Biological effects of low-dose ionizing radiation exposure on interventional cardiologists.

BACKGROUND: Interventional cardiologists (ICs) are likely to receive high radiation exposure as a result of procedures they undertake. AIMS: To assess the effects of low-dose X-ray radiation exposure on chromosomal damage and on selected indices of cellular and humoral immunity in ICs. METHODS: The study population consisted of 37 ICs and 37 clinical physicians as the control group with similar age, sex and duration of employment, without any work-related exposure to ionizing radiation. Cytogenetic studies were performed by chromosome aberration analysis and immunological studies by flow cytometry, enzyme-linked immunosorbent assay and immunodiffusion techniques. RESULTS: The frequencies of aberrant cells, chromosome breaks and dicentrics plus centric rings were significantly higher in the exposed group compared to the control group (P < 0.05; P < 0.01; P < 0.001, respectively), without positive correlation between the frequency of dicentric and centric ring aberrations and the cumulative doses of the ICs (r = 0.24, not significant). A significant increase was observed in the expression of activation marker CD69 on TCD4(+) stimulated cells in serum immunoglobulin G and interleukin (IL)-2 (P < 0.05) and a significant decrease in serum IL-10 (P < 0.05) in the ICs compared with that of the control group. There was no statistical difference between the two groups in terms of number of white blood cells and lymphocytes, CD3(+), CD4(+) and CD8(+) T cells, CD19(+) and CD16(+) 56(+) cells and concentrations of interferon (IFN)-gamma, IL-4, IL-6 and IL-8 cytokines. CONCLUSIONS: While cytogenetic results show higher chromosomal damage, some immune responses are stimulated or modulated immunologically in ICs.

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.

Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis.

Using the epidermis-specific cytokeratin 14 promoter to deliver HGF exclusively from epidermal keratinocytes, we have examined the potential of hepatocyte growth factor (HGF) secreted from the normal environment to control morphogenesis. The transgenic mice displayed a significant increase of the number of melanocytes and their precursors in embryos starting not later than 16.5 dpc, and then after birth an explosive increase of dermal melanocytes started within 1 week, and these melanocytes were maintained throughout the entire life of the mice. Thus, HGF acts as a paracrine agent to promote survival, proliferation and differentiation of melanocyte precursors in vivo, and eventually causes melanocytosis. Loss of E-cadherin expression in dermal melanocyte precursors suggests that HGF caused dermal localization of melanocytes and their precursors by down-regulation of E-cadherin molecules.

ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture.

It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro. Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes. In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions. Their potencies were compared with the potency of alpha-MSH. After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies. By 14 days, cultures contained mostly pigmented melanocytes. The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39). The length of their peptide chains was inversely proportional to the potency. On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes. In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM). These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely. The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.

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.

The proliferation and differentiation of neonatal epidermal melanocytes in F1 hairless mice of HR-1 x HR/De in serum-free culture.

To investigate the characteristics of the proliferation and differentiation of epidermal melanocytes in F1 hairless mice of HR-1 x HR/De parents in vitro, cell suspensions of the neonatal epidermis were cultured in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). The differentiation of melanocytes was induced by treatment with DBcAMP. In contrast, the sustained proliferation of melanoblasts was induced by combined treatment with DBcAMP and bFGF. The melanoblasts could be subcultured in serum-free medium supplemented with the two factors in the presence of keratinocytes, but not in their absence. This is the first report of successful culture of melanoblasts and melanocytes from hairless mice; it is expected to be useful in understanding the mechanism of the development of pigmented spots in the epidermis of (HR-1 x HR/De)F1 mice, which are reported to be induced by repeated exposure to ultraviolet light B.

Effects of low-dose X-irradiation on the development of the mouse cerebellar cortex.

We labeled proliferating cells of the cerebellum of 6-day-old mice with 5-bromo-2'-deoxyuridine (BrdU) followed by a single exposure to 0.5, 1 or 2 Gy of X-rays. We then studied the effects of low-dose irradiation on the migration and survival of granule neurons in the mouse cerebellum. The animals were killed at 4 days, or at 2, 4 or 6 weeks after irradiation. Brains were fixed and BrdU-labeled cells in the cerebella were immunohistochemically analyzed. BrdU was predominantly distributed in the superficial layer of the external granular layer soon after injection. Four days after irradiation with 0.5 or 1 Gy, labeled cells were mainly seen in the inner granular layer, which was also the case in non-irradiated mice. However, following 2 Gy irradiation BrdU was found not only in the inner granular layer, but also in the Purkinje cell layer. This distribution was also seen at 2 and 4-6 weeks after irradiation. In animals irradiated with 1 Gy 4-6 weeks after irradiation, the proportion of labeled cells present in the inner granular layer decreased, while labeled cells in the Purkinje cell layer increased. On the other hand, 0.5 Gy irradiation did not change the distribution of labeled cells, except that the proportion of labeled cells in the inner granular layer decreased at 2 weeks after irradiation. The number of labeled cells in the cerebellar cortex per unit area decreased with time and dose. These results suggest that 2 Gy irradiation induces a migratory delay, abnormal distribution, and cell death of the granule neurons of the mouse cerebellum.

