Comparative Biochemistry and Physiology, Part B 151 (2008) 197–202

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Comparative Biochemistry and Physiology, Part B

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Temporal and spatial expression pattern of the myostatin gene during larvaland juvenile stages of the Chilean flounder (Paralichthys adspersus)

Iselys Delgado a, Eduardo Fuentes a, Sebastián Escobar a, Cristina Navarro a, Tatiana Corbeaux a,1,Ariel E. Reyes b, María Inés Vera a,c, Marco Álvarez a,c, Alfredo Molina a,c,⁎a Laboratorio de Biotecnología Molecular, Universidad Andres Bello, Av. República 217, Santiago, Chileb Laboratorio de Biología del Desarrollo, Facultad de Ciencias de la Salud, Universidad Diego Portales, Av. Ejército Libertador 141, Santiago, Chilec Millennium Institute for Fundamental and Applied Biology, Santiago, Chile

⁎ Corresponding author. Laboratorio de BiotecnologíaBello, Av. República 217, Santiago, Chile. Tel.: +56 2661 8

E-mail address: amolina@unab.cl (A. Molina).1 Present address: Department of Developmental Imm

of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Ge

1096-4959/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.cbpb.2008.07.003

a b s t r a c t

a r t i c l e i n f o

Article history:

The full length cDNA sequen Received 11 March 2008Received in revised form 30 June 2008Accepted 2 July 2008Available online 11 July 2008

Keywords:MyostatinMuscle growthFishChilean flounderWhole mount in situ hybridization

ce of the myostatin gene was cloned from a teleostean fish, the Chilean flounder(Paralichthys adspersus) through RT-PCR amplification coupled with the RACE approach to complete the 5′- and3′-region. The deduced amino acid sequence encodes a protein of 377 amino acid residues, including thestructural domains responsible for its biological activity. Amino acid sequence comparison revealed highsequence conservation, and confirmed that the isolated sequence corresponds to the MSTN1 gene. Geneexpression analysis showed that cfMSTNmRNA is present in awide variety of tissues in juvenile fish. In addition,we assessed the spatial expression pattern of the MSTNmRNA during embryos and larval stages through wholemount in situ hybridization. No expression was observed in embryos, whereas in larvae of 8 and 9 days postfertilization, the notochord, somites, intestine and some discrete territories in the head, such as brain and eye,were positive forMSTNmRNA. Our results contribute to the knowledge of theMSTN system in larval and juvenilestages; inparticular the strongexpressionobserved in thenotochord suggests thatMSTN, in synchronizationwithpositive growth signals, may play an important role in the control of the development of larvae somites.

© 2008 Elsevier Inc. All rights reserved.

1. Introduction

The “double muscle” phenotype described in mammals, whichexhibits an outstanding increase of muscle mass, is a consequence ofloss-of-function of themyostatin gene (MSTN), amember of the TGF-βfamily (McPherron and Lee, 1997; Kambadur et al., 1997; Grobet et al.,1997; Grobet et al., 1998). MSTN negatively regulates skeletal musclegrowth during embryonic and adult development (Lee, 2004). As withother members of the TGF-β superfamily, MSTN is synthesized as aninactive pre-pro-peptide. Proteolytical processing renders a biologi-cally active dimeric peptide with the capacity to bind specificreceptors in its target cells (Anderson et al., 2008).

The MSTN gene sequence has been described in several verte-brates, including mammals, birds and fish. Within commerciallyimportant fish, a number of cDNA and genomic MSTN sequences havebeen reported, such as for rainbow trout, Atlantic salmon, white bass,Mozambique tilapia, striped bass, gilthead seabream, catfish, yel-low catfish, orange spotted grouper, croceine croaker and sea perch

Molecular, Universidad Andres319; fax: +56 2661 8415.

unology, Max-Planck Institutermany.

l rights reserved.

(Maccatrozzo et al., 2001a; Rodgers and Weber, 2001; Rodgers et al.,2001; Roberts and Goetz, 2001; Rescan et al., 2001; Ostbye et al., 2001;Maccatrozzo et al., 2002; Kocabas et al., 2002; Gregory et al., 2004; Koet al., 2006, Xue et al., 2006; Pan et al., 2007; Ye et al., 2007).Furthermore, a novel MSTN form (MSTN2) coded by a second gene,has been described in teleosts (Maccatrozzo et al., 2001b; Biga et al.,2005; Kerr et al., 2005). In addition, four different genes that depicttissue-specific expression and differential processing have beendescribed in salmonids (Garikipati et al., 2007).

