MD, University of Washington, Medicine (1984)
BS, University of California, Irvine, Biology (1977)
PhD, University of Washington, Genetics of Human Disease (1984)
Color variation is one of the most readily apparent differences among closely related animals, and has been studied extensively as a model for Mendelian genetics over the last 100 years. Our laboratory is interested in the mechanisms that give rise to eye, hair, and skin coloration, both as a tool for studying gene action and interaction, and because many signaling pathways used by the pigmentary system play important roles in human development and disease.
All mammals use the same genetic toolbox, and several mouse coat color mutations have human counterparts such as oculocutaneous albinism or Chediak-Higashi syndrome. Applying the genetics of mouse hair color as a model, however, is relevant not only to rare inborn errors but also to common diseases including diabetes and obesity, neurodegeneration, and skin cancer. Production of normal hair and skin color depends on a series of processes--cell migration, stem cell renewal, paracrine regulation of cell physiology--used in many different contexts throughout the body; pigmentation phenotypes are especially well-suited for studying these processes because mutations are efficiently recognized, subtle effects on gene expression are easily detected, and the cell types and tissues involved are amenable to experimental manipulation.
Our original interest in mouse coat color genetics stems from mutations that cause a back-and-forth switch between pigment granules characteristic of red hair, to those characteristic of black, brown, or blond hair. Studies of these pigment type-switching mutations have identified one set of pathways important for body weight regulation, and another set of pathways implicated in neurodegeneration. Several current projects in the laboratory are directed at specific aspects of these pathways.
Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-high-throughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6-8 million reads. EDGE exhibits very little technical noise, reveals a large (10(6)) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions.
View details for DOI 10.1101/gr.122135.111
View details for Web of Science ID 000296696600014
View details for PubMedID 21844123
View details for PubMedCentralID PMC3205575
Mutations in genes encoding ribosomal proteins cause the Minute phenotype in Drosophila and mice, and Diamond-Blackfan syndrome in humans. Here we report two mouse dark skin (Dsk) loci caused by mutations in Rps19 (ribosomal protein S19) and Rps20 (ribosomal protein S20). We identify a common pathophysiologic program in which p53 stabilization stimulates Kit ligand expression, and, consequently, epidermal melanocytosis via a paracrine mechanism. Accumulation of p53 also causes reduced body size and erythrocyte count. These results provide a mechanistic explanation for the diverse collection of phenotypes that accompany reduced dosage of genes encoding ribosomal proteins, and have implications for understanding normal human variation and human disease.
View details for DOI 10.1038/ng.188
View details for Web of Science ID 000258026900012
View details for PubMedID 18641651
View details for PubMedCentralID PMC3979291
Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as beta-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of beta-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.
View details for PubMedID 17947548
Mutations of pigment type switching have provided basic insight into melanocortin physiology and evolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis of red-yellow pheomelanin vs. black-brown eumelanin. However, in domestic dogs, historical studies based on pedigree and segregation analysis have suggested that the pigment type-switching system is more complicated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador x greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment type switching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominance order of black (K(B)) > brindle (k(br)) > yellow (k(y)), whose genetic map position on dog chromosome 16 is distinct from the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic to variation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles being tested. These findings suggest a molecular model for a new component of the melanocortin signaling pathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.
View details for DOI 10.1534/genetics.107.074237
View details for Web of Science ID 000248416300025
View details for PubMedID 17483404
View details for PubMedID 17522405
Normal aging in humans and rodents is accompanied by a progressive increase in adiposity. To investigate the role of hypothalamic neuronal circuits in this process, we used a Cre-lox strategy to create mice with specific and progressive degeneration of hypothalamic neurons that express agouti-related protein (Agrp) or proopiomelanocortin (Pomc), neuropeptides that promote positive or negative energy balance, respectively, through their opposing effects on melanocortin receptor signaling. In previous studies, Pomc mutant mice became obese, but Agrp mutant mice were surprisingly normal, suggesting potential compensation by neuronal circuits or genetic redundancy. Here we find that Pomc-ablation mice develop obesity similar to that described for Pomc knockout mice, but also exhibit defects in compensatory hyperphagia similar to what occurs during normal aging. Agrp-ablation female mice exhibit reduced adiposity with normal compensatory hyperphagia, while animals ablated for both Pomc and Agrp neurons exhibit an additive interaction phenotype. These findings provide new insight into the roles of hypothalamic neurons in energy balance regulation, and provide a model for understanding defects in human energy balance associated with neurodegeneration and aging.
View details for DOI 10.1371/journal.pbio.0030415
View details for Web of Science ID 000233903800012
View details for PubMedID 16296893
Central control of energy balance depends on the ability of proopiomelanocortin (POMC) or agouti-related protein (Agrp) hypothalamic neurons to sense and respond to changes in peripheral energy stores. Leptin and insulin have been implicated as circulating indicators of adiposity, but it is not clear how changes in their levels are perceived or integrated by individual neuronal subtypes. We developed mice in which a fluorescent reporter for PI3K activity is targeted to either Agrp or POMC neurons and used 2-photon microscopy to measure dynamic regulation of PI3K by insulin and leptin in brain slices. We show that leptin and insulin act in parallel to stimulate PI3K in POMC neurons but in opposite ways on Agrp neurons. These results suggest a new view of hypothalamic circuitry, in which the effects of leptin and insulin are integrated by anorexigenic but not by orexigenic neurons.
View details for Web of Science ID 000228145700026
View details for PubMedID 15761497
A new class of dominant dark skin (Dsk) mutations discovered in a screen of approximately 30,000 mice is caused by increased dermal melanin. We identified three of four such mutations as hypermorphic alleles of Gnaq and Gna11, which encode widely expressed Galphaq subunits, act in an additive and quantitative manner, and require Ednrb. Interactions between Gq and Kit receptor tyrosine kinase signaling can mediate coordinate or independent control of skin and hair color. Our results provide a mechanism that can explain several aspects of human pigmentary variation and show how polymorphism of essential proteins and signaling pathways can affect a single physiologic system.
View details for DOI 10.1038/ng1412
View details for Web of Science ID 000223658100022
View details for PubMedID 15322542
Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (de(H)), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (a(t)) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the de(H) mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in de(H) that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in de(H)/de(H) animals. Construction of a targeted allele of Tbx15 confirmed that the de(H) phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.
View details for PubMedID 14737183
View details for PubMedID 14551921
mahoganoid is a mouse coat-color mutation whose pigmentary phenotype and genetic interactions resemble those of Attractin (Atrn). Atrn mutations also cause spongiform neurodegeneration. Here, we show that a null mutation for mahoganoid causes a similar age-dependent neuropathology that includes many features of prion diseases but without accumulation of protease-resistant prion protein. The gene mutated in mahoganoid encodes a RING-containing protein with E3 ubiquitin ligase activity in vitro. Similarities in phenotype, expression, and genetic interactions suggest that mahoganoid and Atrn genes are part of a conserved pathway for regulated protein turnover whose function is essential for neuronal viability.
View details for Web of Science ID 000180687700048
View details for PubMedID 12560552
Chemical mutagenesis in the mouse is a powerful approach for phenotype-driven genetics, but questions remain about the efficiency with which new mutations ascertained by their phenotype can be localized and identified, and that knowledge applied to a specific biological problem. During a global screen for dominant phenotypes in about 30,000 animals, a novel class of pigmentation mutants were identified by dark skin (Dsk). We determined the genetic map location, homozygous phenotype, and histology of 10 new Dsk and 2 new dark coat (Dcc) mutations, and identified mutations in Agouti (Met1Leu, Dcc4), Sox18 (Leu220ter, Dcc1), Keratin 2e (Thr500Pro, Dsk2), and Egfr (Leu863Gln, Dsk5). Cutaneous effects of most Dsk mutations are limited to melanocytes, except for the Keratin 2e and Egfr mutations, in which hyperkeratosis and epidermal thickening precede epidermal melanocytosis by 3-6 wk. The Dsk2 mutation is likely to impair intermediate filament assembly, leading to cytolysis of suprabasal keratinocytes and secondary hyperkeratosis and melanocytosis. The Dsk5 mutation causes increased tyrosine kinase activity and a decrease in steady-state receptor levels in vivo. The Dsk mutations represent genes or map locations not implicated previously in pigmentation, and delineate a developmental pathway in which mutations can be classified on the basis of body region, microscopic site, and timing of pigment accumulation.
View details for DOI 10.1101/gad.1023703
View details for Web of Science ID 000180496600006
View details for PubMedID 12533510
Agouti protein, a paracrine signaling molecule normally limited to skin, is ectopically expressed in lethal yellow (A(y)) mice, and causes obesity by mimicking agouti-related protein (Agrp), found primarily in the hypothalamus. Mouse attractin (Atrn) is a widely expressed transmembrane protein whose loss of function in mahogany (Atrn(mg-3J)/ Atrn(mg-3J)) mutant mice blocks the pleiotropic effects of A(y). Here we demonstrate in transgenic, biochemical and genetic-interaction experiments that attractin is a low-affinity receptor for agouti protein, but not Agrp, in vitro and in vivo. Additional histopathologic abnormalities in Atrn(mg-3J)/Atrn(mg-3J) mice and cross-species genomic comparisons indicate that Atrn has multiple functions distinct from both a physiologic and an evolutionary perspective.
View details for Web of Science ID 000166187900013
View details for PubMedID 11137996
The role of genetics in obesity is twofold. Studying rare mutations in humans and model organisms provides fundamental insight into a complex physiological process, and complements population-based studies that seek to reveal primary causes. Remarkable progress has been made on both fronts, and the pace of advance is likely to accelerate as functional genomics and the human genome project expand and mature. Approaches based on mendelian and quantitative genetics may well converge, and lead ultimately to more rational and selective therapies.
View details for Web of Science ID 000086400100064
View details for PubMedID 10766251
The melanocortin 1 receptor (Mc1r) is encoded by the Extension locus in many different mammals, where a loss-of-function causes exclusive production of red/yellow pheomelanin, and a constitutively activating mutation causes exclusive production of black/brown eumelanin. In the domestic dog, breeds with a wild-type E allele, e. g., the Doberman, can produce either pigment type, whereas breeds with the e allele, e.g., the Golden Retriever, produce exclusively yellow pigment. However, a black coat color in the Newfoundland and similar breeds is thought to be caused by an unusual allele of Agouti, which encodes the physiologic ligand for the Mc1r. Here we report that the predicted dog Mc1r is 317 residues in length and 96% identical to the fox Mc1r. Comparison of the Doberman, Newfoundland, Black Labrador, Yellow Labrador, Flat-coated Retriever, Irish Setter, and Golden Retriever revealed six sequence variants, of which two, S90G and R306ter, partially correlated with a black/brown coat and red/yellow coat, respectively. R306ter was found in the Yellow Labrador, Golden Retriever, and Irish Setter; the latter two had identical haplotypes but differed from the Yellow Labrador at three positions other than R306ter. In a larger survey of 194 dogs and 19 breeds, R306ter and a red/yellow coat were completely concordant except for the Red Chow. These results indicate that the e allele is caused by a common Mc1r loss-of-function mutation that either reoccurred or was subject to gene conversion during recent evolutionary history, and suggest that the allelic and locus relationships for dog coat color genes may be more analogous to those found in other mammals than previously thought.
View details for Web of Science ID 000084344800005
View details for PubMedID 10602988
Expression of Agouti protein is normally limited to the skin where it affects pigmentation, but ubiquitous expression causes obesity. An expressed sequence tag was identified that encodes Agouti-related protein, whose RNA is normally expressed in the hypothalamus and whose levels were increased eightfold in ob/ob mice. Recombinant Agouti-related protein was a potent, selective antagonist of Mc3r and Mc4r, melanocortin receptor subtypes implicated in weight regulation. Ubiquitous expression of human AGRP complementary DNA in transgenic mice caused obesity without altering pigmentation. Thus, Agouti-related protein is a neuropeptide implicated in the normal control of body weight downstream of leptin signaling.
View details for Web of Science ID A1997XZ12400056
View details for PubMedID 9311920
Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.
View details for DOI 10.1038/ng.3475
View details for Web of Science ID 000369043900012
View details for PubMedID 26691985
Morphological variation in natural populations is a genomic test bed for studying the interface between molecular evolution and population genetics, but some of the most interesting questions involve non-model organisms that lack well annotated reference genomes. Many felid species exhibit polymorphism for melanism but the relative roles played by genetic drift, natural selection, and interspecies hybridization remain uncertain. We identify mutations of Agouti signaling protein (ASIP) or the Melanocortin 1 receptor (MC1R) as independent causes of melanism in three closely related South American species: the pampas cat (Leopardus colocolo), the kodkod (Leopardus guigna), and Geoffroy's cat (Leopardus geoffroyi). To assess population level variation in the regions surrounding the causative mutations we apply genomic resources from the domestic cat to carry out clone-based capture and targeted resequencing of 299 kb and 251 kb segments that contain ASIP and MC1R, respectively, from 54 individuals (13-21 per species), achieving enrichment of ~500-2500-fold and ~150x coverage. Our analysis points to unique evolutionary histories for each of the three species, with a strong selective sweep in the pampas cat, a distinctive but short melanism-specific haplotype in the Geoffroy's cat, and reduced nucleotide diversity for both ancestral and melanism-bearing chromosomes in the kodkod. These results reveal an important role for natural selection in a trait of longstanding interest to ecologists, geneticists, and the lay community, and provide a platform for comparative studies of morphological variation in other natural populations.
View details for DOI 10.1371/journal.pgen.1004892
View details for Web of Science ID 000352081800006
View details for PubMedID 25695801
The ?-defensins are a class of small, cationic proteins first recognized as antimicrobial components of the innate and adaptive immune system. More recently, one of the major ?-defensins produced in skin, ?-defensin 3, has been discovered to function as a melanocortin receptor ligand in vivo and in vitro, but its biophysical and pharmacological basis of action has been enigmatic. Here, we report functional and biochemical studies focused on human ?-defensin 3 (HBD3) and melanocortin receptors 1 and 4. Genetic and pharmacologic studies indicate that HBD3 acts as a neutral melanocortin receptor antagonist capable of blocking the action of either stimulatory agonists such as ?-melanocyte stimulating hormone or inhibitory inverse agonists such as Agouti signaling protein (ASIP) and Agouti-related protein (AGRP). A comprehensive structure-function analysis demonstrates that two patches of positively charged residues, located on opposite poles of HBD3 and spatially organized by the compact ?-defensin fold, are primarily responsible for high-affinity binding to melanocortin receptors. These findings identify a distinct mode of melanocortin receptor-ligand interactions based primarily on electrostatic complementarity, with implications for designing ligands that target melanocortin and potentially other seven transmembrane receptors.
View details for DOI 10.1016/j.chembiol.2013.04.015
View details for Web of Science ID 000321167500007
View details for PubMedID 23790489
Variation in human skin and eye color is substantial and especially apparent in admixed populations, yet the underlying genetic architecture is poorly understood because most genome-wide studies are based on individuals of European ancestry. We study pigmentary variation in 699 individuals from Cape Verde, where extensive West African/European admixture has given rise to a broad range in trait values and genomic ancestry proportions. We develop and apply a new approach for measuring eye color, and identify two major loci (HERC2[OCA2] P = 2.3 × 10(-62), SLC24A5 P = 9.6 × 10(-9)) that account for both blue versus brown eye color and varying intensities of brown eye color. We identify four major loci (SLC24A5 P = 5.4 × 10(-27), TYR P = 1.1 × 10(-9), APBA2[OCA2] P = 1.5 × 10(-8), SLC45A2 P = 6 × 10(-9)) for skin color that together account for 35% of the total variance, but the genetic component with the largest effect (~44%) is average genomic ancestry. Our results suggest that adjacent cis-acting regulatory loci for OCA2 explain the relationship between skin and eye color, and point to an underlying genetic architecture in which several genes of moderate effect act together with many genes of small effect to explain ~70% of the estimated heritability.
View details for DOI 10.1371/journal.pgen.1003372
View details for Web of Science ID 000316866700048
View details for PubMedID 23555287
Prion diseases are rare but invariably fatal neurodegenerative disorders. They are associated with spongiform encephalopathy, a histopathology characterized by the presence of large, membrane-bound vacuolar structures in the neuropil of the brain. While the primary cause is recognized as conversion of the normal form of prion protein (PrP(C)) to a conformationally distinct, pathogenic form (PrP(Sc)), the cellular pathways and mechanisms that lead to spongiform change, neuronal dysfunction and death are not known. Mice lacking the Mahogunin Ring Finger 1 (MGRN1) E3 ubiquitin ligase develop spongiform encephalopathy by 9 months of age but do not become ill. In cell culture, PrP aberrantly present in the cytosol was reported to interact with and sequester MGRN1. This caused endo-lysosomal trafficking defects similar to those observed when Mgrn1 expression is knocked down, implicating disrupted MGRN1-dependent trafficking in the pathogenesis of prion disease. As these defects were rescued by over-expression of MGRN1, we investigated whether reduced or elevated Mgrn1 expression influences the onset, progression or pathology of disease in mice inoculated with PrP(Sc). No differences were observed, indicating that disruption of MGRN1-dependent pathways does not play a significant role in the pathogenesis of transmissible spongiform encephalopathy.
View details for DOI 10.1371/journal.pone.0055575
View details for Web of Science ID 000315563800169
View details for PubMedID 23383230
Like obesity, prolonged food deprivation induces severe hepatic steatosis; however, the functional significance of this phenomenon is not well understood. In this study, we show that the fall in plasma leptin concentration during fasting is required for the development of hepatic steatosis in mice. Removal of leptin receptors from AGRP neurons diminishes fasting-induced hepatic steatosis. Furthermore, the suppressive effects of leptin on fasting-induced hepatic steatosis are absent in mice lacking the gene encoding agouti-related protein (Agrp), suggesting that this function of leptin is mediated by AGRP. Prolonged fasting leads to suppression of hepatic sympathetic activity, increased expression of acyl CoA:diacylglycerol acyltransferase-2 in the liver, and elevation of hepatic triglyceride content and all of these effects are blunted in the absence of AGRP. AGRP deficiency, despite having no effects on feeding or body adiposity in the free-fed state, impairs triglyceride and ketone body release from the liver during prolonged fasting. Furthermore, reducing CNS Agrp expression in wild-type mice by RNAi protected against the development of hepatic steatosis not only during starvation, but also in response to consumption of a high-fat diet. These findings identify the leptin-AGRP circuit as a critical modulator of hepatic triglyceride stores in starvation and suggest a vital role for this circuit in sustaining the supply of energy from the liver to extrahepatic tissues during periods of prolonged food deprivation.
View details for DOI 10.1523/JNEUROSCI.0830-13.2013
View details for PubMedID 23864684
Color variation in companion animals has long been of interest to the breeding and scientific communities. Simple traits, like black versus brown or yellow versus black, have helped to explain principles of transmission genetics and continue to serve as models for studying gene action and interaction. We present a molecular genetic review of pigmentary variation in dogs and cats using a nomenclature and logical framework established by early leaders in the field. For most loci in which molecular variants have been identified (nine in dogs and seven in cats), homologous mutations exist in laboratory mice and/or humans. Exceptions include the K locus in dogs and the Tabby locus in cats, which give rise to alternating stripes or marks of different color, and which illustrate the continued potential of coat color genetics to provide insight into areas that transcend pigment cell biology.