Effects of genic substitution at the agouti, brown, albino, dilute, and pink-eyed dilution loci on the proliferation and differentiation of mouse epidermal melanocytes in serum-free culture.

To examine the effects of coat-color genes on the proliferation and differentiation of mouse epidermal melanocytes, we cultured epidermal, cell suspensions derived from neonatal skins of C57BL/10JHir (black) and its congenic mice carrying agouti, brown, albino, dilute, and pink-eyed dilution genes in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate. The proliferative rates of agouti, brown and dilute black melanocytes were similar to that of black melanocytes, while those of albino and pink-eyed black melanocytes were about one-half of that of black melanocytes. The morphology of albino and pink-eyed black melanocytes, though nonpigmented, was similar to black melanocytes; namely, dendritic, polygonal or epithelioid. Dilute black melanocytes also possessed the similar morphology, whereas their melanosomes were accumulated in the perinuclear region. Dopa-melanin depositions after dopa reaction in brown and dilute black melanocytes were greater than in black and agouti melanocytes. Although dopa-melanin depositions were not observed in albino melanocytes, about 8% of pink-eyed black melanocytes were positive to dopa reaction. Silver depositions after combined dopa-premelanin reaction in agouti, brown and dilute black melanocytes were similar to that in black melanocytes. Although albino melanocytes were devoid of silver depositions, about 25% of pink-eyed black melanocytes were positive to the reaction. Pyrrole-2,3,5-tricarboxylic acid (PTCA, degradation product of eumelanin) contents in agouti and dilute black melanocytes were slightly lower than in black melanocytes, while that in brown melanocytes was reduced to one-third. In contrast, PTCA contents in albino and pink-eyed black melanocytes were reduced to less than 0.5%. Aminohydroxyphenylalanine (AHP, degradation product of pheomelanin) contents did not differ among these melanocytes. These results suggest that the coat-color genes exert their influences on the proliferation and differentiation of mouse epidermal melanocytes by affecting tyrosinase activity, melanosome maturation and transport, and eumelanin synthesis.

Effects of continuous low-dose prenatal irradiation on neuronal migration in mouse cerebral cortex.

We investigated the effects of continuous exposure to gamma-rays during corticogenesis on the migration of neuronal cells in developing cerebral cortex. Pregnant mice were injected with 0.5 mg of bromodeoxyuridine (BrdU) on day 14 of gestation to label cells in the S phase. The mice were then exposed to 137Cs gamma-rays (dose rates of 0.1, 0.3, and 0.94 Gy/day) continuously for 3 days. Brains from 17-day-old embryos and from offspring at 3 and 8 weeks after birth were processed immunohistochemically to track the movements of BrdU-labeled cells. Comparative analyses of the distribution pattern of BrdU-labeled cells in the cerebral cortex revealed that (1) the migration of neurons was delayed during the embryonic period in mice irradiated at 0.94 Gy/day, (2) in 3-week-old mice, there was a significant difference in the distribution pattern of BrdU-labeled cells in the cerebral cortex between the mice irradiated prenatally and control, and (3) in 8-week-old mice, there were no differences in the distribution pattern of BrdU-labeled cells between control and animals irradiated with 0.1 and 0.3 Gy/day. In contrast, in the animals irradiated with 0.94 Gy/day, the significant difference in the distribution pattern of the labeled cells relative to control was maintained. These results suggest that the migration of neuronal cells in mouse cerebral cortex is disturbed by continuous prenatal irradiation at low-dose and some modificational process occurred during the postnatal period.

Short- and long-term effects of low-dose prenatal X-irradiation in mouse cerebral cortex, with special reference to neuronal migration.

