- Email: [email protected]
Molecular cloning of chicken prepro-orexin cDNA and preferential expression in the chicken hypothalamus
Biochimica et Biophysica Acta 1577 (2002) 476 – 480 www.bba-direct.com
Short sequence-paper
Molecular cloning of chicken prepro-orexin cDNA and preferential expression in the chicken hypothalamus $ Takeshi Ohkubo a,*, Tim Boswell b, Sophie Lumineau c a
Center for Molecular Biology and Genetics, Mie University, Tsu, Mie 514-8507, Japan b Division of Integrative Biology, Roslin Institute, Roslin, Midlothian EH25 9PS, UK c UMR CNRS 6552 Ethologie – Evolution – Ecologie, Universite de Rennes 1, 35042 cedex, Rennes, France Accepted 6 August 2002
Abstract Chicken prepro-orexin cDNA has been cloned, sequenced and characterized. The predicted amino acid sequence of chicken prepro-orexin cDNA revealed that orexin-A and -B are highly conserved among vertebrate species. In situ hybridization and immunohistochemistry localized orexin-positive cell bodies in the periventricular hypothalamic nucleus extending into the lateral hypothalamic area. Comparisons of orexin gene expression in the brains of 24-h-fasted and ad libitum-fed chickens were made using semi-quantitative RT-PCR. No significant differences in orexin mRNA expression were observed. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Orexin; cDNA cloning; Hypothalamus; Fasting; Chicken
Orexins-A and -B are recently discovered peptides originally isolated from the rat and named hypocretin 1 and 2 in recognition of their hypothalamic localization and amino acid sequence identity with the gut hormone secretin [1]. The same peptides were identified independently as the ligands of two previously orphan G protein-coupled receptors and named orexin-A and -B after a stimulatory (orexigenic) effect of the peptides was observed on food intake [2]. Both peptides are derived from proteolytic processing of a single prepro-orexin precursor peptide encoded by a gene expressed in the hypothalamus, specifically in the lateral hypothalamic area and adjacent regions [1 – 3]. Neurons expressing orexins project to other parts of the hypothalamus and to a variety of extrahypothalamic regions, including the thalamus, cerebral cortex, limbic system, and hind-
Abbreviations: RT-PCR, Reverse transcription polymerase chain reaction $ The nucleotide sequence data will appear in the EMBL/GenBank/ DDBJ nucleotide databases with the accession number AB056748. * Corresponding author. Present address: Department of Bioresource Production, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761-0795, Japan. Tel.: +81-87-891-3057; fax: +81-87-8913021. E-mail address: [email protected] (T. Ohkubo).
brain [4,5]. Expression of the orexin receptors OX1R and OX2R has been demonstrated in these areas [6]. The projection of orexin neurons to multiple brain regions suggests an involvement of orexin-A and -B in varied neural and neuroendocrine functions. Consistent with this, physiological studies have implicated orexin peptides in the control of food and water intake, cardiovascular function, the sleep – wake cycle, and neuroendocrine secretion [7 – 9]. Targeted genetic ablation of the orexin gene in mice produced a phenotype of narcolepsy, hypophagia, and a late-onset obesity [10]. The structure of orexin peptides is highly conserved between different mammalian species [2,11]. In contrast to mammals, little is known about orexin pathways in nonmammalian vertebrates. Bioactivity of mammalian orexins has been reported in the goldfish in the form of increased food intake after central injection [12]. In amphibians, a Xenopus gene has been cloned that shares 56% overall amino acid identity with human orexin, and Xenopus orexin peptides were bioactive in a human orexin receptor in vitro functional assay [13]. However, the orexin gene has not been cloned from representatives of any other vertebrate classes. In birds, information on orexins is limited to the observation that central injection of mammalian orexin peptides did not stimulate food intake in domestic chicks
0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 4 8 3 - 9
T. Ohkubo et al. / Biochimica et Biophysica Acta 1577 (2002) 476–480
477
Fig. 1. The nucleotide sequence of chicken orexin cDNA and the deduced amino acid sequence. Both strands were completely sequenced. Putative orexin-A and -B are boxed. Poly A signal is underlined. The termination codon is marked with an asterisk.
[14]. This suggests either that the orexin peptide sequence does not show strong evolutionary conservation, or that the orexigenic control of feeding behavior differs between birds and mammals. To address this issue, our aims in the present study were to clone orexin cDNA and to localize the sites of orexin expression in the brain of the chicken. Furthermore, we compared fed and fasted chickens to determine whether orexin expression in the hypothalamus is influenced by food deprivation. Total RNA was extracted from chicken hypothalami by using a commercial kit (ISOGEN, Nippon Gene, Japan) to
amplify a fragment of chicken prepro-orexin cDNA by reverse transcription-polymerase chain reaction (RT-PCR). A cDNA fragment of chicken orexin cDNA was amplified by RT-PCR with a pair of primers, primer 1 (sense), 5VCGTCAAAAGACGTGCTCCTGCCGCCTCTA-3Vand primer 2 (antisense), 5V-CATGGTCAGGATGCCCGCTGCGTGGTT-3V, which were designed from conserved nucleotide sequence in mammalian orexins [1,2,11]. The rapid amplification of cDNA ends (RACE) procedure for cloning 5V- and 3V-ends of the chicken prepro-orexin cDNA was carried out using Marathon cDNA amplification kit
Fig. 2. Multiple alignment of the amino acid sequences of orexin from chicken, human, pig, rat and Xenopus. Sequences were aligned using Clustalx [17]. Conserved residues are shaded, and identical residues in the five species are black-boxed.
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T. Ohkubo et al. / Biochimica et Biophysica Acta 1577 (2002) 476–480
acid sequences of chicken orexin are shown in Fig. 1. The chicken orexin cDNA is 658 bp long and encodes 148 amino acids. Overall sequence identity between chicken orexin and mammalian orexin was approximately 55% at amino acid level: the majority of substitutions were in the signal peptide and the C-terminus of the pro-orexin. Chicken orexin-A and -B showed approximately 85% and 65% similarities with the corresponding mammalian sequence at the amino acid level (Fig. 2). The evolutionary conservation of the amino acid sequences of mature orexinA and -B in the chicken is demonstrated in Fig. 2. The conservation of orexin sequences between vertebrate classes suggests that the proteins play a fundamental regulatory role in vertebrates. Distribution of orexin-B-like immunoreactive cell bodies and orexin mRNA in the chicken brain were determined by
Fig. 3. In situ hybridization of 35S end-labelled oligonucleotides to chicken orexin mRNA (b) and localization of orexin-like immunoreactive cell bodies (c) in the periventricular hypothalamic nucleus (PHN) and lateral hypothalamic area (Lhy) of the chicken brain. The distribution of orexin cell bodies (a) is schematically represented by small black dots on a section taken from plane A6.6 of the stereotaxic atlas of the chick brain [18]. Abbreviation VIII = third ventricle. Scale bars represent 1 mm (b) and 0.75 mm (c).
(Clontech) according to the manufacturer’s instruction. The 5V- and 3V-regions of chicken orexin cDNA were amplified with primers 3 (initial primer), 5V-CTCTGGAAGGCTGGCGGGATGCT-3V and 4 (nested primer), 5V-TTCCTCTTGCCCAGCGTGAGGAT-3V, and primers 5 (initial primer), 5V-ACCTCCTGCACGGCATGGGCAACCA-3V and 6 (nested primer), 5V-ATCCTCACGCTGGGCAAGAGGAAGA-3V, respectively. All cDNA fragments were subcloned into pGEM T-easy vector (Promega), and both strands were sequenced by Applied Biosystems 373A automated sequencer. The nucleotide and deduced amino
Fig. 4. Semiquantitative analysis of chicken orexin mRNA in the chicken brain. (A) Chicken orexin mRNA expression measured by RT-PCR in the dissected brain areas. (B) Effect of fasting on orexin mRNA expression in the basal hypothalamus. Values are relative to that of fed birds, which is assigned a value of 1, and given as mean F S.E. of triplicate experiments. The amounts of cDNA amplified from h-actin mRNA were used to normalize the orexin results.
T. Ohkubo et al. / Biochimica et Biophysica Acta 1577 (2002) 476–480
immunocytochemistry with a polyclonal goat anti-human orexin B primary antibody (Santa Cruz Biotechnology) and two 35S-labelled oligonucleotide probes, 5V-TACCGTTTATTGTGAAAGGCCAAGTGTGCCGAGCAGCCTTGGCCA-3Vand 5V-ATGCCGTGCAGGAGGTCGTAGATACGGCAGGAGCAGGTTTTCT-3V, which are complementary to chicken orexin mRNA, respectively. As well as demonstrating evolutionary conservation in structure, the distribution of orexin mRNA and orexin-Blike immunoreactive cells in the chicken brain was also similar to what has been reported in other vertebrates [7,9,13,15]. Orexin cell bodies were detected only in the hypothalamus and not in any other brain area (Fig. 3). The orexin cell bodies were observed in two regions of the chicken hypothalamus: in the periventricular hypothalamic nucleus, and in the lateral hypothalamic area. The pattern of distribution of orexin-B-like cell bodies in the chicken hypothalamus is intermediate between the medial periventricular distribution described for amphibians [13], and the dorsolateral localization reported in rats, mice and primates [7,9,15]. The fact that orexin mRNA and orexin immunoreactivities determined by a mammalian orexin-B antibody showed an identical distribution provides further confirmation that the gene we have cloned is an avian homologue of mammalian orexins. Semiquantitative RT-PCR analysis of orexin gene expression in the chicken hypothalamus and forebrain used chicken-specific primers, primer 5 (sense primer), 5VACCTCCTGCACGGCATGGGCAACCA-3V and primer 7 (antisense primer), 5V-CAGGTCCTTCTCAGCGTGCTCCTGG-3V. Chicken h-actin cDNA was amplified with specific primers to normalize the expression levels of orexin. Amplified cDNA for chicken orexin or h-actin was separated by electrophoresis on a 2% agarose gel, and the gel was stained with ethidium bromide. After the gel staining, image-quantitative analysis of the amplified bands was performed with ImageMaster-CL (Amersham Pharmacia). Semiquantitative RT-PCR demonstrated that orexin mRNA was presented in the basal hypothalamus and the preoptic hypothalamus of laying hens, but no signal was detected in the hyperstriatum accessorium (forebrain) of the chickens (Fig. 4A). There was no significant difference in the amount of orexin mRNA in the basal hypothalamus in 12-week-old female chickens between birds provided food ad libitum or deprived of food for 24 h (Fig. 4B). These semi-quantitative RT-PCR data suggested that a change in orexin gene expression in the hypothalamus is not involved in the regulation of food intake. This is consistent with the observation that intracerebroventricular injection of mammalian orexins fails to affect food intake in the domestic chicken [14]. However, as described above, we detected orexin cell bodies in the periventricular hypothalamic nucleus and lateral hypothalamus. It is noteworthy that electrolytic lesions to the lateral hypothalamic area of the chicken brain result in aphagia [16], and damage to orexin-containing
479
neuronal cell bodies in this region could be predicted to contribute to this effect. Our hypothalamic dissections did not clearly separate the medial and lateral cell groups, so that we may not have been able to detect regional changes in orexin expression after the physiological manipulation. Therefore, we cannot eliminate the possibility that orexin peptides may be involved in the central regulation of food intake in chicken as well as in mammals. Further investigation involving quantification of orexin mRNA in specific cell groups by a technique such as in situ hybridization is required to resolve this issue, and also, a homologous system will make it possible to clarify specific function(s) of orexin in chicken.
Acknowledgements This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists 11760192 from the Japanese Ministry of Education, Science, Sports and Culture (T.O.). Dr. Lumineau was supported by a travel grant from the European Science Foundation BIRD Programme.
References [1] L. de Lecea, T.S. Kilduff, C. Peyron, X.-B. Gao, P.E. Foye, P.E. Danielson, C. Fukuhara, E.L.F. Battenberg, V.T. Gautvik, F.S. Bartlett, W.N. Frankel, A.N. van den Pol, F.E. Bloom, K.M. Gautvik, J.G. Sutclifffe, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 322 – 327. [2] T. Sakurai, A. Amemiya, M. Ishii, I. Matsuzaki, R.M. Chemelli, H. Tanaka, S.C. Williams, J.A. Richardson, G.P. Kozlowski, S. Wilson, J.R.S. Arch, R.E. Buckingham, A.C. Haynes, S.A. Carr, R.S. Annan, D.E. McNulty, W.-S. Liu, J.A. Terrett, N.A. Elshourbagy, D.J. Bergsma, M. Yanagisawa, Cell 92 (1998) 573 – 585. [3] C. Broberger, L. de Lecea, J.G. Sutcliffe, T. Hokfelt, J. Comp. Neurol. 402 (1998) 460 – 474. [4] C. Peyron, D.K. Tighe, A.N. van den Pol, L. de Lecea, H.C. Heller, J.G Sutcliffe, T.S. Kilduff, J. Neurosci. 18 (1998) 9996 – 10015. [5] Y. Date, Y. Ueta, H. Yamashita, H. Yamaguchi, S. Matsukura, K. Kangawa, T. Sakurai, M. Yanagisawa, M. Nakazato, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 748 – 753. [6] P. Trivedi, H. Yu, D.J. MacNeil, L.H. Van der Ploeg, X.M. Guan, FEBS Lett. 438 (1998) 71 – 75. [7] L. de Lecea, J.G. Sutcliffe, Cell. Mol. Life Sci. 56 (1999) 473 – 480. [8] J.J. Hagan, R.A. Leslie, S. Patel, M.L. Evans, T.A. Wattam, S. Holmes, C.D. Benham, S.G. Taylor, C. Routledge, P. Hemmati, R.P. Munton, T.E. Ashmeade, A.S. Shah, J.P. Hatcher, P.D. Hatcher, D.N. Jones, M.I. Smith, D.C. Piper, A.J. Hunter, R.A. Porter, N. Upton, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 10911 – 10916. [9] T. Sakurai, Regulatory Pept. 85 (1999) 25 – 30. [10] J. Hara, C.T. Beuckmann, T. Nambu, J.T. Willie, R.M. Chemelli, C.M. Sinton, F. Sugiyama, K. Yagami, K. Goto, M. Yanagisawa, T. Sakurai, Neuron 30 (2001) 345 – 354. [11] C.J. Dyer, K.J. Touchette, J.A. Carroll, G.L. Allee, R.L. Matteri, Domest. Anim. Endocrinol. 16 (1999) 145 – 148. [12] H. Volkoff, J.M. Bjorklund, R.E. Peter, Brain Res. 846 (1999) 204 – 209. [13] M. Shibahara, T. Sakurai, T. Nambu, T. Takenouchi, H. Iwaasa, S. Egashira, M. Ihara, K. Goto, Peptides 20 (1999) 1169 – 1176.
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[14] M. Furuse, R. Ando, T. Bungo, R. Ao, M. Shimojo, Y. Masuda, Br. Poult. Sci. 40 (1999) 698 – 700. [15] L. Galas, H. Vaudry, B. Braun, A.N. van den Pol, L. de Lecea, J.G. Sutcliffe, N. Chartrel, J. Comp. Neurol. 429 (2001) 242 – 252. [16] W.J. Kuenzel, M.M. Beck, R. Teruyama, J. Exp. Zool. 283 (1999) 348 – 364.
