Proliferation Of Melanoblasts Research Articles

Mutations in gfpt1 and skiv2l2 Cause Distinct Stage-Specific Defects in Larval Melanocyte Regeneration in Zebrafish

The establishment of a single cell type regeneration paradigm in the zebrafish provides an opportunity to investigate the genetic mechanisms specific to regeneration processes. We previously demonstrated that regeneration melanocytes arise from cell division of the otherwise quiescent melanocyte precursors following larval melanocyte ablation with a small molecule, MoTP. The ease of ablating melanocytes by MoTP allows us to conduct a forward genetic screen for mechanisms specific to regeneration from such precursors or stem cells. Here, we reported the identification of two mutants, eartha j23e1 and julie j24e1 from a melanocyte ablation screen. Both mutants develop normal larval melanocytes, but upon melanocyte ablation, each mutation results in a distinct stage-specific defect in melanocyte regeneration. Positional cloning reveals that the eartha j23e1 mutation is a nonsense mutation in gfpt1 (glutamine:fructose-6-phosphate aminotransferase 1), the rate-limiting enzyme in glucosamine-6-phosphate biosynthesis. Our analyses reveal that a mutation in gfpt1 specifically affects melanocyte differentiation (marked by melanin production) at a late stage during regeneration and that gfpt1 acts cell autonomously in melanocytes to promote ontogenetic melanocyte darkening. We identified that the julie j24e1 mutation is a splice-site mutation in skiv2l2 (superkiller viralicidic activity 2-like 2), a predicted DEAD-box RNA helicase. Our in situ analysis reveals that the mutation in skiv2l2 causes defects in cell proliferation, suggesting that skiv2l2 plays a role in regulating melanoblast proliferation during early stages of melanocyte regeneration. This finding is consistent with previously described role for cell division during larval melanocyte regeneration. The analyses of these mutants reveal their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process, but also in late stages of differentiation of the regenerating melanocyte. We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis, and those involved in larval or adult growth.

Ferrous Ferric Chloride Stimulates the Proliferation and Differentiation of Cultured Keratinocytes and Melanocytes in the Epidermis of Neonatal Mouse Skin

Ferrous ferric chloride (FFC) is a special aqueous iron which is ac omplex of ferrous chloride and ferric chloride and has a function in both oxidation and reduction. FFC is thought to stimulate the cellular function of living organisms. Although FFC is reported to stimulate the function of red blood cells, it has not been determined whether FFC stimulates the function of skin cells. To understand the role of FFC in the function of skin cells, we studied its effects on the proliferation and differentiation of keratinocytes and melanoblasts or melanocytes. FFC was added to a serum-free culture of neonatal mouse epidermal cells and its effects on the proliferation and differentiation of keratinocytes and melanoblasts or melanocytes were investigated. FFC stimulated the proliferation and differentiation of keratinocytes and melanoblasts or melanocytes. The proliferation of keratinocytes and that of melanoblasts or melanocytes was stimulatedto the same extent (atwofold increase), suggesting tha tt heproliferation of the two types of cells constituting the epidermis may be equally stimulated by FFC. These results suggest that FFC may activate skin function by promoting cell renewal via th es timulation of the proliferation and differentiation of keratinocytes and melanoblasts or melanocytes.

A modified method for purifying amelanotic melanocytes from human hair follicles

We describe a modified method for establishing long-term pure cultures of amelanotic melanocytes (AMMC) derived from human hair follicles. Normal human corpse scalp (just after death, 1 h) was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. Hair follicle cell suspensions were prepared by 0.50% trypsin treatment for 30 min and cultured in an optimized melanoblast proliferation nature mitogen medium. Cells attached to the substratum were mostly amelanotic melanocytic in character with small, bipolar shapes in the early stage; only a few keratinocytes and rare fibroblasts were observed. Keratinocytes were easily removed by differential trypsinization. After the third passage, the proliferating cells were all amelanotic melanocytes as confirmed by immunostaining with polyclonal antibodies to alphaPEP7h, which recognized the tyrosinase protein located on melanosomes and NKI/beteb, which is a pre-melanosomal antigen against synthetic peptides corresponding to the carboxyl termini of human melanosomal protein GP100. Cultured AMMC were highly positive to L-dopa reactivity after the addition of IBMX to the culture medium for 7 days. Many stage I and II melanosomes and occasional stage III melanosomes without stage IV melanosomes were found in the cytoplasm by transmission electron microscope. This modified technique is potentially more suitable for cultivating amelanotic melanocytes. The availability of pure cultures of hair-follicle amelanotic melanocytes will facilitate investigations of the roles of those cells in migration and differentiation during treatment of vitiligo.

