Agouti-induced anxiety-like behaviour is mediated by central serotonergic pathways in zebrafish.

Overexpression of the agouti-signalling protein (asip1), an endogenous melanocortin antagonist, under the control of a constitutive promoter in zebrafish [Tg(Xla.Eef1a1:Cau.Asip1]iim4] (asip1-Tg) increases food intake by reducing sensitivity of the central satiety systems and abolish circadian activity rhythms. The phenotype also shows increased linear growth and body weight, yet no enhanced aggressiveness in dyadic fights is observed. In fact, asip1-Tg animals choose to flee to safer areas rather than face a potential threat thus suggesting a potential anxiety-like behaviour (ALB). Standard behavioural tests, i.e. the open field test (OFT), the novel object test (NOT) and the novel tank dive test (NTDT) were used to investigate thigmotaxis and ALB in male and female zebrafish. Results showed that the asip1-Tg strain exhibited severe ALB in every test, mainly characterised by pronounced freezing behaviour and increased linear and angular swimming velocities. asip1-Tg animals exhibited low central serotonin (5HT) and dopamine (DA) levels and high turnover rates, thus suggesting that central monoaminergic pathways might mediate melanocortin antagonist-induced ALB. Accordingly, the treatment of asip1-Tg animals with fluoxetine, a selective serotonin reuptake inhibitor (SSRI), reversed the ALB phenotype in NTDT as well as 5-HT turnover. Genomic and anatomical data further supported neuronal interaction between melanocortinergic and serotonergic systems. These results suggest that inhibition of the melanocortin system by ubiquitous overexpression of endogenous antagonist has an anxiogenic effect mediated by serotonergic transmission.Significance statement In this paper, we show that inhibition of the melanocortin system by ubiquitous overexpression of endogenous antagonists has a potent anxiogenic effect mediated by serotonergic transmission in zebrafish. asip1-overexpressing fish (asip1-Tg) also exhibit a severe disruption of the central satiety system, leading to increased feed intake and abolishing circadian locomotor activity patterns. The melanocortin system plays a key role in the control of hunger, and data suggest that the anxiety-like behaviour in asip1-Tg may be related to the feeding anxiety induced by negative energy balance states, which promote constant foraging and thus disrupt activity rhythms. This makes asip1-Tg animals an excellent model to study dieting-induced anxiety, one of the major causes of dieting failure.

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

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

Obesity Impairs Cognitive Function with No Effects on Anxiety-like Behaviour in Zebrafish.

Over the last decade, the zebrafish has emerged as an important model organism for behavioural studies and neurological disorders, as well as for the study of metabolic diseases. This makes zebrafish an alternative model for studying the effects of energy disruption and nutritional quality on a wide range of behavioural aspects. Here, we used the zebrafish model to study how obesity induced by overfeeding regulates emotional and cognitive processes. Two groups of fish (n = 24 per group) were fed at 2% (CTRL) and 8% (overfeeding-induced obesity, OIO) for 8 weeks and tested for anxiety-like behaviour using the novel tank diving test (NTDT). Fish were first tested using a short-term memory test (STM) and then trained for four days for a long-term memory test (LTM). At the end of the experiment, fish were euthanised for biometric sampling, total lipid content, and triglyceride analysis. In addition, brains (eight per treatment) were dissected for HPLC determination of monoamines. Overfeeding induced faster growth and obesity, as indicated by increased total lipid content. OIO had no effect on anxiety-like behaviour. Animals were then tested for cognitive function (learning and memory) using the aversive learning test in Zantiks AD units. Results show that both OIO and CTRL animals were able to associate the aversive stimulus with the conditioned stimulus (conditioned learning), but OIO impaired STM regardless of fish sex, revealing the effects of obesity on cognitive processes in zebrafish. Obese fish did not show a deficiency in monoaminergic transmission, as revealed by quantification of total brain levels of dopamine and serotonin and their metabolites. This provides a reliable protocol for assessing the effect of metabolic disease on cognitive and behavioural function, supporting zebrafish as a model for behavioural and cognitive neuroscience.

Agouti-Signalling Protein Overexpression Reduces Aggressiveness in Zebrafish.