To elucidate the short- and long-term effects of ionizing radiation on cell migration in the developing cerebral cortex, we labeled proliferating cells on day 14 of gestation of mice with bromodeoxyuridine (BrdU) followed by a single exposure to 0.1-1 Gy of X-rays. The brains of embryos on day 17 and offspring at 2, 3 and 8 weeks after birth were processed for BrdU immunohistochemistry to trace the movements of BrdU-labeled cells. The location of BrdU-labeled neurons in the cerebral cortex was quantitatively analyzed between irradiated animals and non-irradiated controls. We have demonstrated that the initial migration of BrdU-labeled cells from the matrix cell zone towards the cortical plate during embryonic periods was decelerated when exposed to X-rays of 0.25, 0.5 and 1 Gy on embryonic day 14, and that aberrantly placed neurons in the cerebral neocortex were noted in younger animals that were irradiated prenatally, whereas such derangement was less pronounced in mature animals. These observations suggest that some modification process might have occurred during the postnatal period.

Hydrocortisone is involved in regulating the proliferation and differentiation of mouse epidermal melanoblasts in serum-free culture in the presence of keratinocytes.

Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free medium (MPM) supplemented with dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). After 12 days, almost all keratinocytes died, and pure and enriched cultures of melanoblasts (approximately 90%) and melanocytes (approximately 10%) could be obtained. In order to clarify the role of hydrocortisone (HC) which is thought to be important for the regulation of melanocyte proliferation and differentiation, the hormone was added to MPM from the initiation of primary culture. The proliferation of melanoblasts was inhibited by HC in a dose-dependent manner. Instead, most of the proliferating melanoblasts were induced to differentiate into melanocytes by HC in a dose-dependent manner. A small number of pure melanoblasts derived from primary cultures at 12 days with MPM depleted of DBcAMP and bFGF were cultured with MPM with or without HC in the presence of secondary keratinocytes that were subcultured from a pure population of primary keratinocytes in a serum-free medium. The inhibition of the proliferation of melanoblasts by HC as well as the stimulation of the differentiation of melanoblasts into melanocytes by HC was observed in the presence of keratinocytes, but not in the absence of keratinocytes. Conditioned media or extracts prepared from pure keratinocytes in serum-free primary culture failed to replace the role of keratinocytes. These results suggest that HC plays an important role in the regulation of the proliferation and differentiation of mouse epidermal melanoblasts in culture in cooperation with factors supplied by keratinocytes.

Chemical characterization of hair melanins in various coat-color mutants of mice.

Mammalian melanins exist in two chemically distinct forms: the brown to black eumelanins and the yellow to reddish pheomelanins. Melanogenesis is influenced by a number of genes, the levels of whose products determine the quantity and quality of the melanins produced. To examine the effects of various coat-color genes on the chemical properties of melanins synthesized in the follicular melanocytes of mice, we have introduced new methods to solubilize differentially pheomelanins and brown-type eumelanins. We applied these and previously developed high-performance liquid chromatography and spectrophotometric methods for assaying eu- and pheomelanins to characterize melanins in various mutant mice: black, lethal yellow, viable yellow, agouti, brown, light, albino, dilute, recessive yellow, pink-eyed dilution, slaty, and silver. It was demonstrated that 1) complete solubilization of melanins in Soluene-350 is a convenient method to estimate the total amount of eu- and pheomelanins, 2) lethal yellow, viable yellow, and recessive yellow hairs contain almost pure pheomelanins, and 3) melanins from brown, light, silver, and pink-eyed black hairs share chemical properties in common that are characterized by partial solubility in strong alkali. We suggest that 1) the brown-type eumelanins have lower degrees of polymerization than the black-type eumelanins, and 2) slaty hair melanin contains a greatly reduced ratio of 5,6-dihydroxyindole-2-carboxylic acid-derived units as compared with black and other eumelanic hair melanins. These results indicate that our methodology, high-performance liquid chromatography and spectrophotometric methods combined, may be useful in chemically characterizing melanin pigments produced in follicular melanocytes.

Structure and function of melanocytes: microscopic morphology and cell biology of mouse melanocytes in the epidermis and hair follicle.

