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Inaugural Article

Pineal-specific agouti protein regulates teleost background adaptation

Chao Zhang, Youngsup Song, Darren A. Thompson, Michael A. Madonna, Glenn L. Millhauser, Sabrina Toro, Zoltan Varga, Monte Westerfield, Joshua Gamse, Wenbiao Chen, and Roger D. Cone
PNAS November 23, 2010 107 (47) 20164-20171; https://doi.org/10.1073/pnas.1014941107
Chao Zhang
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Youngsup Song
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Darren A. Thompson
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Michael A. Madonna
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Glenn L. Millhauser
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Sabrina Toro
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Zoltan Varga
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Monte Westerfield
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Joshua Gamse
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Wenbiao Chen
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Roger D. Cone
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  • For correspondence: roger.cone@vanderbilt.edu
  1. Contributed by Roger D. Cone, October 6, 2010 (sent for review August 30, 2010)

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    Fig. 1.

    agrp2 is expressed in the zebrafish pineal gland and is not regulated by metabolic state. (A) Lateral view of a 96-hpf whole-mount embryo. Whole-mount in situ hybridization was performed with dig-agrp2 antisense probe followed by Nitro blue tetrazolium chloride/ 5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) color development. (B) Dorsal view of a 72-hpf embryo. (C) Frontal view of a 20-μm section from a 96-hpf embryo hybridized as described in A, then embedded in optimal cutting temperature compound and processed using a cryostat. (D) qPCR analysis of agrp2 with tissues from four adult zebrafish (two male and two female). agrp2 mRNA expression was normalized to β-actin mRNA. (E and F) Relative expression levels of agrp and agrp2 by metabolic state as analyzed by qPCR. One-year-old male fish were fed or fasted for indicated times, and the relative mRNA expression levels of agrp and agrp2 normalized to β-actin were determined from whole-brain tissues. Results are expressed as mean ± SEM, and statistical analyses were done by unpaired t test. **P < 0.01; ***P < 0.001. (Scale bars in A–C: 100 μm.)

  • Fig. 2.
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    Fig. 2.

    Pharmacological activity of zebrafish AgRP(83–127) and AgRP2(93–136) peptides. The left column of graphs (A, C, E, G, and I) shows dose–response curves for α-MSH in the presence of 10−6 M (squares, red lines), 10−7 M (triangles, blue lines), 10−8 M (inverted triangles, green lines), or absence (diamonds, black lines) of AgRP(83–127) peptide at the zebrafish melanocortin receptors indicated. The right column of graphs (B, D, F, H, and J) shows dose–response curves for α-MSH in the presence of the same doses of AgRP2(93–136) peptide. α-MSH–stimulated activity of zebrafish melanocortin receptors was monitored using a cAMP-dependant β-galactosidase assay. Data points indicate the averages of triplicate determinations. Experiments were performed in triplicate, and graphs were drawn and analyzed using Graphpad Prism.

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    Fig. 3.

    agrp2 is required for melanosome contraction in zebrafish. Control or morpholino-injected embryos were kept in black-, gray-, or white-bottomed Petri dishes with 14-h/10-h light/dark cycle at 28 °C upon fertilization. (A–C) Dorsal melanocytes of (A) black, (B) gray, or (C) white background-adapted wild-type embryos at 4 dpf. (D–I) Whole-mount in situ hybridization of (D–F) pmch and (G–I) pmchl in black (D and G), gray (E and H), or white (F and I) background-adapted wild-type embryos at 4 dpf. At least 30 embryos for each condition were analyzed. Scale bar: 100 μM. (J) Relative expression levels of pmch and pmchl were analyzed by real-time qPCR. At 2, 3, and 4 dpf, 30 black, gray, or white background-adapted wild-type embryos were divided into three groups and killed for RNA extraction and cDNA synthesis. mRNA expression was normalized to ef1α mRNA. Results are expressed as mean ± SEM, and statistical analysis was done by unpaired t test. *P < 0.05; **P < 0.01. (K–P) MOs designed to inhibit expression of each of the zebrafish agouti proteins were injected into wild-type zebrafish embryos. Dermal melanocytes were examined at 3–5 dpf at 1200 hours. Photographs show the (K and L) dorsal head, (M and N) lateral trunk, and (O and P) yolk melanocytes in inverted control (K, M, and O) or agrp2 (L, N, and P) antisense MO-injected embryos at 4 dpf at 1200 hours. (Q) Melanosome coverage of the lateral trunk was quantified at 4 dpf using ImageJ (National Institutes of Health) on agrp2 ATG MO-injected (28.4%, n = 10), agrp ATG MO-injected (3.6%, n = 10), and asp ATG MO-injected (3.5%, n = 10) embryos compared with inverted control MO-injected embryos (4.4%, n = 10). Error bar indicates ± SEM. Statistical significance tested by unpaired t test. ***P < 0.001.