Feeding motivation plays a crucial role in food intake and growth. It closely depends on hunger and satiation, which are controlled by the melanocortin system. Overexpression of the inverse agonist agouti-signalling protein (ASIP) and agouti-related protein (AGRP) leads to enhanced food intake, linear growth, and weight. In zebrafish, overexpression of Agrp leads to the development of obesity, in contrast to the phenotype observed in transgenic zebrafish that overexpress asip1 under the control of a constitutive promoter (asip1-Tg). Previous studies have demonstrated that asip1-Tg zebrafish exhibit larger sizes but do not become obese. These fish display increased feeding motivation, resulting in a higher feeding rate, yet a higher food ration is not essential in order to grow larger than wild-type (WT) fish. This is most likely attributed to their improved intestinal permeability to amino acids and enhanced locomotor activity. A relationship between high feeding motivation and aggression has been previously reported in some other transgenic species showing enhanced growth. This study aims to elucidate whether the hunger observed in asip1-Tg is linked to aggressive behaviour. Dominance and aggressiveness were quantified using dyadic fights and mirror-stimulus tests, in addition to the analysis of basal cortisol levels. The results indicate that asip1-Tg are less aggressive than WT zebrafish in both dyadic fights and mirror-stimulus tests.

Loss-of-function mutations in melanocortin-1 receptor modulate immune response in teleost fishes.

The melanocortin system is an ancient neuroendocrine system conserved from teleosts to mammals. The melanocortin system is a set of complex neuroendocrine signaling pathways involved in numerous physiological processes, and particularly associated with the hypothalamic-pituitary-interrenal (HPI) axis response. The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in pigmentation in vertebrates, including fish. In order to assess the immune role of MC1R, this study used a homozygous Mc1r knockout zebrafish. Hence, skin cortisol levels, variations in the blood leucocyte population, as well as the expression levels of immune genes in various tissues of wild-type TU strain (Tübingen, Nüsslein-Volhard Lab) (WT) and homozygous mc1r knockout zebrafish (mc1rK.O.) stimulated with LPS was carried out. Results show that the mc1rK.O. mutant fish produce lower levels of cortisol in mucus and fewer macrophages in blood after exposure to LPS compared to control fish. Regarding the expression of immune genes, mutant fish show a significant increase in the expression of the anti-inflammatory interleukin il10. These results suggest that the mc1rK.O. mutant fish may follow an alternative mechanism among the immune responses, where macrophages seem to have an anti-inflammatory function, attenuating nitric oxide (NO) production and providing an advantage through the mitigation of excessive or strong inflammatory reactions. Nonetheless, a lower number of this cell type could imply a reduced phagocytic potential in the face of an infection. At the same time, lower cortisol levels in the mc1rK.O. mutant fish could be an advantage as for the lower susceptibility to stress and the physiological and metabolic consequences of high cortisol levels.

Role of the Melanocortin System in Gonadal Steroidogenesis of Zebrafish.

In teleost, as in other vertebrates, stress affects reproduction. A key component of the stress response is the pituitary secretion of the adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. In zebrafish, Mc2r was identified in male and female gonads, while ACTH has been shown to have a physiological role in modulating reproductive activity. In this study, the hypothesis that other melanocortins may also affect how the zebrafish gonadal function is explored, specifically steroid biosynthesis, given the presence of members of the melanocortin signaling system in zebrafish gonads. Using cell culture, expression analysis, and cellular localization of gene expression, our new observations demonstrated that melanocortin receptors, accessory proteins, antagonists, and agonists are expressed in both the ovary and testis of zebrafish (n = 4 each sex). Moreover, melanocortin peptides modulate both basal and gonadotropin-stimulated steroid release from zebrafish gonads (n = 15 for males and n = 50 for females). In situ hybridization in ovaries (n = 3) of zebrafish showed mc1r and mc4r in follicular cells and adjacent to cortical alveoli in the ooplasm of previtellogenic and vitellogenic oocytes. In zebrafish testes (n = 3), mc4r and mc1r were detected exclusively in germ cells, specifically in spermatogonia and spermatocytes. Our results suggest that melanocortins are, directly or indirectly, involved in the endocrine control of vitellogenesis in females, through modulation of estradiol synthesis via autocrine or paracrine actions in zebrafish ovaries. Adult zebrafish testes were sensitive to low doses of ACTH, eliciting testosterone production, which indicates a potential role of this peptide as a paracrine regulator of testicular function.

Distribution of two isoforms of tryptophan hydroxylase in the brain of rainbow trout (Oncorhynchus mykiss). An in situ hybridization study.

