Dynamic expression pattern of the growth hormone receptor during early development of the Chilean flounder

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Comparative Biochemistry and Physiology, Part B 150 (2008) 93 – 102 www.elsevier.com/locate/cbpb

Dynamic expression pattern of the growth hormone receptor during early development of the Chilean flounder Eduardo Fuentes a , Erika Poblete a , Ariel E. Reyes c , María Inés Vera a,b , Marco Álvarez a,b , Alfredo Molina a,b,⁎ a

c

Laboratorio de Biotecnología Molecular, Universidad Andrés Bello, Av. República 217, Piso 3, Santiago, Chile b Millennium Institute for Fundamental and Applied Biology, Santiago, Chile Laboratorio de Biología del Desarrollo, Facultad de Ciencias de la Salud, Universidad Diego Portales, Av. Ejército Libertador 141, Santiago, Chile Received 29 November 2007; received in revised form 30 January 2008; accepted 30 January 2008 Available online 9 February 2008

Abstract The entire cDNA sequence of the growth hormone receptor (GHR) of the Chilean flounder (Paralichthys. adspersus) was cloned by RT-PCR and RNA ligase rapid amplification of 5′ and 3′ends. The deduced amino acid sequence contains 641 residues and codes for the GHR1 form. The receptor includes all the structural domains and motifs responsible for its interaction with the growth hormone and growth signal transduction. Sequence comparison revealed 95 and 88% identity with other flat fish such as the Japanese flounder and Atlantic halibut respectively, but decreased to 41% with the GHR of other teleosts such as salmon. In addition we performed a phylogenetic analysis of this receptor in teleosts. RTPCR experiments were performed to study the expression of GHR1 mRNA in different tissues of juvenile fish, detecting the transcripts in all tissues investigated with higher expressions in the liver, brain and gonads. Additionally, using whole-mount in situ hybridization in larvae stages, we observed an on and off GHR1 mRNA expression pattern. This novel finding evidences that during early development of a teleost, GHR1 is transiently expressed in somites, a source of muscle, bone and spinal chord precursors cells, suggesting a relevant role of GH in fish development. GHR1 was also temporally detected in the notochord, intestines, brain and retinal layers, before its ubiquitous establishment. © 2008 Elsevier Inc. All rights reserved. Keywords: Chilean flounder; Early development expression pattern; Growth hormone receptor; Whole mount in situ hybridization

1. Introduction In contrast to mammals, in fish GH exhibits pleiotropic functions. In addition to its role in somatic growth, the hormone is involved in sexual maturation, seawater adaptation, immune responses, metabolism of lipids and carbohydrates, and cellular differentiation (for a review see Reinecke et al., 2005). Like prolactin, some interleukins, erythropoietin and other important ligands, GH interacts with a receptor (GHR) belonging to the cytokine/haematopoietin receptor superfamily. This receptor superfamily presents common structural features. In the extra⁎ Corresponding author. Laboratorio de Biotecnología Molecular, Universidad Andrés Bello, Av. República 217, Piso 3, Santiago, Chile. Tel.: +56 2661 8319; fax: +56 2661 8415. E-mail address: [email protected] (A. Molina). 1096-4959/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2008.01.009

cellular hormone-binding domain: i) a fibronectin type III domain; ii) seven cysteine residues playing significant roles to stabilize the GHR tridimensional structure (Fuh et al., 1990); iii) a few putative N-glycosylation sites involved in GH-binding affinity (Harding et al., 1994). In the intracellular domain: iv) some tyrosine residues involved in the phosphorylation of JAK2 (Argetsinger et al., 1993). Additionally, GHR contains the conserved motifs: v) FGDFS, involved in the correct interaction of GH-GHR (Baumgartner et al., 1994); vi) box 1 LLPPVPAP involved in JAK-STAT signaling pathway (Wang et al., 1996) and vii) box 2 EPWVEFIEVD involved in the internalization of the receptor (Ihle et al., 1995). The binding of a single hormone molecule to a GHR monomer induces receptor homodimerization and activation of the tyrosine activity of JAK2 associated kinase, triggering a phosphorylation cascade, undergoing the trophic signal. This signal takes place through activation of the STATs

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family transcription factors, which translocate to the nucleus where they modify transcription of specific genes (Zhu et al., 2001). The GHR cDNA sequence has been described in several vertebrates, including mammals, birds, reptiles, amphibians (Edens and Talamantes, 1998) and recently in teleost fish (Lee et al., 2001; Calduch-Giner et al., 2001, 2003; Tse et al., 2003; Fukada et al., 2004; Nakao et al., 2004; Kajimura et al., 2004; Very et al., 2005; Small et al., 2006; Ozaki et al., 2006a; Hildahl et al., 2007). In vertebrates there have been described a number of mRNA variants originated by alternative processing of the GHR gene; encoding GH-binding proteins (GHBP), which are soluble circulating proteins and truncated forms of the receptor, that are membrane-anchored forms lacking most of the intracellular domain (for a review see Edens and Talamantes, 1998). In fish, these truncated forms have been found in flat fish as turbot, as well as Japanese flounder and Atlantic halibut (Calduch-Giner et al., 2001; Nakao et al., 2004; Hildahl et al., 2007) and probably acts as a negative regulator of GH action (Calduch-Giner et al., 2001). Recently in teleost fish, a second gene coding for a novel form of the receptor called GHR2 has been described (Saera-Vila et al., 2005; Jiao et al., 2006). It is probable that this form, even if it can be activated by the GH binding, is unable to transduce growth signal as the GHR1 form (Jiao et al., 2006; Saera-Vila et al., 2007). In mammals GHR expression is observed mainly in the liver, nevertheless it has been found in several extra-hepatic tissues such as; muscle, adipose tissue, brain, kidney and cartilage, among others (Kopchick and Andry, 2000). In fish, a similar expression pattern has been described (Lee et al., 2001; Tse et al., 2003; Kajimura et al., 2004; Nakao et al., 2004; Fukada et al., 2004; Very et al., 2005; Small et al., 2006; Ozaki et al., 2006a). In general, the trophic signal mediated by GH in the adult mainly requires activation of IGF-I expression in the liver (Moriyama et al., 2000). In early developmental stages this role probably is mediated by direct GH expression in extra-pituitary tissues (Harvey et al., 2000). Several GHR tissue-specific expression studies have been assessed in fish. Only a few studies report GHR expression at larval stages and there is no information about its spatial mRNA expression pattern at these stages. In the eel, GHR1 expression, measured by conventional RT-PCR, is observed even before fecundation, in contrast the GHR2 expression begins only after hatching (Ozaki et al., 2006b). Interestingly, GH mRNA appeared after both forms of the receptor were expressed (Ozaki et al., 2006b). In the Atlantic halibut, GHR1 expression was quantitatively measured finding a peak in expression levels at premetamorphic stages (Hildahl et al., 2007). Considering that GHR1 seems to be the principal transducer of the growth signaling initiated by the pituitary-hypothalamic axis through GH, we focused our interest of study to this form of the receptor in the Chilean flounder (Paralichthys adspersus), currently a new candidate for aquaculture diversification. We identified the GHR1 cDNA and the corresponding mRNA in different tissues of juvenile flounder and while assessing the receptor expression during early development of this teleost we found a unique dynamic spatial expression pattern which suggests that the pleiotropic activity that GH displays in fish demands a distinctive dynamic expression of the corresponding receptor.