View details for DOI 10.1146/annurev-animal-031412-103659
View details for Web of Science ID 000323479900007
View details for PubMedID 25387014
Pigmentation of the skin, hair, and eyes varies both within and between human populations. Identifying the genes and alleles underlying this variation has been the goal of many candidate gene and several genome-wide association studies (GWAS). Most GWAS for pigmentary traits to date have been based on subjective phenotypes using categorical scales. But skin, hair, and eye pigmentation vary continuously. Here, we seek to characterize quantitative variation in these traits objectively and accurately and to determine their genetic basis. Objective and quantitative measures of skin, hair, and eye color were made using reflectance or digital spectroscopy in Europeans from Ireland, Poland, Italy, and Portugal. A GWAS was conducted for the three quantitative pigmentation phenotypes in 176 women across 313,763 SNP loci, and replication of the most significant associations was attempted in a sample of 294 European men and women from the same countries. We find that the pigmentation phenotypes are highly stratified along axes of European genetic differentiation. The country of sampling explains approximately 35% of the variation in skin pigmentation, 31% of the variation in hair pigmentation, and 40% of the variation in eye pigmentation. All three quantitative phenotypes are correlated with each other. In our two-stage association study, we reproduce the association of rs1667394 at the OCA2/HERC2 locus with eye color but we do not identify new genetic determinants of skin and hair pigmentation supporting the lack of major genes affecting skin and hair color variation within Europe and suggesting that not only careful phenotyping but also larger cohorts are required to understand the genetic architecture of these complex quantitative traits. Interestingly, we also see that in each of these four populations, men are more lightly pigmented in the unexposed skin of the inner arm than women, a fact that is underappreciated and may vary across the world.
View details for DOI 10.1371/journal.pone.0048294
View details for Web of Science ID 000310600500094
View details for PubMedID 23118974
View details for PubMedCentralID PMC3485197
Color markings among felid species display both a remarkable diversity and a common underlying periodicity. A similar range of patterns in domestic cats suggests a conserved mechanism whose appearance can be altered by selection. We identified the gene responsible for tabby pattern variation in domestic cats as Transmembrane aminopeptidase Q (Taqpep), which encodes a membrane-bound metalloprotease. Analyzing 31 other felid species, we identified Taqpep as the cause of the rare king cheetah phenotype, in which spots coalesce into blotches and stripes. Histologic, genomic expression, and transgenic mouse studies indicate that paracrine expression of Endothelin3 (Edn3) coordinates localized color differences. We propose a two-stage model in which Taqpep helps to establish a periodic pre-pattern during skin development that is later implemented by differential expression of Edn3.
View details for DOI 10.1126/science.1220893
View details for Web of Science ID 000308912900051
View details for PubMedID 22997338
Animals display incredibly diverse color patterns yet little is known about the underlying genetic basis of these phenotypes. However, emerging results are reshaping our view of how the process of phenotypic evolution occurs. Here, we outline recent research from three particularly active areas of investigation: melanin pigmentation in Drosophila, wing patterning in butterflies, and pigment variation in lizards. For each system, we highlight (i) the function and evolution of color variation, (ii) various approaches that have been used to explore the genetic basis of pigment variation, and (iii) conclusions regarding the genetic basis of convergent evolution which have emerged from comparative analyses. Results from these studies indicate that natural variation in pigmentation is a particularly powerful tool to examine the molecular basis of evolution, especially with regard to convergent or parallel evolution. Comparison of these systems also reveals that the molecular basis of convergent evolution is heterogeneous, sometimes involving conserved mechanisms and sometimes not. In the near future, additional work in other emerging systems will substantially expand the scope of available comparisons.
View details for DOI 10.1111/j.1755-148X.2012.01014.x
View details for Web of Science ID 000305511200006
View details for PubMedID 22578174
Synchronous activation of neural networks is an important physiological mechanism, and dysregulation of synchrony forms the basis of epilepsy. We analyzed the propagation of synchronous activity through chronically epileptic neural networks. Electrocorticographic recordings from epileptic patients demonstrate remarkable variance in the pathways of propagation between sequential interictal spikes (IISs). Calcium imaging in chronically epileptic slice cultures demonstrates that pathway variance depends on the presence of GABAergic inhibition and that spike propagation becomes stereotyped following GABA receptor blockade. Computer modeling suggests that GABAergic quenching of local network activations leaves behind regions of refractory neurons, whose late recruitment forms the anatomical basis of variability during subsequent network activation. Targeted path scanning of slice cultures confirmed local activations, while ex vivo recordings of human epileptic tissue confirmed the dependence of interspike variance on GABA-mediated inhibition. These data support the hypothesis that the paths by which synchronous activity spreads through an epileptic network change with each activation, based on the recent history of localized activity that has been successfully inhibited.
View details for DOI 10.1523/JNEUROSCI.5853-11.2012
View details for Web of Science ID 000300938100009
View details for PubMedID 22378874
For most of the world, human genome structure at a population level is shaped by interplay between ancient geographic isolation and more recent demographic shifts, factors that are captured by the concepts of biogeographic ancestry and admixture, respectively. The ancestry of non-admixed individuals can often be traced to a specific population in a precise region, but current approaches for studying admixed individuals generally yield coarse information in which genome ancestry proportions are identified according to continent of origin. Here we introduce a new analytic strategy for this problem that allows fine-grained characterization of admixed individuals with respect to both geographic and genomic coordinates. Ancestry segments from different continents, identified with a probabilistic model, are used to construct and study "virtual genomes" of admixed individuals. We apply this approach to a cohort of 492 parent-offspring trios from Mexico City. The relative contributions from the three continental-level ancestral populations-Africa, Europe, and America-vary substantially between individuals, and the distribution of haplotype block length suggests an admixing time of 10-15 generations. The European and Indigenous American virtual genomes of each Mexican individual can be traced to precise regions within each continent, and they reveal a gradient of Amerindian ancestry between indigenous people of southwestern Mexico and Mayans of the Yucatan Peninsula. This contrasts sharply with the African roots of African Americans, which have been characterized by a uniform mixing of multiple West African populations. We also use the virtual European and Indigenous American genomes to search for the signatures of selection in the ancestral populations, and we identify previously known targets of selection in other populations, as well as new candidate loci. The ability to infer precise ancestral components of admixed genomes will facilitate studies of disease-related phenotypes and will allow new insight into the adaptive and demographic history of indigenous people.
View details for DOI 10.1371/journal.pgen.1002410
View details for Web of Science ID 000299167900027
View details for PubMedID 22194699
Reduced gene dosage of ribosomal protein subunits has been implicated in 5q- myelodysplastic syndrome and Diamond Blackfan anemia, but the cellular and pathophysiologic defects associated with these conditions are enigmatic. Using conditional inactivation of the ribosomal protein S6 gene in laboratory mice, we found that reduced ribosomal protein gene dosage recapitulates cardinal features of the 5q- syndrome, including macrocytic anemia, erythroid hypoplasia, and megakaryocytic dysplasia with thrombocytosis, and that p53 plays a critical role in manifestation of these phenotypes. The blood cell abnormalities are accompanied by a reduction in the number of HSCs, a specific defect in late erythrocyte development, and suggest a disease-specific ontogenetic pathway for megakaryocyte development. Further studies of highly purified HSCs from healthy patients and from those with myelodysplastic syndrome link reduced expression of ribosomal protein genes to decreased RBC maturation and suggest an underlying and common pathophysiologic pathway for additional subtypes of myelodysplastic syndrome.
View details for DOI 10.1182/blood-2010-11-318584
View details for Web of Science ID 000295359300028
View details for PubMedID 21788341
View details for PubMedCentralID PMC3186336
View details for Web of Science ID 000294019200225
Insulin and leptin intracellular signaling pathways converge and act synergistically on the hypothalamic phosphatidylinositol-3-OH kinase/3-phosphoinositide-dependent protein kinase 1 (PDK1). However, little is known about whether PDK1 in agouti-related peptide (AGRP) neurons contributes to energy homeostasis. We generated AGRP neuron-specific PDK1 knockout (AGRPPdk1(-/-)) mice and mice with selective expression of transactivation-defective Foxo1 (?256Foxo1(AGRP)Pdk1(-/-)). The AGRPPdk1(-/-) mice showed reductions in food intake, body length, and body weight. The ?256Foxo1(AGRP)Pdk1(-/-) mice showed increased body weight, food intake, and reduced locomotor activity. After four weeks of calorie-restricted feeding, oxygen consumption and locomotor activity were elevated in AGRPPdk1(-/-) mice and reduced in ?256Foxo1(AGRP)Pdk1(-/-) mice. In vitro, ghrelin-induced changes in [Ca(2+)](i) and inhibition of ghrelin by leptin were significantly attenuated in AGRPPdk1(-/-) neurons compared to control neurons. However, ghrelin-induced [Ca(2+)](i) changes and leptin inhibition were restored in ?256Foxo1(AGRP)Pdk1(-/-) mice. These results suggested that PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms.
View details for DOI 10.1371/journal.pone.0018324
View details for Web of Science ID 000289238700008
View details for PubMedID 21694754
Agouti-related protein (AgRP) and agouti signaling protein (ASIP) are homologs that play critical roles in energy balance and pigmentation, respectively, by functioning as antagonistic ligands at their cognate melanocortin receptors. Signaling specificity is mediated in part through receptor binding selectivity brought about by alterations in the cysteine-rich carboxy-terminal domains of the ligands. AgRP binds with high affinity to the melanocortin 3 receptor and the melanocortin 4 receptor, but not to the melanocortin 1 receptor (MC1R), whereas ASIP binds with high affinity to all three receptors. This work explores the structural basis for receptor selectivity by studying chimeric proteins developed by interchanging loops between the cysteine-rich domain of ASIP and the cysteine-rich domain of AgRP. Binding data demonstrate that melanocortin 4 receptor responds to all chimeras and is therefore highly tolerant of gross loop changes. By contrast, MC1R responds primarily to those chimeras with a sequence close to that of wild-type ASIP. Further analysis of binding and functional data suggests that the ASIP C-terminal loop (a six-amino-acid segment closed by the final disulfide bond) is essential for high-affinity MC1R binding and inverse agonism. Comparison with previously published molecular models suggests that this loop makes contact with the first extracellular loop of MC1R through a series of key hydrophobic interactions.
View details for DOI 10.1016/j.jmb.2010.08.054
View details for Web of Science ID 000284674000004
View details for PubMedID 20831872
View details for Web of Science ID 000289662200195
Hair color and skin color are frequently coordinated in mammalian species. To explore this, we have studied mutations in two different G protein coupled pathways, each of which affects the darkness of both hair and skin color. In each mouse mutant (Gnaq(Dsk1), Gna11(Dsk7), and Mc1r(e)), we analyzed the melanocyte density and the concentrations of eumelanin (black pigment) and pheomelanin (yellow pigment) in the hair or skin to determine the mechanisms regulating pigmentation. Surprisingly, we discovered that each mutation affects hair and skin color differently. Furthermore, we have found that in the epidermis, the melanocortin signaling pathway does not couple the synthesis of eumelanin with pheomelanin, as it does in hair follicles. Even by shared signaling pathways, hair and skin melanocytes are regulated quite independently.
View details for DOI 10.1111/j.1755-148X.2009.00609.x
View details for Web of Science ID 000270830600017
View details for PubMedID 19627560
PI3K signaling is thought to mediate leptin and insulin action in hypothalamic pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, key regulators of energy homeostasis, through largely unknown mechanisms. We inactivated either p110alpha or p110beta PI3K catalytic subunits in these neurons and demonstrate a dominant role for the latter in energy homeostasis regulation. In POMC neurons, p110beta inactivation prevented insulin- and leptin-stimulated electrophysiological responses. POMCp110beta null mice exhibited central leptin resistance, increased adiposity, and diet-induced obesity. In contrast, the response to leptin was not blocked in p110alpha-deficient POMC neurons. Accordingly, POMCp110alpha null mice displayed minimal energy homeostasis abnormalities. Similarly, in AgRP neurons, p110beta had a more important role than p110alpha. AgRPp110alpha null mice displayed normal energy homeostasis regulation, whereas AgRPp110beta null mice were lean, with increased leptin sensitivity and resistance to diet-induced obesity. These results demonstrate distinct metabolic roles for the p110alpha and p110beta isoforms of PI3K in hypothalamic energy regulation.
View details for DOI 10.1016/j.cmet.2009.09.008
View details for Web of Science ID 000271498700004
View details for PubMedID 19883613
Melanocortin-1 receptor (MC1R) and its ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan-a non agouti (a/a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200-fold increases in the pheomelanin to eumelanin ratio, and a tan-yellow color in pelletted cells. Moreover, ASIP-treated cells showed reduced proliferation and a melanoblast-like appearance, seen also in melanocyte lines from yellow (A(y)/a and Mc1r(e)/ Mc1r(e)) mice. However ASIP-YY, a C-terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP-YY inhibited the cAMP rise induced by alphaMSH analog NDP-MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrn(mg-3J/mg-3J) or Mgrn1(md-nc/md-nc)) also responded to both ASIP and ASIP-YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP-MC1R signaling includes a cAMP-independent pathway through attractin and mahogunin, while the known cAMP-dependent component requires neither attractin nor mahogunin.
View details for DOI 10.1111/j.1755-148X.2009.00582.x
View details for Web of Science ID 000269390800016
View details for PubMedID 19493315
Genome-wide scans for recent positive selection in humans have yielded insight into the mechanisms underlying the extensive phenotypic diversity in our species, but have focused on a limited number of populations. Here, we present an analysis of recent selection in a global sample of 53 populations, using genotype data from the Human Genome Diversity-CEPH Panel. We refine the geographic distributions of known selective sweeps, and find extensive overlap between these distributions for populations in the same continental region but limited overlap between populations outside these groupings. We present several examples of previously unrecognized candidate targets of selection, including signals at a number of genes in the NRG-ERBB4 developmental pathway in non-African populations. Analysis of recently identified genes involved in complex diseases suggests that there has been selection on loci involved in susceptibility to type II diabetes. Finally, we search for local adaptation between geographically close populations, and highlight several examples.
View details for DOI 10.1101/gr.087577.108
View details for Web of Science ID 000265668800016
View details for PubMedID 19307593
View details for PubMedCentralID PMC2675971
Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color variation that contributes to camouflage, species recognition, and morphologic diversity. X-linked determinants of this pattern--recognized by variegation in females but not in males--have been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow (Sly), but are curiously absent from other vertebrate species. Using a comparative genomic approach, we develop molecular markers and a linkage map for the euchromatic region of the Syrian hamster X chromosome that places Sly in a region homologous to the centromere-proximal region of human Xp. Comparison to analogous work carried out for Orange in domestic cats indicates, surprisingly, that the cat and hamster mutations lie in nonhomologous regions of the X chromosome. We also identify the molecular cause of recessively inherited black coat color in hamsters (historically referred to as nonagouti) as a Cys115Tyr mutation in the Agouti gene. Animals doubly mutant for Sly and nonagouti exhibit a Sly phenotype. Our results indicate that Sly represents a melanocortin pathway component that acts similarly to, but is genetically distinct from, Mc1r and that has implications for understanding both the evolutionary history and the mutational mechanisms of pigment-type switching.
View details for DOI 10.1534/genetics.108.095018
View details for Web of Science ID 000270213700022
View details for PubMedID 19189957
Morphological diversity within closely related species is an essential aspect of evolution and adaptation. Mutations in the Melanocortin 1 receptor (Mc1r) gene contribute to pigmentary diversity in natural populations of fish, birds, and many mammals. However, melanism in the gray wolf, Canis lupus, is caused by a different melanocortin pathway component, the K locus, that encodes a beta-defensin protein that acts as an alternative ligand for Mc1r. We show that the melanistic K locus mutation in North American wolves derives from past hybridization with domestic dogs, has risen to high frequency in forested habitats, and exhibits a molecular signature of positive selection. The same mutation also causes melanism in the coyote, Canis latrans, and in Italian gray wolves, and hence our results demonstrate how traits selected in domesticated species can influence the morphological diversity of their wild relatives.
View details for DOI 10.1126/science.1165448
View details for Web of Science ID 000263876700041
View details for PubMedID 19197024
BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown. Here we report frequent somatic mutations in the heterotrimeric G protein alpha-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention.
View details for DOI 10.1038/nature07586
View details for Web of Science ID 000262852200045
View details for PubMedID 19078957
Named originally for their effects on peripheral end organs, the melanocortin system controls a diverse set of physiological processes through a series of five G-protein-coupled receptors and several sets of small peptide ligands. The central melanocortin system plays an essential role in homeostatic regulation of body weight, in which two alternative ligands, alpha-melanocyte-stimulating hormone and agouti-related protein, stimulate and inhibit receptor signaling in several key brain regions that ultimately affect food intake and energy expenditure. Much of what we know about the relationship between central melanocortin signaling and body weight regulation stems from genetic studies. Comparative genomic studies indicate that melanocortin receptors used for controlling pigmentation and body weight regulation existed more than 500 million years ago in primitive vertebrates, but that fine-grained control of melanocortin receptors through neuropeptides and endogenous antagonists developed more recently. Recent studies based on dog coat-color genetics revealed a new class of melanocortin ligands, the beta-defensins, which reveal the potential for cross talk between the melanocortin and the immune systems.
View details for DOI 10.1038/ijo.2008.234
View details for Web of Science ID 000262376700005
View details for PubMedID 19136986
Two known types of leptin-responsive neurons reside within the arcuate nucleus: the agouti gene-related peptide (AgRP)/neuropeptide Y (NPY) neuron and the proopiomelanocortin (POMC) neuron. By deleting the leptin receptor gene (Lepr) specifically in AgRP/NPY and/or POMC neurons of mice, we examined the several and combined contributions of these neurons to leptin action. Body weight and adiposity were increased by Lepr deletion from AgRP and POMC neurons individually, and simultaneous deletion in both neurons (A+P LEPR-KO mice) further increased these measures. Young (periweaning) A+P LEPR-KO mice exhibit hyperphagia and decreased energy expenditure, with increased weight gain, oxidative sparing of triglycerides, and increased fat accumulation. Interestingly, however, many of these abnormalities were attenuated in adult animals, and high doses of leptin partially suppress food intake in the A+P LEPR-KO mice. Although mildly hyperinsulinemic, the A+P LEPR-KO mice displayed normal glucose tolerance and fertility. Thus, AgRP/NPY and POMC neurons each play mandatory roles in aspects of leptin-regulated energy homeostasis, high leptin levels in adult mice mitigate the importance of leptin-responsiveness in these neurons for components of energy balance, suggesting the presence of other leptin-regulated pathways that partially compensate for the lack of leptin action on the POMC and AgRP/NPY neurons.
View details for DOI 10.1210/en.2007-1132
View details for Web of Science ID 000254264100037
View details for PubMedID 18162515
View details for Web of Science ID 000254353800656
View details for Web of Science ID 000255061700038
Nearly all neurodegenerative diseases are associated with abnormal accumulation of ubiquitin (Ub) conjugates within neuronal inclusion bodies. To directly test the hypothesis that depletion of cellular Ub is sufficient to cause neurodegeneration, we have disrupted Ubb, one of four genes that supply Ub in the mouse. Here, we report that loss of Ubb led to a progressive degenerative disorder affecting neurons within the arcuate nucleus of the hypothalamus. This neurodegenerative cytopathology was accompanied by impaired hypothalamic control of energy balance and adult-onset obesity. Ubb was highly expressed in vulnerable hypothalamic neurons and total Ub levels were selectively reduced in the hypothalamus of Ubb-null mice. These findings demonstrate that maintenance of adequate supplies of cellular Ub is essential for neuronal survival and establish that decreased Ub availability is sufficient to cause neuronal dysfunction and death.
View details for DOI 10.1073/pnas.0800096105
View details for Web of Science ID 000253930600064
View details for PubMedID 18299572
Leptin, an adipocyte-derived hormone, acts on hypothalamic neurons located in the arcuate nucleus (ARC) of the hypothalamus to regulate energy homeostasis. One of the leptin-regulated neuronal subtypes in the ARC are agouti-related peptide (AgRP)-expressing neurons, which are involved in the regulation of food intake and are directly inhibited by leptin. Leptin activates the signal transducer and activator of transcription 3 (Stat3), but the role of Stat3 in the regulation of AgRP neurons is unclear. Here we show that mice expressing a constitutively active version of Stat3 selectively in AgRP neurons are lean and exhibit relative resistance to diet-induced obesity. Surprisingly, this phenotype arises from increased locomotor activity in the presence of unaltered AgRP expression. These data demonstrate that Stat3-dependent signaling in AgRP neurons in the ARC controls locomotor activity independently of AgRP regulation.