[17] J.D. Thompson, D.G. Higgins, T.J. Gibson, Nucleic Acids Res. 22 (1994) 4673 – 4680. [18] W.J. Kuenzel, M. Masson, A Stereotaxic Atlas of the Brain of the Chick (Gallus domesticus), Johns Hopkins Univ. Press, Baltimore, 1988.
Short sequence-paper
Molecular cloning of chicken prepro-orexin cDNA and preferential expression in the chicken hypothalamus $ Takeshi Ohkubo a,*, Tim Boswell b, Sophie Lumineau c a
Center for Molecular Biology and Genetics, Mie University, Tsu, Mie 514-8507, Japan b Division of Integrative Biology, Roslin Institute, Roslin, Midlothian EH25 9PS, UK c UMR CNRS 6552 Ethologie – Evolution – Ecologie, Universite de Rennes 1, 35042 cedex, Rennes, France Accepted 6 August 2002
Abstract Chicken prepro-orexin cDNA has been cloned, sequenced and characterized. The predicted amino acid sequence of chicken prepro-orexin cDNA revealed that orexin-A and -B are highly conserved among vertebrate species. In situ hybridization and immunohistochemistry localized orexin-positive cell bodies in the periventricular hypothalamic nucleus extending into the lateral hypothalamic area. Comparisons of orexin gene expression in the brains of 24-h-fasted and ad libitum-fed chickens were made using semi-quantitative RT-PCR. No significant differences in orexin mRNA expression were observed. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Orexin; cDNA cloning; Hypothalamus; Fasting; Chicken
Orexins-A and -B are recently discovered peptides originally isolated from the rat and named hypocretin 1 and 2 in recognition of their hypothalamic localization and amino acid sequence identity with the gut hormone secretin [1]. The same peptides were identified independently as the ligands of two previously orphan G protein-coupled receptors and named orexin-A and -B after a stimulatory (orexigenic) effect of the peptides was observed on food intake [2]. Both peptides are derived from proteolytic processing of a single prepro-orexin precursor peptide encoded by a gene expressed in the hypothalamus, specifically in the lateral hypothalamic area and adjacent regions [1 – 3]. Neurons expressing orexins project to other parts of the hypothalamus and to a variety of extrahypothalamic regions, including the thalamus, cerebral cortex, limbic system, and hind-
Abbreviations: RT-PCR, Reverse transcription polymerase chain reaction $ The nucleotide sequence data will appear in the EMBL/GenBank/ DDBJ nucleotide databases with the accession number AB056748. * Corresponding author. Present address: Department of Bioresource Production, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761-0795, Japan. Tel.: +81-87-891-3057; fax: +81-87-8913021. E-mail address: [email protected] (T. Ohkubo).
brain [4,5]. Expression of the orexin receptors OX1R and OX2R has been demonstrated in these areas [6]. The projection of orexin neurons to multiple brain regions suggests an involvement of orexin-A and -B in varied neural and neuroendocrine functions. Consistent with this, physiological studies have implicated orexin peptides in the control of food and water intake, cardiovascular function, the sleep – wake cycle, and neuroendocrine secretion [7 – 9]. Targeted genetic ablation of the orexin gene in mice produced a phenotype of narcolepsy, hypophagia, and a late-onset obesity [10]. The structure of orexin peptides is highly conserved between different mammalian species [2,11]. In contrast to mammals, little is known about orexin pathways in nonmammalian vertebrates. Bioactivity of mammalian orexins has been reported in the goldfish in the form of increased food intake after central injection [12]. In amphibians, a Xenopus gene has been cloned that shares 56% overall amino acid identity with human orexin, and Xenopus orexin peptides were bioactive in a human orexin receptor in vitro functional assay [13]. However, the orexin gene has not been cloned from representatives of any other vertebrate classes. In birds, information on orexins is limited to the observation that central injection of mammalian orexin peptides did not stimulate food intake in domestic chicks
0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 4 8 3 - 9
T. Ohkubo et al. / Biochimica et Biophysica Acta 1577 (2002) 476–480
477
Fig. 1. The nucleotide sequence of chicken orexin cDNA and the deduced amino acid sequence. Both strands were completely sequenced. Putative orexin-A and -B are boxed. Poly A signal is underlined. The termination codon is marked with an asterisk.
[14]. This suggests either that the orexin peptide sequence does not show strong evolutionary conservation, or that the orexigenic control of feeding behavior differs between birds and mammals. To address this issue, our aims in the present study were to clone orexin cDNA and to localize the sites of orexin expression in the brain of the chicken. Furthermore, we compared fed and fasted chickens to determine whether orexin expression in the hypothalamus is influenced by food deprivation. Total RNA was extracted from chicken hypothalami by using a commercial kit (ISOGEN, Nippon Gene, Japan) to
amplify a fragment of chicken prepro-orexin cDNA by reverse transcription-polymerase chain reaction (RT-PCR). A cDNA fragment of chicken orexin cDNA was amplified by RT-PCR with a pair of primers, primer 1 (sense), 5VCGTCAAAAGACGTGCTCCTGCCGCCTCTA-3Vand primer 2 (antisense), 5V-CATGGTCAGGATGCCCGCTGCGTGGTT-3V, which were designed from conserved nucleotide sequence in mammalian orexins [1,2,11]. The rapid amplification of cDNA ends (RACE) procedure for cloning 5V- and 3V-ends of the chicken prepro-orexin cDNA was carried out using Marathon cDNA amplification kit
Fig. 2. Multiple alignment of the amino acid sequences of orexin from chicken, human, pig, rat and Xenopus. Sequences were aligned using Clustalx [17]. Conserved residues are shaded, and identical residues in the five species are black-boxed.
478
T. Ohkubo et al. / Biochimica et Biophysica Acta 1577 (2002) 476–480
acid sequences of chicken orexin are shown in Fig. 1. The chicken orexin cDNA is 658 bp long and encodes 148 amino acids. Overall sequence identity between chicken orexin and mammalian orexin was approximately 55% at amino acid level: the majority of substitutions were in the signal peptide and the C-terminus of the pro-orexin. Chicken orexin-A and -B showed approximately 85% and 65% similarities with the corresponding mammalian sequence at the amino acid level (Fig. 2). The evolutionary conservation of the amino acid sequences of mature orexinA and -B in the chicken is demonstrated in Fig. 2. The conservation of orexin sequences between vertebrate classes suggests that the proteins play a fundamental regulatory role in vertebrates. Distribution of orexin-B-like immunoreactive cell bodies and orexin mRNA in the chicken brain were determined by
Fig. 3. In situ hybridization of 35S end-labelled oligonucleotides to chicken orexin mRNA (b) and localization of orexin-like immunoreactive cell bodies (c) in the periventricular hypothalamic nucleus (PHN) and lateral hypothalamic area (Lhy) of the chicken brain. The distribution of orexin cell bodies (a) is schematically represented by small black dots on a section taken from plane A6.6 of the stereotaxic atlas of the chick brain [18]. Abbreviation VIII = third ventricle. Scale bars represent 1 mm (b) and 0.75 mm (c).
(Clontech) according to the manufacturer’s instruction. The 5V- and 3V-regions of chicken orexin cDNA were amplified with primers 3 (initial primer), 5V-CTCTGGAAGGCTGGCGGGATGCT-3V and 4 (nested primer), 5V-TTCCTCTTGCCCAGCGTGAGGAT-3V, and primers 5 (initial primer), 5V-ACCTCCTGCACGGCATGGGCAACCA-3V and 6 (nested primer), 5V-ATCCTCACGCTGGGCAAGAGGAAGA-3V, respectively. All cDNA fragments were subcloned into pGEM T-easy vector (Promega), and both strands were sequenced by Applied Biosystems 373A automated sequencer. The nucleotide and deduced amino
Fig. 4. Semiquantitative analysis of chicken orexin mRNA in the chicken brain. (A) Chicken orexin mRNA expression measured by RT-PCR in the dissected brain areas. (B) Effect of fasting on orexin mRNA expression in the basal hypothalamus. Values are relative to that of fed birds, which is assigned a value of 1, and given as mean F S.E. of triplicate experiments. The amounts of cDNA amplified from h-actin mRNA were used to normalize the orexin results.
T. Ohkubo et al. / Biochimica et Biophysica Acta 1577 (2002) 476–480
immunocytochemistry with a polyclonal goat anti-human orexin B primary antibody (Santa Cruz Biotechnology) and two 35S-labelled oligonucleotide probes, 5V-TACCGTTTATTGTGAAAGGCCAAGTGTGCCGAGCAGCCTTGGCCA-3Vand 5V-ATGCCGTGCAGGAGGTCGTAGATACGGCAGGAGCAGGTTTTCT-3V, which are complementary to chicken orexin mRNA, respectively. As well as demonstrating evolutionary conservation in structure, the distribution of orexin mRNA and orexin-Blike immunoreactive cells in the chicken brain was also similar to what has been reported in other vertebrates [7,9,13,15]. Orexin cell bodies were detected only in the hypothalamus and not in any other brain area (Fig. 3). The orexin cell bodies were observed in two regions of the chicken hypothalamus: in the periventricular hypothalamic nucleus, and in the lateral hypothalamic area. The pattern of distribution of orexin-B-like cell bodies in the chicken hypothalamus is intermediate between the medial periventricular distribution described for amphibians [13], and the dorsolateral localization reported in rats, mice and primates [7,9,15]. The fact that orexin mRNA and orexin immunoreactivities determined by a mammalian orexin-B antibody showed an identical distribution provides further confirmation that the gene we have cloned is an avian homologue of mammalian orexins. Semiquantitative RT-PCR analysis of orexin gene expression in the chicken hypothalamus and forebrain used chicken-specific primers, primer 5 (sense primer), 5VACCTCCTGCACGGCATGGGCAACCA-3V and primer 7 (antisense primer), 5V-CAGGTCCTTCTCAGCGTGCTCCTGG-3V. Chicken h-actin cDNA was amplified with specific primers to normalize the expression levels of orexin. Amplified cDNA for chicken orexin or h-actin was separated by electrophoresis on a 2% agarose gel, and the gel was stained with ethidium bromide. After the gel staining, image-quantitative analysis of the amplified bands was performed with ImageMaster-CL (Amersham Pharmacia). Semiquantitative RT-PCR demonstrated that orexin mRNA was presented in the basal hypothalamus and the preoptic hypothalamus of laying hens, but no signal was detected in the hyperstriatum accessorium (forebrain) of the chickens (Fig. 4A). There was no significant difference in the amount of orexin mRNA in the basal hypothalamus in 12-week-old female chickens between birds provided food ad libitum or deprived of food for 24 h (Fig. 4B). These semi-quantitative RT-PCR data suggested that a change in orexin gene expression in the hypothalamus is not involved in the regulation of food intake. This is consistent with the observation that intracerebroventricular injection of mammalian orexins fails to affect food intake in the domestic chicken [14]. However, as described above, we detected orexin cell bodies in the periventricular hypothalamic nucleus and lateral hypothalamus. It is noteworthy that electrolytic lesions to the lateral hypothalamic area of the chicken brain result in aphagia [16], and damage to orexin-containing
479
neuronal cell bodies in this region could be predicted to contribute to this effect. Our hypothalamic dissections did not clearly separate the medial and lateral cell groups, so that we may not have been able to detect regional changes in orexin expression after the physiological manipulation. Therefore, we cannot eliminate the possibility that orexin peptides may be involved in the central regulation of food intake in chicken as well as in mammals. Further investigation involving quantification of orexin mRNA in specific cell groups by a technique such as in situ hybridization is required to resolve this issue, and also, a homologous system will make it possible to clarify specific function(s) of orexin in chicken.
Acknowledgements This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists 11760192 from the Japanese Ministry of Education, Science, Sports and Culture (T.O.). Dr. Lumineau was supported by a travel grant from the European Science Foundation BIRD Programme.
References [1] L. de Lecea, T.S. Kilduff, C. Peyron, X.-B. Gao, P.E. Foye, P.E. Danielson, C. Fukuhara, E.L.F. Battenberg, V.T. Gautvik, F.S. Bartlett, W.N. Frankel, A.N. van den Pol, F.E. Bloom, K.M. Gautvik, J.G. Sutclifffe, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 322 – 327. [2] T. Sakurai, A. Amemiya, M. Ishii, I. Matsuzaki, R.M. Chemelli, H. Tanaka, S.C. Williams, J.A. Richardson, G.P. Kozlowski, S. Wilson, J.R.S. Arch, R.E. Buckingham, A.C. Haynes, S.A. Carr, R.S. Annan, D.E. McNulty, W.-S. Liu, J.A. Terrett, N.A. Elshourbagy, D.J. Bergsma, M. Yanagisawa, Cell 92 (1998) 573 – 585. [3] C. Broberger, L. de Lecea, J.G. Sutcliffe, T. Hokfelt, J. Comp. Neurol. 402 (1998) 460 – 474. [4] C. Peyron, D.K. Tighe, A.N. van den Pol, L. de Lecea, H.C. Heller, J.G Sutcliffe, T.S. Kilduff, J. Neurosci. 18 (1998) 9996 – 10015. [5] Y. Date, Y. Ueta, H. Yamashita, H. Yamaguchi, S. Matsukura, K. Kangawa, T. Sakurai, M. Yanagisawa, M. Nakazato, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 748 – 753. [6] P. Trivedi, H. Yu, D.J. MacNeil, L.H. Van der Ploeg, X.M. Guan, FEBS Lett. 438 (1998) 71 – 75. [7] L. de Lecea, J.G. Sutcliffe, Cell. Mol. Life Sci. 56 (1999) 473 – 480. [8] J.J. Hagan, R.A. Leslie, S. Patel, M.L. Evans, T.A. Wattam, S. Holmes, C.D. Benham, S.G. Taylor, C. Routledge, P. Hemmati, R.P. Munton, T.E. Ashmeade, A.S. Shah, J.P. Hatcher, P.D. Hatcher, D.N. Jones, M.I. Smith, D.C. Piper, A.J. Hunter, R.A. Porter, N. Upton, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 10911 – 10916. [9] T. Sakurai, Regulatory Pept. 85 (1999) 25 – 30. [10] J. Hara, C.T. Beuckmann, T. Nambu, J.T. Willie, R.M. Chemelli, C.M. Sinton, F. Sugiyama, K. Yagami, K. Goto, M. Yanagisawa, T. Sakurai, Neuron 30 (2001) 345 – 354. [11] C.J. Dyer, K.J. Touchette, J.A. Carroll, G.L. Allee, R.L. Matteri, Domest. Anim. Endocrinol. 16 (1999) 145 – 148. [12] H. Volkoff, J.M. Bjorklund, R.E. Peter, Brain Res. 846 (1999) 204 – 209. [13] M. Shibahara, T. Sakurai, T. Nambu, T. Takenouchi, H. Iwaasa, S. Egashira, M. Ihara, K. Goto, Peptides 20 (1999) 1169 – 1176.
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