2. Materials and methods 2.1. Fish The Chilean flounders (Paralichthys adspersus Steindachner, 1867; Paralichthyidae, Pleuronectiformes; FishBase (http:// www.fishbase.org) name: fine flounder) were collected from the Centro de Investigación Marina de Quintay (CIMARQ) (V Region, Valparaíso, Chile). The fish were maintained under natural temperatures and photoperiodic conditions (12 h:12 h and 13°C ±1°C) corresponding to the geographic localization (33°13′S 71°38′W) in spring season and fed twice daily with turbot pellet (Biomar, Chile). Fish were sacrificed through an overdose of anesthetic (3-aminobenzoic acid ethyl ester) (300 mg/L). The liver, kidneys, white muscles, red muscles, stomach, esophagus, intestines, gonads, spleen, gills and brain tissues were collected and directly frozen in liquid nitrogen and stored at − 80 °C. Larvae were obtained after in vitro fertilization of eggs by male broodstock sperm. Embryos were maintained under intensive-culture conditions in conic larval culture tanks at 19°C + 2°C. Larvae at pre-metamorphic stages were collected (8.0, 9.0 and 9.5 days post-fertilization (dpf)), fixed in 4% paraformaldehyde in PBS for 2 h at 4 °C, dehydrated and stored at − 20 °C. 2.2. cDNA cloning Total RNA was extracted from liver tissue using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer instructions. Using 5 μg of total RNA first-strand cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Sense (cfGHRF: 5′-TGATGTCATGGTCAACTGGGAG-3′) and antisense (cfGHRR: 5′-TCCTGCACCACTGTRTAGTCTG-3′) primers were used to amplify a 1300 bp fragment by RT-PCR, which was cloned into the pGEM®-T Easy Vector (Promega, Madison, WI, USA), originating pCFGHR1300 which was completely sequenced. The full-length 3′- and 5′-regions, including the transcription start site, were completed using the First Choice RLM-RACE® kit (Ambion, Austin, TX, USA) according to manufacturer instructions. Essentially, a nested RT-PCR reaction with an adapter primer and two gene-specific primers (cfGHR5′OP: 5′GGCATTTCTCTCTCTGTACTG-3′; cfGHR5′IP: 5′-TATACTCAAGGCGCATCCAG-3′) using a CIP/TAP mRNA as a template in a nested reaction. The 3′ region was obtained by using the gene-specific primers cfGHR3′OP: 5′-CAGTCGAAGATAACCAATCATC-3′ and cfGHR3′IP: 5′-CTTAGATTGTTCCTGACTCTC-3′. All PCR products were cloned into the pGEM®-T Easy Vector (Promega, Madison, WI, USA) and sequenced. 2.3. Structural and phylogenetic analyses Structural sequence prediction as signal peptide (Emanuelsson et al., 2007) and transmembrane domain (Rost et al., 2004) were carried out using bioinformatics tools available in the Center for Biological Sequence Analysis prediction server (www.cbs.dtu.

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dk). Fibronectin type III domain was predicted by BLAST resource (www.ncbi.nlm.nih.go) using CDART software (Geer et al., 2002).

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Twenty teleost GHR1 sequences and one of the African clawed frog (included as outgroup) were obtained from the GenBank nucleotide data base (www.ncbi.nlm.nih.go). Multiple

Fig. 1. Chilean flounder GHR nucleotide and deduced amino acid sequences (GenBank accession no. Genbank: EU004149). Start and stop codons are in bold and underlined. The additional 5′-UTR sequence is highlighted in cursive-bold letters. Putative signal peptide is underlined. Conserved cysteines, potential N-glycosilation sites, conserved tyrosines and tryptophan residues are indicated by circle, box, gray-circle and gray-box respectively. FGDFS, LLPPVPAP and EPWVEFIEVD motifs are highlighted in bold-underlined letters.

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nucleotide sequences alignment was performed using CLUSTALW (Thompson et al., 1994). Poisson-corrected distances were estimated for all possible pairs. The neighbor-joining method was used to reconstruct the phylogenetic tree (Saitou and Nei, 1987). Node robustness was assessed using a bootstrap approach (1000 replicates). All phylogenetic analyses were implemented using MEGA 3.1 software (Kumar et al., 1994).

