred via dendrites to surrounding keratinocytes where keratinocytes arrange them to form a critical protective barrier (known as supranuclear "caps") to shield the DNA from UV radiation. The anatomical relationship between keratinocytes and melanocytes is known as "the epidermal melanin unit" and it has been estimated that each melanocyte is in contact with 36 keratinocytes in the basal and

The amount and type of melanin produced, that is, eumelanin or pheomelanin, as well as its eventual distribution in the epidermis, dramatically affects visible color, which ultimately determines the various functions of the pigment, such as photoprotection. "Normal" pigmen‐ tation is regulated by more than 250 genes and which function during the development, migration, survival, proliferation, and differentiation of melanocytes from their precursors,

The present chapter is proposed to provide morphological and ultra structural details of melanocytes, found in different locations of the body along with their significant functions. Here we will also highlight some miscellaneous functions of melanocytes, other than melanin

well as maintenance of normal visual functions [5].

The embryonic development of melanocytes provides an opportunity to better understand the story of skin pigmentation and its related skin diseases. Melanocytes derived from the pluripotent, NCC are also known as the fourth embryonic layer [8]. In addition to melanocytes, they give rise to neurons and glial cells, adrenal medulla, cardiac cells, and craniofacial tissue.

Originating from the border between the dorsal neural tube and overlying ectoderm, NCC appears following closure of the neural tube during neurulation. Induction of the neural crest population requires the action of several transcription factors including; Msx1, Pax3 (paired-box 3), FoxD3, Zic1, Snail2, AP-2, and Sox10 (sex-determining region Y (SRY)-box 10), microphthalmia induced transcription factor (MITF), endothelin 3, and endothelin receptor B (EDNRB) [9,10]. Expression of these factors is in turn regulated by Wnt and Bone Morphogenic Protein (BMP) signalline [11]. These proteins and signaling pathways provide integrated spatial and temporal signals to create the proper environment for development and migration [12].

NCC migrates extensively around the embryo, during which they differentiate into specialized cell types. The fate of the NCC depends on environmental factors they meet on the migratory pathways. At the trunk, from somite eight to twenty eight, NCC emerge after an epitheliomesenchymal transition (EMT), proliferate extensively and follow two main migration paths; the dorso-lateral and the dorso-ventral pathways. The cells that migrate along the dorso-lateral pathway, between somite and ectoderm, are thought to be the main source for melanocytes while dorso-ventrally migrating cells give rise to the peripheral nervous system and adrenal medulla [13].

Melanoblasts, which are the precursors of melanocytes, migrate, proliferate, differentiate, and spread to their final destinations in the basal layer of epidermis and hair follicles; however, precise distribution of melanocytes varies among various species [14]. Cell type–specific markers are useful tools to demystify the development of certain cell types. Tyrp-2/Dct is reported to be a specific marker of melanoblasts development [15]. It has been reviewed by [16] that the markers for the precursors for melanocytes are: a tyrosinase kinase receptor KIT (ckit) and transcription factors such as MITF, SOX10, Pax3. Melanocytes are usually identified by their expression of melanocyte-specific proteins, for example tyrosinase (TYR), TYRP1, DCT, Pmel17/gp100, MART-1, and/or MITF (Figure 1).

**Figure 1. Embryonic development of melanocytes from NCC**. Melanocytes originate from the pluripotent NCC which in turn develop from dorsal most point of neural tube. Wnt, slug, and notch are the specified marker genes in‐ volved in the development pathway of early NCC. The bipotent stem cells of melanocytes differentiate into specialized cells termed as melanoblast with its specific marker genes. Melanoblasts are the precursor of melanocytes which mi‐ grate, proliferate, differentiate, and spread to their final destination in epidermis and hair follicles, stria vascularis of inner ears, the uveal track of eyes, substantia nigra, and locus coeruleus of brain and finally evolve into the mature melanocytes.

