Agouti-Induced Anxiety-Like Behavior Is Mediated by Central Serotonergic Pathways in Zebrafish.

Overexpression of the agouti-signaling protein (asip1), an endogenous melanocortin antagonist, under the control of a constitutive promoter in zebrafish [Tg(Xla.Eef1a1:Cau.Asip1]iim4] (asip1-Tg) increases food intake by reducing sensitivity of the central satiety systems and abolish circadian activity rhythms. The phenotype also shows increased linear growth and body weight, yet no enhanced aggressiveness in dyadic fights is observed. In fact, asip1-Tg animals choose to flee to safer areas rather than face a potential threat, thus suggesting a potential anxiety-like behavior (ALB). Standard behavioral tests, i.e., the open field test (OFT), the novel object test (NOT), and the novel tank dive test (NTDT), were used to investigate thigmotaxis and ALB in male and female zebrafish. Results showed that the asip1-Tg strain exhibited severe ALB in every test, mainly characterized by pronounced freezing behavior and increased linear and angular swimming velocities. asip1-Tg animals exhibited low central serotonin (5-HT) and dopamine (DA) levels and high turnover rates, thus suggesting that central monoaminergic pathways might mediate melanocortin antagonist-induced ALB. Accordingly, the treatment of asip1-Tg animals with fluoxetine, a selective serotonin reuptake inhibitor (SSRI), reversed the ALB phenotype in NTDT as well as 5-HT turnover. Genomic and anatomical data further supported neuronal interaction between melanocortinergic and serotonergic systems. These results suggest that inhibition of the melanocortin system by ubiquitous overexpression of endogenous antagonist has an anxiogenic effect mediated by serotonergic transmission.

Dark Morph of the Oriental Honey-Buzzard (Pernis ptilorhynchus orientalis) is Attributable to Specific MC1R Haplotypes.

A thorough understanding of the development of complex plumages in birds necessitates the acquisition of genetic data pertaining to the mechanism underlying this phenomenon from various avian species. The oriental honey-buzzard (Pernis ptilorhynchus orientalis), a tropical summer migrant to Northeast Asia, including Japan, exemplifies this aspect owing to the diversity of its ventral coloration and intra-feather barring patterns. However, genetic polymorphism responsible for this diversity has not been identified yet. This study aimed to investigate the link between dark-plumed phenotypes of this subspecies and haplotypes of the melanocortin-1-receptor (MC1R) gene. A draft sequence of MC1R was constructed using next generation sequencing and subsequently amplified using designed polymerase chain reaction (PCR) primers. The genome sequences of 32 honey-buzzard individuals were determined using PCR, and 12 MC1R haplotype sequences were obtained. Among these haplotypes, we found that unique haplotypes with nine non-synonymous substitutions and four or five synonymous substitutions in the coding region had a perfect correlation with the dark-plumed phenotype. The lack of correlation between the genotype of ASIP coding region and plumage phenotype reiterated that the dark morph is attributable to specific MC1R haplotypes. The absence of a correlation between genetic polymorphisms of MC1R and the intra-feather barring patterns, as well as the diversity observed within lighter ground color classes (pale and intermediate), implies the involvement of alternative molecular mechanisms in the manifestation of the aforementioned phenotypes.

Preliminary investigation of potential links between pigmentation variants and opioid analgesic effectiveness in horses during cerebrospinal fluid centesis.

BACKGROUND: The pleiotropic effects of the melanocortin system show promise in overcoming limitations associated with large variations in opioid analgesic effectiveness observed in equine practice. Of particular interest is variation in the melanocortin-1-receptor (MC1R) gene, which dictates pigment type expression through its epistatic interaction with the agouti signalling protein (ASIP) gene. MC1R has previously been implicated in opioid efficacy in other species; however, this relationship is yet to be explored in horses. In this study, analgesic effectiveness was scored (1-3) based on noted response to dura penetration during the performance of cerebrospinal fluid centisis after sedation and tested for association with known genetic regions responsible for pigmentation variation in horses. RESULTS: The chestnut phenotype was statistically significant (P < 0.05) in lowering analgesic effectiveness when compared to the bay base coat colour. The 11bp indel in ASIP known to cause the black base coat colour was not significant (P>0.05); however, six single nucleotide polymorphisms (SNPs) within the genomic region encoding the ASIP gene and one within MC1R were identified as being nominally significant (P<0.05) in association with opioid analgesic effectiveness. This included the location of the known e MC1R variant resulting in the chestnut coat colour. CONCLUSIONS: The current study provides promising evidence for important links between pigmentation genes and opioid effectiveness in horses. The application of an easily identifiable phenotype indicating variable sensitivity presents a promising opportunity for accessible precision medicine in the use of analgesics and warrants further investigation.