View details for DOI 10.1016/j.cmet.2008.01.007
View details for Web of Science ID 000253727300010
View details for PubMedID 18316029
Human genetic diversity is shaped by both demographic and biological factors and has fundamental implications for understanding the genetic basis of diseases. We studied 938 unrelated individuals from 51 populations of the Human Genome Diversity Panel at 650,000 common single-nucleotide polymorphism loci. Individual ancestry and population substructure were detectable with very high resolution. The relationship between haplotype heterozygosity and geography was consistent with the hypothesis of a serial founder effect with a single origin in sub-Saharan Africa. In addition, we observed a pattern of ancestral allele frequency distributions that reflects variation in population dynamics among geographic regions. This data set allows the most comprehensive characterization to date of human genetic variation.
View details for DOI 10.1126/science.1153717
View details for Web of Science ID 000253311700046
View details for PubMedID 18292342
Manipulation of gene expression in melanocytes is an important tool for studying pigment cell biology. We constructed transgenic mice in which Cre recombinase was placed under the control of regulatory elements from the Microphthalmia-associated transcriptional factor (Mitf) gene using bacterial artificial chromosome (BAC). Bacterial artificial chromosome that contained either 50 or 108 kb DNA 5' to the melanocyte-specific (1M) transcriptional start site gave rise to transgenic lines in which Cre is expressed specifically in cells of the melanocyte lineage, as judged by activation of the Gt(Rosa)26(tm1Sor)(R26R) reporter locus. Activation of R26R is first detectable in melanoblasts of midgestation embryos, and completely marks all melanocyte components of the skin in postnatal animals. To test the utility of the MitfCre transgene, we used a loxP-targeted allele of the protein kinase A alpha catalytic subunit (Prkaca), modified such that Cre-mediated recombination activates PKA signaling. On an agouti background, animals carrying both the MitfCre transgene and the targeted Prkaca allele (CalphaR) exhibited a darker coat color than control littermates, due to a shift from pheomelanin to eumelanin synthesis. Our results confirm that PKA signaling is a key component of pigment type-switching, and provide a new tool for studying pigment cell biology.
View details for DOI 10.1111/j.1755-148X.2007.00425.x
View details for Web of Science ID 000252879900010
View details for PubMedID 18353144
Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.
View details for DOI 10.1128/MCB.00771-07
View details for Web of Science ID 000251925300024
View details for PubMedID 17967885
View details for PubMedCentralID PMC2223308
View details for Web of Science ID 000264425300029
Attractin (ATRN) and Attractin-like 1 (ATRNL1) are highly similar type I transmembrane proteins. Atrn null mutant mice have a pleiotropic phenotype including dark fur, juvenile-onset spongiform neurodegeneration, hypomyelination, tremor, and reduced body weight and adiposity, implicating ATRN in numerous biological processes. Bioinformatic analysis indicated that Atrn and Atrnl1 arose from a common ancestral gene early in vertebrate evolution. To investigate the genetics of the ATRN system and explore potential redundancy between Atrn and Atrnl1, we generated and characterized Atrnl1 loss- and gain-of-function mutations in mice. Atrnl1 mutant mice were grossly normal with no alterations of pigmentation, central nervous system pathology or body weight. Atrn null mutant mice carrying a beta-actin promoter-driven Atrnl1 transgene had normal, agouti-banded hairs and significantly delayed onset of spongiform neurodegeneration, indicating that over-expression of ATRNL1 compensates for loss of ATRN. Thus, the two genes are redundant from the perspective of gain-of-function but not loss-of-function mutations.
View details for DOI 10.1002/dvg.20351
View details for Web of Science ID 000252307200003
View details for PubMedID 18064672
Mutations in the transcription factor Foxn1 cause the nude phenotype in mice, which is characterized by a lack of visible hair. New work by Weiner et al. (2007) in this issue of Cell now shows that Foxn1 also contributes to hair color by marking which cells are to receive pigment from melanocytes.
View details for DOI 10.1016/j.cell.2007.08.032
View details for Web of Science ID 000249581500010
View details for PubMedID 17803901
Agouti-related protein encodes a neuropeptide that stimulates food intake. Agrp expression in the brain is restricted to neurons in the arcuate nucleus of the hypothalamus and is elevated by states of negative energy balance. The molecular mechanisms underlying Agrp regulation, however, remain poorly defined. Using a combination of transgenic and comparative sequence analysis, we have previously identified a 760 bp conserved region upstream of Agrp which contains STAT binding elements that participate in Agrp transcriptional regulation. In this study, we attempt to improve the specificity for detecting conserved elements in this region by comparing genomic sequences from 10 mammalian species. Our analysis reveals a symmetrical organization of conserved sequences upstream of Agrp, which cluster into two inverted repeat elements. Conserved sequences within these elements suggest a role for homeodomain proteins in the regulation of Agrp and provide additional targets for functional evaluation.
View details for DOI 10.1371/journal.pone.0000702
View details for Web of Science ID 000207452400006
View details for PubMedID 17684549
Hypothalamic AMP-activated protein kinase (AMPK) has been suggested to act as a key sensing mechanism, responding to hormones and nutrients in the regulation of energy homeostasis. However, the precise neuronal populations and cellular mechanisms involved are unclear. The effects of long-term manipulation of hypothalamic AMPK on energy balance are also unknown. To directly address such issues, we generated POMC alpha 2KO and AgRP alpha 2KO mice lacking AMPK alpha2 in proopiomelanocortin- (POMC-) and agouti-related protein-expressing (AgRP-expressing) neurons, key regulators of energy homeostasis. POMC alpha 2KO mice developed obesity due to reduced energy expenditure and dysregulated food intake but remained sensitive to leptin. In contrast, AgRP alpha 2KO mice developed an age-dependent lean phenotype with increased sensitivity to a melanocortin agonist. Electrophysiological studies in AMPK alpha2-deficient POMC or AgRP neurons revealed normal leptin or insulin action but absent responses to alterations in extracellular glucose levels, showing that glucose-sensing signaling mechanisms in these neurons are distinct from those pathways utilized by leptin or insulin. Taken together with the divergent phenotypes of POMC alpha 2KO and AgRP alpha 2KO mice, our findings suggest that while AMPK plays a key role in hypothalamic function, it does not act as a general sensor and integrator of energy homeostasis in the mediobasal hypothalamus.
View details for DOI 10.1172/JCI31516
View details for Web of Science ID 000248478100040
View details for PubMedID 17671657
Insulin action in the central nervous system regulates energy homeostasis and glucose metabolism. To define the insulin-responsive neurons that mediate these effects, we generated mice with selective inactivation of the insulin receptor (IR) in either pro-opiomelanocortin (POMC)- or agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus. While neither POMC- nor AgRP-restricted IR knockout mice exhibited altered energy homeostasis, insulin failed to normally suppress hepatic glucose production during euglycemic-hyperinsulinemic clamps in AgRP-IR knockout (IR(DeltaAgRP)) mice. These mice also exhibited reduced insulin-stimulated hepatic interleukin-6 expression and increased hepatic expression of glucose-6-phosphatase. These results directly demonstrate that insulin action in POMC and AgRP cells is not required for steady-state regulation of food intake and body weight. However, insulin action specifically in AgRP-expressing neurons does play a critical role in controlling hepatic glucose production and may provide a target for the treatment of insulin resistance in type 2 diabetes.
View details for DOI 10.1016/j.cmet.2007.05.004
View details for Web of Science ID 000247115400007
View details for PubMedID 17550779
A null mutation in the gene encoding the putative E3 ubiquitin-protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.
View details for DOI 10.1091/mbc.E06-09-0787
View details for Web of Science ID 000245443100001
View details for PubMedID 17229889
Genetic variation at the melanocortin 1 receptor (MC1R) is an important risk factor for developing ultraviolet (UV) radiation-induced skin cancer, the most common form of cancer in humans. The underlying mechanisms by which the MC1R defends against UV-induced skin cancer are not known. We used neonatal mouse skin (which, like human skin, contains a mixture of melanocytes and keratinocytes) to study how pigment cells and Mc1r genotype affect the genome-level response to UV radiation. Animals without viable melanocytes (Kit(W-v)/Kit(W-v)) or animals lacking a functional Mc1r (Mc1r(e)/Mc1r(e)) were exposed to sunburn-level doses of UVB radiation, and the patterns of large-scale gene expression in the basal epidermis were compared to each other and to nonmutant animals. Our analysis revealed discrete Kit- and Mc1r-dependent UVB transcriptional responses in the basal epidermis. The Kit-dependent UVB response was characterized largely by an enrichment of oxidative and endoplasmic reticulum stress genes, highlighting a distinctive role for pigmented melanocytes in mediating antioxidant defenses against genotoxic stresses within the basal epidermal environment. By contrast, the Mc1r-dependent UVB response contained an abundance of genes associated with regulating the cell cycle and oncogenesis. To test the clinical relevance of these observations, we analyzed publicly available data sets for primary melanoma and melanoma metastases and found that the set of genes specific for the Mc1r-dependent UVB response was able to differentiate between different clinical subtypes. Our analysis also revealed that the classes of genes induced by UVB differ from those repressed by UVB with regard to their biological functions, their overall number, and their size. The findings described here offer new insights into the transcriptional nature of the UV response in the skin and provide a molecular framework for the underlying mechanisms by which melanocytes and the Mc1r independently mediate and afford protection against UV radiation.
View details for DOI 10.1371/journal.pgen.0030009
View details for Web of Science ID 000243838600005
View details for PubMedID 17222061
With the goal of increasing the number of genetic entry points for studying physiologic processes and human disease, large-scale, systematic, chemical mutagenesis projects in mice have been initiated in several different centers. We have been studying mouse mutants that exhibit dominantly inherited defects in either skin and/or hair color. Here, we describe a bright coat color mutant, Bright coat color 1 (Bcc1), which develops light-colored hair at 4 weeks of age, and when homozygous exhibits oral leukoplakia and blistering, and growth retardation. We identified a missense mutation in mutant animals that predicts an N154S amino-acid substitution in the 1A domain of Keratin 4 (encoded by the Krt2-4 gene), a region known to be mutated in human patients with white sponge nevus (WSN). Bcc1 recapitulates the gross pathologic, histologic, and genetic aspects of the human disorder, WSN.
View details for DOI 10.1038/sj.jid.5700498;
View details for Web of Science ID 000243192200010
View details for PubMedID 16858417
Leptin is an adipocyte-derived hormone that signals body energy status to the brain by acting on multiple neuronal subgroups in the hypothalamus, including those that express proopiomelanocortin (Pomc) and agouti-related protein (Agrp). Signal transducer and activator of transcription 3 (Stat3) is an important intracellular signaling molecule activated by leptin, and previous studies have shown that mice carrying a mutated leptin receptor that abolished Stat3 binding are grossly obese. To determine the extent to which Stat3 signaling in Pomc neurons was responsible for these effects, we constructed Pomc-specific Stat3 mutants using a Cre recombinase transgene driven by the Pomc promoter. We find that Pomc expression is diminished in the mutant mice, suggesting that Stat3 is required for Pomc transcription. Pomc-specific Stat3 female mutant mice exhibit a 2-fold increase in fat pad mass but only a slight increase in total body weight. Mutant mice remain responsive to leptin-induced hypophagia and are not hypersensitive to a high-fat diet; however, mutant mice fail to mount a normal compensatory refeeding response. These results demonstrate a requirement for Stat3 in transcriptional regulation of Pomc but indicate that this circuit is only one of several components that underlie the neuronal response to leptin and the role of Stat3 in that response.
View details for DOI 10.1210/en.2006-1119
View details for Web of Science ID 000242935200010
View details for PubMedID 17023536
Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.
View details for DOI 10.1016/j.chembiol.2006.10.006
View details for Web of Science ID 000243323600008
View details for PubMedID 17185225
Energy homeostasis depends on the regulation of hypothalamic neurons by leptin, an adipocyte hormone whose circulating levels communicate body energy stores. Leptin activates the transcription factor signal transducer and activator of transcription 3 (Stat3) in hypothalamic neurons, including neuronal subtypes producing Agouti-related protein (Agrp), a neuropeptide that stimulates feeding. Previous studies have suggested a model in which high levels of Agrp transcription during fasting represent a default state that is actively repressed by phospho-Stat3 induced by leptin signaling in the fed state. We identify putative Stat3 binding elements in the Agrp promoter that have been highly conserved during vertebrate evolution. Using a reporter assay in transgenic mice that faithfully recapitulates normal regulation of Agrp, we show that these sites are required, but in a way opposite to that predicted by the existing model: mutation of the sites leads to a default state characterized by a low level of Agrp transcription and insensitivity to fasting. We also find that removing activatable Stat3 from Agrp neurons has no detectable effect on steady-state levels of Agrp mRNA in the fed or fasted state. These results suggest a new model for transcriptional regulation of orexigenic neuropeptides in which the default level of expression is low in the fed state, and transcriptional activation in response to fasting is mediated by factors other than Stat3.
View details for DOI 10.1210/me.2006-0107
View details for Web of Science ID 000240785100028
View details for PubMedID 16709597
The capacity to adjust food intake in response to changing energy requirements is essential for survival. Recent progress has provided an insight into the molecular, cellular and behavioural mechanisms that link changes of body fat stores to adaptive adjustments of feeding behaviour. The physiological importance of this homeostatic control system is highlighted by the severe obesity that results from dysfunction of any of several of its key components. This new information provides a biological context within which to consider the global obesity epidemic and identifies numerous potential avenues for therapeutic intervention and future research.
View details for DOI 10.1038/nature05026
View details for Web of Science ID 000240622000036
View details for PubMedID 16988703
Genitopatellar syndrome is a newly described disorder characterized by absent/hypoplastic patellae, lower extremity contractures, urogenital anomalies, dysmorphic features, skeletal anomalies, and agenesis of the corpus callosum. More recently, cardiac anomalies and ectodermal dysplasia have been suggested as additional features of this syndrome. We report on two additional patients with genitopatellar syndrome and expand the spectrum of anomalies to include radio-ulnar synostosis. Since there exists significant overlap in the skeletal phenotype between genitopatellar syndrome and both the nail-patella and short patella syndromes, mutation screening of their causative genes, LMX1B and TBX4, was performed. Although there still does not appear to be an identifiable molecular etiology in genitopatellar syndrome, mutations in these two candidate genes have been excluded in our patients. Since both LMX1B and TBX4 are involved in a common molecular pathway, it is likely that the causative gene of genitopatellar syndrome functions within the same developmental process.
View details for PubMedID 16761293
The melanocortin 1 receptor (Mc1r) plays a central role in cutaneous biology, but is expressed at very low levels by a small fraction of cells in the skin. In humans, loss-of-function MC1R mutations cause fair skin, freckling, red hair, and increased predisposition to melanoma; in mice, Mc1r loss-of-function is responsible for the recessive yellow mutation, associated with pheomelanic hair and a decreased number of epidermal melanocytes. To better understand how Mc1r signaling affects different cutaneous phenotypes, we examined large-scale patterns of gene expression in different skin components (whole epidermal sheets, basal epidermal cells and whole skins) of neonatal (P2.5) normal and recessive yellow mice, starting with a 26K mouse cDNA microarray. From c. 17 000 genes whose levels could be accurately measured in neonatal skin, we identified 883, 2097 and 552 genes that were uniquely expressed in the suprabasal epidermis, basal epidermis and dermis, respectively; specific biologic roles could be assigned for each class. Comparison of normal and recessive yellow mice revealed 69 differentially expressed genes, of which the majority had not been previously implicated in Mc1r signaling. Surprisingly, many of the Mc1r-dependent genes are expressed in cells other than melanocytes, even though Mc1r expression in the skin is confined almost exclusively to epidermal melanocytes. These results reveal new targets for Mc1r signaling, and point to a previously unappreciated role for a Mc1r-dependent paracrine effect of melanocytes on other components of the skin.
View details for DOI 10.1111/j.1600-0749.2006.00305.x
View details for Web of Science ID 000237597600003
View details for PubMedID 16704453
Leptin controls food intake by regulating the transcription of key neuropeptides in the hypothalamus. The mechanism by which leptin regulates gene expression is unclear, however. Here we show that delivery of adenovirus encoding a constitutively nuclear mutant FoxO1, a transcription factor known to control liver metabolism and pancreatic beta-cell function, to the hypothalamic arcuate nucleus of rodents results in a loss of the ability of leptin to curtail food intake and suppress expression of Agrp. Conversely, a transactivation-deficient FoxO1 mutant prevents induction of Agrp by fasting. We also find that FoxO1 and the transcription factor Stat3 exert opposing actions on the expression of Agrp and Pomc through transcriptional squelching. FoxO1 promotes opposite patterns of coactivator-corepressor exchange at the Pomc and Agrp promoters, resulting in activation of Agrp and inhibition of Pomc. Thus, FoxO1 represents a shared component of pathways integrating food intake and peripheral metabolism.
View details for DOI 10.1038/nm1392
View details for Web of Science ID 000238149100037
View details for PubMedID 16604086
Chemical mutagenesis in the mouse has increased the utility of phenotype-driven genetics as a means for studying different organ systems, developmental pathways, and pathologic processes. From a large-scale screen for dominant phenotypes in mice, a novel class of pigmentation mutants was identified by dark skin (Dsk). We describe a Dsk mutant, Dsk12, which models the human disease, epidermolytic hyperkeratosis (EHK). At 2 days of age, mutant animals exhibit intraepidermal blisters and erosions at sites of trauma, and by 2 weeks of age develop significant hyperkeratosis. We identified a missense mutation in mutant animals that predicts an S194P amino acid substitution in the 1A domain of Keratin 1, a known target for human mutations that cause EHK. Dsk12 recapitulates the gross pathologic, histologic, and genetic aspects of the human disorder, EHK.
View details for DOI 10.1038/sj.jid.5700241
View details for Web of Science ID 000238968700016
View details for PubMedID 16528356
View details for Web of Science ID 000242891500897
Multiple hormones controlling energy homeostasis regulate the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus. Nevertheless, inactivation of the genes encoding NPY and/or AgRP has no impact on food intake in mice. Here we demonstrate that induced selective ablation of AgRP-expressing neurons in adult mice results in acute reduction of feeding, demonstrating direct evidence for a critical role of these neurons in the regulation of energy homeostasis.
View details for DOI 10.1038/nn1548
View details for Web of Science ID 000232145900009
View details for PubMedID 16158063
In human obesity, the stroma vascular fraction (SVF) of white adipose tissue (WAT) is enriched in macrophages. These cells may contribute to low-grade inflammation and to its metabolic complications. Little is known about the effect of weight loss on macrophages and genes involved in macrophage attraction. We examined subcutaneous WAT (scWAT) of 7 lean and 17 morbidly obese subjects before and 3 months after bypass surgery. Immunomorphological changes of the number of scWAT-infiltrating macrophages were evaluated, along with concomitant changes in expression of SVF-overexpressed genes. The number of scWAT-infiltrating macrophages before surgery was higher in obese than in lean subjects (HAM56+/CD68+; 22.6 +/- 4.3 vs. 1.4 +/- 0.6%, P < 0.001). Typical "crowns" of macrophages were observed around adipocytes. Drastic weight loss resulted in a significant decrease in macrophage number (-11.63 +/- 2.3%, P < 0.001), and remaining macrophages stained positive for the anti-inflammatory protein interleukin 10. Genes involved in macrophage attraction (monocyte chemotactic protein [MCP]-1, plasminogen activator urokinase receptor [PLAUR], and colony-stimulating factor [CSF]-3) and hypoxia (hypoxia-inducible factor-1alpha [HIF-1alpha]), expression of which increases in obesity and decreases after surgery, were predominantly expressed in the SVF. We show that improvement of the inflammatory profile after weight loss is related to a reduced number of macrophages in scWAT. MCP-1, PLAUR, CSF-3, and HIF-1alpha may play roles in the attraction of macrophages in scWAT.