#### **3. Morpho-anatomical aspects of mammalian melanocytes**

The pigment cells of lower vertebrates/mammals (melanophores/melanocytes) are specialized type of smooth muscle cells, which due to their intracellular movement of melanin granules, control skin color. Several studies have been conducted on the effects of various hormones, factors, and pharmacological agents on vertebrate pigment cells and it has been widely accepted that they are controlled by either nerves alone or by hormones or by a combination of both [17–20]. Several cytokines, growth factors and other receptors have also been estab‐ lished to support the transformation of migrating melanoblasts to differentiated functional melanocytes. Receptors from a long time have evaded scientific investigations and are present on melanophores/melanocytes. These mysterious protein molecules mediate a host of phys‐ iological and pharmacological actions through endogenously as well as exogenously applied drugs. Receptor systems participate in a very coordinated manner in mediation of responses of color change [21]. Cellular receptors of adrenergic, cholinergic, histaminergic, and seroto‐ nergic nature have been found to regulate pigmentation via melanin displacement in various animal melanocyte models [22–24].

A number of researches have revealed that there is a close association between cell shape and function. Cell form can be affected by the topography of the surface, where the cells are fullgrown, each in cell culture as well as in an animal [25]. Depending on the localization, two types of melanocytes can be identified in the skin. These are (a) epidermal melanocytes occurring in the stratum basal of interfollicular epidermis and (b) follicular melanocytes residing within the hair follicle. Various electron microscopic, histochemical studies have been carried out in order to find out the ultrastructural details of melanocytes present in epidermis, hair follicles, uvea of eyes, etc.

#### **3.1. Morpho-Anatomical Details of Epidermal Melanocytes**

#### **a. Morphological Structure of Epidermal Melanocytes**

The epidermis is a stratified squamous epithelium consisting mainly of cells with two different origins: keratinocytes and melanocytes, of which keratinocytes form the 90–95% of epidermis. Melanocytes comprise from 5% to 10% of the cells in the basal layer of epidermis. Epidermal melanocytes are thin, elongated cell with branched structures, consisting of a central cell body and long numerous branches, or dendrites through which they interact with the adjacent keratinocytes of the basal epidermal layer [26]. Kemkemera *et al.* [27] have examined the cell shape of normal melanocytes (M-C) and of melanocytes from the skin of one neurofibromatosis type 1 patient (M-NFS) *in vitro*. They have reported that all cell types are bipolar and orient parallel to the grooves on the structured surface part, while all melanocytes remain distributed randomly. It has been reported by Valia [28] that although the size of melanocytes can vary, they are typically 7 µm in length. In human body variable pigmentation is seen due to variation in melanocyte population in different regions of the body. However there is no sexual or racial variation in the density of melanocytes in the skin. Thus the cellular activity is considered as the main contributor to racial differences in skin pigmentation [29].

#### **b. Ultrastructural Details of Epidermal Melanocytes**

**Figure 1. Embryonic development of melanocytes from NCC**. Melanocytes originate from the pluripotent NCC which in turn develop from dorsal most point of neural tube. Wnt, slug, and notch are the specified marker genes in‐ volved in the development pathway of early NCC. The bipotent stem cells of melanocytes differentiate into specialized cells termed as melanoblast with its specific marker genes. Melanoblasts are the precursor of melanocytes which mi‐ grate, proliferate, differentiate, and spread to their final destination in epidermis and hair follicles, stria vascularis of inner ears, the uveal track of eyes, substantia nigra, and locus coeruleus of brain and finally evolve into the mature

The pigment cells of lower vertebrates/mammals (melanophores/melanocytes) are specialized type of smooth muscle cells, which due to their intracellular movement of melanin granules, control skin color. Several studies have been conducted on the effects of various hormones, factors, and pharmacological agents on vertebrate pigment cells and it has been widely

**3. Morpho-anatomical aspects of mammalian melanocytes**

melanocytes.

264 Muscle Cell and Tissue

At the ultrastructural level the melanocyte is a distinctive cell. It was noticed by some workers that the melanocyte is free of the fibrillar material (tonofibrils, desmosomes, and tonofilaments) typical of the keratinocytes, and that intercellular bridges between them and the other cells of the epidermis are also absent. The nucleus of a melanocyte is smaller and more deeply basophilic than that of a basal keratinocyte. Also the presence of dendrites makes it distin‐ guished from neighboring keratinocytes [30]. Dendrites of melanocytes are revealed more effectively with silver salts that stain the melanin black; they then are seen to arborize in all directions among neighboring keratinocytes and even extend into the uppermost part of the dermis. Unlike the keratinocyte, mitochondria are abundant in melanocytes. Micro fibrils, as distinct from tonofilaments, are seen in the cytoplasm. Unlike tonofilaments they show no tendency to form bundles and are often seen as parallel arrays of fine filaments. The Golgi apparatus is usually prominent and the endoplasmic reticulum is well developed.

The characteristic organelle of the melanocytes is the melanosome. Melanosomes are produced inside the melanocytes, and they pass through several developmental stages, starting in the middle of the melanocyte, migrating to the outer edge of the cell through the dendrites. In 1971, the following four stages (see Figure 2) in the development of the melanosome were recognized by Fitzpatrick *et al.* [31].

**Figure 2. Development and maturation of melanosomes.** The synthesis, maturation, and translocation of melano‐ somes to the keratinocytes are the result of complex regulatory processes involving various sub-cellular organelles, en‐ zymes, proteins, cytoskeleton, intracellular signal transduction pathways, etc. Stage I premelanosomes are ovoid and have poorly defined protein matrix assumed to be originated from the endoplasmic reticulum in which tyrosinase is transferred from Golgi body through vesicular transport. Stage II premelanosomes are tyrosinase positive and have fibrillar matrix but no deposition of melanin takes place in this stage. Stage II premelanosomes matures to Stage III melanosomes in which synthesis of melanin takes place and hence it was seen as partial electron dense structure. Fi‐ nally, stage IV melanosomes evolved as complete electron dense structure in which the fibrilliar matrix become indis‐ tinguishable due to complete deposition of melanin.

**Stage I**. The Stage I melanosomes or premelanosomes likely develop form endoplasmic reticulum (ER). They have an amorphous matrix and display internal vesicles that form as a result of membrane invagination. Premelanosomes already contain the glycoprotein Pmel17 (gp100), but it requires further processing to become a component of the final fibrillar matrix. These filaments have a distinct periodicity of 100 A.

guished from neighboring keratinocytes [30]. Dendrites of melanocytes are revealed more effectively with silver salts that stain the melanin black; they then are seen to arborize in all directions among neighboring keratinocytes and even extend into the uppermost part of the dermis. Unlike the keratinocyte, mitochondria are abundant in melanocytes. Micro fibrils, as distinct from tonofilaments, are seen in the cytoplasm. Unlike tonofilaments they show no tendency to form bundles and are often seen as parallel arrays of fine filaments. The Golgi

The characteristic organelle of the melanocytes is the melanosome. Melanosomes are produced inside the melanocytes, and they pass through several developmental stages, starting in the middle of the melanocyte, migrating to the outer edge of the cell through the dendrites. In 1971, the following four stages (see Figure 2) in the development of the melanosome were

**Figure 2. Development and maturation of melanosomes.** The synthesis, maturation, and translocation of melano‐ somes to the keratinocytes are the result of complex regulatory processes involving various sub-cellular organelles, en‐ zymes, proteins, cytoskeleton, intracellular signal transduction pathways, etc. Stage I premelanosomes are ovoid and have poorly defined protein matrix assumed to be originated from the endoplasmic reticulum in which tyrosinase is transferred from Golgi body through vesicular transport. Stage II premelanosomes are tyrosinase positive and have fibrillar matrix but no deposition of melanin takes place in this stage. Stage II premelanosomes matures to Stage III melanosomes in which synthesis of melanin takes place and hence it was seen as partial electron dense structure. Fi‐ nally, stage IV melanosomes evolved as complete electron dense structure in which the fibrilliar matrix become indis‐

apparatus is usually prominent and the endoplasmic reticulum is well developed.

recognized by Fitzpatrick *et al.* [31].

