Two tyrosine hydroxylase genes in vertebrates New dopaminergic territories revealed in the zebrafish brain Kei Yamamoto a,1 , Jori O. Ruuskanen a,b,1 , Mario F. Wullimann c , Philippe Vernier a, ⁎ a Développement, Evolution, Plasticité du Système Nerveux, Institut de Neurobiologie Alfred Fessard, CNRS Gif-sur-Yvette, France b Department of Neurology, Turku University Central Hospital, and Department of Pharmacology, Drug Development and Therapeutics, University of Turku, Finland c Department Biology II, Neurobiology, Graduate School of Systemic Neurosciences, Ludwig-Maximilians-University, Munich, Germany abstract article info Article history: Received 15 September 2009 Revised 15 January 2010 Accepted 18 January 2010 Available online 1 February 2010 Keywords: Dopamine Catecholamine Gene duplication Forebrain Hypothalamus Evolution Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis, catalyzing transformation of L-tyrosine to L-DOPA. Two TH genes (TH1 and TH2) have been reported to exist in the genome of some teleost fishes, TH1 being orthologous to the mammalian TH gene (Candy and Collet, 2005). Here we show that two TH genes are commonly found in genomes of jawed vertebrates. Our analyses of molecular phylogeny and gene synteny strongly suggest that the two TH genes emerged as a consequence of a whole genome duplication before the divergence of jawed vertebrates, and that TH2 was secondarily lost in eutherians (placental mammals). The distribution of TH1 and TH2 transcripts revealed that TH1 and TH2 are differentially expressed in the zebrafish adult brain, as often observed for duplicated genes. In particular we found that TH2 transcripts were much more abundant than TH1 in the hypothalamus, and that the TH2 cells along the periventricular zone are devoid of TH immunoreactivity, due to the lack of affinity of the available anti-TH antibodies. Although these neurons have been considered to be dopamine-uptaking cells in previous studies, the expression of other monoaminergic markers such as aromatic amino acid decarboxylase (AADC), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) suggests that these TH2 cells are dopamine-synthesizing neurons. © 2010 Elsevier Inc. All rights reserved. Introduction Tyrosine hydroxylase (TH) is a key enzyme required for catechol- amine synthesis. It is a cytoplasmic enzyme, which catalyzes the transformation of L-tyrosine into L-DOPA, a precursor of dopamine (DA). Up to now only one gene encoding TH was known in mammals. However, two non-allelic TH genes (TH1 and TH2) have been discovered by Candy and Collet (2005) in several teleost species. Based on their phylogenetic analysis, TH1 is an orthologue of the mammalian TH gene, and the TH2 sequences form a separate clade from TH1. However, it is not clear whether these two sister genes arose from the large genome duplication which occurred within the teleost lineage (Postlethwait et al., 2004; Yi and Streelman, 2005), or alternatively, the duplication took place much earlier, as a result of one of the whole genome duplication (WGD) which is proposed to have occurred in ancestral vertebrates or craniates (Kuraku et al., 2009; Putnam et al., 2008). Since extensive genome sequence data are now available for many vertebrate species, the first goal of this study was to search for the existence of a second TH gene in different vertebrate groups, in order to revisit the phylogeny of TH genes and to analyze corresponding gene synteny. TH belongs to the aromatic amino acid hydroxylase (AAAH) family, together with tryptophan hydroxylase (TPH) and phenylala- nine hydroxylase (PAH). TPH, but not PAH, is known to possess two isoforms encoded by two different genes (TPH1 and TPH2) in most vertebrates (with an additional duplication of TPH1 in many teleost fishes). The two TPH genes are differentially expressed in the brain and the periphery, both in mammals and fishes (Lillesaar et al., 2007; Molecular and Cellular Neuroscience 43 (2010) 394–402 Abbreviations: AAAH, aromatic amino acid hydroxylase; AADC, aromatic amino acid decarboxylase; ac, anterior commissure; ATN, anterior tuberal nucleus; ca, commissura ansulata; DA, dopamine; DAT, dopamine transporter; DT, dorsal thalamus; Ha, habenula; Hc, caudal zone of periventricular hypothalamus; Hd, dorsal zone of periventricular hypothalamus; IN, intermediate nucleus of hypothalamus (of Rink and Wullimann, 2001); LH, lateral hypothalamic nucleus; lfb, lateral forebrain bundle; llf, lateral longitudinal fascicle; mlf, medial longitudinal fascicle; OB, olfactory bulb; oc, optic chiasm; pc, posterior commissure; PM, magnocellular preoptic nucleus; poc, postoptic commissure; PPa, anterior parvocellular preoptic nucleus; PPp, posterior parvocellular preoptic nucleus; PPp-ad, anterodorsal part of PPp; PPp-p, posterior part of posterior parvocellular preoptic nucleus; PPr, periventricular pretectum; PTN, posterior tuberal nucleus; PVO, paraventricular organ; SC, suprachiasmatic nucleus; Tel, telencephalon; TeO, tectum opticum; TH, tyrosine hydroxylase; TPp, periventricular nucleus of posterior tuberculum; TS, torus semicircularis; Va, valvula cerebelli; VMAT2, vesicular monoamine transporter 2; VT, ventral thalamus; Vp, postcommissural nucleus of the ventral telencephalic area; Vs, supracommissural nucleus of the ventral telencephalic area; Vv, ventral nucleus of the ventral telencephalic area. ⁎ Corresponding author. D.E.P.S.N. (UPR 2197), Institut de Neurobiologie Alfred Fessard, C.N.R.S., Avenue de la Terrasse, 91198 Gif-sur-Yvette, France. Fax: + 33 1 69 82 34 47. E-mail address: vernier@inaf.cnrs-gif.fr (P. Vernier). 1 These authors contributed equally to this work. 1044-7431/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2010.01.006 Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne