New mechanism of melanosomes transport into hair of lambs of different breeds and genotypes.

By light microscopic investigation of skin and wool specimens of newborn lambs, we discovered a previously unknown mechanism for melanosomes transport in the process of dermal papilla melanocytes regular mitosis and migration into the hair shaft. This mechanism plays a great role in hair pigmentation especially in dominant (ED/ED) and recessive (Aa/Aa) black lambs of all investigated breeds. The rate of pigment cell mitosis, proliferation, and migration differs greatly in lambs of investigated color genotypes. In black genotypes the rate of melanocyte mitosis is very high and is approximately the same as in the hair bulb matrix cells, whereas in brown and red genotypes this rate is much lower. Melanocyte mitosis in the light red and tan groups was not found.     

Presence of the Dominant Extension Allele ED in Red and Mosaic Cattle

The melanocyte-stimulating hormone receptor (MC1-R) is a central regulator of mammalian coat colour, encoded by the extension locus. In cattle, the dominant extension allele E^D is associated with the production of black pigment in coloured areas. Genotyping of the MC1-R gene in a bull with mosaic expression of red vs. black pigment verified the existence of the E^D allele, in spite of the fact that the majority of the animal is red coloured. No further mutations were found within the E^D variant of the MC1-R gene, which was inherited from a completely red mother (genotype E^D/e).     

Tyrosinase Activity (TH,DO, PAGE-Defined Isozymes) and Melanin Production in Regenerating Hairbulb Melanocytes of Lethal Yellow (Ay/a), Black (a/a), Agouti (AwJ/AwJ) and Albino (a/a/c2J/c2J) Mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in participate and soluble fractions of hairbulb melanocytes of lethal yellow (A^y/a C/C), nonagouti black (a/a C/C), and albino (a/a c^2J/c^2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, A^y/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (A^wJ/A^wJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and A^y/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and A^y/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in A^y/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

Inheritance of coat colour in the German Pinscher dog

The two primary colour varieties of the German Pinscher breed are the red and the black and tan. Respectively, these are produced by the dominant yellow allele A ^y and the black and tan allele a ^t of the agouti locus. The dilute gene d is present in the breed at a low frequency and produces the rarer isabella and blue and tan colours, with the genotypes A ^y -dd and a ^t dd, respectively.     

Fluorescence spectrophotometric analysis of melanins in the house mouse

We extracted the yellow melanin (phaeomelanin), black melanin (eumelanin), and mixed type of melanin from dorsal hair of dominant yellow (A ^y /a), non-agouti (a/a), and agouti (A/A) mice, respectively. Spectrophotometric and fluorescence spectrophotometric analysis demonstrated that the yellow melanin was qualitatively distinct from the black melanin and that the agouti hair contained both types of pigment.This work was supported by Grant 244004 from the Ministry of Education. Part of this work was presented at the X International Pigment Cell Conference.     

Inheritance of colour and coat in the Belgian Shepherd dog

The several colours and coats of the Belgian Shepherd dog are shown to be due primary to combinations of the following genes: dominant black (A ^s ), dominant yellow (A ^y ), chinchilla (ch), long hair (l) and wire hair (Wh). The gene for black and tan (a ^t ) is or has been present in the breed. All of the dominant yellow dogs exhibit a black facial mask and extensive suffusion of black guard hairs on the body.     

Agouti-Related Maturation and Tissue Distribution of oc-Melanocyte Stimulating Hormone in Wild-Type (AwJ/AwJ) and Mutant (Ay/a,a/a) Mice

Because of ectopic overproduction of agouti protein, yellow alleles (A^y and A^vy) of the murine agouti gene may secondarily modulate the synthesis, maturation (i.e., acetylation), and/or tissue deployment of α-Melanocyte Stimulating Hormone (MSH). We used HPLC to test the hypothesis that A^y/a mice exhibit altered concentrations of desacetyl-, monoacetyl-, and diacetyl-α-MSH in pituitaries, sera, and telogen hair bulbs when compared to black (a/a) mice. We also used RIA to measure total MSH in those same tissues of A^y/a, a/a, and white-bellied agouti (A^wJ/A^wJ) mice (Strain C57BL/6J). We found no evidence that A^y/a mice possessed an imbalance of des-, mono-, and diacetylated α-MSH species. However, radioimmunoassay (RIA) analyses of total MSH suggest that wild-type agouti mice (A^wJ/A^wJ) exhibited significantly decreased (P < 0.05) tissue levels of total α-MSH in pituitaries, sera, and regenerating hair bulbs when compared to those of mutant A^y/a and a/a mice.     