View details for Web of Science ID 000230869500002
View details for PubMedID 16046292
View details for PubMedID 16251053
The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.
View details for DOI 10.1242/dev.01735
View details for Web of Science ID 000228592900011
View details for PubMedID 15728667
View details for Web of Science ID 000228179901442
The type of pigment synthesized in mammalian hair, yellow-red pheomelanin or black-brown eumelanin, depends on the interaction between Agouti protein and the Melanocortin 1 receptor. Although the genetics of pigmentation is broadly conserved across most mammalian species, pigment type-switching in domestic dogs is unusual because a yellow-tan coat with variable amounts of dark hair is thought to be caused by an allele of the Agouti locus referred to as fawn or sable (a(y)). In a large survey covering thirty seven breeds, we identified an Agouti allele with two missense alterations, A82S and R83H, which was present (heterozygous or homozygous) in 41 dogs (22 breeds) with a fawn or sable coat, but was absent from 16 dogs (8 breeds) with a black-and-tan or tricolor phenotype. In an additional 33 dogs (14 breeds) with a eumelanic coat, 8 (German Shepherd Dogs, Groenendaels, Schipperkes, or Shetland Sheepdogs) were homozygous for a previously reported mutation, non-agouti R96C; the remainder are likely to have carried dominant black, which is independent of and epistatic to Agouti. This work resolves some of the complexity in dog coat color genetics and provides diagnostic opportunities and practical guidelines for breeders.
View details for DOI 10.1007/s00335-004-2445-6
View details for Web of Science ID 000229019800005
View details for PubMedID 15965787
Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in beta cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and beta cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced beta cell mass. Overt diabetes did not ensue, because beta cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced beta cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in beta cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis.
View details for Web of Science ID 000228145700025
View details for PubMedID 15841180
Expression of the agouti signaling protein (ASIP) during hair growth produces the red/yellow pigment pheomelanin. ASIP, and its neuropeptide homolog the agouti-related protein (AgRP) involved in energy balance, are novel, paracrine signaling molecules that act as inverse agonists at distinct subsets of melanocortin receptors. Ubiquitous ASIP expression in mice gives rise to a pleiotropic phenotype characterized by a uniform yellow coat color, obesity, overgrowth, and metabolic derangements similar to type II diabetes in humans. Here we report the synthesis and NMR structure of ASIP's active, cysteine-rich, C-terminal domain. ASIP adopts the inhibitor cystine knot fold and, along with AgRP, are the only known mammalian proteins in this structure class. Moreover, ASIP populates two distinct conformers resulting from a cis peptide bond at Pro102-Pro103 and a coexistence of cis/trans isomers of Ala104-Pro105. Pharmacologic studies of Pro-->Ala mutants demonstrate that the minor conformation with two cis peptide bonds is responsible for activity at all MCRs. The loop containing the heterogeneous Ala-Pro peptide bond is conserved in mammals, and suggests that ASIP is either trapped by evolution in this unusual configuration or possesses function outside of strict MCR antagonism.
View details for DOI 10.1016/j.jmb.2004.12.030
View details for Web of Science ID 000227187800010
View details for PubMedID 15701517
The acute-phase proteins, serum amyloid As (SAA), are precursors of amyloid A, involved in the pathogenesis of AA amyloidosis. This work started with the characterisation of systemic AA amyloidosis concurrent with SAA overexpression in the subcutaneous white adipose tissue (sWAT) of an obese patient with a leptin receptor deficiency. In the present study a series of histopathological, cellular and gene expression studies was performed to assess the importance of SAA in common obesity and its possible production by mature adipocytes.Gene expression profiling was performed in the sWAT of two extremely obese patients with a leptin receptor deficiency. Levels of the mRNAs of the different SAA isoforms were quantified in sWAT cellular fractions from lean subjects and from obese subjects before and after a very-low-calorie diet. These values were subsequently compared with serum levels of SAA in these individuals. In addition, histopathological analyses of sWAT were performed in lean and obese subjects.In sWAT, the expression of SAA is more than 20-fold higher in mature adipocytes than in the cells of the stroma vascular fraction (p<0.01). Levels of SAA mRNA expression and circulating levels of the protein are sixfold (p<0.001) and 3.5-fold (p<0.01) higher in obese subjects than in lean subjects, respectively. In lean subjects, 5% of adipocytes are immunoreactive for SAA, whereas the corresponding value is greater than 20% in obese subjects. Caloric restriction results in decreases of 45-75% in levels of the transcripts for the SAA isoforms and in circulating levels of the protein.The results of the present study indicate that SAA is expressed by sWAT, and its production at this site is regulated by nutritional status. If amyloidosis is seen in the context of obesity, it is possible that production of SAA by adipocytes could be a contributory factor.
View details for DOI 10.1007/s00125-004-1654-6
View details for Web of Science ID 000227741800015
View details for PubMedID 15729583
Agouti-related protein (Agrp) encodes a hypothalamic neuropeptide that promotes positive energy balance by stimulating food intake and reducing energy expenditure. Agrp expression in the brain is restricted to neurons within the arcuate nucleus of the hypothalamus, and expression levels are elevated as a consequence of food deprivation. We tested a series of bacterial artificial chromosome reporter constructs with varying amounts of sequence flanking the Agrp transcription unit in transgenic mice to identify and refine a region of DNA capable of recapitulating characteristics of Agrp expression. We report that a 42.5-kb region upstream of Agrp, containing three distinct regions that are evolutionarily conserved between mouse and human, is necessary and sufficient to consistently drive reporter expression specifically within AgRP neurons in a fasting-responsive manner. In addition, we demonstrate that this region allows for the stable expression of Cre recombinase in transgenic mice, providing a genetic tool for studying anabolic neural circuits that control energy balance.
View details for DOI 10.1210/en.2004-0956
View details for Web of Science ID 000225109400046
View details for PubMedID 15345681
Disruption of melanocortin (MC) signaling, such as by ectopic Agouti overexpression, leads to an obesity syndrome with hyperphagia, obesity, and accelerated body weight gain during high-fat diet. To investigate where in the brain disruption of MC signaling results in obesity, long-term Agouti expression was induced after local injections of recombinant adeno-associated viral particles in selected brain nuclei of adult rats. Agouti expression in the paraventricular nucleus, a hypothalamic region with a high density of MC receptors, induced acute onset hyperphagia and rapid weight gain that persisted for at least 6 weeks. In contrast, obesity and hyperphagia developed with a 3 week delay when Agouti was expressed in the dorsal medial hypothalamus. Agouti expression in the lateral hypothalamus (LH) did not affect food intake and body weight during regular diet, despite the presence of MC receptors in this region. However, during exposure to a high-fat diet, animals with Agouti expression in the LH exhibited a marked increase in body weight. Here we show that the LH is important for the protection against diet-induced obesity by controlling caloric intake during consumption of a high-fat diet. Together, this study provides evidence that different aspects of the Agouti-induced obesity syndrome, such as hyperphagia and diet responsiveness, are mediated by distinct brain regions and opens challenging opportunities for further understanding of pathophysiological processes in the development of the obesity syndrome.
View details for DOI 10.1523/JNEUROSCI.3442-04.2004
View details for Web of Science ID 000225022600018
View details for PubMedID 15537888
Adipose tissue produces inflammation and immunity molecules suspected to be involved in obesity-related complications. The pattern of expression and the nutritional regulation of these molecules in humans are poorly understood. We analyzed the gene expression profiles of subcutaneous white adipose tissue from 29 obese subjects during very low calorie diet (VLCD) using cDNA microarray and reverse transcription quantitative PCR. The patterns of expression were compared with that of 17 non-obese subjects. We determined whether the regulated genes were expressed in adipocytes or stromavascular fraction cells. Gene expression profiling identified 100 inflammation-related transcripts that are regulated in obese individuals when eating a 28 day VLCD but not a 2 day VLCD. Cluster analysis showed that the pattern of gene expression in obese subjects after 28 day VLCD was closer to the profile of lean subjects than to the pattern of obese subjects before VLCD. Weight loss improves the inflammatory profile of obese subjects through a decrease of proinflammatory factors and an increase of anti-inflammatory molecules. The genes are expressed mostly in the stromavascular fraction of adipose tissue, which is shown to contain numerous macrophages. The beneficial effect of weight loss on obesity-related complications may be associated with the modification of the inflammatory profile in adipose tissue.
View details for DOI 10.1096/fj.04-2204com
View details for Web of Science ID 000225482100034
View details for PubMedID 15522911
The interaction between two genes, Agouti and Melanocortin-1 receptor ( Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs ( Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98% identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat.
View details for DOI 10.1007/s00335-004-23778-1
View details for Web of Science ID 000224054000005
View details for PubMedID 15520882
The stress hormone epinephrine produces major physiological effects on skeletal muscle. Here we determined skeletal muscle mRNA expression profiles before and during a 6-h epinephrine infusion performed in nine young men. Stringent statistical analysis of data obtained using 43000 cDNA element microarrays showed that 1206 and 474 genes were up- and down-regulated, respectively. Microarray data were validated using reverse transcription quantitative PCR. Gene classification was performed through data mining of Gene Ontology annotations, cluster analysis of regulated genes among 14 human tissues, and correlation analysis of mRNA and clinical parameter variations. Evidence of an autoregulatory control was provided by the regulation of key genes of the cAMP-dependent transcription pathway. Genes with known functional cAMP response elements were regulated by the hormone. The impact on metabolism was illustrated by coordinated regulations of genes involved in carbohydrate and protein metabolisms. Epinephrine had a profound effect on genes involved in immunity and inflammatory response, a previously unappreciated aspect of catecholamine action. Information on 526 mRNAs corresponded to genes of unknown function. These data define the molecular signatures of epinephrine action in human skeletal muscle. They may contribute to the understanding of skeletal muscle alterations observed in pathological conditions characterized by sympathetic nervous system overdrive.
View details for DOI 10.1210/jc.2003-031733
View details for Web of Science ID 000221220100003
View details for PubMedID 15126512
View details for Web of Science ID 000220470700279
View details for Web of Science ID 000188254600316
View details for PubMedID 14534576
Both central alpha-melanocyte-stimulating hormone and corticotropin-releasing hormone (CRH) have been implicated in feeding and neuroendocrine mechanisms. The anatomical overlap and functional similarities between these two neurotransmitter systems led to the hypothesis that CRH might act as one of the mediators of the central actions of the melanocortin system. By double-labeling in situ hybridization, a subpopulation of CRH neurons in the paraventricular nucleus of the hypothalamus (PVN) were shown to contain the melanocortin-4 receptor (MC4R), concentrated in the ventromedial part of the parvicellular PVN (up to 33%). Intracerebroventricular injection of melanocortin agonist MTII to conscious and freely moving rats induced a rapid induction of CRH gene transcription in the PVN. This effect was accompanied by a rise in plasma corticosterone levels in a dose- and time-dependent manner, with the maximum response observed 30 min after MTII injection. MTII (0.5 nmol)-induced increase in plasma corticosterone was attenuated by the selective MC4R antagonist HS014 (0.25-1.0 nmol) and nonselective CRH receptor antagonist alpha-helical-CRH9-41 (0.125-0.5 nmol) in a dose-dependent manner. Moreover, the anorectic effect of MTII was evaluated at 1, 2, and 24 hr after intracerebroventricular injection. Approximately half of the inhibitory effect of MTII (0.5 nmol) on food intake was reversed by pretreatment with alpha-helical-CRH9-41 at 0.25 and 0.5 nmol doses. Collectively, these results provide evidence that CRH acts as a downstream mediator of melanocortin signaling and contributes to the mechanisms by which the central melanocortin system controls feeding and neuroendocrine responses.
View details for Web of Science ID 000185001700018
View details for PubMedID 12944516
Insulin action in target tissues involved precise regulation of gene expression. To define the set of insulin-regulated genes in human skeletal muscle, we analyzed the global changes in mRNA levels during a 3-h hyperinsulinemic euglycemic clamp in vastus lateralis muscle of six healthy subjects. Using 29,308 cDNA element microarrays, we found that the mRNA expression of 762 genes, including 353 expressed sequence tags, was significantly modified during insulin infusion. 478 were up-regulated and 284 down-regulated. Most of the genes with known function are novel targets of insulin. They are involved in the transcriptional and translational regulation (29%), intermediary and energy metabolisms (14%), intracellular signaling (12%), and cytoskeleton and vesicle traffic (9%). Other categories consisted of genes coding for receptors, carriers, and transporters (8%), components of the ubiquitin/proteasome pathways (7%) and elements of the immune response (5.5%). These results thus define a transcriptional signature of insulin action in human skeletal muscle. They will help to better define the mechanisms involved in the reduction of insulin effectiveness in pathologies such as type 2 diabetes mellitus, a disease characterized by defective regulation of gene expression in response to insulin.
View details for PubMedID 12621037
Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.
View details for DOI 10.1016/S0196-9781(03)00104-9
View details for Web of Science ID 000184329600014
View details for PubMedID 12860205
We describe a model of energy homeostasis to better understand neuronal pathways that control energy balance and their regulation by hormonal signals such as insulin and leptin. Catabolic neuronal pathways are those that both reduce food intake and increase energy expenditure (e.g., melanocortin neurons in the hypothalamic arcuate nucleus) and are stimulated by input from insulin and leptin. We propose that in the basal state, catabolic effectors are activated in response to physiological concentrations of leptin and insulin, and that this activation is essential to prevent excessive weight gain. In contrast, anabolic pathways (e.g., neurons containing neuropeptide Y) are those that stimulate food intake and decrease energy expenditure and are strongly inhibited by these same basal concentrations of insulin and leptin. In the basal state, therefore, catabolic effector pathways are activated while anabolic effector pathways are largely inhibited. The response to weight loss includes both activation of anabolic and inhibition of catabolic pathways and is, thus, inherently more vigorous than the response to weight gain (stimulation of already-activated catabolic pathways and inhibition of already-suppressed anabolic pathways). Teleological, molecular, physiological, and clinical aspects of this hypothesis are presented, along with a discussion of currently available supporting evidence.
View details for Web of Science ID 000180891700002
View details for PubMedID 12540591
The domestic dog exhibits a variety of coat colors that encompass a wide range of variation among different breeds. Very little is known about the molecular biology of dog pigmentation; current understanding is based mostly on traditional breeding experiments, which in some cases have suggested genetic interactions that are different from those reported in other mammals. We have examined the molecular genetics of dominant black, a uniform coat color characteristic of black Labrador retrievers or Newfoundlands that has been proposed to be caused by either variation in the melanocortin-1 receptor gene (Mc1r) or by variation in the Agouti gene (A). We identified several coding polymorphisms within Mc1r and several simple sequence repeat polymorphisms closely linked to A, and examined their inheritance in a Labrador retriever x greyhound cross that segregates dominant black. No single Mc1r allele was found consistently in animals carrying dominant black, and neither Mc1r nor A cosegregated with dominant black. These results refine our understanding of mammalian coat color inheritance and suggest that dominant black coat color in dogs is caused by a gene not previously implicated in pigment type switching.
View details for DOI 10.1093/jhered/esg016
View details for Web of Science ID 000182413500012
View details for PubMedID 12692166
Black mask is a characteristic pattern in which red, yellow, tan, fawn, or brindle dogs exhibit a melanistic muzzle which may extend up onto the ears. Melanistic mask is inherited in several breeds as an autosomal dominant trait, and appears to be a fixed trait in a few breeds of dogs. A MC1R nonsense mutation, R306ter, has been shown to cause a completely red or yellow coat color in certain breeds such as Irish setters, yellow Labrador retrievers, and golden retrievers. The amino acid sequence for the melanocortin receptor 1 gene (MC1R) was examined in 17 dogs with melanistic masks from seven breeds, 19 dogs without melanistic masks, and 7 dogs in which their coat color made the mask difficult to distinguish. We also examined nine brindle dogs of four breeds, including three dogs who also had a black mask. No consistent amino acid change was observed in the brindle dogs. All dogs with a melanistic mask had at least one copy of a valine substitution for methionine at amino acid 264 (M264V) and none were homozygous for the premature stop codon (R306ter). These results suggest that black mask, but not brindle, is caused by a specific MC1R allele.
View details for DOI 10.1093/jhered/esg014
View details for Web of Science ID 000182413500011
View details for PubMedID 12692165
Switching from eumelanin to pheomelanin synthesis during hair growth is accomplished by transient synthesis of Agouti protein, an inverse agonist for the melanocortin-1 receptor (Mc1r). The coat color mutations mahogany and mahoganoid prevent hair follicle melanocytes from responding to Agouti protein. The gene mutated in mahogany, which is also known as Attractin (Atrn), encodes a type I transmembrane protein that functions as an accessory receptor for Agouti protein. We have recently determined that the gene mutated in mahoganoid, which is also known as Mahogunin (Mgrn1), encodes an E3 ubiquitin ligase. Like Attractin, Mahogunin is conserved in invertebrate genomes, and its absence causes a pleiotropic phenotype that includes spongiform neurodegeneration.
View details for Web of Science ID 000184303800037
View details for PubMedID 12851328
The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. We recently determined the NMR structure of AGRP(87-132) and demonstrated that a portion of the domain adopts the inhibitor cystine-knot fold. Remarkably, this is the first identification of a mammalian protein with this specific architecture. Further analysis of the structure suggests that melanocortin receptor contacts are made primarily by two loops presented within the cystine knot. (10) To test this hypothesis we designed a 34-residue AGRP analogue corresponding to only the cystine knot. We found that this designed miniprotein folds to a homogeneous product, retains the desired cystine-knot architecture, functions as a potent antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). (26) The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine-knot region possesses the melanocortin receptor contacts. Based on these design and structure studies, we propose that the N-terminal loop of AGRP(87-132) makes contact with a receptor exoloop and helps confer AGRP's selectivity for the central MCRs.
View details for Web of Science ID 000184303800004
View details for PubMedID 12851295
In the present study we examined the diurnal patterns of agouti-related protein (AGRP) and proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus and their relation to circulating glucocorticoids and food intake. Animals were killed at 4-h intervals throughout the 24-h diurnal cycle, and the expression of AGRP and POMC mRNA was evaluated by semiquantitative in situ hybridization analysis. We observed a significant diurnal rhythm in AGRP mRNA expression, with a marked peak at 2200 h (4 h after lights off) and a trough at 1000 h (4 h after lights on), consistent with the overall day-night rhythm of food intake. In contrast, POMC mRNA levels did not show a significant fluctuation across the diurnal cycle, although there was a tendency for levels to decrease after the onset of the dark cycle. Corticosterone secretion temporally coincided with the rising phase of AGRP mRNA expression. Depletion of corticosterone by adrenalectomy abolished the AGRP diurnal rhythm by suppressing the nighttime expression, but did not alter the feeding rhythm. Exposure of adrenalectomized rats to constant corticosterone replacement (10 or 50 mg continuous release corticosterone pellet) resulted in fixed AGRP mRNA expression throughout the 12-h light, 12-h dark cycle. A relatively high level of corticosterone (50 mg) significantly increased AGRP mRNA expression, with a positive correlation between these two measures. These results indicate that 1) the diurnal expression of AGRP mRNA is regulated by corticosterone independently of the light/dark cue; and 2) a normal endogenous corticosterone rhythm is required for generating the diurnal AGRP rhythm.
View details for DOI 10.1210/en.2002-220150
View details for Web of Science ID 000178212800024
View details for PubMedID 12239102
Molecular components of the glomerular filtration mechanism play critical roles in renal diseases. Many of these components are produced during the final stages of differentiation of glomerular visceral epithelial cells, also known as podocytes. While basic domain leucine zipper (bZip) transcription factors of the Maf subfamily have been implicated in cellular differentiation processes, Kreisler (Krml1/MafB), the gene affected in the mouse kreisler (kr) mutation, is known for its role in hindbrain patterning. Here we show that mice homozygous for the kr(enu) mutation develop renal disease and that Kreisler is essential for cellular differentiation of podocytes. Consistent with abnormal podocyte differentiation, kr(enu) homozygotes show proteinuria, and fusion and effacement of podocyte foot processes, which are also observed in the nephrotic syndrome. Kreisler acts during the final stages of glomerular development-the transition between the capillary loop and mature stages-and downstream of the Pod1 basic domain helix-loop-helix transcription factor. The levels of Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome (NPHS2), and Nephrin, the gene mutated in congenital nephrotic syndrome of the Finnish type (NPHS1), are slightly reduced in kr(enu)/kr(enu) podocytes. However, these observations alone are unlikely to account for the aberrant podocyte foot process formation. Thus, Kreisler must regulate other unknown genes required for podocyte function and with possible roles in kidney disease.