MSTN expression in mammals is restricted predominantly toskeletal muscle (McPherron et al., 1997), lower level in adipose tissue(Gonzalez-Cadavid et al., 1998), mammary gland (Ji et al., 1998), andcardiac muscle (Sharma et al., 1999). In contrast, a wider tissueexpression has been described in different fish species. These tissuesinclude muscle, intestine, brain, kidney, gills, heart, eyes, spleen, liver,ovaries, and testis (Rescan et al., 2001; Ostbye et al., 2001; Rodgerset al., 2001; Roberts and Goetz, 2001; Maccatrozzo et al., 2001a,b;Kocabas et al., 2002; Xu et al., 2003; Amali et al., 2004; Ko et al., 2006;Xue et al., 2006; Helterline et al., 2007). This ample expression patternin tissues other than muscle suggests that MSTN may have a widerfunction in fish. In fact, MSTN knockdown in zebrafish, withmorpholino antisense oligonucleotides, led to the enhancement ofmuscle cell linage growth and also induced a significant accelerationof complete embryonic development (Amali et al., 2004). In addition,MSTN gene silencing through dsRNA generates an important adult fish

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body enhancement, mostly due to an increase of the size and numberof muscle fibers (Acosta et al., 2005).

The Chilean flounder (Paralichthys adspersus) and other potentialcommercially important marine fish species, requires longer times toreach commercial size as compared to classical farm fish such assalmonids, increasing the cost of production. This feature seems to beone of the principal difficulties in farming new marine fish species.Viability to farm these species requires new strategies to improve fishgrowth, in addition to advancing further knowledge in other fieldssuch as nutrition. In this regard, taking into account the atrophicfunction of MSTN, the inhibition of its bioactivity or other moleculesimplied in its signaling pathway has been proposed by a number ofauthors as an attractive strategy for genetic improvement ofcommercially important mammals (Roberts and Goetz, 2001; Yanget al., 2001; Wiener et al., 2002; Bellinge et al., 2005) and fish (Rescanet al., 2001; Rodgers and Weber, 2001; Acosta et al., 2005; Ko et al.,2006; Xue et al., 2006). Consequently, as a first step to usingMSTN as atool to enhance growth rates of P. adspersus in farming conditions, wepresent here the cloning of the MSTN gene and a temporal and spatialstudy of its expression during different developmental stages,including those of embryos, larvae and juveniles. In addition, wepresents data suggesting that MSTN in synchronization with positivegrowth signalsmay play a key role in the control of the development ofsomites in larval stages.

2. Material and methods

2.1. Fish

Chilean flounder (P. adspersus) were collected from the Centro deInvestigación Marina de Quintay (CIMARQ) (V Region, Valparaíso,Chile). The fish were maintained under natural temperature andphotoperiod conditions corresponding to geographic localization ofCIMARQ (33°13′S 71°38′W) and feed twice daily with turbot pellet(Biomar, Chile). Juvenile immature fish (120±15 g) were sacrificedthrough an overdose of anesthetic (3-aminobenzoic acid ethyl ester)(300 mg/L). The kidney, gills, intestine, gonads, spleen, liver, stomach,brain, white muscle, esophagus and red muscle tissues were collectedand directly frozen in liquid nitrogen and stored at −80 °C. Embryosand larvae were obtained after in vitro fertilization of eggs by malebroodstock sperm. Embryos were maintained under intensive-cultureconditions in conic larval culture tanks at 19 °C±2 °C.

Embryos and larvae at pre-metamorphic stages were collected,fixed in 4% paraformaldehyde in PBS for 2 h at 4 °C, dehydrated inmethanol 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'sinstructions. Using 5 µg of total RNA, first-strand cDNAwas synthesizedusing M-MLV reverse transcriptase (Invitrogen). Sense cfMyosF (5′-ATCAGCCGAGACATRGTSAAGCAG-3′) and antisense cfMyosR (5′-ATCTTGCCGTAGATGATCTGCTCT-3′) primers were used to amplify byPCR, a 850 bp fragment. This was cloned into the pGEM®-T Easy Vector(Promega, Madison, WI, USA) originating the pCFMSTN850 clone,which was completely sequenced.

