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The Bengal cat breed was developed from intercrosses between the Asian leopard cat, Prionailurus bengalensis, and the domestic cat, Felis catus, with a last common ancestor approximately 6 million years ago. Predicted to derive ∼94% of their genome from domestic cats, regions of the leopard cat genome are thought to account for the unique pelage traits and ornate color patterns of the Bengal breed, which are similar to those of ocelots and jaguars. We explore ancestry distribution and selection signatures in the Bengal breed by using reduced representation and whole-genome sequencing from 947 cats. The mean proportion of leopard cat DNA in the Bengal breed is 3.48%, lower than predicted from breed history, and is broadly distributed, covering 93% of the Bengal genome. Overall, leopard cat introgressions do not show strong signatures of selection across the Bengal breed. However, two popular color traits in Bengal cats, charcoal and pheomelanin intensity, are explained by selection of leopard cat genes whose expression is reduced in a domestic cat background, consistent with genetic incompatibility resulting from hybridization. We characterize several selective sweeps in the Bengal genome that harbor candidate genes for pelage and color pattern and that are associated with domestic, rather than leopard, cat haplotypes. We identify the molecular and phenotypic basis of one selective sweep as reduced expression of the Fgfr2 gene, which underlies glitter, a trait desired by breeders that affects hair texture and light reflectivity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Color variation is a frequent evolutionary substrate for camouflage in small mammals, but the underlying genetics and evolutionary forces that drive color variation in natural populations of large mammals are mostly unexplained. The American black bear, Ursus americanus (U. americanus), exhibits a range of colors including the cinnamon morph, which has a similar color to the brown bear, U. arctos, and is found at high frequency in the American southwest. Reflectance and chemical melanin measurements showed little distinction between U. arctos and cinnamon U. americanus individuals. We used a genome-wide association for hair color as a quantitative trait in 151 U. americanus individuals and identified a single major locus (p < 10 -13 ). Additional genomic and functional studies identified a missense alteration (R153C) in Tyrosinase-related protein 1 (TYRP1) that likely affects binding of the zinc cofactor, impairs protein localization, and results in decreased pigment production. Population genetic analyses and demographic modeling indicated that the R153C variant arose 9.36 kya in a southwestern population where it likely provided a selective advantage, spreading both northwards and eastwards by gene flow. A different TYRP1 allele, R114C, contributes to the characteristic brown color of U. arctos but is not fixed across the range., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Effective conservation and management of threatened wildlife populations require an accurate assessment of age structure to estimate demographic trends and population viability. Epigenetic aging models are promising developments because they estimate individual age with high accuracy, accurately predict age in related species, and do not require invasive sampling or intensive long-term studies. Using blood and biopsy samples from known age plains zebras (Equus quagga), we model epigenetic aging using two approaches: the epigenetic clock (EC) and the epigenetic pacemaker (EPM). The plains zebra EC has the potential for broad application within the genus Equus given that five of the seven extant wild species of the genus are threatened. We test the EC's ability to predict age in sister taxa, including two endangered species and the more distantly related domestic horse, demonstrating high accuracy in all cases. By comparing chronological and estimated age in plains zebras, we investigate age acceleration as a proxy of health status. An interaction between chronological age and inbreeding is associated with age acceleration estimated by the EPM, suggesting a cumulative effect of inbreeding on biological aging throughout life., (© 2021. The Author(s).)

Distinctive colour patterns in dogs are an integral component of canine diversity. Colour pattern differences are thought to have arisen from mutation and artificial selection during and after domestication from wolves but important gaps remain in understanding how these patterns evolved and are genetically controlled. In other mammals, variation at the ASIP gene controls both the temporal and spatial distribution of yellow and black pigments. Here, we identify independent regulatory modules for ventral and hair cycle ASIP expression, and we characterize their action and evolutionary origin. Structural variants define multiple alleles for each regulatory module and are combined in different ways to explain five distinctive dog colour patterns. Phylogenetic analysis reveals that the haplotype combination for one of these patterns is shared with Arctic white wolves and that its hair cycle-specific module probably originated from an extinct canid that diverged from grey wolves more than 2 million years ago. Natural selection for a lighter coat during the Pleistocene provided the genetic framework for widespread colour variation in dogs and wolves., (© 2021. The Author(s).)

Most endangered species exist today in small populations, many of which are isolated. Evolution in such populations is largely governed by genetic drift. Empirical evidence for drift affecting striking phenotypes based on substantial genetic data are rare. Approximately 37% of tigers ( Panthera tigris ) in the Similipal Tiger Reserve (in eastern India) are pseudomelanistic, characterized by wide, merged stripes. Camera trap data across the tiger range revealed the presence of pseudomelanistic tigers only in Similipal. We investigated the genetic basis for pseudomelanism and examined the role of drift in driving this phenotype's frequency. Whole-genome data and pedigree-based association analyses from captive tigers revealed that pseudomelanism cosegregates with a conserved and functionally important coding alteration in Transmembrane Aminopeptidase Q ( Taqpep ), a gene responsible for similar traits in other felid species. Noninvasive sampling of tigers revealed a high frequency of the Taqpep p.H454Y mutation in Similipal (12 individuals, allele frequency = 0.58) and absence from all other tiger populations (395 individuals). Population genetic analyses confirmed few (minimal number) tigers in Similipal, and its genetic isolation, with poor geneflow. Pairwise F ST (0.33) at the mutation site was high but not an outlier. Similipal tigers had low diversity at 81 single nucleotide polymorphisms (mean heterozygosity = 0.28, SD = 0.27). Simulations were consistent with founding events and drift as possible drivers for the observed stark difference of allele frequency. Our results highlight the role of stochastic processes in the evolution of rare phenotypes. We highlight an unusual evolutionary trajectory in a small and isolated population of an endangered species., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Intricate color patterns are a defining aspect of morphological diversity in the Felidae. We applied morphological and single-cell gene expression analysis to fetal skin of domestic cats to identify when, where, and how, during fetal development, felid color patterns are established. Early in development, we identify stripe-like alterations in epidermal thickness preceded by a gene expression pre-pattern. The secreted Wnt inhibitor encoded by Dickkopf 4 plays a central role in this process, and is mutated in cats with the Ticked pattern type. Our results bring molecular understanding to how the leopard got its spots, suggest that similar mechanisms underlie periodic color pattern and periodic hair follicle spacing, and identify targets for diverse pattern variation in other mammals., (© 2021. The Author(s).)

One of the most iconic wild equids, the plains zebra occupies a broad region of sub-Saharan Africa and exhibits a wide range of phenotypic diversity in stripe patterns that have been used to classify multiple subspecies. After decades of relative stability, albeit with a loss of at least one recognized subspecies, the total population of plains zebras has undergone an approximate 25% decline since 2002. Individuals with abnormal stripe patterns have been recognized in recent years but the extent to which their appearance is related to demography and/or genetics is unclear. Investigating population genetic health and genetic structure are essential for developing effective strategies for plains zebra conservation. We collected DNA from 140 plains zebra, including seven with abnormal stripe patterns, from nine locations across the range of plains zebra, and analyzed data from restriction site-associated and whole genome sequencing (RAD-seq, WGS) libraries to better understand the relationships between population structure, genetic diversity, inbreeding, and abnormal phenotypes. We found that genetic structure did not coincide with described subspecific variation, but did distinguish geographic regions in which anthropogenic habitat fragmentation is associated with reduced gene flow and increased evidence of inbreeding, especially in certain parts of East Africa. Further, zebras with abnormal striping exhibited increased levels of inbreeding relative to normally striped individuals from the same populations. Our results point to a genetic cause of stripe pattern abnormalities, and dramatic evidence of the consequences of habitat fragmentation., (© 2020 John Wiley & Sons Ltd.)

Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNFα-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species., Competing Interests: The authors have declared that no competing interests exist.

Background: The lion (Panthera leo) is one of the most popular and iconic feline species on the planet, yet in spite of its popularity, the last century has seen massive declines for lion populations worldwide. Genomic resources for endangered species represent an important way forward for the field of conservation, enabling high-resolution studies of demography, disease, and population dynamics. Here, we present a chromosome-level assembly from a captive African lion from the Exotic Feline Rescue Center (Center Point, IN) as a resource for current and subsequent genetic work of the sole social species of the Panthera clade., Results: Our assembly is composed of 10x Genomics Chromium data, Dovetail Hi-C, and Oxford Nanopore long-read data. Synteny is highly conserved between the lion, other Panthera genomes, and the domestic cat. We find variability in the length of runs of homozygosity across lion genomes, indicating contrasting histories of recent and possibly intense inbreeding and bottleneck events. Demographic analyses reveal similar ancient histories across all individuals during the Pleistocene except the Asiatic lion, which shows a more rapid decline in population size. We show a substantial influence on the reference genome choice in the inference of demographic history and heterozygosity., Conclusions: We demonstrate that the choice of reference genome is important when comparing heterozygosity estimates across species and those inferred from different references should not be compared to each other. In addition, estimates of heterozygosity or the amount or length of runs of homozygosity should not be taken as reflective of a species, as these can differ substantially among individuals. This high-quality genome will greatly aid in the continuing research and conservation efforts for the lion, which is rapidly moving towards becoming a species in danger of extinction.

The β-defensins are a class of small cationic proteins that serve as components of numerous systems in vertebrate biology, including the immune and melanocortin systems. Human β-defensin 3 (HBD3), which is produced in the skin, has been found to bind to melanocortin receptors 1 and 4 through complementary electrostatics, a unique mechanism of ligand-receptor interaction. This finding indicates that electrostatics alone, and not specific amino acid contact points, could be sufficient for function in this ligand-receptor system, and further suggests that other small peptide ligands could interact with these receptors in a similar fashion. Here, we conducted molecular-similarity analyses and functional studies of additional members of the human β-defensin family, examining their potential as ligands of melanocortin-1 receptor, through selection based on their electrostatic similarity to HBD3. Using Poisson-Boltzmann electrostatic calculations and molecular-similarity analysis, we identified members of the human β-defensin family that are both similar and dissimilar to HBD3 in terms of electrostatic potential. Synthesis and functional testing of a subset of these β-defensins showed that peptides with an HBD3-like electrostatic character bound to melanocortin receptors with high affinity, whereas those that were anticorrelated to HBD3 showed no binding affinity. These findings expand on the central role of electrostatics in the control of this ligand-receptor system and further demonstrate the utility of employing molecular-similarity analysis. Additionally, we identified several new potential ligands of melanocortin-1 receptor, which may have implications for our understanding of the role defensins play in melanocortin physiology., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

The β-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 β-defensins produced in skin, β-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 β-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 α-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 β-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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.