Serotonin (5-HT) is one of the principal neurotransmitters in the nervous system of vertebrates. It is initially synthesized by hydroxylation of tryptophan (Trp) by means of tryptophan hydroxylase or TPH which is the rate-limiting enzyme in the production of 5-HT. In most vertebrates, there are two isoforms of TPH present, TPH1 and TPH2, which exhibit different catalytic or substrate specificity as well as different expression domains. Studies carried out in mammals show that only tph2 is expressed in the brain whereas tph1-mRNA is primarily localized in the enterochromaffin cells and pineal gland. A large number of neurons are also considered to be serotonergic or "pseudo-serotonergic" as they accumulate and release 5-HT yet do not produce it as no amine-synthetic enzymes are expressed, yet a combination of 5-HT transporters is observed. Therefore, tph expression is considered to be the only specific marker of 5-HT-producing neurons that can discriminate true 5-HT from pseudo-serotonergic neurons. This work examined in situ hybridization to study the mRNA distribution of one paralogue for tph1 and tph2 in the central nervous system of rainbow trout. Results show a segregated expression for both paralogues that predominantly match previous immunocytochemical studies. This study thus adds valuable information to the scarce analyses focusing on the central distribution of the expression of serotonergic markers, particularly tphs, in the vertebrate brain thus characterizing the true serotonergic brain territories.

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

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

Brain transcriptome profile after CRISPR-induced ghrelin mutations in zebrafish.

Ghrelin (GRL) is a gut-brain hormone with a role in a wide variety of physiological functions in mammals and fish, which points out the ghrelinergic system as a key element for the appropriate biological functioning of the organism. However, many aspects of the multifunctional nature of GRL remain to be better explored, especially in fish. In this study, we used the CRISPR/Cas9 genome editing technique to generate F0 zebrafish in which the expression of grl is compromised. Then, we employed high-throughput mRNA sequencing (RNA-seq) to explore changes in the brain transcriptome landscape associated with the silencing of grl. The CRISPR/Cas9 technique successfully edited the genome of F0 zebrafish resulting in individuals with considerably lower levels of GRL mRNAs and protein and ghrelin O-acyl transferase (goat) mRNAs in the brain, intestine, and liver compared to wild-type (WT) zebrafish. Analysis of brain transcriptome revealed a total of 1360 differentially expressed genes (DEGs) between the grl knockdown (KD) and WT zebrafish, with 664 up- and 696 downregulated DEGs in the KD group. Functional enrichment analysis revealed that DEGs are highly enriched for terms related to morphogenesis, metabolism (especially of lipids), entrainment of circadian clocks, oxygen transport, apoptosis, and response to stimulus. The present study offers valuable information on the central genes and pathways implicated in functions of GRL, and points out the possible involvement of this peptide in some novel functions in fish, such as apoptosis and oxygen transport.

Signal Transduction and Pathogenic Modifications at the Melanocortin-4 Receptor: A Structural Perspective.

The melanocortin-4 receptor (MC4R) can be endogenously activated by binding of melanocyte-stimulating hormones (MSH), which mediates anorexigenic effects. In contrast, the agouti-related peptide (AgRP) acts as an endogenous inverse agonist and suppresses ligand-independent basal signaling activity (orexigenic effects). Binding of ligands to MC4R leads to the activation of different G-protein subtypes or arrestin and concomitant signaling pathways. This receptor is a key protein in the hypothalamic regulation of food intake and energy expenditure and naturally-occurring inactivating MC4R variants are the most frequent cause of monogenic obesity. In general, obesity is a growing problem on a global scale and is of social, medical, and economic relevance. A significant goal is to develop optimized pharmacological tools targeting MC4R without adverse effects. To date, this has not been achieved because of inter alia non-selective ligands across the five functionally different MCR subtypes (MC1-5R). This motivates further investigation of (i) the three-dimensional MC4R structure, (ii) binding mechanisms of various ligands, and (iii) the molecular transfer process of signal transduction, with the aim of understanding how structural features are linked with functional-physiological aspects. Unfortunately, experimentally elucidated structural information is not yet available for the MC receptors, a group of class A G-protein coupled receptors (GPCRs). We, therefore, generated MC4R homology models and complexes with interacting partners to describe approximate structural properties associated with signaling mechanisms. In addition, molecular insights from pathogenic mutations were incorporated to discriminate more precisely their individual malfunction of the signal transfer mechanism.

Evolution of proopiomelanocortin.