Tissue microenvironment and the genetic control of hair pigment patterns in mice

This study was conducted to assess microenvironmental variability within integumental tissue of genetically identical mice with respect to a specific cellular response: cyclic synthesis of yellow and black pigment by hair bulb melanocytes. Crosses were performed within and between inbred strains of mice that were isogenic with the exception of a single gene substitution at the agouti locus. Agouti locus genes included the A^vy, A^w, A, a^td, a^t, a^x, a^m, and a alleles. The pigment patterns of dorsal, flank, and ventral hairs of the first and third hair generations and of hairs growing in special integumentary areas such as the pinna, tail, and hind foot were studied. It was found that the amount of yellow pigment synthesized by hair bulb melanocytes within genetically identical mice is both agedependent and conditioned by the integumentary environment. Furthermore, the special integumentary regions produce hairs with a variety of pigment patterns in which the distribution and relative amounts of black and yellow pigments do not necessarily conform to dominance relationships expected among agouti locus alleles as judged by their effects on the pigmentation of the dorsal pelage. We conclude that within genetically uniform integumental tissues, microenvironmental differences occur and are reflected as alterations in the metabolic pattern of differentiated cells.     

A functional analysis of the crown architecture of tropical forest<Emphasis Type="Italic"> Psychotria</Emphasis> species: do species vary in light capture efficiency and consequently in carbon gain and growth?

The crown architectures of 11 Psychotria species native to Barro Colorado Island, Panama were reconstructed from field measurements of leaf and branch geometry with the three-dimensional simulation model Y-plant. The objective was to assess the role of species differences in architecture in light capture and carbon gain in their natural understory environment. When species were grouped according to their putative light environment preference, the shade tolerant species were found to have a small but significantly higher efficiency of light capture for both diffuse and direct light as compared to the light demanding species. Within each grouping, however, there were few significant differences in light capture efficiency among species. The lower efficiencies of light demanding species was due to slightly higher self-shading and slightly lower angular efficiencies. Simulations of whole plant assimilation showed that light demanding species had greater daily assimilation in both direct and diffuse light due to the significantly greater light availability in the sites where light demanding species were found, as compared to those where shade tolerant species occurred. Among light demanding species, the above ground relative growth rate measured over a 1-year period by applying allometric equations for mass versus linear dimensions, was positively correlated with diffuse PFD and with mean daily assimilation estimated from Y-plant. For the shade tolerant plants, there was no significant correlation between RGR and mean daily assimilation or with any measure of light availability, probably because they occurred over a much narrower range of light environments. Overall, the results reveal a strong convergence in light capture efficiencies among the Psychotria species at lower values than previously observed in understory plants using similar approaches. Constraints imposed by other crown functions such as hydraulics and biomechanical support may place upper limits on light capture efficiency.Abbreviations E_a Efficiency of light absorption (dimensionless) - E_adir Efficiency of direct light absorption (dimensionless) - E_adif Efficiency of diffuse light absorption (dimensionless) - D_E Display efficiency (dimensionless) - P_E Projection efficiency (dimensionless) - CosI Mean cosine of incidence (dimensionless) - aLAR_e Effective leaf area ratio (m² g^–1) - A_tot Daily assimilation (mmol m^–2 day^–1) - A_dir Daily assimilation in direct PFD (mmol m^–2 day^–1) - A_dif Daily assimilation in diffuse PFD (mmol m^–2 day^–1)     

A deletion spanning the promoter and first exon of the hair cycle-specific ASIP transcript isoform in black and tan rabbits

Black and tan animals have tan-coloured ventral body surfaces separated by sharp boundaries from black-coloured dorsal body surfaces. In the a^t mouse mutant, a retroviral 6 kb insertion located in the hair cycle-specific promoter of the murine Asip gene encoding agouti signalling protein causes the black and tan phenotype. In rabbits, three ASIP alleles are thought to exist, including an a^t allele causing a black and tan coat colour that closely resembles the mouse black and tan phenotype. The goal of our study was to identify the functional genetic variant causing the rabbit a^t allele. We performed a WGS-based comparative analysis of the ASIP gene in one black and tan and three wt agouti-coloured rabbits. The analysis identified 75 a^t-associated variants including an 11 kb deletion. The deletion is located in the region of the hair cycle-specific ASIP promoter and thus in a region homologous to the site of the retroviral insertion causing the a^t allele in mice. We observed perfect association of the genotypes at this deletion with the coat colour phenotype in 49 rabbits. The comparative analysis and the previous knowledge about the regulation of ASIP expression suggest that the 11 kb deletion is the most likely causative variant for the black and tan phenotype in rabbits.     