2.4. RT-PCR expression analyses RT-PCR was performed to study GHR1 (cfGHR1) mRNA expression in the Chilean flounder juvenile fish. Total RNA was extracted from several tissues (liver, kidney, white muscle, red muscle, stomach, esophagus, intestine, gonads, spleen, and brain). Reverse transcription reactions were performed using

Fig. 2. Multiple alignment of the amino acid sequences of GHR in different vertebrates species: GenBank accession numbers: human (Homo sapiens) Genbank: NM_000163; mouse (Mus musculus) genbank: BC075720; chicken (Gallus gallus) genbank: M74057; African clawed frog (Xenopus laevis) Genbank: AF193799; Japanese flounder (Paralichthys olivaceus) Genbank: AB058418, Chilean flounder (Paralichthys adspersus) genbank: EU004149 and cherry salmon (Oncorhynchus masou) genbank: AB071216. Identical amino acids residues are indicated by asterisks. Putative signal peptide is underlined. Conserved cysteines, tryptophan and tyrosines residues are designated by open boxes, large dash line and black-shaded boxes, respectively. Putative N-glycosylation sites are indicated by gray-shaded boxes. The FGEFS, DXWVEFIELD and ILPPVPAP motifs are highlighted in bold letters. Transmembrane domain is indicated by dot–dashed line. Fibronectin type III domain is underlined with dashes.

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Fig. 2 (continued ).

1 μg of total RNA previously treated with DNase I. cfGHR1 gene-specific primers were designed to amplify a 100pb fragment (cfGHR1F: 5′-GGAGACATTTCGCTGTTGGT-3′; cfGHR1F: 5′-TGTGTGGTTCACGTCGAAGA-3′). For normalization purposes, gene-specific primers (cfβactF: 5′-AGGGAAATCGTGCGTGACAT-3′; cfβactR: 5′-TCAGGCAGCTCATAGCTCTT-3′) were used to amplify a β-actin 100 bp fragment as constitutive gene expression control. 2.5. Whole-mount in situ hybridization A 794 bp fragment corresponding to the coding sequence of cfGHR1 was amplified by PCR using the gene-specific primers (cfGHRihF: 5′-CGTAGAAGTCTGTGTTGAC-3′; cfGHRihR: 5′-TGATGTCATGGTCAACTGGGAG-3′) and the pCFGHR1300 clone as a template. This fragment was cloned into the pGEM®-T Easy Vector System (Promega, Madison, WI, USA), originating the pCFGHR794 clone, which was linearized with NcoI and NdeI restriction enzymes to synthesize sense (control) and antisense riboprobes DIG-UTP-labeled (Roche Applied Science) using T7 and SP6 RNA polymerases (Promega) respectively. The riboprobes were purified using mini Quick Spin Columns (Roche Diagnostics, Mannheim, Germany) to eliminate unincorporated labeled nucleotides. Whole mount in situ hybridization was performed according to Rojas et al., 2007. Larvae were rehydrated in decreasing methanol series, and were incubated for 5 min in bleach buffer (0.5X SSC, deionized formamide 5% and H2O2 30%). Pre-hybridization was carried out overnight at 60 °C in hybridization buffer (5X SSC, formamide 50%, yeast tRNA 500 μg/mL, heparin 50 μg/mL, Tween-20 0.1%). The larvae were then incubated overnight at 65 °C in hybridization buffer including 50 ng of riboprobe. After hybridization, larvae were washed in a solution with decreasing

formamide concentration (75%, 50%, 25%) in 2X SSC for 10 min at 65 °C each, followed by two wash-steps with SSC 0.2X for 30 min at 65 °C. Larvae were incubated for 4 h in a blocking buffer (5% bovine serum, BSA 2 mg/mL, DMSO 1%) in PBT (PBS/ Tween 20 0.1%) at room temperature. For immunodetection, larvae were incubated overnight at 4 °C with Anti-digoxigenin-AP antibody (Roche), diluted 1:2000. Five washes with PBT for 20 min each at room temperature were carried out to eliminate nonbounded antibodies, followed by three washes with AP-buffer (Tris 1 M pH 9.5, MgCl2 1 M, NaCl 5 M, Tween-20 0.1%). Stains were performed with NBT/BCIP (75 mg/mL and 50 mg/mL, respectively) (Promega) for 6 h at 37 °C. The experiment was performed four times using n = 15 larvae from each developmental stage. 2.6. Histology After in situ hybridization, the larvae were fixed, washed in PBS and mounted in agarose 4% in PBS. Then, the larvae were sectioned (70 μm) in a vibratome (Leica VT1000S) using speed 60 mm/s and 60 Hz of frequency. Sections were mounted in 3aminopropyltriethoxylane treated slides, cover-slipped with glycerol 90% (Merck, Darmstadt, Germany), observed in a Olympus BX-61 microscope and photographed with a Leica DF300 camera. 3. Results 3.1. Chilean flounder GHR1 cDNA sequence We obtained the complete GHR1 cDNA sequence from the teleost fish Paralichthys adspersus. The cDNA contained a single open reading frame that encodes 641 amino acids residues. A 5′ RLM-RACE approach to complete the 5′ cDNA