The full-length 5′-terminal region, including the transcription startsite, was completed using the First Choice RLM-RACE® kit (Ambion,Austin, TX,USA) according to themanufacturer's instructions. Essentially,a RT-PCR with an adapter primer and two gene-specific primers(5cfMyO12OU 5′-ACATCCCTGTTGTCATCTCCCAG-3′ and 5cfMyO12IN5′-CACGTCGTACTGGTCGAGAAGCTG-3′) using a CIP/TAP mRNA as atemplate in a nested reaction. The 3′-region was obtained using thegene-specific primers 3MyO12OU (5′-CACCAAGATGTCGCCCATC-AACATG-3′) and 3MyO12IN (5′-GCTCTACTTTAATCGCAAAGAGCAG-3′)

by using the same kit. All PCR products were cloned into the pGEM®-TEasy Vector (Promega) and sequenced.

2.3. Structural analyses

Signal peptide sequence prediction (Emanuelsson et al., 2007) wascarried out by means of the Center for Biological Sequence AnalysisPrediction Server (www.cbs.dtu.dk). The propeptide and the activepeptide domains were predicted by BLAST resource (www.ncbi.nlm.nih.go) using CDART software (Geer et al., 2002).

2.4. Tissue expression (RT-PCR)

Total RNA was extracted from different tissues (kidney, gills,intestine, gonads, spleen, liver, stomach, brain, esophagus, whitemuscle and red muscle). Reverse transcription reaction was per-formed using 1μg of total RNA previously treated with DNase I. ForcfMSTN, gene-specific primers were designed to amplify a 100pbfragment (cfMSTNF: 5′-GACCACCGTGTTCCTGCAGATC-3′; cfMSTNR:5′-GATAGCGGCAGCACCGGGTCTC-3′). For normalization purposes,gene-specific primers (cfβactF: 5′-AGGGAAATCGTGCGTGACAT-3′;cfβactR: 5′-TCAGGCAGCTCATAGCTCTT-3′) were used to amplify aβ-actin 100 bp fragment as constitutive gene expression control.

2.5. Whole-mount in situ hybridization

A 1165 bp fragment corresponding to the cfMSTN coding sequencewas amplified by PCR using gene-specific primers (cfMSTNihF: 5′-CCAAACCTCCCACCAGAGAAAATG-3′; cfMSTNihR: 5′-TCCGTCCCAACT-CAAGAGCATC-3′) and the same cDNA described before as a template.This fragment was cloned into pGEM®-T Easy Vector System (Promega)originating the pCFMSTNih clone, whichwas linearizedwith SstII or SalIrestriction enzymes to synthesized sense (control) and antisenseriboprobes DIG-UTP-labeled (Roche Diagnostics, Mannheim, Germany)using SP6 and T7 RNA polymerases (Promega) respectively. Theriboprobes were purified using mini Quick Spin Columns (Roche) toeliminate unincorporated labeled nucleotides.

Whole mount in situ hybridization and histology were performedaccording to Fuentes et al., 2008. Briefly, after bleaching treatment,embryos and larvae were pre-hybridized overnight at 60 °C inhybridization buffer and then incubated overnight at 65 °C inhybridization buffer including 50 ng of sense or antisense cfMSTNriboprobes. After hybridization, embryos and larvae were washed in asolutionwith decreasing formamide concentration in 2× SSC, followedby two wash-steps with SSC 0.2× for 30 min at 65 °C. Embryos andlarvae were incubated for 4 h in a blocking buffer at room temp-erature. For immunodetection, samples were incubated overnight at4 °C with Anti-digoxigenin-AP antibody (Roche). After washes withPBT to eliminate non-bounded antibodies and three additional washeswith AP-buffer, stains were performed with NBT/BCIP (75 mg/mL and50 mg/mL, respectively) (Promega) for 6 h at 37 °C. The experimentwas performed four times using n=15 individuals from eachdevelopmental stage. After in situ hybridization, larvaewere sectioned(70 µm), mounted in slides, observed in an Olympus BX-61microscope and photographed with a Leica DF300 camera.