Pax3 transcripts in melanoblast development

The transcription factor encoded by PAX3 is among the first expressed in the embryo, with a key role in development of the melanocytic lineage. Re-expression of PAX3, consistently observed in cutaneous malignant melanoma (CMM) as compared to normal melanocytes, appears linked to progression of CMM. Previous research has identified PAX3d (encoded by exons 1-9) as the predominant isoform present in CMM, together the with an alternate isoform PAX3c (encoded by exons 1-8). We investigated the expression of Pax3c and Pax3d transcripts during mouse development. The reverse transcription-polymerase chain reaction and immunohistochemistry experiments presented here implicate these transcripts in melanoblast development and demonstrate significant spatial and temporal differences in their expression. Differences in expression were also noted during active hair regrowth in adult skin, which is accompanied by proliferation and migration of melanoblasts into the hair cortex to color new hair. Results indicate that the defined spatial and temporal expression of Pax3d may be linked to either melanoblast proliferation or migration during melanogenesis.

Mutations in gfpt1 and skiv2l2 cause distinct stage-specific defects in larval melanocyte regeneration in zebrafish

The establishment of a single cell type regeneration paradigm in the zebrafish provides an opportunity to investigate the genetic mechanisms specific to regeneration processes. We previously demonstrated that regeneration melanocytes arise from cell division of the otherwise quiescent melanocyte precursors following larval melanocyte ablation with a small molecule, MoTP. The ease of ablating melanocytes by MoTP allows us to conduct a forward genetic screen for mechanisms specific to regeneration from such precursors or stem cells. Here, we reported the identification of two mutants, earthaj23e1 and juliej24e1 from a melanocyte ablation screen. Both mutants develop normal larval melanocytes, but upon melanocyte ablation, each mutation results in a distinct stage-specific defect in melanocyte regeneration. Positional cloning reveals that the earthaj23e1 mutation is a nonsense mutation in gfpt1 (glutamine:fructose-6-phosphate aminotransferase 1), the rate-limiting enzyme in glucosamine-6-phosphate biosynthesis. Our analyses reveal that a mutation in gfpt1 specifically affects melanocyte differentiation (marked by melanin production) at a late stage during regeneration and that gfpt1 acts cell autonomously in melanocytes to promote ontogenetic melanocyte darkening. We identified that the juliej24e1 mutation is a splice-site mutation in skiv2l2 (superkiller viralicidic activity 2-like 2), a predicted DEAD-box RNA helicase. Our in situ analysis reveals that the mutation in skiv2l2 causes defects in cell proliferation, suggesting that skiv2l2 plays a role in regulating melanoblast proliferation during early stages of melanocyte regeneration. This finding is consistent with previously described role for cell division during larval melanocyte regeneration. The analyses of these mutants reveal their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process, but also in late stages of differentiation of the regenerating melanocyte. We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis, and those involved in larval or adult growth.

The transcription network regulating melanocyte development and melanoma.

The enormous variety of pigmentation phenotypes in nature reflects a series of remarkable events that begin in the neural crest and end with the manufacture and distribution of pigment by mature melanocytes located in the epidermis and hair follicles. While the origins of melanoblasts from multipotent precursors in the neural crest is striking in itself, yet more so is the fact that these pioneer melanoblasts manage to undertake and survive their long migration, and in doing so proliferate and maintain their identity before ultimately arriving at their destination and undergoing differentiation. With the application of the powerful combination of genetics and molecular and cell biology the mystery surrounding the genesis of the melanocyte lineage is slowly being unravelled. At its heart is the powerful alliance between signal transduction and transcription that coordinates the program of gene expression that confers on a cell its identity, provides its passport for migration, and instructs it in the arts of survival and timely reproduction. The realization that the proliferation and migration of melanoblasts during development resembles closely the proliferation and metastasis of melanoma, a highly dangerous and increasingly common cancer, serves to highlight the value of the melanocyte system as a model for addressing key issues of general significance in both development and cancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the proliferation and differentiation of mouse epidermal melanocytes from pigmented spots induced by ultraviolet radiation B.

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB-induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor-free medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induced the proliferation and differentiation of melanocytes in those keratinocyte-depleted cultures. Moreover, an antibody to GM-CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV-irradiated mice, but not from control mice. Further, the GM-CSF antibody inhibited the proliferation and differentiation of melanocytes co-cultured with keratinocytes derived from UV-irradiated mice, but not from control mice. The quantity of GM-CSF secreted from keratinocytes derived from the pigmented spots of UV-irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM-CSF in keratinocytes derived from the pigmented spots of skin in UV-irradiated mice, but not from normal skin in control mice. These results suggest that GM-CSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB-induced pigmented spots.

Dct::lacZ ES cells: a novel cellular model to study melanocyte determination and differentiation.

Embryonic stem (ES) cells differentiate into various cell lineages in vitro. A procedure was previously designed to promote the differentiation of ES cells towards the melanocyte lineage and to obtain large and reproducible amounts of melanocytes. To elucidate the main events that lead to the development of melanocytes in vitro, we used transgenic Dct::lacZ mouse blastocysts to establish ES cell lines expressing the lacZ reporter gene under the control of the Dct promoter. Dct, a melanoblast marker, is expressed just after melanoblast determination in vivo. We evaluated the importance of recruitment, proliferation and differentiation during melanocyte ontogeny after the in vitro differentiation of Dct::lacZ ES cells into melanocytes. We showed that bFGF and cholera toxin induce precocious melanoblast determination, associated with early melanocyte differentiation. Edn3 induced melanoblast proliferation and long-term melanoblast recruitment, but not precocious determination. The lack of basic Fibroblast Growth Factor (bFGF) and cholera toxin can be partially compensated by Edn3. Thus, Dct::lacZ ES cells can be used as a model to study determination, proliferation and differentiation in the melanocyte lineage in vitro.

Hepatocyte growth factor controls the proliferation of cultured epidermal melanoblasts and melanocytes from newborn mice.

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with hepatocyte growth factor (HGF) from 14 days (keratinocyte depletion). The HGF increased the number of melanoblasts and melanocytes, but not the percentage of differentiated melanocytes in the melanoblast-melanocyte population in the absence of keratinocytes. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and/or G2/M phases of the cell cycle were increased by the treatment with HGF. Moreover, an anti-HGF antibody supplemented to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes. These results suggest that HGF is a keratinocyte-derived factor involved in regulating the proliferation of epidermal melanoblasts and melanocytes from newborn mice in cooperation with cAMP elevators and/or bFGF.

Steel factor controls the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture.

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were then further cultured with MDMD/MDMDF supplemented with steel factor (SLF) (keratinocyte depletion). SLF increased the number of melanoblasts and melanocytes as well as the proportion of differentiated melanocytes in the absence of keratinocytes. Flow cytometric analysis showed that melanoblasts and melanocytes in the S and G2/M phases of the cell cycle were increased by treatment with SLF. Moreover, an anti-SLF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes. These results suggest that SLF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.

IL-06 The overexpression of beta-catenin affects the proliferation and migration of melanoblasts

IL-06 The overexpression of beta-catenin affects the proliferation and migration of melanoblasts

Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture.

Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast/melanocyte-proliferation medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion). LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes. Moreover, anti-LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. These results suggest that LIF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.

Endothelins are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in serum-free primary culture.

Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free melanoblast-defined medium (MDM). After 14 d, almost all keratinocytes that existed predominantly in the early stage of primary culture died, and pure cultures of melanoblasts were obtained. Epidermal melanoblasts dramatically increased in number in MDMDF consisting of MDM supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). Epidermal melanocytes increased in number in MDMD consisting of MDM supplemented with DBcAMP. On the other hand, epidermal melanocytes were induced to differentiate in MDMM consisting of MDM supplemented with alpha-melanocyte-stimulating hormone (MSH). Pure cultured primary melanoblasts or melanocytes in MDMDF or MDMD were further cultured with MDMDF or MDMD supplemented with endothelin (ET)-1, -2, or -3 from 14 d. A dramatic increase in the number of melanoblasts or melanocytes was observed after 7 d; however, no increase in the number of melanoblasts or melanocytes was observed in the absence of ET-1, -2, or -3. The increase in the number of melanoblasts or melanocytes was comparable with that of melanoblasts or melanocytes cocultured with secondary keratinocytes in MDMDF or MDMD. Also, pure cultured primary melanoblasts in MDM were further cultured with MDMM supplemented with ET-1, -2, or -3 from 14 d. A dramatic increase in the percentage of melanocytes in the melanoblast-melanocyte population was observed after 7 d; however, no increase in the percentage of melanocytes was observed in the absence of ET-1, -2, or -3. The increase was comparable with that of melanocytes cocultured with secondary keratinocytes in MDMM. Moreover, anti-ET-1, -2, and -3 antibodies inhibited both the proliferation of melanoblasts or melanocytes in MDMDF or MDMD and the differentiation of melanocytes in MDMM in primary culture. These results suggest that ET-1, -2, and -3 are one member of the keratinocyte-derived factors that are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in primary culture.