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tinguishable due to complete deposition of melanin.

**Stage II**. The organelle is oval and shows numerous membranous filaments, with or without cross linking, having a distinct periodicity. Although no active melanin synthesis takes place in this stage, they already contain the enzyme tyrosinase.

**Stage III**. The internal structure, characteristic of Stage II has become partially obscured by electron-dense melanin due to deposition of melanin in its fibrillar matrix.

**Stage IV**. The oval organelle is electron-opaque without discernible internal structure in routine preparations.

Weiss and Zelickson [32] reported that melanocytes containing melanosomes of several developmental stages were present in the epidermis of 15-day-old C57BL16 mouse embryo and subsequently increased in number. Stage I melanosomes are seen as spherical vesicles near the Golgi apparatus. The other stages are usually seen scattered singly throughout the cytoplasm though there is a preponderance of Stage III and Stage IV melanosomes in the dendritic processes. If preservation is good, a distinct unit membrane can be seen surrounding the internal structure of the organelles.

Later, Hach [33] divided the melanosomes into two basic groups based on their ultrastructure: (1) elliptical (Ovoid) lamellar or fibrilliar melanosomes with a protein matrix arranged into coiled lamella or fibrils or rolled up sheets arranged parallel to the long axis of the organelle; (2) spherical globular melanosomes characterized by a granular appearance possibly due to a sponge-like architecture of their structural proteins with melanin stuffing the empty space. Melanocytes may produce eumelanosomes or pheomelanosomes at different times, switching from one to the other.

Schraermeyer [34] reviewed a comparison between eu- and pheomelanogenesis and further revealed that the earliest form of melanosomes is identical. The vasiculo-globular bodies are involved in melanosomal constituents [35]. In pheomelanogenesis, pheomelanosomes are smaller (~0.7 µm in diameter), ovoid to sub-spherical in shape, and their glycoprotein matrix appears disorganized and loose as lamellae are not formed, but these bodies fuse with each other to form an amorphous matrix on complete differentiation of melanosomes. Mature eumelanosomes in eumelanogenesis are large, (~0.9–0.3 µm), typically elliptical in shape with rounded ends and contain a highly structured fibrillar glycoprotein matrix required for eumelanin synthesis [34]. Melanosomes are numerous in melanocytes of Negroids and Caucasoid individuals, with a dark complexion though they are seen without difficulty in the melanocytes of those with a fair skin. They are ovoid or rod shaped bodies measuring 0.4–1.0 µm in length and 0.1–0.5 µm in diameter.

As lower vertebrates share similar mechanism of skin pigmentation with human beings, they have outstanding potential as phylogenetical tools to demystify and better understand their role in pigmentation biology and its evolutionary significance. Several detailed elucidations have been done on the fine structure of melanophores of various classes of vertebrates like teleosts and lung fishes [36–38], larval amphibians, Indian toad [39], and reptiles [40], which have revealed remarkably consistent fine structural features of these melanosomes synthesiz‐ ing cells in order to explain their role in color changes. In the cytoplasm of the epidermal and sub-epidermal melanophores, well developed nucleus, tubular type of vesicular mitochondria, Golgi body, vesicular endoplasmic reticulum with oval to elliptical melanosomes of various degree of melanization have been found to remain randomly scattered [39], resembling similar ultrastructural features of mammalian melanocytes. From these studies it is concluded that the presence of immature, pre-melanosomes to highly electron dense, mature melanosomes showing progressive degrees of melanization from stage I to stage IV melanosomes, in lower vertebrate melanophores is similar to mammalian melanocytes with same mode of develop‐ ment [41,42]. Hence, the ultrastructure of melanophores of these animal models appears to have a treasure of information, which may have physiological links with the human melano‐ somes from phylogenetic and therapeutic points of view.