View details for DOI 10.1006/dbio.2002.0751
View details for Web of Science ID 000177924600002
View details for PubMedID 12217315
View details for Web of Science ID 000177428100797
The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The NMR structure of this active domain was recently determined and suggested that melanocortin receptor contacts were made primarily by two loops presented by a well-structured cystine knot domain within AGRP(87-132) [McNulty et al. (2001) Biochemistry 40, 15520-15527]. This hypothesis is tested here with NMR structure and activity studies of a 34-residue AGRP analogue designed to contain only the cystine knot domain. The designed miniprotein folds to a homogeneous product, retains the desired cystine knot architecture, functions as an antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine knot region possesses the melanocortin receptor contact points. Moreover, this potent AGRP analogue is synthetically accessible, may serve in the development of therapeutics for the treatment of diseases related to energy balance. and may also find use as a new reagent for probing melanocortin receptor structure and function.
View details for DOI 10.1021/bi012000x
View details for Web of Science ID 000176156700005
View details for PubMedID 12056887
The metalloprotease-disintegrin family, or ADAM, proteins, are implicated in cell-cell interactions, cell fusion, and cell signaling, and are widely distributed among metazoan phyla. Orthologous relationships have been defined for a few ADAM proteins including ADAM10 (Kuzbanian), and ADAM17 (TACE), but evolutionary relationships are not clear for the majority of family members. Human ADAM33 refers to a testis cDNA clone that does not contain a complete open reading frame, but portions of the predicted protein are similar to Xenopus laevis ADAM13.In a 48 kb region of mouse DNA adjacent to the Attractin gene on mouse chromosome 2, we identified sequences very similar to human ADAM33. A full-length mouse cDNA was identified by a combination of gene prediction programs and RT-PCR, and the probable full-length human cDNA was identified by comparison to human genomic sequence in the homologous region on chromosome 20p13. Mouse ADAM33 is 44% identical to Xenopus laevis ADAM13, however a phylogenetic alignment and consideration of functional domains suggests that the two genes are not orthologous. Mouse Adam33 is widely expressed, most highly in the adult brain, heart, kidney, lung and testis.While mouse ADAM33 is similar to Xenopus ADAM13 in sequence, further examination of its embryonic expression pattern, catalytic activity and protein interactions will be required to assess the functional relationship between these two proteins. Adam33 is expressed in the mouse adult brain and could play a role in complex processes that require cell-cell communication.
View details for Web of Science ID 000179726900001
View details for PubMedID 11897009
Pleiotropic effects of melanocortin signaling were first described nearly 100 years ago when mice carrying the lethal yellow (A(y)) allele of the Agouti coat color gene were recognized to develop increased growth and adiposity. Work from our laboratory and others over the last several years has demonstrated that the non-pigmentary effects of A(y) are caused by ectopic expression of Agouti protein, a paracrine signaling molecule whose normal function is to inhibit signaling through the melanocortin 1 receptor (Mc1r), but which can mimic the effects of Agouti-related protein (Agrp), a homologous neuropeptide produced in the medial portion of the arcuate nucleus that acts as a potent antagonist of the Mc3r and Mc4r. Recently we have used the genetics of pigmentation as an in vivo screening system to analyze other mutations in the Agouti-melanocortin pathway, leading to the identification of Attractin (Atrn), a widely expressed type I transmembrane protein that serves as an accessory receptor for Agouti protein. Surprisingly, homologs of Atrn are found in fruitflies and nematodes, even though Agouti and/or Agouti-related protein are found only in vertebrates. Insight into this apparent paradox now comes from studies of different Atrn alleles, in which we find hyperactivity, abnormal myelination, and widespread CNS vacuolation. We suggest that the neurodegenerative phenotype reflects the ancestral function of Atrn to facilitate and/or maintain cell-cell interactions in the nervous system. Expression in neurectodermal cells during vertebrate evolution may have allowed Atrn to be recruited by the Agouti-melanocortin system to control coat color.
View details for DOI 10.1081/RRS-120014588
View details for Web of Science ID 000179631000006
View details for PubMedID 12503608
Thyroid hormones are key regulators of metabolism that modulate transcription via nuclear receptors. Hyperthyroidism is associated with increased metabolic rate, protein breakdown, and weight loss. Although the molecular actions of thyroid hormones have been studied thoroughly, their pleiotropic effects are mediated by complex changes in expression of an unknown number of target genes. Here, we measured patterns of skeletal muscle gene expression in five healthy men treated for 14 days with 75 microg of triiodothyronine, using 24,000 cDNA element microarrays. To analyze the data, we used a new statistical method that identifies significant changes in expression and estimates the false discovery rate. The 381 up-regulated genes were involved in a wide range of cellular functions including transcriptional control, mRNA maturation, protein turnover, signal transduction, cellular trafficking, and energy metabolism. Only two genes were down-regulated. Most of the genes are novel targets of thyroid hormone. Cluster analysis of triiodothyronine-regulated gene expression among 19 different human tissues or cell lines revealed sets of coregulated genes that serve similar biologic functions. These results define molecular signatures that help to understand the physiology and pathophysiology of thyroid hormone action.
View details for Web of Science ID 000173689600008
View details for PubMedID 11827947
The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic (appetite-stimulating) activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), contains five disulfide bonds and exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The three-dimensional structure of this domain has been determined by 1H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel beta sheet, where the last two strands form a beta hairpin. The relative spatial positioning of the disulfide cross-links demonstrates that the ordered region of AGRP(87-132) adopts the inhibitor cystine knot (ICK) fold previously identified for numerous invertebrate toxins. Interestingly, this may be the first example of a mammalian protein assigned to the ICK superfamily. The hairpin's turn region presents a triplet of residues (Arg-Phe-Phe) known to be essential for melanocortin receptor binding. The structure also suggests that AGRP possesses an additional melanocortin-receptor contact region within a loop formed by the first 16 residues of its C-terminal domain. This specific region shows little sequence homology to the corresponding region of the agouti protein, which is an MC1R antagonist involved in pigmentation. Consideration of these sequence differences, along with recent experiments on mutant and chimeric melanocortin receptors, allows us to postulate that this loop in the first 16 residues of its C-terminal domain confers AGRP's distinct selectivity for MC3R and MC4R.
View details for DOI 10.1021/bi0117192
View details for Web of Science ID 000173153300009
View details for PubMedID 11747427
To simplify the analysis of asthma susceptibility genes located at human chromosome 5q23-35, we examined congenic mice that differed at the homologous chromosomal segment. We identified a Mendelian trait encoded by T cell and Airway Phenotype Regulator (Tapr). Tapr is genetically distinct from known cytokine genes and controls the development of airway hyperreactivity and T cell production of interleukin 4 (IL-4) and IL-13. Positional cloning identified a gene family that encodes T cell membrane proteins (TIMs); major sequence variants of this gene family (Tim) completely cosegregated with Tapr. The human homolog of TIM-1 is the hepatitis A virus (HAV) receptor, which may explain the inverse relationship between HAV infection and the development of atopy.
View details for DOI 10.1038/ni739
View details for Web of Science ID 000172473200012
View details for PubMedID 11725301
The melanocyte lineage potentially forms an attractive model system for studies in cell differentiation, developmental genetics, cell signaling, and melanoma, because differentiated cells produce the visible pigment melanin. Immortal lines of murine melanoblasts (melanocyte precursors) have been described previously, but induction of differentiation involved a complex culture system with keratinocyte feeder cells. Here we describe conditions for both growth and induced differentiation of the melanoblast line melb-a, without feeder cells, and analyze factors that directly control proliferation and differentiation of these pure melanoblasts. Several active factors are products of developmental and other coat color genes, including stem cell factor (SCF), melanocyte-stimulating hormone (alphaMSH), and agouti signaling protein (ASP), a natural antagonist at the MSH receptor (melanocortin 1 receptor, MC1R) encoded by the agouti gene. A stable analog of alphaMSH (NDP-MSH) stimulated differentiation and inhibited growth. ASP in excess inhibited both effects of NDP-MSH, that is, ASP could inhibit pigmentation and stimulate growth. These effects provide an explanation for the interactions in mice of melanocyte developmental mutations with yellow agouti and Mc1r alleles, and a role for embryonic expression patterns of ASP.
View details for Web of Science ID 000170149600002
View details for PubMedID 11500974
Mutations of the mouse Attractin (Atrn; formerly mahogany) gene were originally recognized because they suppress Agouti pigment type switching. More recently, effects independent of Agouti have been recognized: mice homozygous for the Atrn(mg-3J) allele are resistant to diet-induced obesity and also develop abnormal myelination and vacuolation in the central nervous system. To better understand the pathophysiology and relationship of these pleiotropic effects, we further characterized the molecular abnormalities responsible for two additional Atrn alleles, Atrn(mg) and Atrn(mg-L), and examined in parallel the phenotypes of homozygous and compound heterozygous animals. We find that the three alleles have similar effects on pigmentation and neurodegeneration, with a relative severity of Atrn(mg-3J) > Atrn(mg) > Atrn(mg-L), which also corresponds to the effects of the three alleles on levels of normal Atrn mRNA. Animals homozygous for Atrn(mg-3J) or Atrn(mg), but not Atrn(mg-L), show reduced body weight, reduced adiposity, and increased locomotor activity, all in the presence of normal food intake. These results confirm that the mechanism responsible for the neuropathological alteration is a loss--rather than gain--of function, indicate that abnormal body weight in Atrn mutant mice is caused by a central process leading to increased energy expenditure, and demonstrate that pigmentation is more sensitive to levels of Atrn mRNA than are nonpigmentary phenotypes.
View details for Web of Science ID 000170603700024
View details for PubMedID 11514456
Transgenic expression in the hypothalamus of syndecan-1, a cell surface heparan sulfate proteoglycan (HSPG) and modulator of ligand-receptor encounters, produces mice with hyperphagia and maturity-onset obesity resembling mice with reduced action of alpha melanocyte stimulating hormone (alphaMSH). Via their HS chains, syndecans potentiate the action of agouti-related protein and agouti signaling protein, endogenous inhibitors of alphaMSH. In wild-type mice, syndecan-3, the predominantly neural syndecan, is expressed in hypothalamic regions that control energy balance. Food deprivation increases hypothalamic syndecan-3 levels several-fold. Syndecan-3 null mice, otherwise apparently normal, respond to food deprivation with markedly reduced reflex hyperphagia. We propose that oscillation of hypothalamic syndecan-3 levels physiologically modulates feeding behavior.
View details for Web of Science ID 000169870300013
View details for PubMedID 11461706
The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with (&agr;)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.
View details for Web of Science ID 000167569000019
View details for PubMedID 11181184
To gain insight into the role of Oa1, the mouse homolog of the human X-linked ocular albinism 1 protein, its properties and subcellular localization were investigated. Antiserum raised against an expressed segment of the Oa1 protein recognized a band of approximately 48 kDa in immunoblots of extracts of cultured mouse melan-a melanocytes, but not of cells of non-melanocyte origin. When melanocyte extracts were treated with glycopeptidase F, a approximately 44 kDa band appeared. Like the melanogenic enzyme tyrosinase, expression of Oa1 was stimulated by alpha-melanocyte stimulating hormone and inhibited by agouti signal protein. Upon density gradient centrifugation of organelles of melan-a cells, Oa1 protein colocalized with the late endosomal/lysosomal marker Lamp1, but only partial overlap was observed with melanosomal proteins in the high density region of the gradient. Immunofluorescence staining revealed that neither endogenous Oa1 nor an Oa1-green fluorescent protein fusion product colocalized with the melanosomal protein tyrosinase related protein-1 in the cell periphery. In contrast, colocalization of Oa1 and Oa1-green fluorescent protein fusion product with Lamp1 was extensive throughout the cell. These results indicate that Oa1 is a melanocyte-specific integral membrane glycoprotein localized to late endosomes/lysosomes but not mature melanosomes. Considering the microscopic findings in patients with X-linked ocular albinism 1, we speculate that Oa1 may play a role in the trafficking of vesicles to developing melanosomes.
View details for Web of Science ID 000167393700013
View details for PubMedID 11180981
The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.
View details for Web of Science ID 000166816400034
View details for PubMedID 11217851
The rat zitter (zi) mutation induces hypomyelination and vacuolation in the central nervous system (CNS), which result in early-onset tremor and progressive flaccid paresis. By positional cloning, we found a marked decrease in Attractin (Atrn) mRNA in the brain of the zi/zi rat and identified zi as an 8-bp deletion at a splice donor site of Atrn. Atrn has been known to play multiple roles in regulating physiological processes that are involved in monocyte-T cell interaction, agouti-related hair pigmentation, and control of energy homeostasis. Rat Atrn gene encoded two isoforms, a secreted and a membrane form, as a result of alternative splicing. The zi mutation at the Atrn locus darkened coat color when introduced into agouti rats, as also described in mahogany (mg) mice, carrying the homozygous mutation at the Atrn locus. Transgenic rescue experiments showed that the membrane-type Atrn complemented both neurological alteration and abnormal pigmentation in zi/zi rats, but that the secreted-type Atrn complemented neither mutant phenotype. Furthermore, we discovered that mg mice exhibited hypomyelination and vacuolation in the CNS associated with body tremor. We conclude from these results that the membrane Atrn has a critical role in normal myelination in the CNS and would provide insights into the physiology of myelination as well as the etiology of myelin diseases.
View details for Web of Science ID 000166485300038
View details for PubMedID 11209055
View details for PubMedCentralID PMC14626
The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.
View details for Web of Science ID 000166430900011
View details for PubMedID 11024027
View details for Web of Science ID 000089876100127
A novel RIA was used to examine the release of agouti-related protein-like immunoreactivity (AGRP-LI) from perfused rat hypothalamic tissue slices and to characterize AGRP-LI in rat serum. A continuous low level basal AGRP-LI release was observed from hypothalami of rats fed ad libitum before the rats were killed. Basal AGRP-LI release was 3-fold greater in rats fasted 48 h. In fasted animals leptin dose-dependently suppressed basal AGRP-LI release. In fed animals no change in basal AGRP-LI release was detected in response to 10(-6) M alpha-MSH, orexin B, melanin-concentrating hormone, or serotonin. HPLC analysis of AGRP-LI in rat serum identified a single peak that eluted in close proximity to synthetic AGRP (87-132) and mouse [Leu127Pro]AGRP and that was identical to the peak seen in hypothalamic and adrenal tissue extracts. The serum concentration of AGRP-LI in rats fed ad libitum was 0.865+/-0.323 nmol/liter (mean +/- SE). Food deprivation resulted in a slow, but statistically significant rise in serum immunoreactivity at 48 h [1.174+/-0.118 nmol/liter (mean +/- SE)]. Bilateral adrenalectomy did not change serum levels of AGRP-LI. These studies demonstrate that in the rat there are different levels of basal hypothalamic AGRP-LI release in fed and fasted states and that in the fasted rat this release can be profoundly suppressed by leptin. These studies also suggest that AGRP is present in the systemic circulation of rats.
View details for Web of Science ID 000088386100007
View details for PubMedID 10830275
Attractin, initially identified as a soluble human plasma protein with dipeptidyl peptidase IV activity that is expressed and released by activated T lymphocytes, also has been identified as the product of the murine mahogany gene with connections to control of pigmentation and energy metabolism. The mahogany product, however, is a transmembrane protein, raising the possibility of a human membrane attractin in addition to the secreted form. The genomic structure of human attractin reveals that soluble attractin arises from transcription of 25 sequential exons on human chromosome 20p13, where the 3' terminal exon contains sequence from a long interspersed nuclear element-1 (LINE-1) retrotransposon element that includes a stop codon and a polyadenylation signal. The mRNA isoform for membrane attractin splices over the LINE-1 exon and includes five exons encoding transmembrane and cytoplasmic domains with organization and coding potential almost identical to that of the mouse gene. The relative abundance of soluble and transmembrane isoforms measured by reverse transcription-PCR is differentially regulated in lymphoid tissues. Because activation of peripheral blood leukocytes with phytohemagglutinin induces strong expression of cell surface attractin followed by release of soluble attractin, these results suggest that a genomic event unique to mammals, LINE-1 insertion, has provided an evolutionary mechanism for regulating cell interactions during an inflammatory reaction.
View details for Web of Science ID 000087318700064
View details for PubMedID 10811918
The mouse mahogany mutation affects melanocortin signaling pathways that regulate energy homeostasis and hair color. The gene mutated in mahogany mice encodes attractin, a large transmembrane protein that is broadly expressed and conserved among multicellular animals. Mouse attractin is likely to have additional roles outside melanocortin signaling, and cloning of the gene provides information that can be used to form testable hypotheses about its biochemical function.
View details for Web of Science ID 000166163000006
View details for PubMedID 11150734
Maf is a basic domain/leucine zipper domain protein originally identified as a proto-oncogene whose consensus target site in vitro, the T-MARE, is an extended version of an AP-1 site normally recognized by Fos and Jun. Maf and the closely related family members Neural retina leucine zipper (Nrl), L-Maf, and Krml1/MafB have been implicated in a wide variety of developmental and physiologic roles; however, mutations in vivo have been described only for Krml1/MafB, in which a loss-of-function causes abnormalities in hindbrain development due to failure to activate the Hoxa3 and Hoxb3 genes. We have used gene targeting to replace Maf coding sequences with those of lacZ, and have carried out a comprehensive analysis of embryonic expression and the homozygous mutant phenotype in the eye. Maf is expressed in the lens vesicle after invagination, and becomes highly upregulated in the equatorial zone, the site at which self-renewing anterior epithelial cells withdraw from the cell cycle and terminally differentiate into posterior fiber cells. Posterior lens cells in Maf(lacZ) mutant mice exhibit failure of elongation at embryonic day 11.5, do not express (&agr;)A- and all of the (beta)-crystallin genes, and display inappropriately high levels of DNA synthesis. This phenotype partially overlaps with those reported for gene targeting of Prox1 and Sox1; however, expression of these genes is grossly normal, as is expression of Eya1, Eya2, Pax6, and Sox2. Recombinant Maf protein binds to T-MARE sites in the (alpha)A-, (beta)B2-, and (beta)A4-crystallin promoters but fails to bind to a point mutation in the (alpha)A-crystallin promoter that has been shown previously to be required for promoter function. Our results indicate that Maf directly activates many if not all of the (beta)-crystallin genes, and suggest a model for coordinating cell cycle withdrawal with terminal differentiation.
View details for Web of Science ID 000085399700010
View details for PubMedID 10603348
Mutations that affect the balance between the synthesis of eumelanin and pheomelanin provide a powerful set of tools with which to understand general aspects of cell signaling. Previous work from our laboratory has demonstrated that pheomelanin synthesis is triggered by the ability of Agouti protein to inhibit signaling through the Melanocortin 1 receptor (Mc1r). In a bioassay based on the Xenopus Mc1r, Agouti protein has two effects, competitive inhibition of receptor occupancy by alpha-MSH and down-regulation of receptor signaling, which are mediated separately by domains in the amino- and carboxy-terminal regions of Agouti protein, respectively. Recently, we have used the genetics of pigmentation as an in vivo system to screen for and analyze other mutations in the Agouti-melanocortin pathway. The pigmentary effects of Agouti are suppressed by the previously existing coat-color mutations mahogany (mg), mahoganoid (md), and Umbrous (U). Double mutant studies, with animals deficient for the Mc1r or those which carry Ay, indicate that mg and md are genetically upstream of the Mc1r, and can suppress the effects of Ay on both pigmentation and body weight. Positional cloning has recently identified the gene mutated in mahogany as a single transmembrane-spanning protein whose ectodomain is orthologous to human Attractin (Atrn).
View details for Web of Science ID 000089714000009
View details for PubMedID 11041357
The cloning and characterization of the human melanocortin-1 receptor (MC1R) and the demonstration that normal human melanocytes respond to the melanocortins, alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotrophic hormone (ACTH), with increased proliferation and eumelanogenesis had put an end to a long-standing controversy about the role of melanocortins in regulating human cutaneous pigmentation. We have shown that alpha-MSH and ACTH bind the human MC1R with equal affinity, and are equipotent in their mitogenic and melanogenic effects on human melanocytes. We also showed that the activation of the MC1R is important for the melanogenic response of human melanocytes to ultraviolet radiation (UVR). The MC1R is also the principal mediator of the inhibitory effects of agouti signaling protein (ASP) on melanogenesis. Expression of the MC1R is subject to regulation by its own ligands alpha-MSH and ACTH, as well as by UVR and endothelin-1. Recent studies that we conducted on the expression of MC1R variants by human melanocytes and the implications of these variants on the function of the MC1R revealed the following. Human melanocytes homozygous for Arg160Trp mutation in the MC1R demonstrated a significantly reduced response to alpha-MSH. Also, this culture responded poorly to ASP and exhibited an exaggerated cytotoxic response to UVR. Another culture, which was homozygous for Val92Met mutation in the MC1R, demonstrated a normal response to alpha-MSH. Heterozygous mutations that are frequently expressed in various melanocyte cultures did not disrupt MC1R function. These results begin to elucidate the significance of MC1R variants in the function of the receptor. Our data emphasize the significance of a normally functioning MC1R in the response of melanocytes to melanocortins, ASP, and UVR.
View details for Web of Science ID 000089714000027
View details for PubMedID 11041375
Insertional mutagenesis based on gene trap vectors that capture endogenous splice sites is a promising tool for functional genomics. Several groups have proposed large-scale gene trap screens, but questions remain as to the type of vectors and their design. We report a set of plasmid-encoded gene trap vectors and the disruption of two novel genes. Our results include a comparison of the relative gene trapping efficiencies of two different splice acceptor sequences in ES cells and an analysis of the structure of several gene trap insertions.
View details for Web of Science ID 000085580100005
View details for PubMedID 10767988
Central administration of neuropeptide Y (NPY) potently induces feeding and its abundance in the hypothalamus increases when energy stores fall. Consequently, NPY is considered to be a physiological effector of feeding behavior. Surprisingly, NPY-deficient (NPY-/-) mice feed and grow normally with ad libitum access to food and manifest a normal hyperphagic response after fasting, suggesting that other feeding effectors may compensate for the lack of NPY. Agouti-related protein (AgRP), a melanocortin receptor antagonist, can also stimulate feeding behavior when administered centrally and is coexpressed in a majority of hypothalmamic NPY-ergic neurons, making AgRP a candidate compensatory factor. To test this possibility, we evaluated AgRP mRNA and protein expression, as well as responsiveness to centrally administered AgRP in NPY-/- mice. These studies demonstrate that hypothalamic AgRP mRNA and immunoreactivity are upregulated with fasting and that these increases are not affected by NPY deficiency. Interestingly, NPY-/- mice are hypersensitive to central administration of AgRP(83-132), yet exhibit a normal response to centrally administered MTII, a melanocortin receptor agonist. These data suggest that if AgRP compensates for the lack of NPY in NPY-/- mice, it is not at the level of AgRP synthesis and may instead involve alterations in the postsynaptic signaling efficacy of AgRP. Moreover, the effects of AgRP are not limited to its actions at the melanocortin-4 receptor (MC4R), because MC4R-deficient (MC4R-/-) mice manifest a significant response to centrally administered AgRP. These data imply that AgRP has additional targets in the hypothalamus.
View details for Web of Science ID 000084420300147
View details for PubMedID 10612698
The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. The present study examined the distribution of Atrn mRNA in the brain and spinal cord by in situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.
View details for Web of Science ID 000083943300020
View details for PubMedID 10580100
With the rise in the field of neuroimmunomodulation research, there is increased recognition of the influence of the nervous system and neuropeptides in peripheral disease. The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuroimmunomodulatory agent that modulates production of proinflammatory cytokines and inhibits peripheral inflammation via actions on CNS receptors. We examined whether central alpha-MSH operates by inhibiting activation of the nuclear factor kappa B (NF-kappaB) that is essential to the expression of proinflammatory cytokines and development of inflammation in the periphery. Electrophoretic mobility shift assays of nuclear extracts from the murine foot pad injected with TNF-alpha demonstrated that centrally administered alpha-MSH does inhibit NF-kappaB activation. Western blot analysis revealed that this inhibition was linked to central alpha-MSH-induced preservation of expression of IkappaBalpha protein in the peripheral tissue. The NF-kappaB and IkappaBalpha effects were inhibited in mice with spinal cord transection. Intraperitoneal (i.p.) injection of the nonspecific beta-adrenergic receptor blocker propranolol, and of a specific beta2-adrenergic receptor antagonist, likewise prevented these effects of central alpha-MSH; blockade of cholinergic, alpha-adrenergic, or beta1-adrenergic receptors did not. Centrally administered alpha-MSH inhibited peripheral NF-kappaB activation and IkappaBalpha degradation even in mice with nonfunctional melanocortin 1 receptors (MC1R). These findings indicate that alpha-MSH can act centrally to inhibit NF-kappaB activation in peripheral acute inflammation via a descending neural pathway. The pathway involves beta2-adrenergic receptors, but does not require activation of MC1R within the brain.
View details for Web of Science ID 000082762900008
View details for PubMedID 10505977
Agouti-related protein (AGRP) is a recently discovered orexigenic neuropeptide that inhibits the binding and action of alpha-melanocyte-stimulating hormone derived from proopiomelanocortin (POMC) at the melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) and has been proposed to function primarily as an endogenous melanocortin antagonist. To better understand the interplay between the AGRP and melanocortin signaling systems, we compared their nerve fiber distributions with each other by immunohistochemistry and their perikarya distribution with MC3R and MC4R by double in situ hybridization. Although deriving from distinct cell groups, AGRP and melanocortin terminals project to identical brain areas. Both AGRP and melanocortin neurons selectively express the MC3R, which provides a neuroanatomical basis for a dual-input circuit with biological amplification and feedback inhibition. These studies highlight a broader complexity in POMC-mediated behavior in the brain.
View details for Web of Science ID 000082539900004
View details for PubMedID 10479719
The cloning of the melanocortin-1 receptor (MC1R) gene from human melanocytes and the demonstration that these cells respond to the melanocortins alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) with increased proliferation and melanogenesis have renewed the interest in investigation the physiological role of these hormones in regulating human pigmentation. Alpha-melanocyte stimulating hormone and ACTH are both synthesized in the human epidermis, and their synthesis is upregulated by exposure to ultraviolet radiation (UVR). Activation of the MC1R by ligand binding results in stimulation of cAMP formation, which is a principal mechanism for inducing melanogenesis. The increase in cAMP is required for the pigmentary response of human melanocytes to UVR, and for allowing them to overcome the UVR-induced G1 arrest. Treatment of human melanocytes with alpha-MSH increases eumelanin synthesis, an effect that is expected to enhance photoprotection of the skin. Population studies have revealed more than 20 allelic variants of the MC1R gene. Some of these variants are overexpressed in individuals with skin type I or II, red hair, and poor tanning ability. Future studies will aim at further exploration of the role of these variants in MC1R function, and in determining constitutive human pigmentation, the response to sun exposure, and possibly the susceptibility to skin cancer.
View details for Web of Science ID 000084348400007
View details for PubMedID 10537004
Members of the MAF family of basic region/leucine zipper transcription factors can affect transcription in either a positive or a negative fashion, depending on their partner protein(s) and the context of the target promoter. The KRML (MAFB) transcriptional regulator plays a pivotal role in regulating lineage-specific hematopoiesis by repressing ETS1-mediated transcription of erythroid-specific genes in myeloid cells. In previous studies, we mapped the human KRML gene within a genomic contig on human chromosome 20, bands q11.2-q13.1. We have isolated the human cDNA containing the full-length predicted open reading frame (ORF). Multiple KRML transcripts of approximately 1.8 and approximately 3 kb, which differ in the length of the 3' untranslated region, are ubiquitously expressed in hematopoietic tissues and encode a protein with 323 amino acids (MW 35,832). The protein has 84% identity and 92% similarity to the murine protein. The ORF of the human KRML gene contains no introns, and the gene spans approximately 3 kb. KRML maps within the smallest commonly deleted segment in malignant myeloid disorders characterized by a deletion of 20q; however, we detected no mutations of KRML in leukemia cells with loss of 20q. Thus, KRML is unlikely to be involved in the pathogenesis of malignant myeloid disorders characterized by abnormalities of chromosome 20.
View details for Web of Science ID 000082181800003
View details for PubMedID 10444328
The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) and its C-terminal tripeptide alpha-MSH11-13 modulate production of proinflammatory cytokines and inhibit inflammation. We examined whether systemic alpha-MSH and alpha-MSH11-13 inhibit activation of the nuclear transcription factor, nuclear factor kappa B (NF-kappaB), a factor that is essential to expression of proinflammatory cytokines, in experimental murine brain inflammation induced by lipopolysaccharide. Electrophoretic mobility shift assays of nuclear extracts demonstrated that parenteral alpha-MSH inhibited NF-kappaB activation. Western blot analysis revealed that this inhibition was linked to alpha-MSH-induced preservation of expression of IkappaBalpha protein in the brain. The effects of alpha-MSH on NF-kappaB and IkappaBalpha were paralleled by pretreatment with alpha-MSH11-13. Similar effects of the two peptides were observed in mice with nonfunctional melanocortin 1 receptors (MC1R), ruling out the possibility that this receptor subtype is essential to the influence on NF-kappaB. These findings indicate that alpha-MSH peptides given systemically can inhibit NF-kappaB activation induced in acute brain inflammation even in the absence of MC1R.
View details for Web of Science ID 000081744800004
View details for PubMedID 10415402
Defects in signaling by leptin, a hormone produced primarily by adipose tissue that informs the brain of the body's energy reserves, result in obesity in mice and humans. However, the majority of obese humans do not have abnormalities in leptin or its receptor but instead exhibit leptin resistance that could result from defects in downstream mediators of leptin action. Recently, two potential downstream mediators, agouti-related protein (Agrp) and its receptor, the melanocortin-4 receptor (Mc4r), have been identified. Agrp and Mc4r are excellent candidates for human disorders of body weight regulation and represent promising targets for pharmacological intervention in the treatment of these disorders.
View details for Web of Science ID 000081619900009
View details for PubMedID 10366820
Agouti protein and Agouti-related protein (Agrp) regulate pigmentation and body weight, respectively, by antagonizing melanocortin receptor signaling. A carboxyl-terminal fragment of Agouti protein, Ser73-Cys131, is sufficient for melanocortin receptor antagonism, but Western blot analysis of skin extracts reveals that the electrophoretic mobility of native Agouti protein corresponds to the mature full-length form, His23-Cys131. To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopus melanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time- and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein.
View details for Web of Science ID 000080560100076
View details for PubMedID 10336487
Agouti-related protein (AGRP) is an orexigenic neuropeptide that acts via central melanocortin receptors, and whose messenger RNA (mRNA) levels are elevated in leptin-deficient mice. Fasting associated with a decline in circulating leptin normally causes a 15-fold elevation of hypothalamic Agrp mRNA levels but has no effect in leptin-deficient mice. Chronic hyperleptinemia associated with the tubby and Cpe(fat) mutations has no effect on Agrp mRNA levels, but short term leptin administration causes a 17% reduction of Agrp mRNA levels in nonmutant mice and a 700% reduction in leptin-deficient mice. In young nonobese animals, melanocortin receptor blockade associated with the Ay mutation causes complete resistance to leptin-induced weight loss. Dual in situ hybridization reveals that Agrp-expressing neurons in the medial portion of the arcuate nucleus constitute a subpopulation different from Pomc-expressing neurons, and that a significant proportion of Agrp-expressing neurons (10-25%) coexpresses the leptin receptor, Lepr-b. Immunocytochemistry confirms distinct locations of AGRP- and POMC-expressing cell bodies, but reveals an overlapping distribution of their terminal fields in the arcuate nucleus, the paraventricular hypothalamus, and the dorsomedial hypothalamus. These results suggest that in the fed state, AGRP is normally suppressed by leptin, and that release of this suppression during fasting leads to increased ingestive behavior.
View details for Web of Science ID 000079801200052
View details for PubMedID 10218993
Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here we use positional cloning to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocortin-receptor signalling and immune function.
View details for Web of Science ID 000079135200045
View details for PubMedID 10086356
Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.
View details for Web of Science ID 000079085400011
View details for PubMedID 10049570
During anteroposterior patterning of the developing hindbrain, the anterior expression of 3' Hox genes maps to distinct rhombomeric boundaries and, in many cases, is upregulated in specific segments. Paralogous genes frequently have similar anterior boundaries of expression but it is not known if these are controlled by common mechanisms. The expression of the paralogous Hoxa3 and Hoxb3 genes extends from the posterior spinal cord up to the rhombomere (r) 4/5 boundary and both genes are upregulated specifically in r5. However, in this study, we have found that Hoxa3 expression is also upregulated in r6, showing that there are differences in segmental expression between paralogues. We have used transgenic analysis to investigate the mechanisms underlying the pattern of segmental expression of Hoxa3. We found that the intergenic region between Hoxa3 and Hoxa4 contains several enhancers, which summed together mediate a pattern of expression closely resembling that of the endogenous Hoxa3 gene. One enhancer specifically directs expression in r5 and r6, in a manner that reflects the upregulation of the endogenous gene in these segments. Deletion analysis localized this activity to a 600 bp fragment that was found to contain a single high-affinity binding site for the Maf bZIP protein Krml1, encoded by the kreisler gene. This site is necessary for enhancer activity and when multimerized it is sufficient to direct a kreisler-like pattern in transgenic embryos. Furthermore the r5/r6 enhancer activity is dependent upon endogenous kreisler and is activated by ectopic kreisler expression. This demonstrates that Hoxa3, along with its paralog Hoxb3, is a direct target of kreisler in the mouse hindbrain. Comparisons between the Krml1-binding sites in the Hoxa3 and Hoxb3 enhancers reveal that there are differences in both the number of binding sites and way that kreisler activity is integrated and restricted by these two control regions. Analysis of the individual sites revealed that they have different requirements for mediating r5/r6 and dorsal roof plate expression. Therefore, the restriction of Hoxb3 to r5 and Hoxa3 to r5 and r6, together with expression patterns of Hoxb3 in other vertebrate species suggests that these regulatory elements have a common origin but have later diverged during vertebrate evolution.
View details for Web of Science ID 000079085700015
View details for PubMedID 9895323
Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action that is thought to play an important role in the hypothalamic control of feeding behavior. The exact mechanism of AGRP and Agouti protein action has been difficult to examine, in part because of difficulties in producing homogeneous forms of these molecules that can be used for direct binding assays. In this report we describe the application of chemical protein synthesis to the construction of two novel AGRP variants. Examination of the biological activity of the AGRP variants demonstrates that a truncated variant, human AGRP(87-132), a 46-amino acid variant based on the carboxyl-terminal cysteine-rich domain of AGRP, is equipotent to an 111-amino acid variant, mouse [Leu127Pro]AGRP (mature AGRP minus its signal sequence), in its ability to dose dependently inhibit alpha-MSH-generated cAMP generation at the cloned melanocortin receptors. Furthermore, deletion of the amino-terminal portion of the full-length variant did not alter the MCR subtype specificity of AGRP(87-132). Finally, iodination of human AGRP(87-132) provided a useful reagent with which the binding properties of AGRP could be analyzed. In both conventional and photoemulsion binding studies [125I]AGRP(87-132) was observed only to bind to cells expressing melanocortin receptors MC3R, MC4R, and MC5R. These results demonstrate that the residues critical for receptor binding, alpha-MSH inhibition, and melanocortin receptor subtype specificity are all located in the carboxyl terminus of the molecule. Because [Nle4, D-Phe7] (NDP)-MSH displaces the binding of [125I]AGRP(87-132) to MCRs and AGRP(87-132) displaces the binding of [125I]NDP-MSH, we conclude that these molecules bind in a competitive fashion to melanocortin receptors.
View details for Web of Science ID 000077819100013
View details for PubMedID 9892020
View details for Web of Science ID 000084261200035
Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation and may also have a role in ageing and acquired diseases of old age. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA. Using a loxP-flanked Tfam allele (TfamloxP) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.
View details for Web of Science ID 000077960700025
View details for PubMedID 9916807
alpha-Melanocyte stimulating hormone (alpha-MSH) modulates all forms of inflammation by acting on peripheral inflammatory cells, glial inflammatory cells, and on CNS receptors that activate descending antiinflammatory neural pathways. The multiple actions of this ancient peptide suggest that there is no singular biochemical mechanism through which it exerts its antiinflammatory activity. However, research on IL-10 deficient and Agouti protein hypersecreting mice provide new insights into the actions of the peptide in living animals. Studies of cultured human astrocytes, whole murine brain, and human monocyte/macrophages indicate that a primary effect of the peptide is modulation of activation of the nuclear transcription factor kappa B. The latter influence may underlie the established reduction of gene expression and production of proinflammatory peptides and inducible nitric oxide by alpha-MSH peptides.
View details for Web of Science ID 000084366500014
View details for PubMedID 10816650
Agouti protein and Agouti-related protein (Agrp) are paracrine signaling molecules that act by antagonizing the effects of melanocortins, and several alternatives have been proposed to explain their mechanisms of action. Genetic crosses in a sensitized background uncover a phenotypic difference between overexpression of Agouti and loss of Mc1r function, demonstrate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. In vitro, Agouti protein inhibits melanocortin action by two mechanisms: competitive antagonism that depends on the carboxyterminus of the protein, and downregulation of melanocortin receptor signaling that depends on the aminoterminus. Our findings provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.
View details for Web of Science ID 000084366500011
View details for PubMedID 10816647
Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin-concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), alpha-melanin-stimulating hormone (alpha-MSH), and agouti-related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual-label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP- and NPY-immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas alpha-MSH-immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY-, AgRP-, and alpha-MSH-immunoreactive fibers. Projections from populations of leptin-responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight.
View details for Web of Science ID 000077534100002
View details for PubMedID 9862320
Molecular and biochemical mechanisms that modulate the production of eumelanin or pheomelanin pigments involve the opposing effects of two intercellular signaling molecules, alpha-melanocyte stimulating hormone (MSH) and agouti signal protein (ASP). ASP is an antagonist of MSH signaling through the melanocyte-specific MSH receptor, although its mechanism(s) of action is controversial. We previously have reported significant down-regulation of all known melanogenic genes during the eumelanin to pheomelanin switch in murine hair follicle melanocytes and in cultured melanocytes treated with recombinant ASP. To identify factors that might be involved in the switch to pheomelanogenesis, we screened ASP-treated melanocytes by using differential display and identified three up-regulated genes: a DNA replication control protein, a basic helix-loop-helix transcription factor, and a novel gene. We have simultaneously identified six down-regulated genes in ASP-treated melanocytes; two of those encode tyrosinase and TRP2, melanogenic genes known to be down-regulated during pheomelanogenesis, which provide good internal controls for this approach. These results suggest that there are complex mechanisms involved in the switch to pheomelanin production, and that these modulated genes might be involved in the pleiotropic changes seen in yellow mice, including the change in coat color.