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region showed two different 5′-UTR sequences (133 bp and 184 bp), giving 2338 bp and 2389 bp cDNAs, which are only differentiated by their length (Fig. 1). The deduced amino acid sequence includes: a signal peptide composed of 27 amino acid residues, an extracellular hormone-binding domain comprising of 225 amino acid residues, a single hydrophobic transmembrane domain of 22 residues, and an intracellular signaling domain of 365 amino acid residues (Fig. 2). The cfGHR1 amino acid sequence displayed high identity (95%) with the GHR1 of another fish of the Paralichthys genus, the Japanese flounder. Other flat fish GHR1 amino acid sequences, like turbot and Atlantic halibut also show high degrees of identity with the Chilean flounder GHR1 (79% and 88% respectively). GHR1 of other teleosts like Japanese eel and common carp, show notably smaller identities, comprising between 45% and 49%. The Chilean flounder GHR1 exhibited even fewer identities with

other vertebrates, showing values of 37% with chicken, 35% with mouse, 33% with African clawed frog and 32% with human. The cfGHR1 amino acid sequence has several features common to the GHR of other vertebrates, including: a 89 amino acid residue fibronectin type III domain (Asp142 to Thr230) located in the extracellular domain, seven cysteine residues, six potential N-glycosylation sites, both in the extracellular hormone-binding domain, and five tyrosine residues in the intracellular domain (Fig. 2). Moreover, cfGHR1 contains specific conserved motifs as FGDFS (Phe233 to Ser237) situated in the extracellular domain; LLPPVPAP (Leu285 to Pro292) located in the box 1 and the EPWVEFIEVD (Glu332 to Asp341) located in the box 2, both belonging to the intracellular domain (Fig. 2). GHR1 cDNA sequences from different teleost fish were used to create a fish GHR1 phylogenetic tree including the African

Fig. 3. Phylogenetic tree based in the cDNA sequence of GHR1 in teleosts and in the African clawed frog (included as outgroup), based in the neighbor-joining method. Numbers of each tree node refers to percent bootstrap values after 1000 replicates, the scale bar refer to phylogenetic distances of 0.1 nucleotide substitution per site. GenBank accession numbers: channel catfish (Ictalurus punctatus) (Genbank:DQ103502); rohu (Labeo rohita) (Genbank:AY691177); catla (Catla catla) (Genbank:AY691178); Mrigal carp (Cirrhinus mrigala) (Genbank:AY691179); carp (Cyprinus carpio) (Genbank:AY732491); goldfish (Carassius auratus) (Genbank:AF293417); grass carp (Ctenopharyngodon idella) (Genbank:AY283778); Nile tilapia (Oreochromis niloticus) (Genbank:AY973232); Mozambique tilapia (Oreochromis mossambicus) (Genbank:AB115179); turbot (Scophthalmus maximus) (Genbank:AF352396); Atlantic halibut (Hippoglossus hippoglossus) (Genbank: DQ062814); Japanese flounder (Paralichthys olivaceus) (Genbank:AB058418); Chilean flounder (Paralichthys adspersus) (Genbank:EU004149); cherry salmon (Oncorhynchus masou) (Genbank:AB071216); coho salmon isoform 1 (IS1) (Oncorhynchus kisutch) (Genbank:AF403539) coho salmon isoform 2 (IS2) (Genbank: AF403540); rainbow trout isoform 1 (IS1) (Oncorhynchus mykiss) (Genbank:AY861675); rainbow trout isoform 2 (IS2) (Genbank:AY751531); Wami tilapia (Oreochromis urolepis hornorum) (Genbank:EF371466); orange-spotted grouper (Epinephelus coioides) (Genbank:EF052273); black seabream (Acanthopagrus schlegelii) (Genbank:AF502071); gilthead seabream (Sparus aurata) (Genbank:AF438176); Atlantic salmon isoform 1 (IS1) (Salmo salar) (Genbank:AY462105); Atlantic salmon IS2 (Genbank:DQ163908); cherry salmon somatolactin receptor (SLR) (Genbank:AB121047); rainbow trout growth hormone receptor 2 (GHR2) (Genbank:AY573600) and African clawed frog (Xenopus laevis) (Genbank:AF193799).