  • Fig. 4.
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    Fig. 4.

    agrp2 regulates the expression of pmch and pmchl genes in the zebrafish. (A and B) Relative expression levels of pmch and pmchl were analyzed by qPCR. Two hundred wild-type zebrafish zygotes were injected with inverted agrp2 ATG control MO, agrp2 ATG MO, agrp2 5′ UTR MO, agrp ATG MO, asp ATG MO, and pomca ATG MO at day 0. Embryos were kept in egg water, changed daily, with 14-h/10-h light/dark cycle at 28 °C. Thirty embryos from each condition were divided into three groups and killed at 4 dpf (96 h) for RNA extraction and cDNA synthesis. Results are expressed as mean ± SEM, and statistical analysis was done by one-way ANOVA followed by Tukey posttest. **P < 0.01; ***P < 0.001. (C) Whole-mount in situ hybridization for pmch (Bottom) or pmchl (Top) at 4 dpf after injection with inverted agrp2 ATG control MO, agrp2 ATG MO, agrp2 5′ UTR MO, or agrp ATG MO. After BM Purple AP staining, embryos were mounted in 2% methyl cellulose, and pictures were taken using AxionVision 3.1 software with a Lumar V12 stereo microscope (Carl Zeiss). At least 20 embryos for each condition were analyzed. (Scale bar: 50 μm.) (D and E) Numbers of pmch- and pmchl-expressing neurons at 4 dpf from fish injected with morpholinos described in C were counted with a stereomicroscope. Results are expressed as mean ± SEM, and statistical analysis was done by one-way ANOVA followed by Tukey posttest. *P < 0.05; **P < 0.01; ***P < 0.001. Numbers of fish analyzed and represented in each bar from left to right in D and E are 16, 14, 16, 15, 22, 19, 22, and 22, respectively.

  • Fig. 5.
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    Fig. 5.

    pmch, pmchl, and agrp2 are decreased in floating head (flh) mutants. flh+/− fish were crossed, and 400 zygotes were collected at 0 dpf, with 25% expected to be flh−/−. Embryos were kept in egg water, changed daily, with 14-h/10-h light/dark cycle at 28 °C. Phenotypically wild-type or flh−/− embryos were fixed for whole-mount in situ hybridization (4 dpf) or killed for qPCR analysis (3 dpf). (A and B) Dorsal melanocytes of (A) white background-adapted sibling wild-type or (B) flh−/− embryos at 4 dpf. (C–L) Whole-mount in situ hybridization of (C and D) agrp2, (E and F) agrp, (G and H) pmch, (I and J) pmchl, and (K and L) pomca in white background-adapted sibling wild-type (C, E, G, I, and K) or flh−/− embryos (D, F, H, J, and L) at 4 dpf. At least 15 embryos for each condition were analyzed. (M) Relative expression levels of agrp, agrp2, pmch, and pmchl were analyzed by qPCR. Thirty flh−/− and 30 phenotypically wild-type embryos (flh+/− or flh+/+) were divided into three groups and killed at 3 dpf for RNA extraction and cDNA synthesis. mRNA expression was normalized to pomca mRNA, and each expression level was further normalized to wild-type expression levels. Results are expressed as mean ± SEM, and statistical analysis was done by unpaired t test. ***P < 0.001. Scale bar: 100 μM.

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    Fig. 6.

    mc1r is expressed in zebrafish hypothalamus. (A) Peripheral tissues and organs indicated were dissected from three 4-mo-old wild-type adults. Hypothalamus and brain tissue lacking hypothalamus were collected from nine 4-mo-old animals. Total RNA (600 ng) was extracted from each sample and used for cDNA synthesis. Expression level of mc1r mRNA, normalized to ef1α, was examined by qPCR. (B) Schematic view of neuroendocrine axes controlling backgound adaptation. Retina and pineal send information derived from photic signals to hypothalamic nuclei such as SCN. SCN and/or other hypothalamic nuclei then participate in relaying this information to pituitary and NLT to control the synthesis and/or release of α-MSH and MCH, respectively. Blockade of MC1R signaling by AgRP2 protein, after exposure to a white background, up-regulates pmch and pmchl mRNA levels in the NLT. Secretion of AgRP2 into the peripheral circulation (dashed lines, not yet tested) could also block the ability of MSH to stimulate melanosome dispersion by directly antagonizing the MC1R on melanocytes. Release of MCH and MCHL peptides results in melanosome aggregation; release of pituitary MSH results in melanosome dispersion. MCHR, MCH receptor; MT, microtubule.

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Pineal-specific agouti protein regulates teleost background adaptation
Chao Zhang, Youngsup Song, Darren A. Thompson, Michael A. Madonna, Glenn L. Millhauser, Sabrina Toro, Zoltan Varga, Monte Westerfield, Joshua Gamse, Wenbiao Chen, Roger D. Cone
Proceedings of the National Academy of Sciences Nov 2010, 107 (47) 20164-20171; DOI: 10.1073/pnas.1014941107

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Pineal-specific agouti protein regulates teleost background adaptation
Chao Zhang, Youngsup Song, Darren A. Thompson, Michael A. Madonna, Glenn L. Millhauser, Sabrina Toro, Zoltan Varga, Monte Westerfield, Joshua Gamse, Wenbiao Chen, Roger D. Cone
Proceedings of the National Academy of Sciences Nov 2010, 107 (47) 20164-20171; DOI: 10.1073/pnas.1014941107
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