Proopiomelanocortin (POMC) belongs to the opioid/orphanin gene family whose peptide precursors include either opioid (YGGF) or the orphanin/nociceptin core sequences (FGGF). In addition to POMC the family includes the proenkephalin (PENK), prodynorphin (PDYN), and nociceptin/proorphanin (PNOC) precursors. The opioid core sequence in POMC is incorporated by the ß-endorphin that occupies the C-terminal region but this propeptide also exhibits at least two "alien" melanocortin core sequences (HFRW). An ACTH/MSH fragment merged into the opioid fragment not earlier than the two tetraploidizations of the vertebrate genome. Therefore, POMC exhibit a complex "evolutionary life" since the gene has coevolved together with two different receptor systems, i.e., opioid and melanocortin following a horse trading system. In this article, we summarize the different evolutionary hypotheses proposed for POMC evolution.

Loss-of-function mutations in the melanocortin 1 receptor cause disruption of dorso-ventral countershading in teleost fish.

The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in vertebrate pigmentation. Mutations in this gene cause variations in coat coloration in amniotes. Additionally, in mammals MC1R is the main receptor for agouti-signaling protein (ASIP), making it the critical receptor for the establishment of dorsal-ventral countershading. In fish, Mc1r is also involved in pigmentation, but it has been almost exclusively studied in relation to melanosome dispersion activity and as a putative genetic factor involved in dark/light adaptation. However, its role as the crucial component for the Asip1-dependent control of dorsal-ventral pigmentation remains unexplored. Using CRISPR/Cas9, we created mc1r homozygous knockout zebrafish and found that loss-of-function of mc1r causes a reduction of countershading and a general paling of the animals. We find ectopic development of melanophores and xanthophores, accompanied by a decrease in iridophore numbers in the ventral region of mc1r mutants. We also reveal subtle differences in the role of mc1r in repressing pigment cell development between the skin and scale niches in ventral regions.

Countershading in zebrafish results from an Asip1 controlled dorsoventral gradient of pigment cell differentiation.

Dorso-ventral (DV) countershading is a highly-conserved pigmentary adaptation in vertebrates. In mammals, spatially regulated expression of agouti-signaling protein (ASIP) generates the difference in shading by driving a switch between the production of chemically-distinct melanins in melanocytes in dorsal and ventral regions. In contrast, fish countershading seemed to result from a patterned DV distribution of differently-coloured cell-types (chromatophores). Despite the cellular differences in the basis for counter-shading, previous observations suggested that Agouti signaling likely played a role in this patterning process in fish. To test the hypotheses that Agouti regulated counter-shading in fish, and that this depended upon spatial regulation of the numbers of each chromatophore type, we engineered asip1 homozygous knockout mutant zebrafish. We show that loss-of-function asip1 mutants lose DV countershading, and that this results from changed numbers of multiple pigment cell-types in the skin and on scales. Our findings identify asip1 as key in the establishment of DV countershading in fish, but show that the cellular mechanism for translating a conserved signaling gradient into a conserved pigmentary phenotype has been radically altered in the course of evolution.

Melanocortin Receptor Accessory Protein 2-Induced Adrenocorticotropic Hormone Response of Human Melanocortin 4 Receptor.

Melanocortin 4 receptor (MC4R), a canonical melanocyte-stimulating hormone receptor, is the main responsible for monogenic obesity in humans. Previous studies in fish and avian species showed that MC4R becomes an ACTH receptor after interaction with the melanocortin receptor accessory protein 2 (MRAP2). We show that human MC4R behaves in a similar way through its interaction with MRAP2. This evolutionary conservation of MRAP2-induced ligand selectivity supports a physiological role for the interaction with MC4R. Both proteins are coexpressed in the same hypothalamic neurons, providing an anatomical substrate and molecular mechanism for the central therapeutic actions of ACTH in the treatment of infantile spasms. These neurons may link the effects of stress on the energy balance independently of glucocorticoid secretion. The complex MC4R-MRAP2 throws light on the action of ACTH and, by extension, on the relay of stress-related information to additional biological systems.

Central regulation of food intake in fish: an evolutionary perspective.

Evidence indicates that central regulation of food intake is well conserved along the vertebrate lineage, at least between teleost fish and mammals. However, several differences arise in the comparison between both groups. In this review, we describe similarities and differences between teleost fish and mammals on an evolutionary perspective. We focussed on the existing knowledge of specific fish features conditioning food intake, anatomical homologies and analogies between both groups as well as the main signalling pathways of neuroendocrine and metabolic nature involved in the homeostatic and hedonic central regulation of food intake.

Expression of genes for melanotropic peptides and their receptors for morphological color change in goldfish Carassius auratus.

To evaluate the association of the melanotropic peptides and their receptors for morphological color change, we investigated the effects of changes in background color, between white and black, on xanthophore density in the scales and expression levels of genes for hormonal peptides and corresponding receptors (MCH-R2, MC1R, and MC5R) in goldfish (Carassius auratus). The xanthophore density in both dorsal and ventral scales increased after transfer from a white to black background. However, xanthophore density in dorsal scales increased after transfer from a black to white background, and that of ventral scales decreased after transfer from a black to black background, which served as the control. In the white-reared fish, melanin-concentrating hormone (mch) mRNA content in the brain was higher than that in black-reared fish, whereas proopiomelanocortin a (pomc-a) mRNA content in the pituitary was lower than that in the black-reared fish. Agouti-signaling protein (asp) mRNA was detected in the ventral skin but not in the dorsal skin. No difference was observed in the asp mRNA content between fish reared in white or black background, suggesting that ASP might not be associated with background color adaptation. In situ hybridization revealed that both mc1r and mc5r were expressed in the xanthophores in scales. The mRNA content of mc1r in scales did not always follow the background color change, whereas those of mc5r decreased in the white background and increased in the black background, suggesting that mc5r might be a major factor reinforcing the function of MSH in morphological color changes. White backgrounds increased mch mRNA content in the brain, but decreased mch-r2 mRNA content in the scales. These altered expression levels of melanotropin receptors might affect reactivity to melanotropins through long-term adaptation to background color.

Effects of acute handling stress on short-term central expression of orexigenic/anorexigenic genes in zebrafish.

Physiological mechanisms driving stress response in vertebrates are evolutionarily conserved. These mechanisms involve the activation of both the hypothalamic-sympathetic-chromaffin cell (HSC) and the hypothalamic-pituitary-adrenal (HPA) axes. In fish, the reduction of food intake levels is a common feature of the behavioral response to stress but the central mechanisms coordinating the energetic response are not well understood yet. In this work, we explore the effects of acute stress on key central systems regulating food intake in fish as well as on total body cortisol and glucose levels. We show that acute stress induced a rapid increase in total body cortisol with no changes in body glucose, at the same time promoting a prompt central response by activating neuronal pathways. All three orexigenic peptides examined, i.e., neuropeptide y (npy), agouti-related protein (agrp), and ghrelin, increased their central expression level suggesting that these neuronal systems are not involved in the short-term feeding inhibitory effects of acute stress. By contrast, the anorexigenic precursors tested, i.e., cart peptides and pomc, exhibited increased expression after acute stress, suggesting their involvement in the anorexigenic effects.

Data on the density of xanthophores in a whole scale of goldfish acclimated to white or black background color.

The data presented in this article are related to the research article entitled "Expression of genes for melanotropic peptides and their receptors for morphological color change in goldfish Carassius auratus" (Mizusawa et al., In press) [1]. This article describes data on the density of xanthophores in the scales of goldfish acclimated to white or black background color. To determine the effects of acclimation history during long-term background color adaptation, fish were transferred from a white tank to a white or black tank and vice versa halfway through the acclimation process. To observe xanthophores, the iridophore layer was scraped from the scale and the pteridine/carotenoid pigments were aggregated. The number of xanthophores was calculated after image processing.

Fish pigmentation and the melanocortin system.

The melanocortin system is a complex neuroendocrine signaling mechanism involved in numerous physiological processes in vertebrates, including pigmentation, steroidogenesis and metabolic control. This review focuses at one of its most fascinating function in fish, its regulatory role in the control of pigmentation, in which the melanocortin 1 receptor (Mc1r), its agonist α-melanocyte stimulating hormone (α-Msh), and the endogenous antagonist agouti signaling protein (Asip1) are the main players. Functional control of Mc1r, which is highly expressed in fish skin and whose activation stimulates melanin production and melanosome dispersion in fish melanophores, is considered a key mechanism for vertebrate pigment phenotypes. The α-Msh peptide, the most documented Mc1r agonist involved in pigmentation, is produced in the pituitary gland, activating melanin synthesis by binding to Mc1r in fish melanophores. Finally, Asip1 is the putative factor for establishing the evolutionarily conserved dorso-ventral pigment pattern found across vertebrates. However, we are just starting to understand how other melanocortin system components are acting in this complex regulatory network.

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

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