Revisiting the hormonal control of sexual dimorphism in chicken feathers.

Sexual dimorphism in plumage is widespread among avian species. In chickens, adult females exhibit countershading, characterized by dull-colored round feathers lacking fringe on the saddle, while adult males display vibrant plumage with deeply fringed bright feathers. This dimorphism is estrogen-dependent, and administering estrogen to males transforms their showy plumage into cryptic female-like plumage. Extensive studies have shown that estrogen's role in female plumage formation requires thyroid hormone; however, the precise mechanisms of their interaction remain unclear. In this study, we investigated the roles of estrogen and thyroid hormone in creating sexual dimorphism in the structure and coloration of saddle feathers by administering each hormone to adult males and observing the resulting changes in regenerated feathers induced by plucking. RT-PCR analysis revealed that the expression of type 3 deiodinase (DIO3), responsible for thyroid hormone inactivation, correlates with fringing. Estrogen suppressed DIO3 and agouti signaling protein (ASIP) expression while stimulating BlSK1, a marker of barbule cells, resulting in female-like feathers with mottled patterns and lacking fringes. Administration of thyroxine (T4) stimulated BlSK1 and proopiomelanocortin (POMC) expression, with no effect on ASIP, leading to the formation of solid black feathers lacking fringes. Triiodothyronine (T3) significantly increased POMC expression in pulp cells in culture. Taken together, these findings suggest that estrogen promotes the formation of solid vanes by suppressing DIO3 expression, while also inducing the formation of mottled patterns through inhibition of ASIP expression and indirect stimulation of melanocortin expression via changes in local T3 concentration. This is the first report describing molecular mechanism underlying hormonal crosstalk in creating sexual dimorphism in feathers.

Differential expression of ASIP transcripts reveals genetic mechanism underpinning black-tail independence from body plumage in yellow-bodied chickens.

The genetic foundation of chicken body plumage color has been extensively studied. However, little attention has been paid to the inheritance patterns and molecular mechanisms underlying the formation of distal feather colors (tail and wingtip). Differences in these colors are common; for example, the Chinese Huiyang Beard chicken has black tail feathers, but yellow body plumage. Here, the hybrid offspring of Huiyang Beard and White Leghorn chickens were used to study the inheritance patterns of tail-feather color. The expression levels of pigment genes in differently colored feather follicles were analyzed using quantitative real-time PCR. The results showed that genetic regulation of tail-feather color was independent of body-plumage color. The Dominant White locus inhibited eumelanin synthesis in tail feathers without affecting the formation of yellow body plumage, whereas the Silver locus had the opposite effect. The expression of agouti signaling protein (ASIP) gene class 1 transcripts was significantly lower in black tail-feather follicles than in yellow body follicles, whereas tyrosinase-related protein 1 (TYRP1) gene expression was significantly higher in black tail feathers. These differentially expressed genes were confirmed to exert an effect on eumelanin and pheomelanin formation in feathers, thus influencing the regulation of chicken tail-feather color. In conclusion, this study lays the foundation for further research on the genetic mechanisms of regional differences in feather color, contributing to a better understanding of plumage pigmentation in chickens.

Functional relation of agouti signaling proteins (ASIPs) to pigmentation and color change in the starry flounder, Platichthys stellatus.