#### **c. Intracellular Site of Melanin Synthesis in Epidermal Melanocytes**

In 1976, it was reported by Hunter [43], that the information gathered from electron microscopy, electron microscopic cytochemistry, autoradiography, and cell particle fractionation supports the view that tyrosinase is synthesized on the ribosomes. It is then transferred via the rough endoplasmic reticulum (RER) to the Golgi apparatus, from where it is channeled via tubular elements to a focal dilatation of the smooth endoplasmic reticulum in which the coiled melanosomal matrix has independently formed. Melanisa‐ tion of the structural protein can then take place and once this is completed, the connec‐ tion with tubular system is severed [44,45].

#### **3.2. Morpho-anatomical details of hair follicular melanocytes**

#### **a. Morphological Description of Hair Follicular Melanocytes**

Hair bulb matrix is the principal site for the fully developed follicular melanocytes, which differs from epidermal melanocytes by a larger size, larger dendrites containing more developed Golgi and RER and producing two to four fold larger melanosomes [46]. Also, they have a supplement of fewer keratinocytes in contrast to epidermal melanocytes (30–40 keratinocytes) [47]. These melanogenically active melanocytes along with the neighboring immature pre cortical keratinocyte constitute the follicular melanin unit, which usually consists of one melanocyte for every five keratinocytes in the hair bulb region, but in the basal epithelial layer next to the dermal papilla the ratio is 1:1 [48]. Pigmentation of hair is regulated by this follicular melanin unit [10].

Hair structure is divided into three morphological components: the multicellular cuticle sheath, the fibrous cortex, and the medulla. Follicular melanocytes in the fully developed hair follicle (HF) are localized in distinct anatomic compartments based on their differentiation status. Formation of hair begins with the inward folding of epidermis into the dermis. The dermis forms thickening from below and the edge of the inward folding comes to enclose it. These dermal thickenings mature into the dermal papilla, while the adjacent parts of inward folding forms the hair bulb (Figure 3). The hair follicular melanocytes derived from epidermal melanocytes are highly dendritic cells and colonize the hair matrix, lower half of the hair bulb. Hair bulb melanocytes secrete stage IV melanosomes into keratinocytes consisting of hair bulb and as a result hair cortex and medulla develops [49].

role in pigmentation biology and its evolutionary significance. Several detailed elucidations have been done on the fine structure of melanophores of various classes of vertebrates like teleosts and lung fishes [36–38], larval amphibians, Indian toad [39], and reptiles [40], which have revealed remarkably consistent fine structural features of these melanosomes synthesiz‐ ing cells in order to explain their role in color changes. In the cytoplasm of the epidermal and sub-epidermal melanophores, well developed nucleus, tubular type of vesicular mitochondria, Golgi body, vesicular endoplasmic reticulum with oval to elliptical melanosomes of various degree of melanization have been found to remain randomly scattered [39], resembling similar ultrastructural features of mammalian melanocytes. From these studies it is concluded that the presence of immature, pre-melanosomes to highly electron dense, mature melanosomes showing progressive degrees of melanization from stage I to stage IV melanosomes, in lower vertebrate melanophores is similar to mammalian melanocytes with same mode of develop‐ ment [41,42]. Hence, the ultrastructure of melanophores of these animal models appears to have a treasure of information, which may have physiological links with the human melano‐

In 1976, it was reported by Hunter [43], that the information gathered from electron microscopy, electron microscopic cytochemistry, autoradiography, and cell particle fractionation supports the view that tyrosinase is synthesized on the ribosomes. It is then transferred via the rough endoplasmic reticulum (RER) to the Golgi apparatus, from where it is channeled via tubular elements to a focal dilatation of the smooth endoplasmic reticulum in which the coiled melanosomal matrix has independently formed. Melanisa‐ tion of the structural protein can then take place and once this is completed, the connec‐