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Fig. 4. mRNA distribution of the Chilean flounder GHR1 in different tissues assessed by RT-PCR. A 100 bp β-actin fragment was amplified as constitutive expression control (the figure represents an example of four different experiments).

clawed frog GHR as an outgroup (Fig. 3). Phylogenetic analysis showed that teleost fish are divided into two large clades. Most of the sequences are included into a clade comprising Cypriniformes, Pleuronectiformes and Perciformes, which is subsequently subdivided in two additional clades, isolating Pleuronectiformes and Perciformes in a single clade. An additional clade contained GHR1 and GHR2 from Salmoniformes. In addition we included the cherry salmon somatolactin receptor (SLR), which is grouped with the Pleuronectiformes and Perciformes orders as a single branch.

3.2. Juvenile and larvae GHR1 mRNA expression RT-PCR experiments were performed to study the expression of cfGHR1 mRNA in different tissues. The transcript was detected in all investigated tissues (Fig. 4). Higher expression levels were found in the liver, brain and gonads compared with other tissues. We also studied the expression pattern of cfGHR1 mRNA using whole mount in situ hybridization in Chilean flounder larvae from 8.0, 9.0 and 9.5 dpf. The cfGHR1 mRNA was detected at 8.0 dpf. At this stage the transcript was strongly

Fig. 5. Whole-mount in situ hybridization for GHR1 in Chilean flounder larvae. Expression of GHR1 mRNA was analyzed at the indicated stages of Chilean flounder larvae with sense and antisense probes. A, larvae at 8 dpf showed strong expression in somites and weak expression in the notochord. C, at 9 dpf larvae showed strong GHR1 mRNA expression in the head, eye (left inset), notochord and (middle inset). Vibratome slices observed at high magnifications shows a higher expression of GHR1 in the notochord, although weak mRNA expression was also detected in somites and intestine (arrow and arrowhead in the right inset in C respectively). Later in development, at 9.5 dpf, the expression of GHR1 mRNA was weaker and ubiquitously detected in the whole larvae. It was also possible to identify positive signals in the optic tectum in the brain. We did not detect positive signals when the larvae were incubated with the sense probe (B, D and F). Pictures are representatives of four independent experiments. Abbreviations: s, somites; h, head; e, eye; i, intestine; nc, notochord; r, retina and ot, optic tectum.