The role of melanocyte-stimulating hormone (MSH) receptor in bovine coat color determination

The melanocyte-stimulating hormone (MSH) receptor has a major function in the regulation of black (eumelanin) versus red (phaeomelanin) pigment synthesis within melanocytes. We report three alleles of the MSH-receptor gene found in cattle. A point mutation in the dominant allele E ^D gives black coat color, whereas a frameshift mutation, producing a prematurely terminated receptor, in homozygous e/e animals, produces red coat color. The wild-type allele E ⁺ produces a variety of colors, reflecting the possibilities for regulating the normal receptor. Microsatellite analysis, RFLP studies, and coat color information were used to localize the MSH-receptor to bovine Chromosome (Chr) 18.     

Temporal variation in coat colour (genotypes) supports major changes in the Nordic cattle population after Iron Age

Variation in coat colour genotypes of archaeological cattle samples from Finland was studied by sequencing 69 base pairs of the extension locus (melanocortin 1‐receptor, MC1R) targeting both a transition and a deletion defining the three main alleles, such as dominant black (E^D), wild type (E⁺) and recessive red (e). The 69‐bp MC1R sequence was successfully analysed from 23 ancient (1000–1800 AD) samples. All three main alleles and genotype combinations were detected with allele frequencies of 0.26, 0.17 and 0.57 for E^D, E⁺ and e respectively. Recessive red and dominant black alleles were detected in both sexes. According to the best of our knowledge, this is the first ancient DNA study defining all three main MC1R alleles. Observed MC1R alleles are in agreement with calculated phenotype frequencies from historical sources. The division of ancient Finnish cattle population into modern Finnish breeds with settled colours was dated to the 20th century. From the existing genotyped populations in Europe (43 breeds, n = 2360), the closest match to ancient MC1R genotype frequencies was with the Norwegian native multicoloured breeds. In combined published genotype data of ancient (n = 147) and genotypes and phenotypes of modern Nordic cattle (n = 738), MC1R allele frequencies showed temporal changes similar to neutral mitochondrial DNA and Y‐chromosomal haplotypes analysed earlier. All three markers indicate major change in genotypes in Nordic cattle from the Late Iron Age to the Medieval period followed by slower change through the historical periods until the present.     

Influence of Maternal Phenotype on Metabolic Differentiation of Agouti Locus Mutants in the Mouse

The results of extensive breeding experiments indicate that the phenotypic differentiation of embryos carrying the viable yellow, A ^vy, or mottled, a^m, mutations is influenced to a major extent by the agouti locus genotype and the strain genome of the dam. The A^vy/a and a^m/a genotypes are each expressed in a spectrum of coat color phenotypes. These can be grouped into two classes, mottled and pseudoagouti.—In a reciprocal cross of C57BL/6JNIcrWf and AM/Wf-a^m/a^m mice, 29.5% of the offspring of C57BL/6 dams were of the pseudoagouti phenotype, whereas no pseudoagouti offspring were produced by AM strain dams.—Mottled yellow A^vy/a mice become obese and tumor formation is enhanced in these mice in comparison with the lean pseudoagouti A^vy/a siblings.—In two different reciprocal crosses using four different inbred strains, the proportion of pseudoagouti A^vy/a offspring differed according to the strain of the dam. Regardless of strain, mottled yellow A ^vy/a dams produced significantly fewer pseudoagouti A ^vy/a offspring than did black a/a dams.—The data suggest that metabolic differentiation of A^vy/a zygotes into phenotypic classes with different susceptibilities to obesity and tumor formation is influenced to a considerable degree by the metabolic characteristics of the oviductal and uterine environment of the dam.     

Agouti Alleles Influence Thiol Concentrations in Hair Follicles and Extrafollicular Tissues of Mice (Ay/a,AwJ/AwJ,a/a)

Agouti protein (AP) expression in the wild-type agouti mouse (A^wJ/A^wJ) coincides with a switch in hair follicle melanogenesis from black (eumelanin) to yellow (pheomelanin). Ectopic overexpression of AP in the lethal yellow (A^y/a) mouse cause a pure yellow coat and the lethal yellow syndrome. Thiol concentrations may control the conversion of dopaquinone to pheomelanin in hair follicle melanocytes. Glutathione (GSH) also plays important roles in cellular health and protection. Using HPLC, cysteine and GSH were measured in 1) hair follicles, liver and serum of A^y/a, A^wJ/A^wJ, and a/a (black) mice, and 2) adipose and spleen tissues of A^y/a and a/a mice on day 9 of regenerating hair growth (late pheomelanin phase). Agouti locus alleles influence thiol metabolism in hair follicles and in other systemic tissues. A^y/a hair follicles and serum showed highest cysteine and lowest GSH levels. A^wJ/A^wJ mice showed intermediate levels, while a/a hair follicles and serum had lowest cysteine and highest GSH concentrations. In the hair follicle, cysteine (likely derived from enzymatic degradation of GSH) appears to be the primary pheomelanogenic thiol. Agouti locus alleles may also directly or indirectly affect thiol concentrations in systemic tissues like liver and spleen. Cysteine in spleen extracts showed A^y/a > a/a (P > 0.01). An A^y-induced imbalance of thiol metabolism (altering GSH concentrations in multiple tissues) may contribute to the pleiotropic defects of the lethal yellow syndrome.     

Morphology of Hair Pigmentation in Wild Red Foxes, Silver Foxes, and Their Hybrids

The effects of dominant allele A ^r of locus Agoution the morphology of hair pigmentation were described in foxes. The A ^r allele was shown to determine the type of melanin and its content in hair with no effect on the morphology of pigment granules and their distribution throughout a hair. Using the method of electron spin resonance (ESR), the types of melanin (eumelanin and pheomelanin) and their content in the hair of red (A ^r A ^r EE) and silver (aaEE) foxes and their hybrids (A ^r aEE) were determined. In silver foxes, only one type of melanin (eumelanin) was found. In red foxes and their hybrids (which are phenotypically similar but darker than red foxes), both types of melanin (eu- and pheomelanin) were found. The highest melanin content was detected in the coat of silver foxes. In the hybrids, the total melanin content was lower than in silver foxes, but significantly higher than in red foxes. In red foxes, the contribution of pheomelanin to the total hair melanin content was twice as large as in the hybrids.     

Generation and suppression of singlet oxygen in hair by photosensitization of melanin

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen (¹O₂). Irradiation of hair shafts (λ_ex > 400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by ¹H NMR. After 532-nm excitation, all hair shafts presented the characteristic ¹O₂ emission (λ_em = 1270 nm), whose intensity varied inversely with the melanin content. ¹O₂ lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a ¹O₂ suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for ¹O₂ in the solvents in which the hair shafts were suspended, indicating that ¹O₂ is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress ¹O₂, with similar efficiencies. The higher amount of ¹O₂ generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of ¹O₂ in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin.     

Inheritance of coat colour in the Anatolian Shepherd dog

The predominant colour of the Anatolian Shepherd dog varies from a dark fawn to light red, with a variable black muzzle and face (mask). Evidence is presented that the colour is due to the dominant yellow allele (A ^y) of the agouti locus. Two other frequent colours are white spotting, due to the piebald allele (s ^p), and the chinchilla allele (ch). Two rarer colours are the agouti wolf-grey wild type (A ⁺) and a light fawn with a blue facial mask, due to the dilution allele (d).     

Mutations in the agouti (ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus)

Coat color genetics, when successfully adapted and applied to different mammalian species, provides a good demonstration of the powerful concept of comparative genetics. Using cross-species techniques, we have cloned, sequenced, and characterized equine melanocortin-1-receptor (MC1R) and agouti-signaling-protein (ASIP), and completed a partial sequence of tyrosinase-related protein 1 (TYRP1). The coding sequences and parts of the flanking regions of those genes were systematically analyzed in 40 horses and mutations typed in a total of 120 horses. Our panel represented 22 different horse breeds, including 11 different coat colors of Equus caballus. The comparison of a 1721-bp genomic fragment of MC1R among the 11 coat color phenotypes revealed no sequence difference apart from the known chestnut allele (C901T). In particular, no dominant black (E ^D) mutation was found. In a 4994-bp genomic fragment covering the three putative exons, two introns and parts of the 5′- and 3′-UTRs of ASIP, two intronic base substitutions (SNP-A845G and C2374A), a point mutation in the 3′-UTRs (A4734G), and an 11-bp deletion in exon 2 (ADEx2) were detected. The deletion was found to be homozygous and completely associated with horse recessive black coat color (A ^a /A ^a ) in 24 black horses out of 9 different breeds from our panel. The frameshift initiated by ADEx2 is believed to alter the regular coding sequence, acting as a loss-of-function ASIP mutation. In TYRP1 a base substitution was detected in exon 2 (C189T), causing a threonine to methionine change of yet unknown function, and an SNP (A1188G) was found in intron 2. Received: 22 November 2000 / Accepted: 07 February 2001