View details for Web of Science ID 000074436400026
View details for PubMedID 9636156
The regulation of mitochondrial DNA (mtDNA) expression is crucial for mitochondrial biogenesis during development and differentiation. We have disrupted the mouse gene for mitochondrial transcription factor A (Tfam; formerly known as m-mtTFA) by gene targetting of loxP-sites followed by cre-mediated excision in vivo. Heterozygous knockout mice exhibit reduced mtDNA copy number and respiratory chain deficiency in heart. Homozygous knockout embryos exhibit a severe mtDNA depletion with abolished oxidative phosphorylation. Mutant embryos proceed through implantation and gastrulation, but die prior to embryonic day (E)10.5. Thus, Tfam is the first mammalian protein demonstrated to regulate mtDNA copy number in vivo and is essential for mitochondrial biogenesis and embryonic development.
View details for Web of Science ID 000072325000020
View details for PubMedID 9500544
The genes for ocular albinisim type 1 (OA1) and the Xenopus laevis-like apical protein (APXL) map between amelogenin (AMELX) and the pseudoautosomal boundary in the distal region of the human X chromosome short arm. The mouse homologues, Oa1 and Apxl, have recently been shown to lie proximal to their expected locations on the mouse X chromosome, but their positions with respect to critical gene loci in the vicinity have not been defined. By analyzing recombination events from (Mus musculus x Mus spretus) x M. musculus backcrosses, we have constructed a detailed mouse genetic map that encompasses Oa1, five other genes, and 13 microsatellite loci. The order of genes and evolutionary breakpoints (EB) is defined as centromere-(EB)-(DXHXS674, DXHXS679)-Smcx-(EB)-Oa1-(EB)-Phex (3'-->5')-Pdha1-telomere. Thus Oa1 lies in a region between two previously characterized conserved segments.
View details for Web of Science ID 000072304800017
View details for PubMedID 9503026
Agouti protein and Agouti-related protein (Agrp) are paracrine-signaling molecules that normally regulate pigmentation and body weight, respectively. These proteins antagonize the effects of alpha-melanocyte-stimulating hormone (alpha-MSH) and other melanocortins, and several alternatives have been proposed to explain their biochemical mechanisms of action. We have used a sensitive bioassay based on Xenopus melanophores to characterize pharmacologic properties of recombinant Agouti protein, and have directly measured its cell-surface binding to mammalian cells by use of an epitope-tagged form (HA-Agouti) that retains biologic activity. In melanophores, Agouti protein has no effect in the absence of alpha-MSH, but its action cannot be explained solely by inhibition of alpha-MSH binding. In 293T cells, expression of the Mc1r confers a specific, high-affinity binding site for HA-Agouti. Binding is inhibited by alpha-MSH, or by Agrp, which indicates that alpha-MSH and Agouti protein bind in a mutually exclusive way to the Mc1r, and that the similarity between Agouti protein and Agrp includes their binding sites. The effects of Agouti and the Mc1r in vivo have been examined in a sensitized background provided by the chinchilla (Tyrc-ch) mutation, which uncovers a phenotypic difference between overexpression of Agouti in lethal yellow (Ay/a) mice and loss of Mc1r function in recessive yellow (Mc1re/Mc1re) mice. Double and triple mutant studies indicate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. These results resolve questions regarding the biochemical mechanism of Agouti protein action, and provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein or Agrp function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.
View details for Web of Science ID 000071942300004
View details for PubMedID 9450927
The vertebrate hindbrain is subdivided into a series of rhombomeres whose segmental organization serves to pattern the architecture and innervation of the developing head. The zebrafish gene valentino is required cell-autonomously in the development of rhombomeres 5 and 6, and valentino mutants lack visible hindbrain segmentation caudal to the r3/4 boundary (Moens, C. B., Yan, Y.-L., Appel, B., Force, A. G., and Kimmel, C. B. (1996) Development 122, 3981-3990). Here we show that valentino is the zebrafish homologue of the mouse segmentation gene kreisler, which encodes a bZip transcription factor. The valentino gene is expressed in a manner consistent with its proposed role in subdividing rhombomeres 5 and 6 from their common precursor 'proto-segment' in the presumptive hindbrain, a process that we also demonstrate is reflected in the normal order of appearance of rhombomere boundaries. As well as having similar phenotypes with respect to visible hindbrain segmentation and patterns of marker gene expression, valentino and kreisler mutants have similar pharyngeal arch and inner ear defects, consistent with a conserved role for this gene in hindbrain segmentation and in patterning of the head periphery.
View details for Web of Science ID 000072350300005
View details for PubMedID 9425134
View details for Web of Science ID 000071446001489
The mouse mutations mahogany (mg) and mahoganoid (md) are negative modifiers of the Agouti coat color gene, which encodes a paracrine signaling molecule that induces a swithc in melanin synthesis from eumelanin to pheomelanin. Animals mutant for md or mg synthesize very little or no pheomelanin depending on Agouti gene background. The Agouti protein is normally expressed in the skin and acts as an antagonist of the melanocyte receptor for alpha-MSH (Mc1r); however, ectopic expression of Agouti causes obesity, possibly by antagonizing melanocortin receptors expressed in the brain. To investigate where md and mg lie in a genetic pathway with regard to Agouti and Mc1r signaling, we determined the effects of these mutations in animals that carried either a loss-of-function Mc1r mutation (recessive yellow, Mc1re) or a gain-of-function Agouti mutation (lethal yellow, Ay). We found that the Mc1re mutation suppressed the effects of md and mg, but that md and mg suppressed the effects of Ay on both coat color and obesity. Plasma levels of alpha-MSH and of ACTH were unaffected by md or mg. These results suggest that md and mg interfere directly with Agouti signaling, possibly at the level of protein production or receptor regulation.
View details for Web of Science ID A1997XM83300017
View details for PubMedID 9258683
View details for PubMedCentralID PMC1208084
Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, alpha-melanocyte-stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan-a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome-like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time- and dose-dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase-related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.
View details for Web of Science ID A1997XG52000017
View details for PubMedID 9218796
View details for Web of Science ID A1997XH77400048
Previous studies have suggested that angiotensin II (Ang II) modulates cardiac contractility, rhythm, metabolism, and structure. However, it is unclear whether the cardiac effects are due to direct actions of Ang II on the myocardium or if they are due to secondary effects mediated through the hemodynamic actions of Ang II. In this study, we used the alpha-myosin heavy chain (alphaMHC) promoter to generate transgenic mice overexpressing angiotensin II type 1 (AT1a) receptor selectively in cardiac myocytes. The specificity of transgene expression in the transgenic offspring was confirmed by radioligand binding studies and reverse transcription-PCR. The offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first weeks after birth. Thus, direct activation of AT1 receptor signaling in cardiac myocytes in vivo is sufficient to induce cardiac myocyte growth and alter electrical conduction.
View details for Web of Science ID A1997XD84400077
View details for PubMedID 9177228
In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.
View details for Web of Science ID A1997XB83600001
View details for PubMedID 9182807
Hox genes control regional identity during segmentation of the vertebrate hindbrain into rhombomeres. Here we use transgenic analysis to investigate the upstream mechanisms for regulation of Hoxb-3 in rhombomere(r)5. We identified enhancers from the mouse and chick genes sufficient for r5-restricted expression. Sequence comparisons revealed two blocks of similarity (of 19 and 45 base pairs), which each contain in vitro binding sites for the kreisler protein (Kmrl1), a Maf/b-Zip protein expressed in r5 and r6 (ref. 4). Both sites are required for r5 activity, suggesting that Hoxb-3 is a direct target of kreisler. Multimers of the 19-base-pair (bp) block recreate a Krml1-like pattern in r5/r6, but the 45-bp block mediates expression only in r5. Therefore elements within the 45-bp block restrict the response to Krml1. We identified additional sequences that contain an Ets-related activation site, required for both the activation and restriction to r5. These studies demonstrate that Krml1 directly activates expression of Hoxb-3 in r5 in combination with an Ets-related activation site, and suggest that kreisler plays a primary role in regulating segmental identity through Hox genes.
View details for Web of Science ID A1997WX94500057
View details for PubMedID 9144291
Mouse agouti protein is a paracrine signaling molecule that has previously been demonstrated to be an antagonist of melanocortin action at several cloned rodent and human melanocortin receptors. In this study we report the effects of agouti-signaling protein (ASIP), the human homolog of mouse agouti, on the action of alpha-MSH or ACTH at the five known human melanocortin receptor subtypes (hMCR 1-5). When stably expressed in L cells (hMC1R, hMC3R, hMC4R, hMC5R) or in the adrenocortical cell line OS3 (hMC1R, hMC2R, hMC4R), purified recombinant ASIP inhibits the generation of cAMP stimulated by alpha-MSH (hMC1R, hMC3R, hMC4R, hMC5R) or by ACTH (hMC2R). However, dose-response and Schild analysis indicated that the degree of ASIP inhibition varied significantly among the receptor subtypes; ASIP is a potent inhibitor of the hMC1R, hMC2R, and hMC4R, but has relatively weak effects at the hMC3R and hMC5R. These analyses also indicated that the apparent mechanism of ASIP antagonism varied among receptor subtypes, with characteristics consistent with competitive antagonism observed only at the hMC1R, and more complex behavior observed at the other receptors. ASIP inhibition at these latter receptors, nonetheless, can be classified as surmountable (hMC3R, hMC4R and hMC5R) or nonsurmountable (hMC2R). Recombinant ASIP also inhibited binding of radiolabeled melanocortins, [125I-Nle4, D-Phe7] alpha-MSH and [125I-Phe2, Nle4]ACTH 1-24, to the hMCR 1-5 receptors, with a relative efficacy that paralleled the ability of ASIP to inhibit cAMP accumulation at the hMC1R, hMC2R, hMC3R, and hMC4R. These results provide new insight into the biochemical mechanism of ASIP action and suggest that ASIP may play an important role in modulating melanocortin signaling in humans.
View details for Web of Science ID A1997WK71000002
View details for PubMedID 9058374
Mitochondrial transcription factor A (mtTFA) is a key activator of mitochondrial transcription in mammals. It also has a role in mitochondrial DNA (mtDNA) replication, since transcription generates an RNA primer necessary for initiation of mtDNA replication. In the mouse, testis-specific mtTFA transcripts encode a protein isoform that is imported to the nucleus rather than into mitochondria of spermatocytes and elongating spermatids. We now report molecular characterization of human mtTFA (h-mtTFA) expression in somatic tissues and male germ cells. Similarly to the mouse, analysis of cDNAs and Northern blots identified abundant testis-specific transcript isoforms generated by use of alternate transcription initiation sites. However, unlike the mouse, none of the testis-specific transcripts predicts a nuclear protein isoform, and Western blot analysis identified only the mitochondrial form of h-mtTFA in human testis. Immunohistochemistry and in situ were used to compare the distribution of mtTFA protein, testis-specific mtTFA transcripts, mtDNA and mtRNA in sections of human testis. Our results show that the mtTFA protein and mtDNA exhibit parallel gradients with high levels in undifferentiated male germ cells and low levels or an absence in different male germ cells. Testis-specific transcripts exhibit the opposite pattern, suggesting that in both humans and mice, these testis-specific mtTFA transcripts down-regulate mtTFA protein levels in mammalian mitochondria. Our findings demonstrate that mtTFA does not have a critical role in the nucleus, suggest a mechanism for reducing mtDNA copy number during spermatogenesis and have implications for the understanding of maternal transmission of mtDNA.
View details for Web of Science ID A1997WH78900005
View details for PubMedID 9063738
Manipulations of the murine genome that alter cardiovascular function have created the need for methods to study cardiovascular physiology in genetically altered animals in vivo. We adapted chronic physiological measurement techniques to the nonanesthetized, nonrestrained murine model, established strain-specific cardiovascular and metabolic norms, and evaluated responses to anesthesia, exercise, and adrenergic stimulation. Anesthesia resulted in alterations in heart rate (HR), blood pressure (BP), and O2 consumption (V(O2)) and CO2 production (V(CO2)) for up to 6 h postoperatively. There were significant interstrain differences in resting values of HR and BP Graded treadmill exercise resulted in linear increases in HR, V(O2), V(CO2), and respiratory exchange ratio (RER) similar to those seen in larger species. Response to beta-adrenergic stimulation showed a classic sigmoidal dose-response curve; however, there was very little tachycardiac response to vagal blockade, indicating low resting vagal tone. This study demonstrates the feasibility of performing chronic cardiovascular measurements in nonanesthetized mice and stresses the importance of allowing for anesthetic recovery and strain variability. Murine cardiovascular responses to exercise can be reliably measured and are qualitatively similar to those in humans.
View details for Web of Science ID A1997WJ80900057
View details for PubMedID 9124413
alpha 2-Adrenergic receptors (alpha 2-ARs) regulate many physiological functions and are targets for clinically important antihypertensive and anesthetic agents. Three human and mouse genes encoding alpha 2-AR subtypes (alpha 2A, alpha 2B, and alpha 2C) have been cloned. We investigated the involvement of the alpha 2C-AR in alpha 2-adrenergic pharmacology by applying molecular genetic techniques to alter the expression of alpha 2C-AR in mice. The effects of dexmedetomidine, a subtype-nonselective alpha 2-AR agonist, on monoamine turnover in brain and on locomotor activity were similar in mice with targeted inactivation of the alpha 2C-AR gene and in their controls, but the hypothermic effect of the alpha 2-AR agonist was significantly attenuated by the receptor gene inactivation. Correspondingly, another strain of transgenic mice with 3-fold overexpression of alpha 2C-AR in striatum and other brain regions expressing alpha 2C-AR showed normal reductions in brain monoamine metabolism and locomotor activity after dexmedetomidine, but their hypothermic response to the alpha 2C-AR agonists was significantly accentuated. The hypothermic effect of alpha 2-AR agonists thus seems to be mediated in part by alpha 2C-AR. Some small but statistically significant differences between the strains were also noted in brain dopamine metabolism. Lack of alpha 2C-AR expression was linked with reduced levels of homovanillic acid in brain, and mice with increased alpha 2C-AR expression had elevated concentrations of the dopamine metabolite compared with their controls.
View details for Web of Science ID A1997WC87200006
View details for PubMedID 9016344
View details for Web of Science ID A1997XM28500214
Ocular albinism type 1 (OA1) is an X-linked human genetic disorder that affects retinal pigment cells and, to a lesser degree, neural crest-derived melanocytes. The OA1 gene is located close to the pseudoautosomal region and predicts a novel protein whose function is unknown. However, histologic studies of affected patients have suggested a potential role in melanosome biogenesis. Here we report the isolation and characterization of the mouse homolog of the human OA1 gene, termed Moa1. Two Moa1 isoforms were isolated from a melanoma cDNA library and predicted to encode proteins of 405 and 249 amino acids with six and two transmembrane-spanning regions, respectively. Interspecific backcross mapping yielded a map order and distances (cM) of cen-Moa1-3.1 +/- 1.8-Piga-2.1 +/- 1.5-Amel, indicating that Moa1 is located much farther away from the pseudoautosomal region than its human homolog. In adult tissues, both Moa1 isoforms were detected in the eye by Northern hybridization. In neonatal tissues, Moa1 RNA was detected in both skin and eyes by Northern hybridization and was not affected by the absence of pigment in mice carrying the albino mutation, or by the type of pigment synthesized, i.e., eumelanin vs pheomelanin, in mice carrying the black-and-tan mutation. Expression of Moa1 RNA was not detected in embryonic tissues by Northern analysis or by in situ hybridization despite the active synthesis of ocular pigment by E16.5. These results provide insight into the structure and possible function of the OA1 protein and suggest a more complex relationship between the human and mouse X chromosomes than was previously thought to exist.
View details for Web of Science ID A1996VN66700010
View details for PubMedID 8921399
The mouse agouti protein is a paracrine signaling molecule that causes yellow pigment synthesis. A pale ventral coloration distinguishes the light-bellied agouti (AW) from the agouti (A) allele, and is caused by expression of ventral-specific mRNA isoforms with a unique 5' untranslated exon. Molecular cloning demonstrates this ventral-specific exon lies within a 3.1-kb element that is duplicated in the opposite orientation 15-kb upstream to produce an interrupted palindrome and that similarity between the duplicated elements has been maintained by gene conversion. Orientation of the palindrome is reversed in A compared to AW, which suggests that mutation from one allele to the other is caused by intrachromosomal homologous recombination mediated by sequences within the duplicated elements. Analysis of 15 inbred strains of laboratory and wild-derived mice with Southern hybridization probes and closely linked microsatellite markers suggests six haplotype groups: one typical for most strains that carry AW (129/SvJ, LP/J, CE/J, CAST/Ei), one typical for most strains that carry A (Balb/cJ, CBA/J, FVB/N, PERA/Rk, RBB/Dn); and four that are atypical (MOLC/Rk, MOLG/Dn, PERA/Ei, PERC/Ei, SPRET/Ei, RBA/Dn). Our results suggest a model for molecular evolution of the agouti locus in which homologous recombination can produce a reversible switch in allelic identity.
View details for Web of Science ID A1996VE25200025
View details for PubMedID 8878692
alpha2-Adrenergic receptors (alpha2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific alpha2AR subtypes (alpha2a, alpha2b, and alpha2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either alpha2b or alpha2c receptors. Stimulation of alpha2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating alpha2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the alpha2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective alpha2AR drugs.
View details for Web of Science ID A1996VB42900045
View details for PubMedID 8670422
Genes that control mammalian pigmentation interact with each other in intricate networks that have been studied for decades using mouse coat color mutations. Molecular isolation of the affected genes and the ability to study their effects in a defined genetic background have led to surprising new insights into the potential interaction between tyrosine kinase and G-protein-coupled signaling pathways. Recent developments show that homologous genes in humans are responsible not only for rare diseases, such as albinism and piebaldism, but also for common phenotypic variations, such as red hair and fair skin.
View details for Web of Science ID A1996VA71300008
View details for PubMedID 8783939
At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.
View details for Web of Science ID A1996UW79200098
View details for PubMedID 8693001
Mitochondrial transcription factor A (mtTFA) is a key regulator of mammalian mitochondrial DNA transcription. We report here that a testis-specific isoform of mouse mtTFA lacks the mitochondrial targeting sequence and is present in the nucleus of spermatocytes and elongating spermatids, thus representing the first reported mammalian gene encoding protein isoforms targeted for the mitochondria or the nucleus. The presence of the mitochondrial transcriptional activator in the nucleus raises the possibility of a role for this protein in both genetic systems. Mutations in the nuclear mtTFA gene may therefore exhibit phenotypic consequences due to altered function in either or both genetic compartments.
View details for Web of Science ID A1996UU28300017
View details for PubMedID 8673128
Angiotensin II is a potent regulator of cardiovascular homeostasis and binds to two different G-protein-coupled receptors. While the type 1 receptor (AT1) mediates the cardiovascular actions of angiotensin II, the function of the recently cloned type 2 receptor (AT2) remains unknown. We have cloned the mouse AT2 receptor gene (Agtr2) and determined its map position by linkage analysis using an interspecific backcross (C57BL/6J x Mus spretus).Agtr2 is located on the proximal mouse X chromosome between DXMit85 and DXMit49, in a region of conserved synteny with a part of the human X chromosome implicated in inherited forms of premature ovarian failure. The mapping of Agtr2 may expand a region of conserved synteny with human Xq26 that includes Hprt.
View details for Web of Science ID A1995TG44700033
View details for PubMedID 8586443
Angiotensin II, a potent regulator of blood pressure and of water and electrolyte balance, binds to two different G-protein-coupled receptors. The type-1 receptor (AT1) mediates the vasopressive and aldosterone-secreting effects of angiotensin II, but the function of the type-2 receptor (AT2) is unknown, although it is expressed in both adult and embryonic life. To address this question, we have generated mice lacking the gene encoding the AT2 receptor. Mutant mice develop normally, but have an impaired drinking response to water deprivation as well as a reduction in spontaneous movements. Their baseline blood pressure is normal, but they show an increased vasopressor response to injection of angiotensin II. Thus, although the AT2 receptor is not required for embryonic development, it plays a role in the central nervous system and cardiovascular functions that are mediated by the renin-angiotensin system.
View details for Web of Science ID A1995TB46900060
View details for PubMedID 7477266
Expression of the agouti gene from two different promoters, one active at the midpoint of the hair cycle and the other specific for the ventrum, is responsible for generating a range of mammalian pigmentation patterns. We demonstrate that in postnatal mice transcripts from both promoters are confined to the dermal papilla of hair follicles, as predicted by classical transplantation experiments. Transcripts from the hair cycle promoter are detected in the embryonic whisker plate but not in other regions of the body before birth, whereas ventral-specific transcripts are detected in the ventral trunk of the embryo as well as ventral whisker plate. To investigate further the embryonic origins of adult pigmentation patterns, we carried out a detailed analysis of agouti expression in the embryo. The ventral-specific agouti isoform is first expressed at E10.5 in neural crest-derived ventral cells of the second branchial arch, in anterior regions of the forelimb buds and in a narrow stripe of ventral mesenchyme. By E14.5 a continuous layer of expression is observed in the upper cells of the dermis, including cells of the developing dermal papillae, and covering the entire ventral surface of the head and trunk and dorsal surfaces of the distal forelimb and hindlimb. This expression pattern reflects the domain of yellow coloration evident in adult animals and suggests that the agouti gene is regulated in part by factors responsible for establishing differences between the dorsal and ventral surfaces of the body during embryogenesis. To test the hypothesis that agouti is a paracrine signaling molecule that can influence pigment production by hair follicle melanocytes when expressed by either dermis or epidermis, as suggested by recombination and transplantation experiments, we created transgenic animals in which agouti is expressed in basal cells of the epidermis. These animals display stripes of yellow hairs corresponding to regions of epidermal agouti expression, confirming that agouti signals melanocytes to synthesize yellow pigment and providing direct evidence that it functions in a paracrine manner with a restricted radius of action.
View details for Web of Science ID A1995RZ75600010
View details for PubMedID 7588057
alpha 2-Adrenergic receptors (alpha 2-ARs) regulate a wide range of physiological functions and are targets for clinically important antihypertensive and anesthetic agents. Three genes encoding alpha 2-AR subtypes have been cloned in humans and mice, but the physiological significance of each subtype has not been completely characterized. The available agonist and antagonist compounds are not sufficiently subtype selective to allow the unambiguous dissection of these receptors in vivo. As an alternative approach, we have used gene targeting in embryonic stem cells to disrupt the Adra2c gene, which encodes the alpha 2c-AR subtype in mice. Adra2c-/Adra2c- animals do not express a functional alpha 2c-AR transcript, as detected by Northern blotting or reverse transcription-polymerase chain reaction analysis. In addition, these mice have markedly reduced [3H]rauwolscine binding in their caudate putamen and in other brain regions normally expressing Adra2c binding sites. Adra2c-/Adra2c- mice, however, are viable and fertile and appear grossly normal. Expression levels of Adra2a and Adra2b mRNA in brain and kidney are not altered by the Adra2c knockout. These data suggest that up-regulation of Adra2a or Adra2b does not compensate for the Adra2c deficiency and that the receptor encoded by Adra2c is not required for normal mouse development or for survival in a laboratory environment.
View details for Web of Science ID A1995RK03200007
View details for PubMedID 7623774
View details for Web of Science ID A1995QP08200135
View details for Web of Science ID A1995QP08200185
The mouse agouti coat color gene encodes a novel paracrine signaling molecule whose pulsatile expression produces a characteristic pattern of banded pigment in individual hairs. Several spontaneous agouti alleles produce adult-onset obesity and diabetes, and have provided important single-gene animal models for alterations in energy metabolism. Utilizing linkage groups conserved between mice and humans, we have cloned the human homolog of the mouse agouti gene from a human chromosome 20 yeast artificial chromosome known to contain S-adenosyl homocysteine hydrolase (AHCY). The human agouti gene, named Agouti Signaling Protein (ASP), encodes a 132 amino acid protein, the mRNA for which is expressed in testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin. As predicted by the interactions of mouse agouti with the extension gene (which encodes the melanocyte receptor for alpha-melanocyte stimulating hormone [alpha-MSH]), expression of ASP in transgenic mice produces a yellow coat, and expression of ASP in cell culture blocks the alpha-MSH-stimulated accumulation of cAMP in mouse melanoma cells. The localization of ASP relative to other loci on chromosome 20 excludes it as a candidate for the MODY1 locus, a gene responsible for one form of early-onset non-insulin-dependent diabetes mellitus or maturity-onset diabetes of the young. The expression of ASP in human tissues suggests a function for agouti homologs in species that do not exhibit the characteristic phenotype of banded hairs.
View details for Web of Science ID A1995QH63400012
View details for PubMedID 7757071
The mouse kreisler (kr) mutation causes segmentation abnormalities in the caudal hindbrain and defective inner ear development. Based on an inversion discovered in the original kr allele, we selected a candidate cDNA highly expressed in the developing caudal hindbrain. This cDNA encodes a basic domain-leucine zipper (bZIP) transcription factor and was confirmed to represent the kr gene by analysis of a second kr allele, generated by chemical mutagenesis, in which a serine is substituted for an asparagine residue conserved in the DNA-binding domain of all known bZIP family members. The identity, expression, and mutant phenotype of kr indicate an early role in axial patterning and provide insights into the molecular and embryologic mechanisms that govern hindbrain and otic development.
View details for Web of Science ID A1994PY08600012
View details for PubMedID 8001130
View details for Web of Science ID A1994PN41700829
Several dominant mutations of the mouse agouti coat colour gene have pleiotropic effects that include obesity and a yellow coat. The Ay allele is caused by a large deletion that affects the expression of several contiguous genes. We show that three other obesity-associated agouti mutations, Aiy, Asy and Avy, are due to different molecular alterations that result in ubiquitous expression of a chimaeric RNA that encodes a normal agouti protein. The Aiy and Avy alleles are caused by insertion of an intracisternal A particle element 1 kb or 100 kb, respectively, upstream of agouti coding sequences. These results provide a model for other genes that show allele-specific imprinting, and demonstrate that molecular mechanisms typically responsible for activation of proto-oncogenes can also lead to other disease phenotypes.
View details for Web of Science ID A1994PE15700016
View details for PubMedID 7987393
The agouti coat color gene encodes a paracrine signaling molecule that controls the production of yellow and black pigment by melanocytes within hair follicles. Some agouti alleles affect the dorsum and ventrum independently, which has provided the basis for speculation that agouti gene action in different regions of the body is controlled by distinct genetic loci that are closely linked. Using a combination of cDNA cloning and RNA expression studies, we find that alternative isoforms of agouti mRNA contain different noncoding first exons located 100 kb apart, whose patterns of expression indicate independent control by regulatory elements that are either ventral specific or hair cycle specific. These results demonstrate that the apparent genetic complexity of the agouti locus is explained by the existence of multiple regulatory elements exerting control over a single coding sequence and provide a conceptual basis for understanding differences in dorsal and ventral hair coloration in many mammalian species. The ventral-specific agouti isoform represents an example of a transcript whose expression is restricted to ventral skin and provide an approach to investigate the mechanisms by which dorsal-ventral differences in gene expression are established and maintained.
View details for Web of Science ID A1994NR27500093
View details for PubMedID 8202545
Heterozygosity for the mouse lethal yellow (Ay) mutation leads to obesity, increased tumor susceptibility and increased activity of the agouti coat color gene; homozygosity for Ay results in embryonic death around the time of implantation. Although these pleiotropic effects have not been separated by recombination, previous studies have suggested that the dominant and recessive effects result from distinct genetic lesions. Here we use a combination of genomic and cDNA cloning experiments to demonstrate that the Ay mutation is caused by a 120 kb deletion which lies centromere-proximal to the agouti coat color gene. The deletion removes coding but not 5' untranslated sequences for a ubiquitously expressed gene predicted to encode a protein similar in sequence to an RNA-binding protein, which we named Merc, for maternally expressed hnRNP C-related gene, but have renamed Raly, since the gene is nearly identical to one reported recently by Michaud et al. (Gene Dev. 7, 1203-1213, 1993). The Ay deletion results in the splicing of Merc/Raly 5' untranslated sequences to agouti protein-coding sequences, which suggests that ectopic expression of the normal agouti protein by the Ay fusion RNA is responsible for the pleiotropic effects associated with heterozygosity for Ay. We find that Merc/Raly RNA is present in the unfertilized egg and is also transcribed in preimplantation embryos. Using a PCR-based assay to determine the genotype of individual embryos from an Ay/a x Ay/a intercross, we show that, in the absence of zygotic Merc/Raly expression, Ay/Ay embryos develop to the blastocyst stage, but do not hatch from the zona pellucida or form trophoblastic outgrowths. Injection of a Merc/Raly antisense oligonucleotide into non-mutant embryos blocks development prior to the blastocyst stage, and can be rescued by coinjection of a Merc/Raly transgene. These results suggest that maternal expression of Merc/Raly plays an important role in preimplantation development and that its deletion of is sufficient to explain Ay-associated embryonic lethality.
View details for Web of Science ID A1994NT55500031
View details for PubMedID 8050375
The lethal nonagouti (a(x)) mutation is a hypomorphic allele of the agouti coat color locus which, when homozygous, also leads to embryonic death around the time of implantation. To understand the molecular basis of these phenotypes, we identified and cloned a deletion breakpoint junction present in the ax chromosome. Long range restriction mapping demonstrated a simple deletion of approximately 100 kb, which does not affect agouti coding sequences, but begins only 4 kb 3' of the last exon, and thus may affect coat color by removing an agouti 3' enhancer. The Ahcy gene, which codes for the enzyme S-adenosylhomocysteine hydrolase (SAHase), is contained within a 20 kb region within the a(x) deletion. SAHase RNA and protein were detectable in early blastocysts and in embryonic stem cells, respectively, and analysis of embryos derived from an a(x)/a x a(x)/a embryo intercross indicated that a(x)/a embryos die between the late blastocyst and early implantation stages. Treatment of cultured embryos with an SAHase inhibitor, 3-deazaaristeromycin, or with metabolites that can result in elevated levels of cellular SAH, resulted in an inhibition of inner cell mass development, suggesting that loss of SAHase activity in a(x)/a(x) embryos is sufficient to explain their death around the time of implantation.
View details for Web of Science ID A1994NH08200006
View details for PubMedID 8168479
View details for Web of Science ID A1994NF02001247
View details for Web of Science ID A1994NG77900184
View details for Web of Science ID A1994MV41200538
In a previous survey of endogenous proviruses among inbred mouse strains, the Xmv-10 provirus was found only in strains that carried the non-agouti (a) mutation (Frankel et al. J. Virol. 63: 1763-1774, 1989). To determine whether insertion of Xmv-10 caused the a mutation, we cloned a portion of Xmv-10 and its insertion site. Using a fragment of flanking cellular DNA as a Southern hybridization probe, we found that the Xmv-10 provirus was still present in revertant alleles of a to a(t) or AW. A restriction fragment length variant (RFLV) in cellular DNA at the Xmv-10 insertion site was found to be correlated with the presence or absence of the provirus among inbred strains of laboratory mice regardless of their agouti allele. This correlation did not extend to wild mice, however, in which none of the samples contained Xmv-10, yet one, Mus domesticus poschiavinus, contained the insertion site RFLV correlated with Xmv-10 in laboratory mice. Analysis of an intersubspecific backcross with RFLVs at the insertion sites of Xmv-10 and Emv-15 (an endogenous provirus associated with Ay) revealed the following genetic map information: cen-A-0.31 +/- 0.31 cM-Emv-15-0.62 +/- 0.27 cM-Xmv-10-tel. Haplotype analysis of inbred strains in which a was not associated with Xmv-10 and in which Ay was not associated with Emv-15 demonstrated that these "exceptions" were explained most simply by a single recombination that disturbed the linkage relationships evident in most inbred strains.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1994MQ37400001
View details for PubMedID 8111126
alpha-2 adrenergic receptors can be subdivided into three related subtypes which are conserved in humans, rats, and mice. In the mouse, these receptors are encoded by three genes (Adra-2a, Adra-2b, Adra-2c). To gain insight into the evolution of this multigene family and to investigate whether these genes are candidates for previously identified mouse mutations, we have determined the map positions of the Adra-2b and Adra-2c genes. The Adra-2a gene has been previously mapped to mouse Chromosome (Chr) 19 (Oakey et al. Genomics 10, 338-344, 1991). Using segregation among recombinant inbred strains of a single-stranded conformational polymorphism specific for alleles of Adra-2b and Adra-2c, we present map positions for these genes on mouse Chrs 2 and 5, respectively. In the case of Adra-2b, these results have been confirmed by an analysis of somatic cell hybrids. In addition, we generate AKXD recombinant inbred strain distribution patterns for 11 previously defined SSLP microsatellite markers, further refining the haplotype maps for these chromosomes. Finally, several candidate mouse mutations that map close to Adra-2b and Adra-2c are discussed.
View details for Web of Science ID A1993MF57400005
View details for PubMedID 8281014
Expression of the homeobox fusion gene E2A-PBX1 under control of the immunoglobulin heavy chain enhancer efficiently induced malignancies in transgenic mice. All animals died before 5 months of age with lymphomas that demonstrated phenotypes consistent with transitional intermediate thymocytes (CD4+/CD8+/CD3med). E2A-PBX1 also markedly altered lymphoid development in pretumorous animals, reducing the number of thymocytes and bone marrow B lineage progenitors to 20% of normal levels. In spite of the observed reductions in lymphoid cells, premalignant animals contained significantly increased numbers of cycling thymocytes, but a higher proportion was also undergoing apoptosis, suggesting that increased cell death resulted in the marked lymphopenias. These data indicate that the chimeric homeodomain protein E2A-PBX1 paradoxically induces both proliferation and apoptosis in lymphoid cells, suggesting an in vivo association between nuclear oncogene-induced cell cycle progression and programed cell death.
View details for Web of Science ID A1993LX29200009
View details for PubMedID 8104101
The mouse agouti gene controls the deposition of yellow and black pigment in developing hairs. Several dominant alleles, including lethal yellow (Ay), result in the exclusive production of yellow pigment and have pleiotropic effects that include obesity and increased tumor susceptibility. In an interspecific backcross, we established genetic limits for the agouti gene and found that the Ay and the lethal non-agouti (ax) allele were not separated from a previously identified probe at the breakpoint of the Is1GsO chromosomal rearrangement. Using the Is1GsO probe, we isolated the agouti gene, and find that it has the potential to code for a secreted protein expressed in hair follicles and the epidermis, and that the level of expression correlates with the synthesis of yellow pigment. In the Ay mutation, there is a chromosomal rearrangement that results in the production of a chimeric RNA expressed in nearly every tissue of the body. The 5' portion of this chimeric RNA contains highly expressed novel 5' sequences, but the 3' portion retains the protein-coding potential of the nonmutant allele. We speculate that dominant pleiotropic effects of Ay are caused by ectopic activation of a signaling pathway similar to that used during normal hair growth.
View details for Web of Science ID A1993KR21800011
View details for PubMedID 8449404
We have examined the subcellular distribution of three subtypes of adrenergic receptor by immunocytochemical localization of wild-type and epitope-tagged proteins expressed in Cos-7 and K293 cells. Two subtypes (beta 2 and M alpha 2-10H) are localized in the plasma membrane at steady state in untreated cells, while another subtype (M alpha 2-4H) is found both in the plasma membrane and in a population of intracellular vesicles. Within 15 min following the addition of adrenergic agonists, beta 2 and M alpha 2-10H receptors are differentially sorted; beta 2 receptors are selectively internalized to intracellular vesicles, which are distinct from those containing M alpha 2-4H receptors, while M alpha 2-10H receptors remain in the plasma membrane. Subtype-specific sorting suggests a new class of functional properties that may differentiate the signaling and regulation of homologous G protein-coupled receptors.
View details for Web of Science ID A1993KG07700003
View details for PubMedID 7678260
Three subtypes of alpha 2 adrenergic receptors have been identified in the human and rat. The subtype located on human chromosome 2 (alpha 2-C2) is unique in that it is expressed mainly in the peripheral tissues and lacks sites for N-linked glycosylation. We isolated the gene encoding the mouse homolog of the human alpha 2-C2 adrenergic receptor (M alpha 2-2H). The deduced amino acid sequence of the M alpha 2-2H shows 82% and 96% identity to the human alpha 2-C2 and the rat RNG alpha 2 adrenergic receptors, respectively. Southern blot analysis demonstrated that the M alpha 2-2H was encoded by a single copy gene and was distinct from the mouse homologs of the alpha 2-C4 and alpha 2-C10 adrenergic receptors. When expressed in COS-7 cells, the M alpha 2-2H exhibited a pharmacological profile similar to the human alpha 2-C2 and rat RNG alpha 2 receptors.
View details for Web of Science ID A1992JJ80300014
View details for PubMedID 1354956
TAPA-1 is a member of a new family of evolutionarily conserved transmembrane proteins which may be involved in regulation of cell growth and/or cell signalling. We have examined the temporal pattern of TAPA-1 RNA expression during mouse development. Using a sensitive reverse transcription/polymerase chain reaction assay, we show that TAPA-1 RNA is present in oocytes, fertilized eggs and cleavage stage embryos.
View details for Web of Science ID A1992JJ80300003
View details for PubMedID 1380797
Molecular cloning and ligand binding studies have shown the alpha 2 class of adrenergic receptor (alpha 2-AR) to be a family of at least three related subtypes in humans. These studies have not, however, identified distinct subtype-specific functions for these receptors in vivo. It should be possible to extend the analysis of alpha 2-AR subtype function to the animal level through the use of experimental mammalian embryology in mice. To begin this process, we have isolated two mouse genomic clones encoding alpha 2-AR subtypes and expressed these genes in COS-7 cells for binding studies. Sequence homology and ligand binding data allow the assignment of one clone (M alpha 2-4H) as the mouse homolog of the human alpha 2-C4 subtype. The other clone (M alpha 2-10H) closely resembles the human alpha 2-C10 subtype in sequence but binds with significantly lower affinity to yohimbine and rauwolscine, members of a distinct class of bulky alpha 2-selective antagonists commonly used to evaluate alpha 2-AR function in vivo. To define the domain(s) responsible for this unusual binding property, we constructed a series of M alpha 2-10H/human alpha 2-C10 chimeric receptors. Analysis of these receptors identified a conservative Cys201 to Ser201 change in the fifth transmembrane domain of M alpha 2-10H as being responsible for the low affinity of the mouse receptor for yohimbine.
View details for Web of Science ID A1992JE03200004
View details for PubMedID 1353249
The region surrounding the agouti coat color locus on mouse Chromosome 2 contains several genes required for peri-implantation development, limb morphogenesis, and segmentation of the nervous system. We have applied radiation hybrid mapping, a somatic cell genetic technique for constructing long-range maps of mammalian chromosomes, to eight molecular markers in this region. Using a mathematical model to estimate the frequency of radiation-induced breakage, we have constructed a map that spans approximately 20 recombination units and 475 centirays8000. The predicted order of markers, Prn-p-Pygb-Emv-13-Psp-Xmv-10-Emv-15-Src-Ada, is consistent with a previously derived multipoint meiotic map for six of the eight markers and suggests that Xmv-10 may lie relatively close to one or more of the agouti recessive lethal mutations. The resolution of our map is approximately 40-fold higher than the meiotic map, but the median retention frequency of mouse DNA in hybrid cells, 0.12, is 4-fold lower than similar experiments with human chromosomes. From one of the radiation hybrid lines that contained a minimum amount of mouse DNA, 25 independent cosmids were isolated with a mouse-specific hybridization probe. Single-copy fragments from two of these cosmids were shown to originate from mouse Chromosome 2, and the meiotic map position of one was found to be within 10 recombination units of the region of interest. Our results indicate more precise map positions for Pygb and Xmv-10, demonstrate that radiation hybrid mapping can provide high-resolution map information for the mouse genome, and establish a new method for isolating large fragments of DNA from a specific subchromosomal region.
View details for Web of Science ID A1992HY38800034
View details for PubMedID 1639401