We investigated the involvement of agouti-signaling proteins (ASIPs) in morphological pigmentation and physiological color change in flatfishes. We isolated ASIP1 and 2 mRNAs from the skin of starry flounder (Platichthys stellatus), and compared their amino acid (aa) structures to those of other animals. Then, we examined the mRNA expression levels of two ASIPs (Sf-ASIPs) in the pigmented ocular body and in the unpigmented blind body, as well as in the ordinary skin and in albino skin, in flatfishes. To investigate the role of Sf-ASIPs in physiological color change (color camouflage), we compared the expression of the two genes in two background colors (dark-green and white). Sf-ASIP1 cDNA had a 375-bp open reading frame (ORF) that encoded a protein consisting of 125 aa residues, and Sf-ASIP2 cDNA had a 402-bp ORF that encoded a protein consisting of 132 aa residues. RT-PCR revealed that the strongest Sf-ASIP1 and Sf-ASIP2 expression levels were observed in the eye and blind-skin, respectively. In Sf-ASIP1, the gene expression did not differ between the ocular-side skin and blind-side skin, nor between ordinary skin and abnormal skin of the fish. However, in Sf-ASIP2, the expression level was significantly higher in blind-side skin, compared to ocular-side skin, suggesting that the ASIP2 gene is related to the countershading body pigment pattern of the fish. In addition, the Sf-ASIP2 gene expression level was lower in the pigmented spot regions than in the unpigmented spot regions of the malpigmented pseudo-albino skins on the ocular side, implying that ASIP2 is responsible for the ocular-side pseudo-albino. Additionally, ASIP2 gene expression in the blind-side skin of ordinary fish was enhanced by a white tank, implying that a bright background color could inhibit hypermelanosis in the blind-side skin of cultured flounder by increasing the activity of the Sf-ASIP2 gene. However, we did not find any relationship of ASIPs with camouflage color changes. In conclusion, the ASIP2 gene is related to the morphological pigmentation (countershading and malpigmentation) of the skin in starry flounder, but not with physiological color changes (color camouflage) in the ocular-side skin.

Unveiling the molecular mechanisms of pigmentation control in Queen Loach, Botia dario (Hamilton, 1822): Insights from sesame seed and marigold-induced antityrosinase effects.

Fish pigmentation study can reveal understandings in dermatological research based on functional genomics. Cultured ornamental fish becomes dull coloured and antityrosinase activity through sesame seed may enhance skin colour, which has not been studied. Botia dario is an indigenous fish, having ornamental and aesthetic value and can be studied as a model for fish pigmentation genetics. In this study, fish specimens were fed with 15% marigold petal meal along with 5, 10 and 15% w/w sesame seed in diet. Pigmentation genes, that is, tyr, tyrp1a, asip1, gnaq, kitlga, mc1r, mitf, pax7a, rab38, slc7a11, sox9a, sox10, csf1r, bcdo2 and gsta2 in skin and immunogens, that is, il20, nramp, tlr9 and trail in kidney were studied. Gene expression in tissues revealed enhanced pigmentation and immunity as well as the role of tyr, tyrp1a and asip1 in pigmentation. Immunogenes and blood parameters confirmed the best pigmentation diet. Colorimetric analysis also showed the enhancement of pigmentation. Insights from sesame seed and marigold-induced antityrosinase effects will be applied in aquaculture to develop natural, dietary formulations that will enhance pigmentation in ornamental fish, leading to improved skin colour and market value.

A Cre knockin mouse reveals specific expression of Agouti gene in mesenchymal lineage cells in multiple organs and provides a unique tool for conditional gene targeting.

The mouse Agouti gene encodes a paracrine signaling factor which promotes melanocytes to produce yellow instead of black pigment. It has been reported that Agouti mRNA is confined to the dermal papilla after birth in various mammalian species. In this study, we created and characterized a knockin mouse strain in which Cre recombinase was expressed in-frame with endogenous Agouti coding sequence. The Agouti-Cre mice were bred with reporter mice (Rosa26-tdTomato or Rosa26-ZsGreen) to trace the lineage of Agouti-expressing cells during development. In skin, the reporter was detected in some dermal fibroblasts at the embryonic stage and in all dermal fibroblasts postnatally. It was also expressed in all mesenchymal lineage cells in other organs/tissues, including eyes, tongue, muscle, intestine, adipose, prostate and testis. Interestingly, the reporter expression was excluded from epithelial cells in the above organs/tissues. In brain, the reporter was observed in the outermost meningeal fibroblasts. Our work helps to illustrate the Agouti expression pattern during development and provides a valuable mouse strain for conditional gene targeting in mesenchymal lineage cells in multiple organs.

A 13.42-kb tandem duplication at the ASIP locus is strongly associated with the depigmentation phenotype of non-classic Swiss markings in goats.

BACKGROUND: The pigmentation phenotype diversity is rich in domestic goats, and identification of the genetic loci affecting coat color in goats has long been of interest. Via the detections of selection signatures, a duplication upstream ASIP was previously reported to be a variant affecting the Swiss markings depigmentation phenotype in goats. RESULTS: We conducted a genome-wide association study using whole-genome sequencing (WGS) data to identify the genetic loci and causal variants affecting the pigmentation phenotype in 65 Jintang black (JT) goats (i.e., 48 solid black vs. 17 non-classic Swiss markings). Although a single association peak harboring the ASIP gene at 52,619,845-72,176,538 bp on chromosome 13 was obtained using a linear mixed model approach, all the SNPs and indels in this region were excluded as causal variants for the pigmentation phenotype. We then found that all 17 individuals with non-classic Swiss markings carried a 13,420-bp duplication (CHI13:63,129,198-63,142,617 bp) nearly 101 kb upstream of ASIP, and this variant was strongly associated (P = 1.48 × 10- 12) with the coat color in the 65 JT goats. The copy numbers obtained from the WGS data also showed that the duplication was present in all 53 goats from three European breeds with Swiss markings and absent in 45 of 51 non-Swiss markings goats from four other breeds and 21 Bezoars, which was further validated in 314 samples from seven populations based on PCR amplification. The copy numbers of the duplication vary in different goat breeds with Swiss markings, indicating a threshold effect instead of a dose-response effect at the molecular level. Furthermore, breakpoint flanking repeat analysis revealed that the duplication was likely to be a result of the Bov-B-mediated nonallelic homologous recombination. CONCLUSION: We confirmed that a genomic region harboring the ASIP gene is a major locus affecting the coat color phenotype of Swiss markings in goats. Although the molecular genetic mechanisms remain unsolved, the 13,420-bp duplication upstream of ASIP is a necessary but not sufficient condition for this phenotype in goats. Moreover, the variations in the copy number of the duplication across different goat breeds do not lead to phenotypic heterogeneity.

Genomic Analysis Revealed a Convergent Evolution of LINE-1 in Coat Color: A Case Study in Water Buffaloes (Bubalus bubalis).

Visible pigmentation phenotypes can be used to explore the regulation of gene expression and the evolution of coat color patterns in animals. Here, we performed whole-genome and RNA sequencing and applied genome-wide association study, comparative population genomics and biological experiments to show that the 2,809-bp-long LINE-1 insertion in the ASIP (agouti signaling protein) gene is the causative mutation for the white coat phenotype in swamp buffalo (Bubalus bubalis). This LINE-1 insertion (3' truncated and containing only 5' UTR) functions as a strong proximal promoter that leads to a 10-fold increase in the transcription of ASIP in white buffalo skin. The 165 bp of 5' UTR transcribed from the LINE-1 is spliced into the first coding exon of ASIP, resulting in a chimeric transcript. The increased expression of ASIP prevents melanocyte maturation, leading to the absence of pigment in white buffalo skin and hairs. Phylogenetic analyses indicate that the white buffalo-specific ASIP allele originated from a recent genetic transposition event in swamp buffalo. Interestingly, as a similar LINE-1 insertion has been identified in the cattle ASIP gene, we discuss the convergent mechanism of coat color evolution in the Bovini tribe.

Comparative ontogenetic and transcriptomic analyses shed light on color pattern divergence in cichlid fishes.

