1

Sample manuscript 1 2 Manuscript must be submitted to CBP as a single pdf file, including text, figures, tables and 3 supplementary material 4

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Characterisation and expression of myogenesis regulatory factors during in vitro 6

myoblast development and in vivo fasting in the gilthead sea bream (Sparus 7

aurata) 8

9

Daniel García de la serranaa,b,#, Marta Codinaa,c,#, Encarnación Capillaa, Vanesa 10

Jiménez-Amilburua, Isabel Navarroa, Shao-Jun Duc, Ian A. Johnstonb, Joaquim 11

Gutiérreza* 12

13 aDepartament de Fisiologia i Immunologia, Facultat de Biologia, Barcelona Knowledge Campus, 14

Universitat de Barcelona, 08028, Barcelona, Spain 15 bScottish Oceans Institute, School of Biology, University of St Andrews, Fife KY16 8LB, St 16

Andrews, Scotland, UK 17 cInstitute of Marine and Environmental Technology, University of Maryland School of Medicine, 18

USA 19

20

# These authors contributed equally to the study 21

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Running title: Myogenic factors expression in Sparus aurata muscle cells. 23

24

ms. has 47 pages, 7 figures, 2 tables, 5 suppl. files 25

26

*Corresponding author: 27

Dr. Joaquim Gutiérrez 28

Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona 29

Av. Diagonal, 643 30

08028 Barcelona, Spain 31

Tel: +34 934021532; Fax: +34 934110358 32

jgutierrez@ub.edu 33

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Abstract (maximum 250 words) 35

The aim of this study was to characterise a primary cell culture isolated from fast skeletal muscle of 36

the gilthead sea bream. Gene expression profiles during culture maturation were compared with 37

those obtained from a fasting-refeeding model that is widely used to modulate myogenesis in vivo. 38

Myogenesis is controlled by numerous extracellular signals together with intracellular 39

transcriptional factors whose coordinated expression is critical for the appropriate development of 40

muscle fibres. Full-length cDNAs for the transcription factors Myf5, Mrf4, Pax7 and Sox8 were 41

cloned and sequenced for gilthead sea bream. Pax7, sox8, myod2 and myf5 levels were up-regulated 42

during the proliferating phase of the myogenic cultures coincident with the highest expression of 43

proliferating cell nuclear antigen (PCNA). In contrast, myogenin and mrf4 transcript abundance 44

was highest during the differentiation phase of the culture when myotubes were present, and was 45

correlated with increased myosin heavy chain (mhc) and desmin expression. In vivo, 30 days of 46

fasting resulted in muscle fibre atrophy, a reduction in myod2, myf5 and igf1 expression, lower 47

number of Myod-positive cells, and decreased PCNA protein expression, whereas myogenin 48

expression was not significantly affected. Myostatin1 (mstn1) and pax7 expression were up-49

regulated in fasted relative to well-fed individuals, consistent with a role for Pax7 in the reduction 50

of myogenic cell activity with fasting. The primary cell cultures and fasting-feeding experiments 51

described provide a foundation for the future investigations on the regulation of muscle growth in 52

gilthead sea bream. 53

54

Key words (maximum 10): Mrf4, Myf5, Myod, myogenesis, Myogenin, Pax7, primary cell 55

cultures, Sox8. 56

57

1. Introduction 58

59

Gilthead sea bream (Sparus aurata, Sparidae) is widely distributed from the Mediterranean Sea to 60

the eastern coast of the North Atlantic Ocean. This species is intensively farmed in Mediterranean 61

countries including Greece, Turkey, France, Spain and Italy. Because of its commercial interest 62

gilthead sea bream has been extensively studied, although relatively very little is known about the 63

regulation of muscle development and growth in this species. In vertebrates, the process of 64

myogenesis involves a series of events in which precursor cells are committed into the myogenic 65

lineage and become myoblasts which proliferate prior to exiting the cell cycle and fusing to form 66

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multinucleated myotubes as the differentiation program is initiated (Chen and Goldhamer, 2003; 67

Johnston, 2006). Myogenesis is controlled by numerous extracellular signals together with 68

intracellular factors. Myogenic regulatory factors (MRFs), show conserved sequence identity 69

among vertebrates including teleosts and play an essential role in specification and determination of 70

the muscle cell lineage (Holterman and Rudnicki, 2005). The MRFs (Myod, Myf5, Myogenin and 71

Mrf4), belong to a larger group of transcription factors, which share a highly conserved central 72

region termed the basic helix-loop-helix (bHLH) domain, containing a basic DNA-binding motif 73

and a helix-loop-helix dimerization domain. bHLH transcription factors dimerize with E-proteins 74

and bind to E-box elements (CANNTG) present in the promoter sequences of many muscle specific 75

genes (Holterman and Rudnicki, 2005). 76

During myogenesis, each MRF is expressed in a sequential-manner (Cornelison et al., 2000). Myod 77

and Myf5 are required for the initial specification of the myogenic lineage, while Myogenin and 78

Mrf4 are activated during myoblast differentiation and cell fusion (Cornelison et al., 2000). Studies 79

of gene knockout in mice showed that lack of myod and myf5 results in failure of myoblast 80

formation, and a consequent lack of both head and trunk skeletal muscle (Rudnicki et al., 1993). 81

Knockdown studies in zebrafish revealed that myod and myf5 are both required to drive myogenesis 82

in slow muscle (Hinits et al., 2009). However, knockdown of myod or myf5 alone had little effect. 83

This was further confirmed by a null zebrafish myf5 mutant that produced no embryonic phenotype, 84

but failed to develop into an adult fish (Hinits et al., 2009). In the myogenin knockout mice, 85

myoblasts formed in the correct location but failed to fuse into muscle fibres (Hasty et al., 1993); 86

however, other studies have shown that Myogenin inefficiently induced skeletal muscle 87

differentiation in cell culture (Roy et al., 2002). The function of Mrf4 is less clear and appears to be 88

involved in both differentiation (Hinits et al., 2007) and proliferation (Jin et al., 2007). 89

Myod, myf5, myogenin and mrf4 have orthologues in all fish species studied, and paralogues may be 90

present as a result of whole genome duplication (WGD) events in some fish lineages (Jailon et al., 91

2004; Macqueen and Johnston, 2008). Gilthead sea bream retained two paralogues of myod (myod1 92

and myod2) due to the early teleost WGD (Tan and Du, 2002). The situation in Atlantic salmon 93