3. Results

3.1. Chilean flounder myostatin cDNA sequence

Using RT-PCR amplification coupled with the RLM-RACE approachto complete the 5′- and 3′-regions, the MSTN full length cDNAsequence from the teleost fish P. adspersus was obtained. The entirecDNA sequence has 2006 bp, with a 5′-UTR of 95 bp and a 3′-UTR of765 bp. The cDNA contains a single open reading frame of 1131 bp,encoding 377 amino acid residues. The cfMSTN deduced amino acid

Fig. 1. cfMSTN nucleotide and deduced amino acid sequences (GenBank accession no. EU443627). Start and stop codons are underlined. Putative signal peptide is depicted in italic.Conserved cysteines and the cleavage sequence RVRR are indicated by gray-boxes. The active peptide is highlighted in bold letters.

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sequence includes a signal peptide composed of 22 amino acidresidues, a propeptide in the N-terminal region that comprises 247amino acid residues, the TGF-β active peptide in the C-terminal regionof 109 residues, and between both domains, the conserved RVRRputative proteolytic processing sequence. In addition, the cfMSTNsequence also exhibits nine conserved cysteine residues, located in theC-terminal domain (Fig. 1).

The cfMSTN amino acid sequence displayed an extraordinarily highidentity (99%) with the MSTN1 of other fish belonging to the Para-lichthys genus, the bastard halibut (P. olivaceus) that displays only threeamino acid residue changes in the propeptide domain. In general,cfMSTN shows high degrees of identity withMSTN1 of other bony fish,comprising between 94 and 99% within Pleuronectiformes, and 83%with Salmoniformes like the Atlantic salmon MSTN1a. The cfMSTNexhibited fewer identities with other vertebrates, showing values of65% with chicken, 63% with mouse, and 64% with human. Never-theless, most of the amino acid changes are in the propeptide domain,whereas the C-terminal active peptide domain reaches identitieshigher than 88% comparing cfMSTN with mammalian MSTN, andbetween 95 and 100% within teleosts' MSTN (Fig. 2).

3.2. Juvenile and larvae cfMSTN mRNA expression

RT-PCR experiments were performed to study the expression ofcfMSTN mRNA in different tissues of juvenile fish using β-actin asconstitutive expression control. The transcript was detected in allinvestigated tissues (Fig. 3). In addition, we studied the expressionpattern of cfMSTN mRNA using whole mount in situ hybridization inChilean flounder embryos from 16, 40 and 42 hpf and larvae from 8.0and 9.0 dpf. No expression was detected in all examined embryostages (Fig. 4A, C and D). Larvae at 8 dpf showed strong expression in

the notochord and head while weak expression was observed insomites (Fig. 4G). cfMSTN mRNAwas also detected in the pectoral fins(Fig. 4H). Transversal sections of these larvae showed staining in thebrain (Fig. 4g), somites and notochord (Fig. 4g' and g"). In contrast, noexpression was observed in the neural tube (Fig. 4g' and g"). Later inthe development at 9 dpf, larvae exhibited strong cfMSTN mRNAexpression in the intestine and in discrete territories of the head: theoptic tectum, eye, jaw, and notochord (Fig. 4J and K). Transversalsections of the 9 dpf larvae show a localized expression in the brainand the otic capsule (Fig. 4j). Posterior slices show expression in thenotochord and intestine, although weak expression of cfMSTN wasdetected in the somites compared with the notochord (Fig. 4j' and j").Sense probe was included as a negative control in all in situhybridization experiments. No signal was detected, showing thanRNA hybridization was specific (Fig. 4B, D, F, I and L).

4. Discussion

We obtained the complete cDNA sequence of the MSTN from theflatfish Chilean flounder, an emergent species for aquaculture. ThecfMSTN deduced amino acid sequence includes the classical structuralfeatures of the TGF-β superfamily, including the N-terminal signalpeptide followed by the propeptide, which is responsible for forming alatent complex with the active peptide located in the C-terminalregion, and between both domains, a RVRR putative proteolyticprocessing sequence that matches with the TGF-β consensus RXRRsequence (Rodgers and Weber, 2001). Furthermore, the cfMSTNsequence contains nine conserved cysteine amino acid residues,located in the active peptide. These residues form a cysteine knotmotif involved in the formation of disulfide bound linkage betweenboth monomers that is an important property of the TGF-β

Fig. 2. MSTN1 amino acid sequences alignment in different vertebrates species: GenBank accession numbers: human AAH74757; mouse NP_034964; chicken AAK18000; bastardhalibut ABK91834, Chilean flounder EU443627; European seabass AAW29442; sea perch AAX82169; gilthead seabream AAX82169; Atlantic salmon 1a CAC51427; and zebrafishNP_571094. Identical amino acids residues are indicated by asterisks.

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superfamily members and is crucial for the correct structureformation of the biologically active peptide dimer (Avsian-Kretchmerand Hsueh, 2004).

The cfMSTN C-terminal active peptide amino acid sequencedisplays a remarkably high identity within the MSTN1 of bony fishand other vertebrates, whereas the propeptide is the most divergentdomain. This conspicuously high conservation of the active peptidesequence reveals an important evolutionary pressure for structuralconservation of the protein, most likely related to its biologicalfunction vis a vis the interaction with receptors in target cells.Moreover, phylogenetic analysis (data not shown) demonstrated thatcfMSTN sequence is grouped with teleostean MSTN1 in a single cladeseparated from MSTN2, a second MSTN coding gene previouslydescribed in zebrafish (Biga et al., 2005; Kerr et al., 2005) and inseabream (Maccatrozzo et al., 2001b). This divergent partitionbetween both genes (MSTN1 and MSTN2) suggests an early duplica-tion in the bony fish linage (Kerr et al., 2005). Until now we have been

Fig. 3. Juvenile Chilean flounder myostatin mRNA distribution in different tissuesassessed by RT-PCR. Kidney (1); gills (2); intestine (3); gonads (4); spleen (5); liver(6); stomach (7); brain (8); esophagus (9); white muscle (10); red muscle (11); negativecontrol (without DNA) (12). A 100 bp β-actin fragment was amplified as constitutiveexpression control (the figure is representative of three different individuals).

unable to isolate the cDNA sequence of the MSTN2 in the Chileanflounder (data not shown).

In the Chilean flounder, as well as in other commercially importantfish, the growth rates in juvenile stages are critical to maximize thebiomass yield in farming conditions. Gene expression analysis showedthat cfMSTN mRNA is present in a wide variety of tissues in juvenilefish. This extensive cfMSTN gene expression has been described in allteleosts studied, as compared to the restricted expression observed inmammals (McPherron et al., 1997; Gonzalez-Cadavid et al., 1998; Jiet al., 1998; Sharma et al., 1999), suggesting that the biologicalfunction of MSTN in fish is not exclusively restricted to the negativegrowth control of muscle tissue, but may also regulate the growth ofother tissues (Ostbye et al., 2001; Rodgers et al., 2001; Maccatrozzoet al., 2001a,b; Kocabas et al., 2002; Amali et al., 2004; Ko et al., 2006;Xue et al., 2006; Helterline et al., 2007).

Even if MSTN expression has been described during embryonic orlarval stages in several teleosts (Rescan et al., 2001; Maccatrozzo et al.,2001a,b; Kocabas et al., 2002; Xu et al., 2003; Amali et al., 2004; Koet al., 2006; Helterline et al., 2007), only a few studies have exploredthe MSTN spatial expression pattern by using immunohistochemistryor in situ hybridization (Radaelli et al., 2003; Amali et al., 2004;Patruno et al., 2007). Consequently, in addition to evaluating cfMSTNmRNA contents in juvenile fish, we studied the cfMSTN mRNAdistribution during embryonic and larval stages by whole mount insitu hybridization. No expressionwas perceived in any embryos stagesexamined. In contrast, in larvae cfMSTN mRNA expression wasobserved in a large variety of different tissues, particularly whenlarvae begin to feed themselves (9 dpf). The expression patternobserved in the Chilean flounder is consistent with the MSTN

Fig. 4. Whole-mount in situ hybridization for MSTN in Chilean flounder embryos and larvae. Expression of cfMSTN mRNA was analyzed from 16 hpf to 9 dpf of Chilean flounderembryos and larvae with antisense and sense probes. A and B, hybridized embryos with antisense (A) or sense (B) probes against miostatin at 16 hpf. C and D, show hybridizedembryos with miostatin antisense (C) or sense (D) probes. E and F, embryos hybridized with antisense (E) or sense (F) probes. Non signal was detected at all embryos stages analyzed.G, larvae at 8 dpf showed strong expression in the notochord and head; weak expressionwas observed in somites. H, Dorsal view shows expression of cfMSTNmRNA in the pectoralfins. Transversal sections of these larvae show staining in the brain (g), somites and notochord (g' and g"), and on the contrary, the neural tube was negative for myostatin mRNA(g' and g"). J and K, later in the development at 9 dpf, larvae show strong cfMSTNmRNA expression in the brain (optic tectum), eye, jaw, intestine and notochord. Transversal sectionsof the 9 dpf larvae show a localized expression in discrete territories in the brain and otic capsule (j). Posterior slices show expression in the notochord and intestine, although weakexpression of cfMSTN was detected in the somites compared with the notochord (j' and j"). We did not detect positive signals in the matched larvae incubated with the sense probe(I and L). Beelines in G and J indicate the respective sections in g, g', g" and j, j', j", respectively. Pictures are representatives of four independent experiments. Abbreviations: pf,pectoral fin; nc, notochord; e, eye; b, brain; s, somites; nt, neural tube; ot optic tectum; j, jaw; i, intestine; oc, otic capsule.

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expression recently reported in sea bass,meanwhile,MSTN is observedonly after 25 days post hatchery (Patruno et al., 2007). In both larvalstages studied, the notochord, somites and some discrete territories inthe head (brain and eyes) were positives forMSTN. In this regard, it hasbeen suggested that MSTN may play a role in the development of thecentral nervous system (Maccatrozzo et al., 2001a) and the expressionin the eye may also indicate a role in the development of longitudinaland circular muscle growth (Amali et al., 2004). Moreover, particularlyinteresting is the expression observed in the notochord, which is anessential structure for differentiation of adjacent territories such as,the neuroectoderm, heart and paraxial mesoderm, as well as inducingand maintaining the ventral fates (Pourquie, 2001). Furthermore, theno-tail mutant zebrafish, which lacks the notochord, exhibits anabnormal development of somites (Halpern et al., 1993). In addition,MSTN knockdown in zebrafish induces an enlargement of the somites(Amali et al., 2004) and a hyperplasic and hypertrophic effect onmuscle fibers (Acosta et al., 2005). On the other hand, we recentlyreported the expression of the GH receptor in the notochord in the

same larval stages (Fuentes et al., 2008) and we also observedexpression of IGF-I (Fuentes E., personal communication). Both genesare involved in the trophic signal mediated by GH (Reinecke et al.,2005). Moreover, transgenic salmon overexpressing the GH showslower levels of theMSTN transcript and protein compared towild typefish (Roberts et al., 2004). It is possible to hypothesize that in thenotochord there is an established balance between positive andnegative growth signals that determine the correct differentiation ofthe adjacent somites, source of muscle, bone and spinal chordprecursor cells. Further functional approaches have to be performedto elucidate this theory.

In summary, the complete cDNA sequence of the MSTN gene wascloned from the Chilean flounder. The deduced protein sequenceincludes all of the structural domains responsible for its biologicalactivity. In addition, for the first time we described the spatialexpression pattern of the MSTN gene during larvae stages in a flat fish.In this regard, the strong expression observed in the notochordsuggests that MSTN, in synchronization with positive growth signals,

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may play an important role in the control of the development ofsomites. Indeed, our results contribute to the knowledge of the MSTNsystem in the larvae and juvenile stages, both of which are crucialperiods for developing successful farming of the Chilean flounder.

Acknowledgements

This work was supported by Grants N°1050272 from theFONDECYT and 15-03/28-04/13-06I from the UNAB Research Fundto A.M and FONDECYT N°1060441 to A.E.R. We would like to thank Dr.Manuel Krauskopf for critical reading of the manuscript. We thankJuan Manuel Estrada for technical assistance and animal care in theCentro de Investigación Marina de Quintay (CIMARQ).

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