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present in somites (Fig. 5A), although weaker mRNA expression was detected in the head. Later in development at 9.0 dpf, the strong expression of cfGHR1 mRNA was restricted to discrete territories such as the head, eye and notochord, although weak expression was present in somites (Fig. 5C). Interestingly, at higher magnifications it was possible to detect specific expression in the retinal layers (Fig. 5 left inset in C), and in the intestine (in the middle inset in C). After in situ hybridization, larvae slices showed specific staining into the notochord and weak staining in somites, compared with no expression in the neural tube (Fig. 5C, right inset). Later in development at 9.5 dpf larvae show ubiquitous expression of the cfGHR1, present in almost the whole larvae including structures as the optic tectum in the brain, intestines, eyes, somites and the notochord (Fig. 5E). At higher magnifications in 9.5 dpf it was also possible to detect expression in some layers of the retina (left inset in E) and in the notochord, somites and intestines (right inset in E). We included a negative control sense probe in all in situ hybridization assays; these controls follow the same treatment that the larvae incubated with the antisense probe. No signal was detected, showing than RNA hybridization was specific (Fig. 5B, D and F). 4. Discussion The entire cDNA sequence of the cfGHR1 was obtained, which codes for a 641 amino acids residue. Interestingly, by using RLM-RACE technique we found two 5′-UTR sequences, which were only differentiated by their length. In the Japanese flounder, another flat fish, by means of RNAse protection assays and also using liver RNA as a template, multiple transcription start sites in the GHR1 gene had been demonstrated (Nakao et al., 2004). This feature is characteristic of mammalian GHR genes, where multiple 5′-UTR have been described (Edens and Talamantes, 1998). It is possible that these heterogeneous 5′-sequences could be involved in differential tissue-specific expression, allowing a fine post-transcriptional control mechanism. Truncated forms have been found in other Pleuronectiformes fish such as the turbot, Japanese flounder and Atlantic halibut (Calduch-Giner et al., 2001; Nakao et al., 2004; Hildahl et al., 2007) probably acting as a negative regulator of GH action (Calduch-Giner et al., 2001). Nevertheless, until now we have been unable to find these forms in the Chilean flounder (data not shown). A comparison of the amino acid sequences revealed high conservation in fish GHR1 sequences belonging to the Pleuronectiformes order. Nevertheless, the identity decreased notably with other fish GHR1 and even more compared to mammals. This phenomenon seems to be almost a constant in fish, which as a taxonomic group show high sequence homology variation and are considered as a very divergent group with respect to other vertebrates (Tse et al., 2003). However, as well as we previously showed about the prolactin receptor (San Martín et al., 2004), the cfGHR1 exhibits the same structural features present in other members of the cytokine/haematopoietin receptor superfamily. Even if these characteristics are well conserved in every GHR sequence described until now, probably due to a strong evolutionary pressure to conserve important

domains involved in the GHR biological function, multiple amino acid sequence alignments denote that there are some sequence- and positional-differences between higher vertebrates and fish or within fish from different orders. In this regard, most GHRs display eight tyrosine residues located in the intracellular domain. In rat GHR, the first and second tyrosine residues (Y333 and Y338) are involved in some cellular responses to GH including lipogenesis and protein synthesis (Lobie et al., 1995). However, cfGHR1 lacks the first residue, present in almost all species studied until now, including the closely related Japanese flounder. A similar case is reported in salmonids, which additionally lack the third tyrosine residue (Fukada et al., 2004). The biological significance of the lacking tyrosine in some fish species remains unclear. cfGHR shows seven putative Nglycosilation sites located at N68, N96, N106, N122, N148, N152 and N192. The first one (N68) is present in every teleost fish GHR sequence reported until now and also in amphibians and avians, but is absent in mammalian GHRs. The second putative site (N96) was found only in flat fishes excepting the turbot, which belongs to the Scophthalmidae family. Its phylogenetic relationships are less than the Pleuronectidae (Atlantic halibut) and Paralichthyidae (Japanese flounder and Chilean flounder) families. To clarify the role and functionality of these exclusive GHR putative N-glycosilation sites, additional experimental data should be assayed. Nevertheless, as GHR glycosilation is necessary to increase and maintain high affinity for the GH (Harding et al., 1994), additional N-glycosilation could be affecting the GH-GHR interaction in teleost fish. The GHR1 amino acid sequence based on the phylogenetic tree shows a consistent pattern with teleost fish evolution and phylogeny. All fish GHR sequences form a monophyletic group with a good bootstrap support (85%). The phylogenetic tree isolates bony fish in two large clades. The first one includes Cypriniformes, Perciformes and Pleuronectiformes and it is subsequently subdivided in two additional clades, isolating Pleuronectiformes and Perciformes in a single clade separate from the Cypriniformes. Both orders belong to higher fishes of Actinopterygii class, specifically the superorder Acanthopterygii, Percomorpha series. Inside the clade grouping Pleuronectiformes, the Chilean flounder is closely related with the Japanese flounder, isolating both species from other flat fishes with a solid support in the bootstrap (100%). The turbot forms an additional clade separated of the Chilean flounder, the Japanese flounder and the Atlantic halibut. This fact is in agreement with the additional N-glycosilation site N96 which was found only in these three species. It is interesting that the described salmon somatolactin receptor appears to be more related to the GHR1 form of Pleuronectiformes and Perciformes as compared to other salmonid GHR sequences. In this regard, it has been suggested that most of the GHR sequences of non salmonid species described until now could correspond to the somatolactin receptor (Fukamachi et al., 2005). Further functional approaches have to be performed to clarify this intriguing point. The cfGHR mRNA expression analysis showed a wide distribution in all studied tissues in juvenile fish. This observation agrees with the extensive GHR expression described previously in other fish (Lee et al., 2001; Tse et al., 2003; Fukada et al., 2004;