Melanocytes characterized by their tyrosinase activity, melanosomes and dendrites locate in the basal layer of epidermis and hair bulb in the skin of mice. Melanocytes differentiate from undifferentiated melanoblasts derived from embryonic neural crest. Melanocyte-stimulating hormone plays an important role in the regulation of the differentiation of mouse melanocytes in the epidermis and hair bulb by inducing tyrosinase activity, melanosome formation, transfer of melanosomes and increased dendritogenesis. The proliferative activity of differentiating epidermal melanocytes of newborn mice during the healing of skin wounds is regulated by semidominant genes, suggesting that the genes are involved in regulating the proliferative activity of epidermal melanocytes during differentiation. The morphology and differentiated functions of mouse melanocytes are shown to be influenced by environmental factors such as ultraviolet and ionizing radiations. From the results of serum-free culture of mouse epidermal melanoblasts, basic fibroblast growth factor is shown to stimulate the sustained proliferation of melanoblasts in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate and keratinocytes. In contrast, melanocyte differentiation in serum-free culture is induced by melanocyte-stimulating hormone in the presence of keratinocytes. These results suggest that the structure and function of mouse melanocytes in the epidermis and hair bulb are controlled by both genetic factors and local tissue environment, such as hormones and growth factors.

Effects of gamma-irradiation on the yield of mid-ventral white spots in mice in different genetic backgrounds and at different times during development.

Pregnant females C57BL/10JHir-p/p mice crossed with C57BL/10JHir males were whole-body irradiated with a single acute dose of 60Co-gamma-rays to investigate the effect of gamma-radiation on embryonic melanoblasts. The effect was studied by scoring changes in the cutaneous coats of F1 offspring 25 days after birth. White spots were found in mid-ventrum of the animals. Melanoblasts and melanocytes were not observed in the spotted skin. The frequency of the spots increased in a dose-dependent manner. White spots were found in mid-ventrum of (C57BL/6J female x C3H/HeJmsHir male) F1 exposed to gamma-rays. However, the frequency of the spots in (C57BL/6J female x C3H/HeJmsHir male) F1 were extremely lower than that in (C57BL/10JHir-p/p female x C57BL/10JHir male) F1, suggesting the possibility that the frequency of mid-ventral white spots are genetically controlled. Moreover, the highest frequency was found in (C57BL/10JHir-p/p female x C57BL/10JHir male) F1 irradiated at 8.5 days of gestation. This stage corresponds to the stage of initiation of neural-crest cell migration. These results indicate that gamma-radiation affects the differentiation of melanocytes in the skin both with genetical control and with greater effects seen at the stage of initiation of neural-crest cell migration.

Effects of activators (SC-9 and OAG) and inhibitors (staurosporine and H-7) of protein kinase C on the proliferation of mouse epidermal melanoblasts in serum-free culture.

Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast proliferation medium containing dibutyryl adenosine 3':5'-cyclic monophosphate and basic fibroblast growth factor. After 12 days, almost all of the keratinocytes died and pure cultures of melanoblasts (approximately 80%) and melanocytes (approximately 20%) could be obtained. No further proliferation of melanoblasts was observed in the melanoblast proliferation medium. In order to clarify the role of protein kinase C, which is important for the regulation of cellular proliferation, activators or inhibitors of protein kinase C were added to the culture of the quiescent melanoblasts at 12 days. The proliferation of melanoblasts was induced by an activator of protein kinase C, N-(6-phenylhexyl)-5-chloro-1-naphthalene-sulfonamide or 1-oleoyl-2-acetyl-glycerol. It was also induced by an inhibitor of protein kinase C, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine. However, the melanoblasts failed to proliferate in the melanoblast proliferation medium supplemented with both the activator and the inhibitor. These results suggest that the proliferation of mouse epidermal melanoblasts in culture is regulated by activating or inhibiting the activity of protein kinase C.

Keratinocytes are involved in regulating the developmental changes in the proliferative activity of mouse epidermal melanoblasts in serum-free culture.

When epidermal cell suspensions derived from 0.5-, 2.5-, and 4.5-day-old mice were plated onto uncoated polystyrene dishes and cultured with serum-free medium supplemented with dibutyryl cyclic adenosine 3',5'-monophosphate and basic fibroblast growth factor, melanoblasts proliferated dramatically around keratinocyte colonies and after 12-14 days pure and enriched cultures of melanoblasts (ca. 75%) and melanocytes (ca. 25%) were obtained. In contrast, when epidermal cell suspensions derived from 7.5-, 20.5-, and 60.5-day-old mice were cultured similarly, keratinocytes failed to attach to the dish and melanoblasts did not proliferate at all. However, when epidermal cell suspensions of older mice were plated onto type I collagen-coated dishes and cultured similarly, keratinocytes attached well to the dish and melanoblasts proliferated dramatically around keratinocyte colonies. Moreover, pure melanoblasts and melanocytes derived from primary cultures of young and old mice could be subcultured on collagen-coated dishes with the medium in the presence of secondary keratinocytes that were subcultured from a pure population of primary keratinocytes. No differences were observed in the proliferative activity of secondary melanoblasts between young and old mice. These results suggest that keratinocytes are involved in regulating the proliferation of mouse epidermal melanoblasts and that the developmental changes in the proliferative activity of epidermal melanoblasts in culture are due to the developmental changes in the substrate attachment and proliferation of keratinocytes, rather than to intrinsic changes in melanoblasts.