Stripe patterns are a striking example for a repeatedly evolved color pattern. In the African adaptive radiations of cichlid fishes, stripes evolved several times independently. Previously, it has been suggested that regulatory evolution of a single gene, agouti-related-peptide 2 (agrp2), explains the evolutionary lability of this trait. Here, using a comparative transcriptomic approach, we performed comparisons between (adult) striped and nonstriped cichlid fishes of representatives of Lake Victoria and the two major clades of Lake Malawi (mbuna and non-mbuna lineage). We identify agrp2 to be differentially expressed across all pairwise comparisons, reaffirming its association with stripe pattern divergence. We therefore also provide evidence that agrp2 is associated with the loss of the nonstereotypic oblique stripe of Mylochromis mola. Complementary ontogenetic data give insights into the development of stripe patterns as well as vertical bar patterns that both develop postembryonically. Lastly, using the Lake Victoria species pair Haplochromis sauvagei and Pundamilia nyererei, we investigated the differences between melanic and non-melanic regions to identify additional genes that contribute to the formation of stripes. Expression differences-that most importantly also do not include agrp2-are surprisingly small. This suggests, at least in this species pair, that the stripe phenotype might be caused by a combination of more subtle transcriptomic differences or cellular changes without transcriptional correlates. In summary, our comprehensive analysis highlights the ontogenetic and adult transcriptomic differences between cichlids with different color patterns and serves as a basis for further investigation of the mechanistic underpinnings of their diversification.

Golden cats: A never-ending story!

In the British feline breed a golden coat modification, called light-gold, akita or copper, was reported by breeders during the 2010s. This modification restricted eumelanin to the tip of the tail and hairs showed a wideband modification. Pedigree analyses revealed an autosomal recessive inheritance pattern. A single candidate region was identified using a genome-wide association study. Within that region, we identified CORIN (Corin, serine peptidase) as the strongest candidate gene, since two CORIN variants have previously been identified in Siberian cats with a golden phenotype. A homozygous CORIN:c.2425C>T nonsense variant was identified in copper British cats. Segregation of the variant was consistent with recessive inheritance. This nonsense CORIN:c.2425C>T variant, located in CORIN exon 19, was predicted to produce a truncated CORIN protein - CORIN:p.(Arg809Ter) - that would lack part of the scavenger receptor domain and the trypsine-like serine protease catalytic domain. All 30 copper cats were T/T homozygous for the variant, which was also found in 20 C/T heterozygous British control cats but was absent in 340 cats from the 99 Lives dataset. Finally, genotyping of 218 cats from 12 breeds failed to identify carriers in cats from other breeds. We propose that this third CORIN:c.2425C>T variant represents the wbBSH (British recessive wideband) allele in the domestic cat.

Instructive role of melanocytes during pigment pattern formation of the avian skin.

Animal skin pigment patterns are excellent models to study the mechanism of biological self-organization. Theoretical approaches developed mathematical models of pigment patterning and molecular genetics have brought progress; however, the responsible cellular mechanism is not fully understood. One long unsolved controversy is whether the patterning information is autonomously determined by melanocytes or nonautonomously determined from the environment. Here, we transplanted purified melanocytes and demonstrated that melanocytes could form periodic pigment patterns cell autonomously. Results of heterospecific transplantation among quail strains are consistent with this finding. Further, we observe that developing melanocytes directly connect with each other via filopodia to form a network in culture and in vivo. This melanocyte network is reminiscent of zebrafish pigment cell networks, where connexin is implicated in stripe formation via genetic studies. Indeed, we found connexin40 (cx40) present on developing melanocytes in birds. Stripe patterns can form in quail skin explant cultures. Several calcium channel modulators can enhance or suppress pigmentation globally, but a gap junction inhibitor can change stripe patterning. Most interestingly, in ovo, misexpression of dominant negative cx40 expands the black region, while overexpression of cx40 expands the yellow region. Subsequently, melanocytes instruct adjacent dermal cells to express agouti signaling protein (ASIP), the regulatory factor for pigment switching, which promotes pheomelanin production. Thus, we demonstrate Japanese quail melanocytes have an autonomous periodic patterning role during body pigment stripe formation. We also show dermal agouti stripes and how the coupling of melanocytes with dermal cells may confer stable and distinct pigment stripe patterns.

Comparative analysis of inbreeding parameters and runs of homozygosity islands in 2 Italian autochthonous cattle breeds mainly raised in the Parmigiano-Reggiano cheese production region.

Reggiana and Modenese are autochthonous cattle breeds, reared in the North of Italy, that can be mainly distinguished for their standard coat color (Reggiana is red, whereas Modenese is white with some pale gray shades). Almost all milk produced by these breeds is transformed into 2 mono-breed branded Parmigiano-Reggiano cheeses, from which farmers receive the economic incomes needed for the sustainable conservation of these animal genetic resources. After the setting up of their herd books in 1960s, these breeds experienced a strong reduction in the population size that was subsequently reverted starting in the 1990s (Reggiana) or more recently (Modenese) reaching at present a total of about 2,800 and 500 registered cows, respectively. Due to the small population size of these breeds, inbreeding is a very important cause of concern for their conservation programs. Inbreeding is traditionally estimated using pedigree data, which are summarized in an inbreeding coefficient calculated at the individual level (FPED). However, incompleteness of pedigree information and registration errors can affect the effectiveness of conservation strategies. High-throughput SNP genotyping platforms allow investigation of inbreeding using genome information that can overcome the limits of pedigree data. Several approaches have been proposed to estimate genomic inbreeding, with the use of runs of homozygosity (ROH) considered to be the more appropriate. In this study, several pedigree and genomic inbreeding parameters, calculated using the whole herd book populations or considering genotyping information (GeneSeek GGP Bovine 150K) from 1,684 Reggiana cattle and 323 Modenese cattle, were compared. Average inbreeding values per year were used to calculate effective population size. Reggiana breed had generally lower genomic inbreeding values than Modenese breed. The low correlation between pedigree-based and genomic-based parameters (ranging from 0.187 to 0.195 and 0.319 to 0.323 in the Reggiana and Modenese breeds, respectively) reflected the common problems of local populations in which pedigree records are not complete. The high proportion of short ROH over the total number of ROH indicates no major recent inbreeding events in both breeds. ROH islands spread over the genome of the 2 breeds (15 in Reggiana and 14 in Modenese) identified several signatures of selection. Some of these included genes affecting milk production traits, stature, body conformation traits (with a main ROH island in both breeds on BTA6 containing the ABCG2, NCAPG, and LCORL genes) and coat color (on BTA13 in Modenese containing the ASIP gene). In conclusion, this work provides an extensive comparative analysis of pedigree and genomic inbreeding parameters and relevant genomic information that will be useful in the conservation strategies of these 2 iconic local cattle breeds.

Functional conservation and divergence of color-pattern-related agouti family genes in teleost fishes.

While color patterns are highly diverse across the animal kingdom, certain patterns such as countershading and stripe patterns have evolved repeatedly. Across vertebrates, agouti-signaling genes have been associated with the evolution of both patterns. Here we study the functional conservation and divergence by investigating the expression patterns of the two color-pattern-related agouti-signaling genes, agouti-signaling protein 1 (asip1) and agouti-signaling protein 2b (asip2b, also known as agrp2) in Teleostei. We show that the dorsoventral expression profile of asip1 and the role of the "stripe repressor" asip2b are shared across multiple teleost lineages and uncover a previously unknown association between stripe-interstripe patterning and both asip1 and asip2b expression. In some species, including the zebrafish (Danio rerio), these two genes show complementary and overlapping expression patterns in line with functional redundancy. Our results thus suggest how conserved and novel functions of agouti-signaling genes might have shaped the evolution of color patterns across teleost fishes.

Siberian cats help in solving part of the mystery surrounding golden cats.

