fi Pineal-speci c agouti protein regulates teleost INAUGURAL ARTICLE background adaptation Chao Zhanga, Youngsup Songb, Darren A. Thompsonc, Michael A. Madonnac, Glenn L. Millhauserc, Sabrina Torod, Zoltan Vargad, Monte Westerfieldd, Joshua Gamsee, Wenbiao Chena, and Roger D. Conea,1 aDepartment of Molecular Physiology and Biophysics, The Vanderbilt University School of Medicine, Nashville, TN 37232; bThe Salk Institute for Biological Sciences, La Jolla, CA 92037; cDepartment of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064; dInstitute of Neuroscience, University of Oregon, Eugene, OR 97403; and eDepartment of Biological Sciences, The Vanderbilt University Medical Center, Nashville, TN 37232 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2010. Contributed by Roger D. Cone, October 6, 2010 (sent for review August 30, 2010) Background adaptation is used by teleosts as one of a variety of An important mechanism in the pigmentary response to photic camouflage mechanisms for avoidance of predation. Background information involves the dispersion or aggregation of pigment adaptation is known to involve light sensing by the retina and granules, or melanophores, in dermal melanocytes via the actions subsequent regulation of melanophore dispersion or contraction of the pituitary peptide α-MSH (melanocyte-stimulating hormone) in melanocytes, mediated by α-melanocyte–stimulating hormone and hypothalamic peptide MCH (melanocyte-concentrating hor- and melanin-concentrating hormone, respectively. Here, we dem- mone), respectively (8, 9). The secretory activity of α-MSH– onstrate that an agouti gene unique to teleosts, agrp2, is specifi- expressing cells of the pituitary increases in response to a black α cally expressed in the pineal and is required for up-regulation of background (10), and serum -MSH levels increase as well (11). hypothalamic pmch and pmchl mRNA and melanosome contraction MCH in teleosts is found in secretory neurophysiotrophic neurons fl in the lateral tuberal nuclei (NLT) of the hypothalamus, an arcuate in dermal melanocytes in response to a white background. oating α head, a mutant with defective pineal development, exhibits defec- nucleus equivalent. In contrast to regulation of -MSH, exposure to a white background elevates MCH in the NLT in a variety of fish tive up-regulation of mch mRNAs by white background, whereas fi nrc, a blind mutant, exhibits a normal response. These studies iden- species (12), including the zebra sh (13), and can produce a mea- surable increase in serum MCH. Thus, background adaptation NEUROSCIENCE tify a role for the pineal in background adaptation in teleosts, involves a classic neuroendocrine pathway. a unique physiological function for the agouti family of proteins, fi In nonmammalian vertebrates, the pineal gland is not only an and de ne a neuroendocrine axis by which environmental back- endocrine gland releasing melatonin but also a photosensory or- ground regulates pigmentation. gan, with secondary afferent neurons that innervate a variety of brain regions, comparable to the retinal ganglion cells (14). In- fl fi pigmentation | camou age | zebra sh deed, in the zebrafish, pineal and retinal neurons send projections to a number of nuclei in common, including sites in the anterior amouflage mechanisms are widely used across all orders of the hypothalamus, such as the SCN (15). SCN neurons, in turn, are Canimal kingdom. Although mammals have evolved coat pat- hypophysiotrophic in teleosts, and thus the pineal, through its terning to aid in escaping predation or stalking prey, regulated projections to the SCN, provides a second neuroanatomical path- camouflage, such as the seasonal pelage change in the arctic hare, way through which light can regulate pigmentation. occurs slowly because shedding and growth of a new coat is re- The agouti proteins are small secreted molecules (16–19) that quired. In contrast, reptiles, amphibians, and teleosts can more act as endogenous antagonists of the melanocortin receptors (18, rapidly alter skin color to match their surroundings. 20, 21), a family of G protein–coupled receptors. Two agouti genes One neural circuit involved in nonvisual phototransduction is are found in mammals, agouti (ASP) (16, 17), which regulates the the retinohypothalamic tract. A class of intrinsically photosensi- eumelanin–pheomelanin switch in mammalian pelage, and agouti- tive retinal ganglion cells (ipRGCs) project to anterior hypotha- relatedprotein (AgRP), whichis expressed in theCNS and regulates lamic nuclei, such as the suprachiasmatic nucleus (SCN) (1). This energy homeostasis (18, 19). A third agouti gene, agrp2, has been fi fi fi non–image-forming photoreceptive pathway is independent of identi ed now in several sh species, including zebra sh, trout, fi tetraodon, and torafugu (22). We show here that teleost agrp2 is rod and cone photoreceptors and is suf cient to provide the photic fi input necessary to entrain the circadian clock to the environ- expressed speci cally in the pineal gland, regulates the hypotha- lamic proMCH and proMCH-like genes (pmch and pmchl), and is mental light–dark cycle (2). Blind transgenic mice lacking rods required for melanosome aggregation in response to environmental and cones retain normal photic entrainment of circadian rhythms, background, thus elucidating a pineal–hypothalamic neuroendo- for example (2, 3). Melanopsin seems to be the primary photo- crine pathway by which light regulates pigmentation. pigment in mammalian ipRGCs involved in nonvisual photore- ception regulating circadian entrainment, the sleep–wake cycle, Results fl and the pupillary light re ex (4, 5). agrp2 Is Expressed in the Pineal Gland and Is Not Regulated by In mammals, both visual and nonvisual photoreception re- Metabolic State. To determine the physiological functions of the quires the retina, and the pineal gland receives photic input indi- teleost-specific agouti protein AgRP2, we first examined the rectly, via projections from the SCN that receive direct retinal inputs. In contrast, in nonmammalian vertebrates photoreceptors are found in both the pineal and the skin; melanopsin was origi- fi Author contributions: C.Z., Y.S., G.L.M., M.W., J.G., W.C., and R.D.C. designed research; nally identi ed, for example, in dermal melanocytes of Xenopus C.Z., Y.S., D.A.T., M.A.M., S.T., and Z.V. performed research; G.L.M., M.W., and J.G. con- laevis (6). A variety of opsin molecules are found in the pineal tributed new reagents/analytic tools; G.L.M., M.W., J.G., W.C., and R.D.C. analyzed data; organ of nonmammalian vertebrates, including exo-rhodopsin, and R.D.C. wrote the paper. pinopsin, parapinopsin, and vertebrate ancient opsin (5). In many The authors declare no conflict of interest. species of teleosts background adaptation is regulated in part by Freely available online through the PNAS open access option. the pineal gland: shining light specifically on the pineal causes skin 1To whom correspondence should be addressed. E-mail: [email protected]. lightening, whereas covering the area of the pineal with India ink This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. causes skin darkening (7). 1073/pnas.1014941107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1014941107 PNAS Early Edition | 1of8 Downloaded by guest on October 1, 2021 distribution of agrp2 mRNA in zebrafish embryos [72–96 h post- cubated with serial dilutions of α-MSH in the presence or absence fertilization (hpf)], viewed laterally (Fig. 1A) or dorsally (Fig. 1B) of the folded, cysteine-rich C-terminal domain of AgRP(83–127) or by in situ hybridization to whole mounts, or viewed dorsally in thin AgRP2(93–136), which are comparable to the biologically active hu- sections (Fig. 1C); agrp2 mRNA is detected exclusively in the man AgRP(87–132) (25). AgRP(83–127) blocked MC1R, MC3R, and pineal gland. Real-time quantitative PCR (qPCR) analysis with MC4R, and less potently, MC5Ra and MC5Rb (Fig. 2 A, C, E, G, fi fi adult zebra sh tissues con rmed that agrp2 mRNA is also ex- and I). On the other hand, AgRP2(93–136) was a potent antagonist of pressed most strongly in brain, with lower levels seen in skin, the MC1R but showed little activity at MC4R (Fig. 2 B and F) and no muscle, and testis (Fig. 1D). antagonist activity at MC3R (Fig. 2D). Together with the data on Both mammalian and teleost agrp are regulated by metabolic regulation of agrp2 gene expression, these findings imply distinct state (23), thus we examined whether agrp2 was under similar reg- roles for AgRP and AgRP2. ulatory control. Compared with the control-fed group, a 15-d fast fi signi cantly up-regulated agrp mRNA expression by eightfold in Agrp2 Expression Is Required for Up-Regulation of mch and mchl and the brain of male fish (Fig. 1E), comparable to results previously fi fi Melanosome Contraction Induced by a White Background. To ex- published for the female sh (23). In contrast, no signi cant change amine the functional consequences of agrp2 expression directly, in agrp2 mRNA levels was observed (Fig. 1F). Consistent with the we first characterized the background adaptation response of pineal-specific expression of agrp2,weidentified pineal-specific enhancer elements, such as the photoreceptor-conserved element (PCE), E-box, and pineal expression-promoting element (PIPE), upstream of the agrp2 coding sequence (Fig. S1A). The devel- opmental expression of agrp2 by both whole-mount in situ hybrid- ization and qPCR was also examined. Both procedures detected agrp2 mRNA by 2 d postfertilization (dpf) (Fig. S1 B–F). AgRP2 Is a Competitive Antagonist of the Zebrafish MC1-R. In mammals, AgRP is a selective antagonist of the melanocortin re- ceptors MC3R and MC4R, expressed primarily in the CNS (18, 21). Using HEK293 cells expressing recombinant zebrafish melano- cortin receptors, we showed previously that mouse AgRP(82–131) can competitively decrease α-MSH–mediated cAMP production, with high efficacy at MC1R, MC3R, and MC4R (24).