Review: ligands for receptor tyrosine kinases expressed in the skin as environmental factors for melanocyte development.

Skin is the major tissue where melanocytes develop, and skin keratinocytes provide the necessary micro-environment for melanocyte survival, proliferation, differentiation, and migration. In this paper, we will discuss the ligands for receptor tyrosine kinases produced as environmental cues to support melanocyte development in the skin.

Agouti signaling protein and other factors modulating differentiation and proliferation of immortal melanoblasts.

The melanocyte lineage potentially forms an attractive model system for studies in cell differentiation, developmental genetics, cell signaling, and melanoma, because differentiated cells produce the visible pigment melanin. Immortal lines of murine melanoblasts (melanocyte precursors) have been described previously, but induction of differentiation involved a complex culture system with keratinocyte feeder cells. Here we describe conditions for both growth and induced differentiation of the melanoblast line melb-a, without feeder cells, and analyze factors that directly control proliferation and differentiation of these pure melanoblasts. Several active factors are products of developmental and other coat color genes, including stem cell factor (SCF), melanocyte-stimulating hormone (alphaMSH), and agouti signaling protein (ASP), a natural antagonist at the MSH receptor (melanocortin 1 receptor, MC1R) encoded by the agouti gene. A stable analog of alphaMSH (NDP-MSH) stimulated differentiation and inhibited growth. ASP in excess inhibited both effects of NDP-MSH, that is, ASP could inhibit pigmentation and stimulate growth. These effects provide an explanation for the interactions in mice of melanocyte developmental mutations with yellow agouti and Mc1r alleles, and a role for embryonic expression patterns of ASP.

MGF (KIT Ligand) Is a Chemokinetic Factor for Melanoblast Migration into Hair Follicles

Melanoblasts, the precursors of the pigment-producing cells of the skin and hair, are derived from the neural crest and migrate to the skin around 12 days of gestation in the mouse. In adult mice almost all the melanoblasts are confined to the hair follicles except for the epidermal layers of nonhairy skin. The receptor tyrosine kinase, KIT, is necessary for the survival, proliferation, and migration of melanoblasts. We have utilised an organ culture for embryonic skin taken from Dct-lacZ transgenic mice. The early patterning of the follicles and developing skin layers is retained within the cultures and the lacZ reporter allows visualisation of the melanoblasts within their native tissue environment. Soon after initiation of hair follicle development, melanoblasts localise in the follicles. Inhibition of follicle formation demonstrates that this localisation is an active process; in the absence of follicles, the melanoblasts proliferate but remain associated with the basement membrane. Implantation of beads releasing MGF, the ligand of KIT, does not result in melanoblast migration towards the bead, rather their localisation to the follicles is accelerated. Addition of soluble MGF induces the same effect; KIT therefore promotes melanocyte movement and acts as a chemokinetic, or motogenic, receptor. The melanoblasts must be guided to their correct location by other chemotactic signals or move at random and locate by ceasing movement when the follicle is engaged.

Mitf from neural crest to melanoma: signal transduction and transcription in the melanocyte lineage