Golden cats have been appreciated since the beginning of the cat fancy. Golden is a modification of the tabby coat. In the Siberian breed, a specific golden phenotype, named sunshine, has been described. Sunshine tabby cats exhibit a warm tone of tabby, a pink nose lacking the black lining and a large light cream area around the nose. Pedigree analyses revealed an autosomal recessive inheritance pattern. A single candidate region was identified by genome-wide association study (GWAS) and homozygosity mapping. Within that region, we identified CORIN (Corin, serine peptidase) as a strong candidate gene, since CORIN variants have been identified in mice and tigers with a golden phenotype and CORIN has been described as a modifier of the ASIP (Agouti Signaling Protein) pathway. A homozygous CORIN:c.2383C>T missense variant was identified in sunshine tabby cats. Segregation of the variant was consistent with recessive inheritance. The variant was also found in three Kurilian bobtail cats and in two ToyBob cats from the 99 Lives dataset but genotyping of 106 cats from 13 breeds failed to identify carriers in cats from other breeds. The CORIN:c.2383C>T variant was predicted to change an arginine to a cysteine at position 795 in the protein: CORIN:p.(Arg795Cys). Finally, hair observation in Siberian cats was consistent with elongated ASIP signaling as golden hair showed a large yellow band instead of the short subapical one usually observed in agouti hair. These results support an association of the Siberian sunshine modification with the CORIN:c.2383C>T variant. The Siberian cat has helped us to decipher one of the golden phenotypes observed in cats and we propose that the CORIN:c.2383C>T variant represents the wbSIB (Siberian recessive wideband) allele in the domestic cat.

Impact of white-spotting alleles, including W20, on phenotype in the American Paint Horse.

The American Paint Horse Association (APHA) records pedigree and performance information for their breed, a stock-type horse valued as a working farm or ranch horse and as a pleasure horse. As the name implies, the breed is also valued for its attractive white-spotting patterns on the coat. The APHA utilizes visual inspections of photographs to determine if coat spotting exceeds threshold anatomical landmarks considered characteristic of desirable patterns. Horses with sufficient white patterning enter the 'Regular' registry, rather than the 'Solid Paint-Bred' division, providing a threshold modeled phenotype. Genetic studies previously defined sequence variants corresponding to 35 alleles for white spotting in the horse. Here, we calculate the allele frequencies for nine common white-spotting alleles in the American Paint Horse using a sample of 1054 registered animals. The APHA spotting phenotype is altered by additive interactions among spotting loci, and epistatically by the MC1R and ASIP genes controlling pigment production. The W20 allele within the KIT gene, independent of other known spotting alleles, was strongly associated with the APHA-defined white-spotting phenotype (P = 1.86 × 10-18 ), refuting reports that W20 acts only as a modifier of other underlying white-spotting patterns. The parentage of an individual horse, either American Paint or American Quarter Horse, did not alter the likelihood of its entering the APHA Regular Registry. An empirical definition of the action of these genetic loci on the APHA-defined white-spotting phenotype will allow more accurate application of genome-assisted selection for improving color production and the marketability of APHA horses.

Multiple Conserved Elements Structuring Inverted Repeats in the Mammalian Coat Color-Related Gene Asip.

In the agouti signaling gene protein (Asip) of the house mouse (Mus musculus), inverted repeat (IR) arrays are known to exist in a non-coding region adjacent to the ventral-specific promoter region and the accompanying two exons (exons 1A and 1A'), which are around 100 kb upstream from the amino acid coding regions of exons 2, 3, and 4. To determine the gene structure of mammalian Asip and to elucidate trends in its evolution, non-coding sequences of six rodent (mouse, rat, Chinese hamster, squirrel, guinea pig, and naked mole rat) and three non-rodent (rabbit, human, and cow) species were retrieved from databases and compared. Our homology search analyses revealed the presence of three to five highly conserved non-coding elements (CNE). These CNEs were found to form IRs in rodents and lagomorphs. Combinations of IRs were further shown to build symmetric, long IR arrays. Intra- and inter-specific comparisons of the sequences of three universal CNEs showed homogeneity between CNE pairs within species. This implies that certain evolutionary constraints maintained the IR structure in the rodent and rabbit species.

Left-right pigmentation pattern of Japanese flounder corresponds to expression levels of melanocortin receptors (MC1R and MC5R), but not to agouti signaling protein 1 (ASIP1) expression.

Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.