(Salmo salar) is even more complicated with the loss of myod2 and the presence of three myod1 94

paralogues myod1a, myod1b and myod1c) (Macqueen et al., 2007), each with distinct expression 95

patterns during embryogenesis and following maturation of primary myogenic cell cultures (Bower 96

and Johnston, 2010a). To-date only a single copy of the myf5, myogenin and mrf4 genes has been 97

described in teleosts (Rescan et al., 1995; Macqueen et al., 2007; Codina et al., 2008a). 98

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Other transcription factors that regulate myogenesis are Pax7 and Sox8. Pax7 is a member of the 99

PAX superfamily implicated in organogenesis (Lang et al., 2007). Pax7 knockout studies in mouse 100

have demonstrated that embryogenesis is not affected, but mutants do not have satellite cells after 101

birth and the skeletal muscle does not develop normally (Seale et al., 2000; Relaix et al., 2006). 102

Studies in adult mice indicated that Pax7 has an important role in controlling myogenesis through a 103

direct action on Myf5 and Myod (Olguin et al., 2007; McKinnell et al., 2008). In experiments 104

conducted with salmon myoblasts, the expression of pax7 was significantly increased during serum 105

or amino acid deprivation concomitant with the down-regulation of the myod1c paralog, suggesting 106

a role for Pax7 in the repression of myogenesis (Bower and Johnston, 2010a). Sox8 is a member of 107

a large family of proteins known as SOX, with a common “High Mobility Group” DNA binding 108

domain (HMG). Sox8 is part of the Sox subgroup E (including Sox9 and Sox10), which is involved 109

in organogenesis (Guth and Wegner, 2008). Sox8 has been proposed as a molecular marker of 110

mammalian satellite cells and it is important for maintaining their quiescent state (Schmidt et al., 111

2003). 112

In addition to the myogenic transcription factors, many extracellular protein molecules are involved 113

in the regulation of muscle growth including Myostatin (Mstn), which belongs to the transforming 114

growth factor beta (Tgf-ß) superfamily of signalling molecules. Mstn is a complex regulator of 115

muscle growth, with two copies described in gilthead sea bream (Nadjar-Bojer and Funkestein, 116

2011). Mstn appears to prevent myoblast hyperplasia in mammals by inhibiting cell cycle 117

progression, and its effects on cell differentiation are controversial, although the most recent model 118

seems to indicate that Mstn inhibits proliferation but not differentiation (Seiliez et al., 2012). Mstn-119

induced cellular quiescence is reversible and associated with reduced expression of the MRFs pax3, 120

myod and myf5 (Amthor et al., 2006). 121

The insulin-like growth factor (Igf) system regulates myoblast proliferation and the balance 122

between protein synthesis and breakdown in muscle (Seiliez et al., 2011). In gilthead sea bream, an 123

increase in plasma levels of Igf1 has been observed in periods of increased growth (Dyer et al., 124

2004). More recently, Igf2 has been postulated as a growth promoting factor throughout the whole 125

life cycle of gilthead sea bream (Benedito-Palos et al., 2008). In addition, cell culture studies in 126

trout and gilthead sea bream have revealed that both Igf1 and Igf2 have a role in muscle cell 127

proliferation and metabolism (Castillo et al., 2004; Montserrat et al., 2007; Codina, et al., 2008b; 128

Rius-Francino et al., 2011. 129

In the present study on gilthead sea bream we used quantitative real-time PCR (qPCR), 130

immunocytochemistry and western blotting to investigate gene and protein expression in in vitro 131

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and in vivo models of myogenesis. The aim of the study was to characterise the main myogenic 132

factors in gilthead sea bream and to analyse them during in vitro myoblast culture and in in vivo 133

fasting to better understand the regulation of muscle development and growth in this species. 134

135

2. Material and methods 136

137

2.1 Ethical procedures 138

All animal handling procedures were approved by the Ethics and Animal Care Committee of the 139

University of Barcelona, following the EU, Spanish and Catalan Government-established norms 140

and procedures. 141

142

2.2 Myf5, Mrf4, Pax7 and Sox8 isolation and characterization 143

Myf5, mrf4, pax7 and sox8 fish sequences were obtained from Genbank 144

(www.ncbi.nlm.nih.gov/Genbank), with the following accession numbers: myf5 from Takifugu 145

rubripes (NP001027942), Danio rerio (AAF70195), Salmo salar (NP001117116), Micropterus 146

salmoides (ACB45872), Lateolabrax japonicus (AAZ76912); mrf4 from T. rubripes (CAC39207), 147

Tetraodon nigroviridis (AAS88438), S. salar (NP001117079), Sternopygus macrurus 148

(AAY53906), Danio rerio (AAQ67704); pax7 sequences from S. salar (NP001117167), Salvelinus 149

alpinus (CAG25718), Sternopygus macrurus (ACC86107) and Danio rerio (AAI63580); sox8 150

from S. salar (NP001117071), Danio rerio (AAX73357), T. nigroviridis (AAT42231). Sequences 151

were aligned using ClustalW from BioEdit sequence alignment editor 152

(www.mbio.ncsu.edu/BioEdit/bioedit.html). Degenerate primers were designed over the most 153

conserved regions of the aligned sequences to amplify the whole coding sequence. In the case of 154

pax7 different nested primers were designed. Netprimer software 155

(www.premierbiosoft.com/netprimer) was used to estimate the melting temperature and the 156

presence of potential hairpins, primer dimers and cross dimers between them. The degenerated 157

primers used are summarized in Table 1. cDNA from gilthead sea bream myoblasts at day 5 of 158

culture was used to clone the target molecules. The PCR protocol was performed with the designed 159

primers using a standard PCR kit (BioTaq™ DNA polymerase kit; Bioline, London, UK) with the 160

following PCR protocol: 10 min at 95 ºC; 35 cycles of 30 s at 95 ºC, 30 s at 60 ºC and 1 min at 72 161

ºC; and a final extension of 7 min at 75 ºC. PCR products were visualized in a 1% (m/v) TBE-162

agarose gel with ethidium bromide. Bands of the expected size were extracted (QIAquick® Gel 163

extraction kit; QIAGEN, Sussex, UK) and carried over a second PCR, using the same primers. The 164

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different PCR products were cloned in TOPO® vectors and introduced in E.coli PC10 competent 165

cells following manufacturer’s instructions (Invitrogen, Paisley, UK). Clones with the right insert 166

size were analysed at the Sequencing Service of the University of Dundee, UK. Identities of the 167

sequences were checked using the Blast tool from the NCBI. Prior to pax7 blast analysis, the 168

nested-PCR fragments were ensembled to generate a unique cDNA molecule using the CAP3 169

option from BioEdit. Conserved domains were determined using Conserved domains Databases 170

from NCBI (www.ncbi.nlm.nih.gov/Structure/cdd/) and Prosite (http://www.expasy.ch/prosite/). To 171

establish sequence identity ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used with 172

different vertebrate sequences. 173

174

2.3 Cell cultures 175

Gilthead sea bream fingerlings (Sparus aurata; 6g) obtained from a fish farm in Northen Spain 176

were maintained at the facilities of Servei d’Estabulari in the Faculty of Biology at the University of 177