Perhaps the most obvious manifestation of the complex interplay between environmental cues, signal transduction pathways, and transcription factors underlying the genesis and differentiation of a cell lineage is provided by the enormous diversity of genetically determined pigmentation patterns. Because melanocytes, the cells responsible for skin, hair, and eye color, are dispensable for viability, the highly visible consequences of mutations affecting the melanocyte lineage (Fig. 1) has allowed over 90 independent genes to be implicated in the genesis of mouse coat color (Mouse Genome Informatics: http:// www.informatics.jax.org/). The 30 or so of these genes that have been cloned to date (http://www.cbc.umn. edu/ifpcs/micemut.htm) not only encode proteins required for the manufacture of the pigment melanin and the function of the melanosome, but also encode signaling molecules and transcription factors that play critical roles in the development of the melanocyte lineage. This unusually large genetic resource which extends to a wide variety of species has made the regulation of melanocyte development an attractive system for understanding how the coordination of gene expression required for the commitment and differentiation of a specific cell lineage is achieved. Melanocytes originate as nonpigmented precursors termed melanoblasts in the mouse neural crest at around embryonic day 10.5 (E10.5), and following migration and proliferation reach the limb buds by E12 and enter the epidermis at the level of the lateral trunk by E13/E14 (Mayer 1973). Melanocytes are found as mature pigment cells in the skin and hair follicles, as well as a range of other sites where their role as pigment-producing cells is less understood. Thus, melanocytes are present in the choroid layer of the eye, the Harderian gland, the anal canal, and in the stria vascularis of the inner ear where they are crucial for hearing because they play a vital role in the endolymph-controlled generation of action potentials (Steel and Barkway 1989; Tachibana 1999). The effects of mutations affecting genes playing a key role in the melanocyte lineage may therefore be manifest at several levels: Mutations may affect genes required for differentiation-specific functions such as the genesis of pigment, or more interestingly from the developmental perspective, any step from the specification and commitment to the melanocyte lineage, to the survival, proliferation, and migration of melanoblasts from the neural crest. Interest in the melanocyte system is also afforded by the impact on melanocytes of ultraviolet (UV) light. In humans, epidermal melanocytes respond to UV irradiation by increasing the synthesis of melanin in membrane-bound structures termed melanosomes that are transferred to surrounding keratinocytes, a process known as the tanning response. At least one role for melanin is likely to be in protection against UV-induced DNA damage (Hill 1992). The incidence of UV-induced DNA damage, however, correlates with the risk of transformation of the melanocyte to a malignant melanoma, an aggressive and increasingly common form of cancer for which a proportion of the population have a genetic predisposition. The difficult path towards understanding the molecular and genetic basis of the progression from a melanocyte to a malignant melanoma has been made easier with recent observations that link the genesis of melanoma to deregulation of pathways operating in melanocyte development. The aim of this review is to provide an overview of how gene expression in neural crest-derived melanocytes is controlled by the interplay between specific transcription factors and signal transduction pathways. Because of limitations of space, the review focuses on the regulation of the expression and function of the Microphthalmia-associated transcription factor, Mitf. For the same reason, the development of the retinal pigment epithelium (RPE), the pigmented layer of cells lying immediately behind the retina which arises via differentiation of the posterior part of the optic cup, is not discussed.

Genetic and Epigenetic Control of the Proliferation and Differentiation of Mouse Epidermal Melanocytes in Culture

Serum-free culture of epidermal cell suspensions from neonatal skin of mice of strain C57BL/10JHir (B10) showed that alpha-melanocyte-stimulating hormone (alpha-MSH) was involved in regulating the differentiation of melanocytes by inducing tyrosinase activity, melanosome formation, and dendritogenesis. Dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) similarly induced the differentiation of melanocytes. On the other hand, DBcAMP induced the proliferation of epidermal melanocytes in culture in the presence of keratinocytes. Basic fibroblast growth factor (bFGF) was also shown to stimulate the sustained proliferation of undifferentiated melanoblasts in the presence of DBcAMP and keratinocytes. These results suggest that the proliferation and differentiation of mouse epidermal melanoblasts and melanocytes in culture are regulated by the three factors; namely, cAMP, bFGF, and keratinocyte-derived factors. Moreover, serum-free primary culture of mouse epidermal melanocytes derived from B10 congenic mice, which carry various coat color genes, showed that the coat color genes were involved in regulating the proliferation and differentiation of mouse epidermal melanocytes by controlling the proliferative rate, melanosome formation and maturation, and melanosome distribution.

STAT Protein Recruitment and Activation in c-Kit Deletion Mutants

Stem cell factor (SCF) and its tyrosine kinase receptor, c-Kit, play a crucial role in regulating migration and proliferation of melanoblasts, germ cells, and hemopoietic cell progenitors by activating a number of intracellular signaling molecules. Here we report that SCF stimulation of myeloid cells or fibroblasts ectopically expressing c-Kit induces physical association with and tyrosine phosphorylation of three signal transducers and activators of transcription (STATs) as follows: STAT1alpha, STAT5A, and STAT5B. Other STAT proteins are not recruited upon SCF stimulation. Recruitment of STATs leads to their dimerization, nuclear translocation, and binding to specific promoter-responsive elements. Whereas STAT1alpha, possibly in the form of homodimers, binds to the sis-inducible DNA element, STAT5 proteins, either as STAT5A/STAT5B or STAT5/STAT1alpha heterodimers, bind to the prolactin-inducible element of the beta-casein promoter. The tyrosine kinase activity of Kit appears essential for STAT activation since a kinase-defective mutant lacking a kinase insert domain was inactive in STAT signaling. However, another mutant that lacked the carboxyl-terminal region retained STAT1alpha activation and nuclear translocation but was unable to fully activate STAT5 proteins, although it mediated their transient phosphorylation. These results indicate that different intracellular domains of c-Kit are involved in activation of the various STAT proteins.

Endothelin signalling in the development of neural crest-derived melanocytes

In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest-derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early Dct+ cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+ melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-alpha-methylleucyl-D -1-methoxycarbonyltryptophanyl-D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+ melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation.