Role of melanocortin system in the locomotor activity rhythms and melatonin secretion as revealed by agouti-signalling protein (asip1) overexpression in zebrafish.

Temporal signals such as light and temperature cycles profoundly modulate animal physiology and behaviour. Via endogenous timing mechanisms which are regulated by these signals, organisms can anticipate cyclic environmental changes and thereby enhance their fitness. The pineal gland in fish, through the secretion of melatonin, appears to play a critical role in the circadian system, most likely acting as an element of the circadian clock system. An important output of this circadian clock is the locomotor activity circadian rhythm which is adapted to the photoperiod and thus determines whether animals are diurnal or nocturnal. By using a genetically modified zebrafish strain known as Tg (Xla.Eef1a1:Cau.asip1)iim04, which expresses a higher level of the agouti signalling protein 1 (Asip1), an endogenous antagonist of the melanocortin system, we observed a complete disruption of locomotor activity patterns, which correlates with the ablation of the melatonin daily rhythm. Consistent with this, in vitro experiments also demonstrated that Asip1 inhibits melatonin secretion from the zebrafish pineal gland, most likely through the melanocortin receptors expressed in this gland. Asip1 overexpression also disrupted the expression of core clock genes, including per1a and clock1a, thus blunting circadian oscillation. Collectively, these results implicate the melanocortin system as playing an important role in modulating pineal physiology and, therefore, circadian organisation in zebrafish.

Lethal Yellow Mutation Causes Anxiety, Obsessive-compulsive Behavior and Affects the Brain Melanocortin System in Males and Females of Mice.

BACKGROUND: The brain melanocortin system regulates numerous physiological functions and kinds of behavior. The agouti protein inhibits melanocortin receptors in melanocytes. The lethal yellow (AY) mutation puts the Agouti gene under the control of the Raly gene promotor and causes the agouti protein expression in the brain. In the present article, we investigated the effects of the AY mutation on brain mRNA levels of Agouti, Raly, and melanocortin-related genes such as Agrp, Pomc, Mc3r, Mc4r, and their relationship to behavior. METHODS: The experiment was performed on 6-month-old males and females of AY/a and a/a (control) mice. Anxiety and obsessive-compulsive behavior were studied in elevated plus-maze and marble- burying tests. The mRNA levels were quantified by qPCR. RESULTS: AY mutation caused anxiety in males and obsessive-compulsive behavior in females. Positive correlation between Agouti and Raly genes mRNA levels were shown in the hypothalamus, hippocampus, and frontal cortex in AY/a mice. Reduced RNA concentrations of Mc3r and Mc4r genes were found respectively in the hypothalamus and frontal cortex in AY/a males. The Raly gene expression positively correlates with mRNA concentrations of the Mc3r gene in the hypothalamus and the Mc4r gene in the hypothalamus and frontal cortex. CONCLUSION: Possible association of obsessive-compulsive behavior with reduced Raly, Mc3r, or Mc4r gene expression is suggested.

Aberrant expression of agouti signaling protein (ASIP) as a cause of monogenic severe childhood obesity.

Here we report a heterozygous tandem duplication at the ASIP (agouti signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female patient with extreme childhood obesity. The mutation places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase promoter, driving the generation of ASIP in patient-derived native and induced pluripotent stem cells for all germ layers and hypothalamic-like neurons. The patient's phenotype of early-onset obesity, overgrowth, red hair and hyperinsulinemia is concordant with that of mutant mice ubiquitously expressing the homolog nonagouti. ASIP represses melanocyte-stimulating hormone-mediated activation as a melanocortin receptor antagonist, which might affect eating behavior, energy expenditure, adipocyte differentiation and pigmentation, as observed in the index patient. As the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig Childhood Obesity cohort of 1,745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity.

An enhancer of Agouti contributes to parallel evolution of cryptically colored beach mice.

Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the oldfield mouse (Peromyscus polionotus), which occurs in the southeastern United States, where it exhibits considerable color variation. Dorsal coats range from dark brown in mainland mice to near white in mice inhabiting sandy beaches; this light pelage has evolved independently on Florida's Gulf and Atlantic coasts as camouflage from predators. To facilitate genomic analyses, we first generated a chromosome-level genome assembly of Peromyscus polionotus subgriseus. Next, in a uniquely variable mainland population (Peromyscus polionotus albifrons), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We find that pigment variation is strongly associated with an ∼2-kb region ∼5 kb upstream of the Agouti signaling protein coding region. Using a reporter-gene assay, we demonstrate that this regulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this Agouti region indicate that the light allele experienced recent and strong positive selection. Notably, this same light allele appears fixed in both Gulf and Atlantic coast beach mice, despite these populations being separated by >1,000 km. Together, our results suggest that this identified Agouti enhancer allele has been maintained in mainland populations as standing genetic variation and from there, has spread to and been selected in two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.

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.

Investigating DNA methylation as a potential mediator between pigmentation genes, pigmentary traits and skin cancer.

Pigmentation characteristics are well-known risk factors for skin cancer. Polymorphisms in pigmentation genes have been associated with these traits and with the risk of malignancy. However, the functional relationship between genetic variation and disease is still unclear. This study aims to assess whether pigmentation SNPs are associated with pigmentary traits and skin cancer via DNA methylation (DNAm). Using a meta-GWAS of whole-blood DNAm from 36 European cohorts (N = 27,750; the Genetics of DNA Methylation Consortium, GoDMC), we found that 19 out of 27 SNPs in 10 pigmentation genes were associated with 391 DNAm sites across 30 genomic regions. We examined the effect of 25 selected DNAm sites on pigmentation traits, sun exposure phenotypes and skin cancer and on gene expression in whole blood. We uncovered an association of DNAm site cg07402062 with red hair in the Avon Longitudinal Study of Parents and Children (ALSPAC). We also found that the expression of ASIP and CDK10 was associated with hair colour, melanoma and basal cell carcinoma. Our results indicate that DNAm and expression of pigmentation genes may play a role as potential mediators of the relationship between genetic variants, pigmentation phenotypes and skin cancer and thus deserve further scrutiny.

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.

Growth Performance After Agouti-Signaling Protein 1 (Asip1) Overexpression in Transgenic Zebrafish.

The melanocortin system is a key structure in the regulation of energy balance. Overexpression of inverse agonists, agouti-signaling protein (ASIP), and agouti-related protein (AGRP) results in increased food intake, linear growth, and body weight. ASIP regulates dorsal-ventral pigment polarity through melanocortin 1 receptor (MC1R) and overexpression induces obesity in mice by binding to central MC4R. Asip1 overexpression in transgenic zebrafish (asip1-Tg) enhances growth, yet experiments show fish overexpressing Asip1 do not develop obesity even under severe feeding regimes. Asip1-Tg fish do not need to eat more to grow larger and faster; thus, increased food efficiency can be observed. In addition, asip1-Tg fish reared at high density are able to grow far more than wild-type (WT) fish reared at low density, although asip1-Tg fish seem to be more sensitive to crowding stress than WT fish, thus making the melanocortin system a target for sustainable aquaculture, especially as the U.S. Food and Drug Association has recently approved transgenic fish trading.

Skin transcriptome profiles associated with black- and white-coated regions in Boer and Macheng black crossbred goats.

To increase the current understanding of the gene-expression profiles in different skin regions associated with different coat colors and identify key genes for the regulation of color patterns in goats, we used the Illumina RNA-Seq method to compare the skin transcriptomes of the black- and white-coated regions containing hair follicles from the Boer and Macheng Black crossbred goat, which has a black head and a white body. Six cDNA libraries derived from skin samples of the white-coated region (n = 3) and black-coated region (n = 3) were constructed from three full-sib goats. On average, we obtained approximately 76.5 and 73.5 million reads for skin samples from black- and white-coated regions, respectively, of which 75.39% and 76.05% were covered in the genome database. A total of 165 differentially expressed genes (DEGs) were detected between these two color regions, among which 110 were upregulated and 55 were downregulated in the skin samples of white- vs. black-coated regions. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that some of these DEGs may play an important role in controlling the pigmentation of skin or hair follicles. We identified three key DEGs, i.e., Agouti, DCT, and TYRP1, in the pathway related to melanogenesis in the different skin regions of the crossbred goat. DCT and TYRP1 were downregulated and Agouti was upregulated in the skin of the white-coated region, suggesting a lack of mature melanocytes in this region and that Agouti might play a key developmental role in color-pattern formation. All data sets (Gene Expression Omnibus) are available via public repositories. In addition, MC1R was genotyped in 200 crossbred goats with a black head and neck. Loss-of-function mutations in MC1R as well as homozygosity for the mutant alleles were widely found in this population. The MC1R gene did not seem to play a major role in determining the black head and neck in our crossbred goats. Our study provides insights into the transcriptional regulation of two distinct coat colors, which might serve as a key resource for understanding coat color pigmentation in goats. The region-specific expression of Agouti may be associated with the distribution of pigments across the body in Boer and Macheng Black crossbred goats.

Screening and analysis of agouti signaling protein interaction partners in Pelodiscus sinensis suggests a role in lipid metabolism.

Agouti signaling protein (ASP) is a secreted paracrine protein that has been widely reported to function in melanogenesis and obesity and could potentially be a core protein that regulates the color and fatty phenotype of P. sinensis. In this study, we screened out interacting proteins of ASP by combined co-immunoprecipitation mass spectrometry (CoIP-MS), yeast two hybrid (Y2H) analysis, and computational predictions. We performed docking of ASP with its well-known receptor melanocortin receptor 4 (MC4R) to predict the binding capacity and to screen out actual ASP interacting proteins, CoIP-MS was performed where identified 32 proteins that could bind with ASP and Y2H confirmed seven proteins binding with ASP directly. CoIP-MS and Y2H screening results including PPI prediction revealed that vitronectin (VTN), apolipoprotein A1 (APOA1), apolipoprotein B (APOB), and filamin B (FLNB) were the key interacting proteins of ASP. VTN, APOA1, and APOB are functional proteins in lipid metabolism and various skin disorders, suggesting ASP may function in lipid metabolism through these partners. This study provided protein-protein interaction information of ASP, and the results will promote further research into the diverse roles of ASP, as well as its binding partners, and their function in different strains of P. sinensis.

Nested retrotransposition in the East Asian mouse genome causes the classical nonagouti mutation.

Black coat color (nonagouti) is a widespread classical mutation in laboratory mouse strains. The intronic insertion of endogenous retrovirus VL30 in the nonagouti (a) allele of agouti gene was previously reported as the cause of the nonagouti phenotype. Here, we report agouti mouse strains from East Asia that carry the VL30 insertion, indicating that VL30 alone does not cause the nonagouti phenotype. We find that a rare type of endogenous retrovirus, ß4, was integrated into the VL30 region at the a allele through nested retrotransposition, causing abnormal splicing. Targeted complete deletion of the ß4 element restores agouti gene expression and agouti coat color, whereas deletion of ß4 except for a single long terminal repeat results in black-and-tan coat color. Phylogenetic analyses show that the a allele and the ß4 retrovirus originated from an East Asian mouse lineage most likely related to Japanese fancy mice. These findings reveal the causal mechanism and historic origin of the classical nonagouti mutation.

Two new structural mutations in the 5' region of the ASIP gene cause diluted feather color phenotypes in Japanese quail.

BACKGROUND: In quail, two feather colour phenotypes i.e. fawn-2/beige and yellow are associated with the ASIP locus. The aim of our study was to characterize the structural modifications within this locus that explain the yellow mutation (large deletion) and the fawn-2/beige mutation (assumed to be caused by a different structural modification). RESULTS: For the yellow phenotype, we identified a complex mutation that involves a 141,162-bp long deletion. For the fawn-2/beige phenotype, we identified a 71-kb tandem duplication that comprises one unchanged copy of ASIP and one copy present in the ITCH-ASIP fusion gene, which leads to a transcript coding for a normal ASIP protein. Although this agrees with previous reports that reported an increased level of ASIP transcripts in the skin of mutant animals, we show that in the skin from fawn-2/beige embryos, this level is higher than expected with a simple duplication of the ASIP gene. Thus, we hypothesize that the 5' region of the ITCH-ASIP fusion gene leads to a higher transcription level than the 5' region of the ASIP gene. CONCLUSIONS: We were able to conclude that the fawn-2 and beige phenotypes are caused by the same allele at the ASIP locus. Both of the associated mutations fawn-2/beige and yellow lead to the formation of a fusion gene, which encodes a transcript for the ASIP protein. In both cases, transcription of ASIP depends on the promoter of a different gene, which includes alternative up-regulating sequences. However, we cannot exclude the possibility that the loss of the 5' region of the ASIP gene itself has additional impacts, especially for the fawn-2/beige mutation. In addition, in several other species including mammals, the existence of other dominant gain-of-function structural modifications that are localized upstream of the ASIP coding sequences has been reported, which supports our hypothesis that repressors in the 5' region of ASIP are absent in the fawn-2/beige mutant.