Hair bulb matrix is the principal site for the fully developed follicular melanocytes, which differs from epidermal melanocytes by a larger size, larger dendrites containing more developed Golgi and RER and producing two to four fold larger melanosomes [46]. Also, they have a supplement of fewer keratinocytes in contrast to epidermal melanocytes (30–40 keratinocytes) [47]. These melanogenically active melanocytes along with the neighboring immature pre cortical keratinocyte constitute the follicular melanin unit, which usually consists of one melanocyte for every five keratinocytes in the hair bulb region, but in the basal epithelial layer next to the dermal papilla the ratio is 1:1 [48]. Pigmentation of hair is regulated

Hair structure is divided into three morphological components: the multicellular cuticle sheath, the fibrous cortex, and the medulla. Follicular melanocytes in the fully developed hair follicle (HF) are localized in distinct anatomic compartments based on their differentiation status. Formation of hair begins with the inward folding of epidermis into the dermis. The dermis forms thickening from below and the edge of the inward folding comes to enclose it.

somes from phylogenetic and therapeutic points of view.

268 Muscle Cell and Tissue

tion with tubular system is severed [44,45].

by this follicular melanin unit [10].

**3.2. Morpho-anatomical details of hair follicular melanocytes a. Morphological Description of Hair Follicular Melanocytes**

**c. Intracellular Site of Melanin Synthesis in Epidermal Melanocytes**

**Figure 3. Developmental stages of mammalian hair follicles.** Formation of hair begins with the inward folding of epi‐ dermis into the dermis. The dermis forms thickening from below and the edge of the inward folding comes to enclose it. These dermal thickenings mature into the dermal papilla, while the adjacent parts of inward folding forms the hair bulb. Hair bulb melanocytes secrete stage IV melanosomes into keratinocytes consisting of hair bulb and as a result hair cortex and medulla develops.

In mice, the process of morphogenesis of hair follicle structures is cyclic [50] and called hair growth cycle [47]. The hair growth cycle consists of three stages: resting (telogen); growth (anagen); and regression (catagen). Anagen phase is divided into six sub stages (anagen I–VI). Melanogenesis is strictly coupled to the growth phase of the hair growth cycle (anagen III–VI). Melanogenesis ceases early in catagen and is completely absent in telogen [51]. During early anagen, melanocyte stem cells residing in the lower permanent portions of the hair follicle are recruited for the regeneration of melanogenically active follicular melanocytes. This melano‐ cyte stem cell progeny on activation migrated out from niche, amplifies and differentiates into pigmented melanocytes. The vacant niches are again repopulated by the melanocyte stem cells and remain quiescent till the next early anagen [52]. Towards the end of anagen the number of identifiable melanocytes decreases and melanocytes lose their dendrites, shrink and become less pigmented, and then they disappear in catagen [47]. However, a small number of dedif‐ ferentiated cells (melanoblasts) positive to the combined dopa-premelanin reaction still remain in the hair bulbs.

Melanocytes occurring in hair follicles outer root sheath (ORS) express tyrosinase related protein-2 (TRP-2) and relatively weak tyrosinase related protein-1 (TRP-1). Unlike the bulge melanocytes, they display proliferative activity during the early and mid-anagen stage of hair cycle. They represent the differentiating melanocytes. Melanocytes, which actively produce melanin in the hair follicle, are located in the hair matrix above the dermal papilla. They express all melanogenic enzymes – tyrosinase (TYR), TRP1, and TRP2 – and proliferate only during mid-anagen [53].