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Nakao et al., 2004; Kajimura et al., 2004; Very et al., 2005; Small et al., 2006; Ozaki et al., 2006a). This suggests that GHR could regulate tissue-specific physiological processes not only related with somatic growth, a fact which agrees with the pleiotropic function described for the GH in key events in the life cycle of fish, such as growth, reproductive functions, osmoregulation and metabolism. To our knowledge this is the first description of GHR1 expression pattern in teleost whole larvae. A dynamic expression of the cfGHR1 mRNAwas observed at different stages in tissues corresponding to the notochord, somites, intestines, brain and retinal layers. The presence of GH-binding sites during larval developmental stages has been demonstrated in gilthead seabream (Martí-Palanca and Pérez-Sánchez, 1994). More recently, it has been shown that GHR1 mRNA is expressed during early development in the Japanese eel and in Atlantic halibut (Ozaki et al., 2006b; Hildahl et al., 2007), even though they did not determine tissue distribution during development. The cfGHR1 transcript is expressed in tissues such as the somites; a source of muscle, bone and spinal chord precursor cells (Richardson et al., 1998), and supports the key role of GHR1 in muscle and bone development. The cfGHR1 mRNA was also detected in the notochord of Chilean flounder larvae, an essential structure for the correct differentiation of adjacent territories such as the neuroectoderm, heart and paraxial mesoderm, inducing and maintaining the ventral fates (Pourquie, 2001). Previous studies using immunohistochemistry showed the presence of GH and GHR in chick embryos (previous to the somatotrophs pituitary secretion of GH), in tissues like somites, notochord, neural tube, cartilage and stomach (Harvey et al., 2000). Suggesting roles of GH in early embryonic growth or differentiation, supported by the expression of GHR at the same stages (Harvey et al., 2000). Additionally, studies using mice bearing a disrupted GHR gene showed that the homozygous knockout (GHR−/−) had severe postnatal growth retardation, and proportional dwarfism, but did not report significant differences in body sizes or weight at birth (Zhou et al., 1997). However, other studies have reported than in pregnant GHR−/− knockout females the fetal weight, crown-rump length and IGF-I levels were decreased (List et al., 2001). Suggesting GH, GHR and IGF-I play a role in fetal growth retardation. In zebrafish, the expression pattern of IGF-I and its receptor have been accessed by whole mount in situ hybridization showing a ubiquitous expression from embryonic to early larvae stages (Maures et al., 2002). In addition, IGF-I larvae expression has also been described in tilapia, rabbitfish, rainbow trout and carp (Greene and Chen, 1999; Tse et al., 2002; Ayson et al., 2002; Berishvili et al., 2006). Together with our findings, these observations suggest that the GH/IGF-I axis is involved in fish development. We report here cfGHR1 mRNA expression in the retinal layers and optic tectum in Chilean flounder larvae. It was recently demonstrated that GH and GHR are present in the retina, extra retinal tissues and optic tectum of developing chick visual system at E7-8 embryos, suggesting than these tissues are likely to be autocrine and/or paracrine sites of GH action (Baudet et al., 2007). Hence, our results agree with the expression patterns reported for superior vertebrates like the mouse and chick, suggesting that GHR1 could have important roles in fish development.

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