On association of the lethal yellow (AY) mutation in the agouti gene with the alterations in mouse brain and behavior.

Lethal yellow (AY) mutation causes obesity and type-2 diabetes in mice. Here we studied the effect of the AY mutation on the brain and behavior. The experiments were carried out on adult (11-12 weeks old) males of AY/a mice and their wild-type littermates (a/a). Mice of AY/a and a/a genotypes did not differ in their home cage activity, sleep, food and water consumption, learning ability in the Morris water maze, anxiety in the open field and elevated plus-maze, as well as in the level of monoamines, metabolites and some genes expression in the brain. At the same time, the fat mass, depressive-like immobility in the forced swim and tail suspension tests were significantly increased in AY/a mice compared with a/a ones. Magnetic resonance imaging revealed a significant reduction of cortex volume in AY/a mice. The level of mRNA of Ptpn5 gene encoding striatal enriched tyrosine phosphatase in the frontal cortex of AY/a mice was significantly elevated compared with their wild-type littermates. This is the first report on the alterations in the brain and behavior in the AY/a mouse line. It is tempting to speculate that this mouse line can serve as a new and useful preclinical model to study neurobehavioral complications associated with obesity and type-2 diabetes.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts.

Behind melanocortin antagonist overexpression in the zebrafish brain: A behavioral and transcriptomic approach.

Melanocortin signaling is regulated by the binding of naturally occurring antagonists, agouti-signaling protein (ASIP) and agouti-related protein (AGRP) that compete with melanocortin peptides by binding to melanocortin receptors to regulate energy balance and growth. Using a transgenic model overexpressing ASIP, we studied the involvement of melanocortin system in the feeding behaviour, growth and stress response of zebrafish. Our data demonstrate that ASIP overexpression results in enhanced growth but not obesity. The differential growth is explained by increased food intake and feeding efficiency mediated by a differential sensitivity of the satiety system that seems to involve the cocaine- and amphetamine- related transcript (CART). Stress response was similar in both genotypes. Brain transcriptome of transgenic (ASIP) vs wild type (WT) fish was compared using microarrays. WT females and males exhibited 255 genes differentially expressed (DEG) but this difference was reduced to 31 after ASIP overexpression. Statistical analysis revealed 1122 DEG when considering only fish genotype but 1066 and 981 DEG when comparing ASIP males or females with their WT counterparts, respectively. Interaction between genotype and sex significantly affected the expression of 97 genes. Several neuronal systems involved in the control of food intake were identified which displayed a differential expression according to the genotype of the fish that unravelling the flow of melanocortinergic information through the central pathways that controls the energy balance. The information provided herein will help to elucidate new central systems involved in control of obesity and should be of invaluable use for sustaining fish production systems.

Inhibition of melanocortin 1 receptor slows melanoma growth, reduces tumor heterogeneity and increases survival.

Melanoma risk is increased in patients with mutations of melanocortin 1 receptor (MC1R) yet the basis for the increased risk remains unknown. Here we report in vivo evidence supporting a critical role for MC1R in regulating melanoma tumor growth and determining overall survival time. Inhibition of MC1R by its physiologically relevant competitive inhibitor, agouti signaling protein (ASIP), reduced melanin synthesis and morphological heterogeneity in murine B16-F10 melanoma cells. In the lungs of syngeneic C57BL/6 mice, mCherry-marked, ASIP-secreting lung tumors inhibited MC1R on neighboring tumors lacking ASIP in a dose dependent manner as evidenced by a proportional loss of pigment in tumors from mice injected with 1:1, 3:1 and 4:1 mixtures of parental B16-F10 to ASIP-expressing tumor cells. ASIP-expressing B16-F10 cells formed poorly pigmented tumors in vivo that correlated with a 20% longer median survival than those bearing parental B16-F10 tumors (p=0.0005). Mice injected with 1:1 mixtures also showed survival benefit (p=0.0054), whereas injection of a 4:1 mixture showed no significant difference in survival. The longer survival time of mice bearing ASIP-expressing tumors correlated with a significantly slower growth rate than parental B16-F10 tumors as judged by quantification of numbers of tumors and total tumor load (p=0.0325), as well as a more homogeneous size and morphology of ASIP-expressing lung tumors. We conclude that MC1R plays an important role in regulating melanoma growth and morphology. Persistent inhibition of MC1R provided a significant survival advantage resulting in part from slower tumor growth, establishing MC1R as a compelling new molecular target for metastatic melanoma.

Agouti signalling protein (ASIP) gene: molecular cloning, sequence characterisation and tissue distribution in domestic goose.

Agouti signalling protein (ASIP) is an endogenous antagonist of melanocortin-1 receptor (MC1R) and is involved in the regulation of pigmentation in mammals. The objective of this study was to identify and characterise the ASIP gene in domestic goose. The goose ASIP cDNA consisted of a 44-nucleotide 5'-terminal untranslated region (UTR), a 390-nucleotide open-reading frame (ORF) and a 45-nucleotide 3'-UTR. The length of goose ASIP genomic DNA was 6176 bp, including three coding exons and two introns. Bioinformatic analysis indicated that the ORF encodes a protein of 130 amino-acid residues with a molecular weight of 14.88 kDa and an isoelectric point of 9.73. Multiple sequence alignments and phylogenetic analysis showed that the amino-acid sequence of ASIP was conserved in vertebrates, especially in the avian species. RT-qPCR showed that the goose ASIP mRNA was differentially expressed in the pigment deposition tissues, including eye, foot, feather follicle, skin of the back, as well as in skin of the abdomen. The expression level of the ASIP gene in skin of the abdomen was higher than that in skin of the back. Those findings will contribute to further understanding the functions of the ASIP gene in geese plumage colouring.

Chemotherapy-induced gut toxicity and pain: involvement of TLRs.

PURPOSE: Chemotherapy-induced gut toxicity is associated with significant pain, and pain influences gut function. Toll-like receptors (TLRs) that regulate gut homeostasis are activated by tissue damage and microbes, and their altered expression following chemotherapy may change cellular responses. This study examined the interaction between chemotherapy-induced gut toxicity and pain and related these to gut TLR and glial fibrillary acidic protein (GFAP) expression. METHODS: Female tumor bearing Dark Agouti rats received irinotecan (175 mg/kg, n = 34) or vehicle (n = 5) and were assessed over 120 h for gut toxicity (diarrhea, weight loss), pain (facial), and GFAP, TLR2, 4, 5, and 9 gut expression. RESULTS: Irinotecan caused diarrhea (72 % of animals grade ≥ 1), weight loss (11.1 ± 6.6 %, P < 0.0001), and pain (5 (0-5), P < 0.0001) all peaking at 72 h. Higher pain scores were observed in rats with diarrhea versus those without: median (range) of 2.0 (0-5) versus 0 (0-5), P = 0.01. Irinotecan also caused a decrease in TLR4 and 5, and an increase in GFAP expression in jejuna crypt at 96 and 120 h (all P < 0.05); with lower TLR4 expression associated with lower pain (P = 0.012). CONCLUSIONS: The association between gut toxicity and pain suggests these toxicities are linked, possibly via TLR-mediated inflammatory pathways. Further, as TLR4 and 5 expression was absent during recovery in the jejuna and GFAP expression was increased in the jejuna, this implies expression of these may be critical in the healing phase following chemotherapy. Detailed studies of gut TLRs and GFAP are now warranted.