#### **b. Ultrastructural Details of Hair Follicular Melanocytes**

The ultra structure of amelanotic melanocytes from human hair follicles is different from that of epidermal melanocytes, and these characteristics determine the functional nature of amelanotic melanocytes. The fine-structure observation of the cultured melanocytes revealed round or elliptical shape, single huge nucleus and the karyotheca were double layered. The clear euchromosomes and meager heterochromosomes were observed within the nucleus. The cytoplasm was furnished with several organelles, such as unit membranous melanosomes of similar size, RER, ribosomes, as well as mitochondria were also present. While the Golgi apparatus in the cells was not seen easily, the electron dense melanin granules were concen‐ trically deposited within the melanosomes [54]. The hair melanosomes are two to four times larger than the epidermal melanosomes. It has been reviewed by Ortonne and Paul [55] that the different stages of hair cycle of hair melanocytes have their own unique ultrastructure features. In hair follicles of C57 black mice, early anagen (growth) stage of hair cycle extensive growth occurred in melanocytes where cytoplasm volume increased, with increase in den‐ dricity, elaborate RER, and Golgi zone along with increase in size and number of melanosomes. However, during catagen and telogen stage of hair cycle some degenerative changes takes place in melanocyte, with only a few small premelanosomes. The volume of cytoplasm decreases with less well developed Golgi complex and RER as well as nuclei with prominent heterochromatin patterns. Hair melanocytes transfer melanosomes to follicular epithelial cells. Medullary cells receive their melanin from melanocytes in the upper part of the hair bulbs similar to the way described for epidermal melanin unit. Melanosomes are also transferred to immature corticle cells. Due to their larger size, melanosomes are usually distributed singly, whatever the ethnic background.

Many studies have been carried out to demystify the ultrastructural aspects of hair melanin pigmentation. Earlier, it was revealed by Jimbow *et al.* [56] that human red hair, specified as pheomelanic, its melanocytes contain spherical melanosomes with vasiculo-globular and proteinaceous matrices on which melanin deposition is spotty and granular. Melanin granules are smaller and less numbered in blonde than in dark-hair human subjects [57]. Melanosomes are not fully melanized even in the dendritic processes of melanocytes. Whereas ultrastructure of follicular melanocytes of black hair is identical to the melanocytes in the epidermis of negroids as they observed to contain typical ellipsoidal melanosomes, at various stages of melanization and large melanosomes are transferred singly to the neighboring keratinocytes [58]. In brown hair, the follicular melanocytes also contain all the developmental stages of melanosomes. Lighter brown hairs have smaller melanosomes [59]. In the melanocytic zone of the senile gray hair bulb, the number of melanocytes appears normal or reduced [60]. These cells show little melanogenic activity and contain very few melanosomes.

#### **3.3. Morpho-anatomical aspects of ocular melanocytes**

melanocytes, they display proliferative activity during the early and mid-anagen stage of hair cycle. They represent the differentiating melanocytes. Melanocytes, which actively produce melanin in the hair follicle, are located in the hair matrix above the dermal papilla. They express all melanogenic enzymes – tyrosinase (TYR), TRP1, and TRP2 – and proliferate only during

The ultra structure of amelanotic melanocytes from human hair follicles is different from that of epidermal melanocytes, and these characteristics determine the functional nature of amelanotic melanocytes. The fine-structure observation of the cultured melanocytes revealed round or elliptical shape, single huge nucleus and the karyotheca were double layered. The clear euchromosomes and meager heterochromosomes were observed within the nucleus. The cytoplasm was furnished with several organelles, such as unit membranous melanosomes of similar size, RER, ribosomes, as well as mitochondria were also present. While the Golgi apparatus in the cells was not seen easily, the electron dense melanin granules were concen‐ trically deposited within the melanosomes [54]. The hair melanosomes are two to four times larger than the epidermal melanosomes. It has been reviewed by Ortonne and Paul [55] that the different stages of hair cycle of hair melanocytes have their own unique ultrastructure features. In hair follicles of C57 black mice, early anagen (growth) stage of hair cycle extensive growth occurred in melanocytes where cytoplasm volume increased, with increase in den‐ dricity, elaborate RER, and Golgi zone along with increase in size and number of melanosomes. However, during catagen and telogen stage of hair cycle some degenerative changes takes place in melanocyte, with only a few small premelanosomes. The volume of cytoplasm decreases with less well developed Golgi complex and RER as well as nuclei with prominent heterochromatin patterns. Hair melanocytes transfer melanosomes to follicular epithelial cells. Medullary cells receive their melanin from melanocytes in the upper part of the hair bulbs similar to the way described for epidermal melanin unit. Melanosomes are also transferred to immature corticle cells. Due to their larger size, melanosomes are usually distributed singly,

Many studies have been carried out to demystify the ultrastructural aspects of hair melanin pigmentation. Earlier, it was revealed by Jimbow *et al.* [56] that human red hair, specified as pheomelanic, its melanocytes contain spherical melanosomes with vasiculo-globular and proteinaceous matrices on which melanin deposition is spotty and granular. Melanin granules are smaller and less numbered in blonde than in dark-hair human subjects [57]. Melanosomes are not fully melanized even in the dendritic processes of melanocytes. Whereas ultrastructure of follicular melanocytes of black hair is identical to the melanocytes in the epidermis of negroids as they observed to contain typical ellipsoidal melanosomes, at various stages of melanization and large melanosomes are transferred singly to the neighboring keratinocytes [58]. In brown hair, the follicular melanocytes also contain all the developmental stages of melanosomes. Lighter brown hairs have smaller melanosomes [59]. In the melanocytic zone of the senile gray hair bulb, the number of melanocytes appears normal or reduced [60]. These

cells show little melanogenic activity and contain very few melanosomes.

**b. Ultrastructural Details of Hair Follicular Melanocytes**

mid-anagen [53].

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whatever the ethnic background.

Ocular melanocytes are found in the uveal tract, which consist of cells of the choroid, iris, ciliary body [61]. The RPE is a monolayer that lies between photoreceptors and choroid. Melanin is produced in RPE and choroid of eyes. Melanocytes and RPE cells have a different embryonic origin and development [62]: While RPE cells emerge from the neural tube, melanocytes are neural crest–derived cells. Consequently some differences exist between melanocyte and RPE melanogenesis [63]. Ocular melanocytes are in contact with only each other, and they do not transfer their melanosomes. The RPE is a monolayer of pigmented cells of neural tube origin in the background of the eye. Together with the endothelial cells of the chorio-capillaris it forms the blood-retinal barrier [64]. RPE cells are cubical and highly polarized. Basal infoldings and apical microvilli serve as enlargement of the surface. The shape of mature RPE melanosomes differs from that of melanocytes in being oval [65].

The typical oval shaped melanosomes of the RPE are located in the microvilli, and round shaped melanosomes in the cytoplasm near the nucleus. In spite of this RPE cells and the epidermal melanocytes have been described to follow the standard pathway of melanogenesis. It was reported that they exploit the similar melanogenic proteins and the fine structural characterization of both the epidermal melanocytes and pre-natal RPE cells confirmed the presence of distinct melanosomes of various stages (I–IV) of development [66]. Schraemeyer [67] reported that the formation of classical premelanosomes in RPE have been induced by illumination. Ocular melanin content differs among species. Stages II–IV melanosomes have been found in the RPE of squirrels [68]. Although melanogenesis in RPE and choroid seems to follow a common pathway, the RPE of adult hamsters and probably all the vertebrates, contains unique melanosomes never present in the melanosomes of choroid. These are large, spherical melanosomes with loosely packed melanofilaments. Whereas the high numbers of melanin granules are spindle shaped in the RPE, they are smaller and more spherical in the choroid. The large type melanosomes often fuse with earlier-stage melanosomes and have been described as late immature melanosomes in the prenatal RPE of humans. In the RPE melano‐ somes contain melanofilament ordered concentrically as are the membrane in phagosomes [69]. In the RPE of chicken, parallel filaments which appeared to have the same structure as those forming the framework of melanosomes were frequently found in phagosomes [70]. Ultrastructural similarities between phagosomes and melanosomes have also been reported. In the RPE of cattle, phagosomes were found that contain an electron dense melanin-like material that was not autofluroscent and therefore not lipofuscin. Additionally electron dense vasiculo-globular bodies (10–100 nm) were found in phagosomes during disk membrane degradation as well as within melanin granules [71].