Melanocyte stimulating hormone induces the differentiation of mouse epidermal melanocytes in serum-free culture.

In serum-free primary culture of dissociated mouse epidermal cells, alpha-melanocyte stimulating hormone (alpha-MSH) and dibutyryl cyclic AMP (DBcAMP) induced the differentiation of melanocytes. Moreover, the proliferation of melanocytes was also induced in the dishes cultured with DBcAMP, but not with alpha-MSH. In order to clarify the role of keratinocytes in melanocyte proliferation and differentiation, pure cultures of keratinocytes were established in serum-free medium. Subconfluent primary keratinocytes were trypsinized and seeded into pure primary melanoblasts cultured with serum-free medium that did not contain alpha-MSH or DBcAMP. Melanoblasts were cultured with alpha-MSH or DBcAMP in the presence or absence of keratinocytes. alpha-MSH failed to induce melanocyte differentiation in the absence of keratinocytes. DBcAMP failed to induce melanocyte proliferation in the absence of keratinocytes, although it induced melanocyte differentiation even in the absence of keratinocytes. These results suggest that keratinocyte-derived factors are required not only for the induction of melanocyte differentiation by alpha-MSH but also for the induction of melanocyte proliferation by DBcAMP.

Control of melanocyte proliferation and differentiation in the mouse epidermis.

Melanocyte-stimulating hormone plays an important role in the regulation of melanocyte differentiation in the mouse epidermis by inducing tyrosinase activity, melanosome formation, translocation of melanosomes, and increased dendritogenesis. The proliferative activity of differentiating epidermal melanocytes of newborn mice during the healing of skin wounds is regulated by semi-dominant genes, suggesting that the genes are involved in regulating the proliferative activity of epidermal melanocytes during differentiation. From the results of serum-free culture of epidermal cell suspensions from neonatal mouse skin, basic fibroblast growth factor is shown to stimulate the sustained proliferation of melanoblasts in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate and keratinocyte-derived factors. Moreover, each step of melanocyte differentiation is controlled by numerous coat color genes. These genes control melanocyte differentiation by regulating the differentiation of neural crest cells into melanoblasts in embryonic skin, or by regulating the differentiation of neural crest cells into melanoblasts in embryonic skin, or by regulating transcription and/or translation of the tyrosinase gene in the differentiating melanocytes. These results suggest that melanocyte proliferation and differentiation in the mouse epidermis are controlled by both genetic factors and local tissue environment.

Basic fibroblast growth factor stimulates the sustained proliferation of mouse epidermal melanoblasts in a serum-free medium in the presence of dibutyryl cyclic AMP and keratinocytes.

Basic fibroblast growth factor (bFGF) stimulated the sustained proliferation of mouse epidermal melanoblasts derived from epidermal cell suspensions in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). The melanoblasts could be subcultured in the serum-free medium supplemented with the two factors in the presence of keratinocytes, but not in the absence of keratinocytes. In these conditions, some melanoblasts proliferated without differentiating for more than 20 days including a subculture. This is the first report of a successful culture of melanoblasts from mammalian skin. This culture system is expected to clarify further markers for melanoblasts and requirements for their proliferation and differentiation.

Developmental interactions in the pigmentary system of the tip of the mouse tail: effects of coat-color genes on the expression of a tail-spotting gene.

The tails of agouti C3H/HeJmsHir mice are completely pigmented, whereas the tails of black C57BL/10JHir animals possess unpigmented tips. Genetic analysis indicates that white tail-tipping is due to an autosomal recessive gene, with incomplete penetrance, that segregates independently from the gene for agouti with a maternal influence in the F1 generation. To analyze the influence of specific coat-color genes on the expression of tail-spotting in mice, five congenic lines of C57BL/10JHir with different coat colors were prepared. No influence was observed on the occurrence of tail-spotting in agouti (A/A) or dilute (d/d) mice or in F1 mice from crosses between black and albino (c/c), or in F1 mice from crosses between black and pink-eyed dilution (p/p). However, the frequency of tail-spotting was dramatically decreased in brown (b/b) mice. These results suggest that the mutant allele (b) at the brown locus is involved in determining the extent of pigmented areas in the tail tips of mice through an interaction with the tail-spotting gene.