Barcelona, in a closed-water flow circuit in 400L tanks with controlled water temperature (21±0.5 178

ºC), pH (7.5-8), salinity (31-38‰), oxygen saturation (>80%) and photoperiod (12 h light:12 h 179

dark). Animals were fed ad libitum with a commercial diet (Excel, Skretting, Burgos, Spain) and 180

fasted 24 h prior to myoblast extraction. 181

Myogenic cells were isolated, pooled and cultured following the procedure described previously for 182

trout (Rescan et al., 1995; Castillo et al., 2004) and adapted for gilthead sea bream (Montserrat et 183

al., 2007). All media and reagents were obtained from Sigma-Aldrich (Tres Cantos, Spain) unless 184

stated otherwise. Cells were seeded for expression analyses at a density of 1.5-2 x 106 cells per well 185

in 6-well plastic plates (9.6 cm2/well) or at a density of 1 x 106 cells per well in cover-slips placed 186

in 12-well plastic plates (4 cm2/well) for immunofluorescence studies (Nunc, Labclinics, Barcelona, 187

Spain) and maintained at 22 ºC in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) 188

fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin, which allowed the spontaneous 189

differentiation of the cells from myoblast to myotubes. 190

The SAF-1 cell line, generated from gilthead sea bream fibroblast-like cells that was spontaneously 191

immortalized (Béjar et al., 1997), was kindly provided by Dr J.J. Borrego from the University of 192

Málaga (Spain). Cells were cultured at 22 ºC in Leibovitz’s (L-15) medium supplemented with 2% 193

L-glutamine, 1% penicillin–streptomycin and 10% FBS. 194

195

2.4 In vivo fasting-feeding experiment 196

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Gilthead sea bream juveniles (100g) were obtained from Piscimar fish farm (Ametlla de Mar, 197

Spain) and maintained at the facilities of Servei d’Estabulari in the Faculty of Biology. Fish were 198

maintained in tanks (200L) in two independent systems, supplied with biological sand filtered (15 199

µm) and UV-treated seawater, and under controlled conditions of temperature (22 ± 0.5 ºC), pH 200

(7.5-8), salinity (31-38‰), oxygenation (>80% saturation) and photoperiod (12 h of light and 201

darkness), and adapted during one month fed 1.5% body weight with a commercial diet (Excel, 202

Skretting, Burgos, Spain). 203

After adaptation, the animals were randomly redistributed in 4 tanks per condition. Control fish 204

(n=8) were fed normally with 1.5% body weight ratio and a second group (n=8) were fasted for 30 205

days. Animals were sacrificed and sampled 30 days after the beginning of the experimental trial. 206

Prior to sampling, fish were anesthetized with Tricaine methanesulfonate (MS222; 100 ppm) 207

diluted in seawater. Animals were sacrificed by decapitation and white (fast) muscle from the 208

middle/dorsal region of the fish was extracted using RNAase-free instrumental (pre-treated with 1 209

M NaOH) and frozen immediately in liquid nitrogen. Muscle was stored at -80 ºC. 210

211

2.5 RNA extraction and cDNA synthesis 212

Total RNA was extracted from myoblasts in culture at different stages of differentiation using the 213

RNeasy® Mini Kit (QIAGEN, Sussex, UK); and from different tissues (fast and slow muscle, 214

spleen, liver, adipose, kidney, heart and brain) of gilthead sea bream fingerlings (6g) and from fast 215

muscle of control and fasted animals using TriReagent (Ambion, UK) following the manufacturers’ 216

instructions. RNA quality was checked on a 1% TBE-agarose gel with ethidium bromide. 217

Concentration and purity of the samples were evaluated with a ND-1000 Nanodrop (Labtech Int., 218

East Sussex, UK). Only samples with 260/280 and 260/230 ratios over 1.9 were used. For cDNA 219

synthesis Verso™ cDNA kit (ABgene, UK) was used following manufacturer’s instructions, 220

starting from the same quantity of RNA for each sample. During the reverse transcription (RT)-221

PCR assay we used/analysed a negative control for the cDNA synthesis without the retro-222

transcriptase enzyme (RT-). 223

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2.6 RACE-PCR 225

To obtain the final portion of mrf4 and sox8 sequences we performed a RACE-PCR, but only sox8 226

end was successfully amplified. The primers for the 3’ end of the molecule are shown in Table 1. 227

We used TRACE a modification of the RACE-PCR protocol using dUTPs/dNTPs for the cDNA 228

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synthesis and a UDG treatment after the asymmetric PCR as described previously (Bower and 229

Johnston, 2010b). 230

231

2.7 Conventional PCR 232

In our aim to find a molecule to help us to define the identity of muscle cells and also to confirm the 233

absence of fibroblasts in our primary culture, we evaluated the expression of desmin, a well-known 234

marker for cells of the myogenic lineage. A specific forward primer and a degenerated reverse 235

primer were designed to amplify a 232bp region of desmin, flanking an intron to avoid genomic 236

amplification. Amplified fragments were analysed in a 1% (m/v) agarose gel. Specificity of the 237

reaction was verified by sequencing the amplified fragment. 238

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2.8 Flow cytometry (FACS) 240

SAF-1 cells and myocytes at different days of culture were washed with PBS and collected. Cells 241

were then fixed with paraformaldehyde 4% (m/v) in PBS for 20 min at 4 ºC, followed by a 242

treatment with a permeabilisation solution (1x PBS, 5% FBS, 0.05% Saponin). Cells were stained 243

using an anti-desmin monoclonal antibody (Sigma D1033, Tres Cantos, Spain) at a 1/100 dilution 244

in permeabilisation solution, for 40 min at 4 ºC, followed by an Alexa Fluor 488 anti-mouse 245

(Invitrogen A11017, Alcobendas, Spain) at a 1/300 dilution. For negative control, the staining was 246

performed in the absence of primary antibody. Cells were then washed and analysed on a FACScan 247

flow cytometer, using CELLQuest software (Becton Dickinson). Further analysis was performed 248

using FlowJo software (Tree Star, Inc. Oregon, USA). 249

250

2.9 Quantitative real-time PCR (qPCR) 251

The following procedures were performed to comply with the Minimum Information for 252

Publication of Quantitative Real-Time PCR experiments, MIQE guidelines (Bustin et al., 2009). 253

Primers for myf5, myod2, myogenin, mrf4, pax7 and sox8 were designed using Primer3 254

(http://biotools.umassmed.edu/bioapps/primer3_www.cgi) to have a Tm of 60 ºC, and when 255

possible, to cross an exon-exon junction to avoid amplification of genomic DNA. To determine 256

exon-intron junction sites, genomic sequences for orthologous genes from Danio rerio, 257

Gasterosteus aculeatus, Oryzias latipes, Takifugu rubripes and Tetraodon nigroviridis were 258

retrieved from Ensembl (http://ensembl.org/), and compared to the Sparus aurata CDS sequences 259

using the Spidey NCBI tool (http://www.ncbi.nlm.nih.gov/spidey/). 260

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Primers for the proliferative cell nuclear antigen (PCNA) were designed using known sequences of 261

Danio rerio, while primers for myosin heavy chain (mhc), myod2, myogenin, mstn1 and elongation 262

factor 1 alpha (ef1α) were designed based on gilthead sea bream sequences. Specific primers for 263

igf1 and igf2 were made as described by Benedito-Palos et al., (2008). Primers were designed 264

flanking an intron to prevent genomic amplification and the PCR products were between 100-200 265

bp. qPCR was performed using an iCycler IQ Real time detector (BioRad). Diluted RT product was 266

used for PCR reactions in 25 µL final volume. Each PCR-well contained SYBR Green Master Mix 267

(BioRad, El Prat de Llobregat, Spain) with specific primers for each target or reference gene (Table 268

2). 269

The efficiency of qPCR reactions for target and reference genes was determined using standard 270

curves generated by serial dilutions of a pool of RT reactions. It ranged from 90% to 99% based on 271

the equation E=10(-1/slope)-1, with R2>0.98. Specificity of the reactions was verified by 272

electrophoresis and by direct sequencing of the product. Reactions were performed in duplicate and 273

fluorescence data acquired during the extension phase was normalized using ef1α as reference gene. 274

qPCR results were expressed as relative values using the Pfaffl method (Pfaffl, 2001). Permutmatrix 275

software (Caraux and Pinloche, 2005) was used for visualization of the gene expression profiles 276

analysed along cell culture differentiation. 277

278

2.10 Western blot 279

To analyse the protein levels of desmin, cells at different days of culture were washed in PBS and 280

lysed with a RIPA buffer containing 1% (v/v) NP-40, 1 mM sodium orthovanadate, 50 mM Tris-281

HCl, 150 mM NaCl, 1 mM EDTA, 1 mM sodium fluoride, 1 mM PMSF, 0.25% sodium 282

deoxycholate and a 1/500 dilution of protease inhibitors (Sigma P8340), at pH 7.4. Total protein 283

was quantified by the Bradford method using BSA as a standard, and 20 µg of protein were loaded 284

on a 10% (m/v) polyacrylamide gel and subjected to electrophoresis (SDS-PAGE) and then, 285

samples were transferred to a PVDF membrane. The membrane was then blocked for 1 h at room 286

temperature in non-fat milk 5% (v/v) buffer, and incubated overnight at 4 ºC with the primary 287

antibody (Sigma D1033) diluted in washing buffer to detect the presence of desmin. The membrane 288

was then washed and incubated for 1 h at room temperature with the corresponding secondary 289

antibody linked to HRP at 1/10000 dilution. The same procedure was performed with muscle 290

samples from the fasting experiment to analyse AKT-p (Cell Signaling #9271), mTOR-p (Cell 291

Signaling #2971) and c-Met (Santa Cruz Biotechnology sc-162). Once these films were developed, 292

the membranes were stripped and blotted again following the same procedure with AKT (Cell 293

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Signaling #9272), mTOR (Sigma T2949) and actin (Sigma A2066) antibodies. Immunoreactive 294

bands were visualized by ECL; films developed in an automatic film processor (Fujifilm) and 295

quantified using the ImageJ software. 296

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2.11 Immunocytochemistry 298

Gilthead sea bream fast skeletal muscle blocks were dissected from the anterior-axial region of the 299

animal (six animals per condition). Blocks were immediately frozen in liquid nitrogen-cooled 2-300

methylbutane for 30 s and stored in liquid nitrogen until further analysis. Six µm thick fast skeletal 301

muscle sections were cut in a Leica cryomicrotome at -20 ºC, plated on Poly-Lysine pre-coated 302

slides (Sigma, P0425) and stored at -80 ºC for further analysis. Samples were fixed and treated, as 303

previously described for salmon (Johnston et al., 1999). For Myod detection, we used a custom 304

made rabbit polyclonal antibody developed against Atlantic salmon Myod1 and previously used to 305

detect Myod in Artic charr as described in Johnston et al., (2004). Sections were co-stained with 306

Mayer’s hematoxylin to facilitate identification of positive nuclei. Five different regions of each 307

sample were captured using a 10x objective with an Olympus camera coupled to a microscope. Per 308

sample 500-900 fibres covering an area of between 3500-5500 µm2 were analysed. Positive cells 309

were counted using image analysis software (analySYS®, Soft Imaging System). 310

311

2.12 Immunofluorescence 312

Cells at days 4 or 8 of culture were fixed with 4% (m/v) paraformaldehyde for 30 min and rinsed 313

with PBS. Then, immunofluorescence was performed in the presence of 0.2% Triton X-100 to 314

detect Myod and Myogenin using monoclonal antibodies (sc-760 and sc-576, respectively, Santa 315

Cruz Biotechnology, Heidelberg, Germany) followed by an Alexa-568 anti-mouse secondary 316

antibody. Nuclei were counterstained using Hoechst at a 1:1000 dilution. Images were obtained by 317

laser confocal microscopy with a LeicaDMIRE2 microscope and quantification analyses of signal 318

intensity were performed using ImageJ (NIH). 319

320

2.13 Statistical analysis 321

Data was plotted using SPSS version 13 for Windows and represented as Mean ± standard error. All 322

parameters were tested for normalization using the non-parametric test for normal distribution, 323

Shapiro-Wilk. Statistical differences between groups were analysed by One-way analysis of 324

variance (ANOVA), followed by a Tukey post-hoc test when possible. When values did not follow 325

a normal distribution Kruskal-Wallis one-way analysis of variance on ranks was applied. For the 326

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fasting experiment a student’s t-test was used. Statistical differences were considered significant 327

when p-values < 0.05. 328

329

3. Results 330

331

3.1 Sequence analysis of Myf5, Mrf4, Pax7 and Sox8 from gilthead sea bream 332

To better understand myogenic gene expression and function during gilthead sea bream myogenesis 333

in vitro, we isolated and characterised the full-length or major part of the myf5, mrf4, pax7 and sox8 334

coding sequences (Supplementary File 1). The sequences were submitted to GenBank [myf5 335

(JN034420), mrf4 (JN034421), pax7 (JN034418), sox8 (JN034419)]. Sequence analysis was 336

performed by comparing the translated amino acid sequences with their respective orthologues from 337

other species. We found that the protein sequences of main functional domains for each gene family 338

were conserved, e.g. the myogenic domain and bHLH-binding domains in Myf5 and Mrf4; and the 339

Paired box domain and the HMG domain in Pax7 and Sox8, respectively (Supplementary File 2). 340

All these transcription factors showed a high degree of conservation compared to other fish species 341

(ranging from 70 to 90% identity) (Supplementary File 3). The percentage of identity of the whole 342

sequence decreased when compared with mouse and frog, but remained high for the functional 343

domains. 344

345

3.2 Myf5, mrf4, pax7 and sox8 tissue expression 346

Myf5 and mrf4 showed similar patterns of muscle-specific expression. Both myf5 and mrf4 were 347

highly expressed in slow and fast skeletal muscle (thousands of times more than in any other tissue) 348

(Supplementary File 4). Similarly, pax7 and sox8 also appeared to be highly expressed in skeletal 349

muscles. However, unlike myf5 and mrf4, strong expression of pax7 and sox8 was also detected in 350

the brain compared with other tissues (Supplementary File 5). 351

352

3.3 Cultured muscle cells progression and PCNA and mhc expression 353

To characterise the pattern of gene expression involved in myoblast proliferation and differentiation 354

during myogenesis in vitro, we analysed the expression of PCNA and mhc in primary muscle cell 355

cultures. We have previously characterised in detail the morphological events associated with 356

maturation of primary cell cultures in gilthead seabream (Montserrat et al., 2007). We showed that 357

on day 2, the cells comprised mononucleated myoblasts undergoing active proliferation. These cells 358

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subsequently differentiated and fused to form small myotubes by days 6-8 and finally became large 359

myotubes by day 15, although some large myotubes can be already observed at day 12. 360

PCNA and mhc expression was detected by qPCR and PCNA was also studied by 361

immunocytochemistry. The results showed abundant PCNA-positive cells during the proliferative 362

phase (Figure 1). Moreover, the PCNA levels were high at the beginning of the culture at the 363

myoblast stage and decreased drastically by 4 to 6-fold during myotube formation (Figure 1). PCNA 364

protein expression was down-regulated during differentiation (data not shown). In contrast, mhc 365

expression significantly increased after day 7 concomitant with the formation of myotubes, with 366

values 4 to 10-fold higher than at day 1 (Figure 1). Together, these data indicated that PCNA and 367

mhc expression correlated well with the proliferation and differentiation phases of primary 368

myogenic cultures. 369

370

3.4 Desmin mRNA and protein expression in cultured muscle cells 371

To further characterise the myogenic progression of muscle cells in culture, we analysed the 372

expression of desmin by RT-PCR at different days after seeding. The mRNA expression of desmin 373

was clearly detected at all three days analysed with stronger expression at days 4 and 9 compared 374

with day 1. In contrast, no desmin expression was detected in SAF-1 cells (Figure 2A). The 375

temporal pattern of desmin expression was further confirmed at the protein levels by western blot 376

analysis (Figure 2B). 377

To further characterise the dynamic pattern of desmin expression during the progression of the 378

culture, we analysed cells at different days after seeding by flow cytometry. We found that the 379

number of Desmin-positive cells increased in parallel to differentiation, reaching a maximum at day 380

8, when 91.5±1.3% of the cells expressed desmin (Figure 2C), indicating that the majority of the 381

cells in culture were from the muscle lineage. The rest of the cells could be either quiescent 382

myogenic progenitor cells, or possibly fibroblasts. No Desmin staining was observed in SAF-1 cells 383

(data not shown). These data indicate that primary muscle cells rapidly undergo myogenic 384

differentiation in culture. 385

386

3.5 Pax7 and sox8 expression in cultured muscle cells 387

Next, we characterized pax7 and sox8 mRNA expression by qPCR. Pax7 expression increased 388

from day 1 to day 4 and as the culture matured the levels of this transcription factor decreased to 389

very low levels (Figure 3A). Sox8 had maximal expression between days 1 and 2, but then the 390

levels decreased consistently until it became almost undetectable at day 12 (Figure 3B). These data 391

13

are consistent with the idea that pax7 and sox8 are expressed in muscle progenitor cells and play a 392

role in their maintenance. 393

394

3.6 MRFs expression in cultured muscle cells 395

MRFs expression was evaluated at various time points in cultured muscle cells from myoblasts at 396

day 1 to myotubes at day 12. The results showed that myf5 had maximal expression at day 2, which 397

was 2-fold higher than at day 1. Then, myf5 expression decreased to very low levels in the latter 398

stages of the culture (Figure 4A). Similarly, a peak of myod2 expression was found at day 3, when 399

the expression was between 3 to 5-fold higher than the expression at day 1. Later expression 400

decreased until day 12, when the transcript was barely detectable (Figure 4B). 401

In contrast, myogenin expression remained very low until day 7 and was followed by a peak of 402

expression at day 8. The levels of the transcript at day 8 were between 3 to 7-fold higher than the 403

levels at day 2 (Figure 4C). Mrf4 expression showed a similar pattern of expression, being almost 404

undetectable at the beginning of the culture before abruptly increasing after day 7 with levels during 405

myotube formation reaching 10-fold of that found on day 1 (Figure 4D). Consistent with the data 406

from qPCR studies, immunofluorescence analysis showed stable Myod protein expression during 407

culture progression, while Myogenin expression increased significantly between days 4 and 8 of 408

culture (1.3-fold increase, P=0.018) (Figure 5). 409

410

3.7 Myogenic and growth factors expression in gilthead sea bream fast skeletal muscle after fasting 411

To determine the effect of fasting on myogenic gene expression in skeletal muscles, whole animal, 412

liver and fillet weight were monitored in gilthead sea bream before and after 30 days of food 413

deprivation. Total animal weight was reduced by 20%, the hepatosomatic index by 70% and the 414

muscle somatic index by 10%. Histological analysis also showed a significant decrease in muscle 415

fibre cross-sectional area of fasted fish (29% reduction) and immunocytochemistry analysis 416

revealed a significant decrease in the number of Myod-positive cells (Figure 6 inset). PCNA and 417

other markers like c-Met, mTOR and AKT determined by means of western blot were also 418

significantly depressed in agreement with the 30 days-fasting producing a depression of protein 419

synthesis and myogenic cell activity (Figure 7A, 7B, 7C). 420

All the genes analysed in cell culture plus mstn1, igf1 and igf2 were analysed in gilthead sea bream 421

fast skeletal muscle after 30 days of fasting (fasted) and compared to well-fed animals (Figure 6). 422

Myod2, myf5 and igf1 expression decreased 65-70% after 30 days of fasting compared to well-fed 423

fish. In contrast, pax7 and mstn1 expression significantly increased 3.5 and 10-fold, respectively 424

14

after fasting. Moreover, myogenin, mrf4, sox8 and igf2 expression was not affected after 30 days of 425

food deprivation in gilthead sea bream muscle. 426

427

4. Discussion 428

429

In this study, we characterised gene and protein expression during the maturation of a primary 430

myogenic cell culture and compared the results with a fasting-refeeding model widely used to 431

manipulate myogenic activity in vivo in teleosts. The morphological events associated with 432

maturation of primary myogenic cultures in vitro, was similar to that previously described in the 433

same species (Montserrat et al., 2007). Cells differentiated from mononucleated myoblasts to form 434

multinucleated myotubes as the culture progressed with up-regulation of desmin, myod and 435

myogenin and down-regulation of PCNA concomitant with differentiation. The purity of the 436

myoblast cell culture used in the present study was investigated by the analysis of desmin 437

expression. In our system, the percentage of cells immunoreactive for Desmin increased from 80% 438

at day 2 to 90% after 6 days in culture, indicating their myogenic origin. Similar or lower 439

percentages of Desmin-positive cells were found in other primary culture systems from mammals 440

(Machida et al., 2004; Grohmann et al., 2005). Furthermore in fish, desmin is expressed in cultured 441

post-mitotic myoblasts of carp (Koumans et al., 1991), and muscle explants of gilthead sea bream 442

(Funkenstein et al., 2006). Together with desmin, mhc expression also increased during culture 443

development as myotubes differentiated and initiated myofibrillargenesis. 444

Genes expressed from the early steps of culture development until middle stages included myod2, 445

myf5, pax7, PCNA and sox8. Other genes increased their expression from the middle stages of the 446

culture until myotube formation including myogenin, mrf4 and mhc. Myf5 and myod2 up-regulation 447

was coincident with the proliferating phase of the culture, as demonstrated by PCNA expression 448

(Rius-Francino et al., 2011), and a parallel up-regulation at the beginning of the culture was also 449

observed for myod1 in similar experiments with the same species (Jiménez-Amilburu et al., 450

unpublished data). Myogenin and mrf4 were highly expressed during myoblast fusion to form 451

myotubes, coincident with the expression of mhc, in agreement with previous findings in zebrafish 452

(Hinits et al., 2007). The expression profile of the MRFs fits with the model proposed by 453

Cornelison et al., (2000), in which myf5 and myod are expressed early in the myogenic program and 454

myogenin and mrf4 appear later. Previous studies have suggested that Myod and Myf5 are essential 455

for the initiation of the myogenic program and Myogenin and Mrf4 for myotube differentiation 456

(Cornelison et al., 2000). This is also in agreement with findings in rainbow trout, Atlantic salmon 457

15

(Rescan et al., 1995; Bower and Johnston, 2010a) and giant Danio (Frochlich et al., 2013) using the 458

same model of myoblast primary culture. In addition to the changes on gene expression, we have 459

confirmed using immunofluorescence that Myod and Myogenin protein expression also followed 460

this same inverse pattern, with Myod being highly expressed at day 4 and Myogenin increasing 461

later, at day 8. 462

To better understand the function of these molecules in vivo, we analysed their expression in 463

gilthead sea bream fast skeletal muscle after 30 days of fasting, which was sufficient to result in 464

muscle wasting and the mobilisation of protein as evidenced by a decrease in fibre cross-sectional 465

area. Different authors have used fasting as a model to investigate the response of muscle myogenic 466

factors (Chauvigné et al., 2003; Montserrat et al., 2006; Terova et al., 2006; Rescan et al., 2007; 467

Alami-Durante et al., 2010). Although there are differences between the species and the 468

experimental conditions used, in general fasting results in significant changes in the expression of 469

MRFs. In our study, fasting resulted in decreased protein expression for Myod, PCNA, c-Met, 470

mTOR and AKT. Our results showed also that myod2 and myf5 transcripts were strongly reduced 471

after fasting, but not mrf4 or myogenin. Similarly, in trout treated with an experimental diet that 472

caused a significant decrease in the mean and median diameters of muscle fibres, a significant 473

decrease in the expression of myod was observed, whereas no significant changes occurred in 474

myogenin expression (Alami-Durante et al., 2010). Montserrat et al., (2006) observed that muscle 475

myod expression did not change in fasted trout, while myogenin first decreased, but then it 476

rebounded after 4 weeks of fasting. Conversely, Chauvigné et al., (2003) found that myogenin 477

mRNA expression was unchanged 4 days post-refeeding, while after 12 days the levels increased 6-478

fold versus those observed in fasted trout. All these results point out the different roles of MyoD 479

and Myogenin in fasting and refeeding. Moreover, in vitro experiments have suggested that Mrf4 is 480

related to myotube formation (Delgado et al., 2003). Because its expression, together with that of 481

myogenin, was not affected after fasting in our study, this suggests that final myotube 482

differentiation was not affected under these conditions, but further investigation at the protein level 483

is necessary to confirm this hypothesis. 484

Furthermore, fasting reduced igf1 expression, but increased that of mstn1. In vivo it is known that 485

Mstn negatively regulates muscle growth and inhibits proliferation (McCroskery et al., 2003; 486

Seiliez et al., 2012), whereas Igf1 is well known to stimulate in vitro cell proliferation in fish, and 487

thus muscle growth (Castillo et al., 2004; Montserrat et al., 2007; Rius-Francino et al., 2011; Seiliez 488

et al., 2011). In agreement with our study, Terova et al., (2006) showed that the nutritional status 489

significantly influenced mstn expression levels in muscle, with up-regulation induced during fasting 490

16

and down-regulation during recovery. Also, studies in chicken myoblasts suggested that Mstn 491

down-regulates myod and myf5 (Yang et al., 2005). Comparative studies of well-fed and fasted 492

gilthead sea bream transcriptomes support this relationship (Garcia de la serrana et al., 2012). 493

During our experiment, fed control animals grew normally, but fasted animals decreased in weight. 494

After 30 days, mstn1 expression was significantly higher in fasted than well-fed fish, coincident 495

with a significant reduction on igf1, myod2 and myf5 expression. The increased expression of mstn1 496

and reduced myod2 and myf5 expression does not necessarily infer a direct relationship, but it may 497

rather be a consequence of the same process of growth reduction. Interestingly in our cells, very 498

low levels of mstn1 expression are detected at all-times during culture progression (García de la 499

serrana et al., unpublished data), in agreement with proper cell development and growth. 500

Pax7 expression patterns in vitro and in vivo suggested a key regulatory function for this 501

transcription factor during myogenesis in fish. The variations in the expression of pax7 observed 502

during culture maturation, seemed to be in agreement with the model proposed by Olguin et al., 503

(2007), in which Pax7 has a pivotal role regulating Myod and Myogenin. It was proposed that Pax7 504

inhibits Myod function at an early stage. Our results support this model, in both cell culture and 505

fasting animals, where pax7 increased at the same time that myod2 was down-regulated. Similarly, 506

in vitro studies in Atlantic salmon myocytes have shown that pax7 increased after being incubated 507

with an amino acid-free media, coincident with a reduction in myod1c expression (Bower and 508

Johnston, 2010a). However, an interaction between Pax7 and Myogenin is not so clear in our 509

experiment and it could be consequence of the moment of sampling or the fasting condition itself. 510

Furthermore, sox8 levels consistently decreased during cell culture development. Schmidt et al., 511

(2003) proposed that Sox8 inhibits myoblast differentiation, and in agreement with this idea the 512

decrease of sox8 in our culture coincided with the differentiation process, together with a decrease 513

of PCNA and an increase of mhc, desmin, myogenin and mrf4 expression. 514

In summary, in the present work we have obtained total or partial sequences of gilthead sea bream 515

Myf5, Mrf4, Pax7 and Sox8, which were highly expressed in muscle and highly conserved between 516

species. Highest levels of expression for myf5, myod2, pax7 and sox8 were found at the beginning 517

of the culture, suggesting that they have major roles during myoblast proliferation. On the other 518

hand, myogenin and mrf4 were up-regulated during the later stages of culture development 519

concomitant with the formation of myotubes. The decrease of igf1, myf5 and myod2 following 30 520

days of fasting suggested a reduction in myoblast activation leading to reduced muscle growth, 521

which is supported by an observed increase in mstn1 expression. 522

523

17

Acknowledgments. The authors would like to thank Carlos Mazorra from Tinamenor S.L. 524

(Cantabria, Spain) for the sea bream used in this study, Dr J.J. Borrego from the University of 525

Málaga (Spain) for kindly providing the SAF-1 cell line, Dr Joan Sánchez-Gurmaches for his help 526

in cell cultures preparation, Parnaz Nik for her help with the western blots and Esmail Lutfi and 527

Emilio Vélez for performing the immunofluorescence analyses. This work was partially supported 528

by the following financing: FPU grant (AP2004-2004) from the Spanish “Ministerio de Educación 529

y Ciencia” (MEC); AGL2009-12427 from the Spanish “Ministerio de Ciencia e Innovación” 530

(MCINN), “Acciones Integradas” HF2008-0019 from the MCINN; and the “Generalitat de 531

Catalunya” through the “Xarxa de Referència d’R+D+I en Aqüicultura” and SGR2009-00402; and 532

by funds from the European Union through the project LIFECYCLE (EU-FP7 222719). 533

534

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687 Legends 688

689

21

Figure 1. Expression of PCNA and mhc in gilthead sea bream cultured muscle cells. 690

Representative images of sea bream muscle cells at different days in culture (2, 4, 8 and 12) 691

immunostained for PCNA showing the different stages of development from myoblasts to 692

myotubes and the amount of PCNA-positive cells at each stage. The transversal bar represents 693

schematically the progression of the culture. PCNA (cracked line) and mhc (full black line) 694

expression was evaluated in myoblasts from day 1 to day 12 of culture by qPCR. Results present 695

mean ± SEM of 3 independent experiments. Values not sharing a common letter are significantly 696

different (p<0.05). (a.u.): arbitrary units. 697

698

Figure 2. Expression of desmin in gilthead sea bream cultured muscle cells. Desmin expression 699

in myoblasts at different days of culture development: (A) 1% (m/v) agarose gel showing the single 700

232bp product amplified by RT-PCR. Fibroblast-like cells (SAF-1 cell line) were used as a negative 701

control. (B) Representative SDS-PAGE showing the single band of 50-55 kDa of Desmin (n=3). 702

(C) Percentage of Desmin-positive cells after incubation with an anti-desmin monoclonal antibody 703

and FACS analysis. Results represent mean ± SEM of 3 independent experiments. Values not 704

sharing a common letter are significantly different (p<0.05). 705

706

Figure 3. Expression of pax7 and sox8 in gilthead sea bream cultured muscle cells. Expression 707

of (A) pax7 and (B) sox8 was determined in myoblast culture from day 1 to 12 by qPCR. Results 708

are presented as mean ± SEM of 4 independent experiments. Values not sharing common letter are 709

significantly different (p<0.05). (a.u.): arbitrary units. 710

711

Figure 4. Expression of MRFs in gilthead sea bream cultured muscle cells. MRFs expression 712

was evaluated in myoblasts from day 1 to day 12 of culture by qPCR. (A) myf5, (B) myod2, (C) 713

myogenin and (D) mrf4. Results present the mean ± SEM of 4 independent experiments. Values not 714

sharing a common letter are significantly different (p<0.05). (a.u.): arbitrary units. 715

716

Figure 5. Protein expression of Myod and Myogenin in gilthead sea bream cultured muscle 717

cells. Representative images and histogram from 3 independent experiments of Myod and 718

Myogenin expression in myoblast cells in culture at days 4 and 8 detected by immunofluorescence 719

using a secondary Alexa-568 antibody. Nuclei were counterstaining with Hoechst dye. The scale 720

bar represents 100µm. Asterisk shows significant differences (p<0.05) between culture days. (a.u.): 721

arbitrary units. 722

22

723

Figure 6. Myogenic-related factors expression in response to fasting in gilthead sea bream fast 724

skeletal muscle. The expression of myod2, myf5, myogenin, mrf4, pax7, sox8, mstn1, igf1 and igf2 725

was analysed in control fish continuously fed (grey bars) or fasted fish (black bars) for 30 days. 726

Inset: Immunohistochemical detection of Myod-positive cells in fast skeletal muscle from fish 727

continuously fed or fasted for 30 days. Results are presented as mean ± SEM (n=8) fish. Asterisks 728

show significant differences (p<0.05) between conditions. (a.u.): arbitrary units. 729

730

Figure 7. AKT, mTOR and c-Met protein expression in response to fasting in gilthead sea 731

bream fast skeletal muscle. SDS-PAGE and quantification of the expression of (A) p-AKT/AKT, 732

(B) p-mTOR/mTOR and (C) c-Met/Actin analysed in control fish continuously fed (grey bars) or 733

fasted fish (black bars) for 30 days. Pictures show bands of two representative animals. Bar chart 734

results are presented as mean ± SEM (n=5) fish. Asterisks show significant differences (p<0.05) 735

between conditions. 736 737

Legends to supplementary files 738

Supplementary file 1. Sparus aurata Pax7, Sox8, MRF4 and Myf5 nucleotide sequences. 739 740 Supplementary file 2. Sparus aurata Pax7, Sox8, MRF4 and Myf5 amino acid sequences and 741 conserved domains. Nucleotides sequences were translated using virtual ribosome 742 (http://www.cbs.dtu.dk/services/VirtualRibosome/) and conserved domains were identified using 743 NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/). 744 745 Supplementary file 3. Sparus aurata Pax7, Sox8, Myf5 and MRF4 matrix of identity and 746 alignment compared with other teleost sequences, Gallus, Xenopus and Mus. Pax7 identity was 747 compared against Salmo salar (NP_001117167), Salvelinus alpinus (CAG25718), Sternopygus 748 macrurus (ACC86106), Danio rerio (NP_571400), Xenopus laevis (NP_001088995), Gallus gallus 749 (NP_990396), Mus musculus (NP_055169) and Orechromis niloticus (XP_003459869). Sox 8 was 750 compared against Danio rerio (NP_001020636), Takifugu rubripes (AAQ18505), Oryzias latipes 751 (NP_001158342), Salmo salar (NP_001117071), Xenopus laevis (NP_001083964), Gallus gallus 752 (NP_990062) and Mus musculus (NP_035577). Myf5 was compared against Micropterus salmoides 753 (ACB45872), Lateolabrax japonicus (AAZ76912), Epinephelus coiodes (ADJ95347), Takifugu 754 rubripes (NP_001027942), Salmo salar (NP_01117116), Oncorhynchus mykiss (NP_001118001), 755 Danio rerio (CAH68981), Gallus gallus (NP_001025534), Xenopus laevis (CAE18109) and Mus 756

23

musculus (NP_032682). MRF4 was compared against Takifugu rubripes (CAC39207), Salmo salar 757 (NP_001117079), Sternopygus macrurus (AAY53906), Danio rerio (NP_001003982), Xenopous 758 laevis (NP_001081477) and Mus musculus (NP_032683). Identity was calculated after ClustalW 759 aligment using JalView Pairwise aligment tool. 760 761 Supplementary file 4. Sparus aurata Myf5 and MRF4 tissue distribution in spleen, kidney, liver, 762 white muscle, red muscle, heart, brain and adipose tissue. Analysis was done by qPCR using tissue 763 from three different animals. Nested graphs represents expression of the tissue in scale excluding 764 white and red muscle. 765 766 Supplementary file 5. Sparus aurata Pax7 and Sox8 tissue distribution in spleen, kidney, liver, 767 white muscle, red muscle, heart, brain and adipose tissue. Analysis was done by qPCR using tissue 768 from three different animals. Nested graph in Pax7 represents expression of the tissue in scale 769 excluding brain, white and red muscle. 770

Figure 1

Proliferation Differentiation

Day 4 Day 8 Day 12 Day 2

100 µm

Figure 2

A

C

2 3 4 6 7 8 9 6 0

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d1 d4 d9 -CSAF-1

myocytes

d1 d4 d9 -CSAF-1

myocytes

d1 d4 d9 SAF-

1 myocytes

B

d5 d8 d11

myocytes

Figure 3

Figure 4

Figure 5

Day 4

Day 8

Day 4

Day 8

Myod

Myogenin

GAR 568 Hoechst Merge

*

Figure 6

Figure 7

Akt-p

0

20

40

60

80

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Akt

-p/A

KT

rela

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essi

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Fed Fasted

*

A

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50

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et/a

ctin

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Fed Fasted

*

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Fed Fasted

mTOR-p

mTOR

Fed Fasted

c-Met

Actin

Fed Fasted

Table1. Primers used for amplification of pax7, myf5, mrf4 and sox8. Primers were designed comparing known sequences using the BioEdit software and selecting the most conservative regions. Viability and Tm of primers were evaluated using NetPrimer program.

Gene Forward (5’-3’) Reverse (5’-3’)

Pax7 ATGKCKWCTYTRCMVGGAMC TCAGTAGGCCTGTCCYGTCTCC

CCMTCCWCCATGCAYCAGGG GGGCCATCTCCACKATCTTGT

GRCACAAGATMGTGGAGATGGC GCCTSCCGTTGATGAASAC

GGWRTCRGAGTAGCTGGAGAA

Myf5 ATGGACGTCTTCTCGACATCC TCACAGGACGTGGTAGACGG

Mrf4 ATGATGGACCTTTTTGAGACC AGGASAGRCRSAGGAGGCT

Sox8 ATGTTRAAAATGACMGARGAGCA YYAAGGYCGRGACAGVGTGGTRT

Sox8 RACE TCTTACGCTTCCTCTTACGG

GCAGGATCTGAAGCACGAGG

Table 2. Quantitative PCR primer sequences, PCR efficiencies, and correlation coefficients of standard curves for genes.

Gene Forward (5’-3’) Reverse (5’-3’) Product (bp)

Tm (Cº)

E (%)

R2 Annealing T (ºC)

Accession No.

Pax7 ATGAACACTGTCGGCAACG AGGCTGTCCACACTCTTGATG 189bp 87.5 98 0.99 64 JN034418

Myf5 CTACGAGAGCAGGTGGAGAACT TGTCTTATCGCCCAAAGTGTC 185bp 85 90 0.98 64 JN034420

Mrf4 CATCCCACAGCTTTAAAGGCA GAGGACGCCGAAGATTCACT 150bp 84.5 94 0.99 60 JN034421

Sox8 TCTCAGCTCCTCAAGGGATACG GCCCAAACCATGAACGCAT 116bp 88 96 0.98 64 JN034419

Mhc AGCAGATCAAGAGGAACAGCC GACTCAGAAGCCTGGCGATT 166bp 87 101 0.98 58 NM131404

Myogenin CAGAGGACTGCCCAAGGTGGAG CAGGTGCTGCCCGAACTGGGCTC 182bp 83.3 91 0.97 68 EF462191.1

Myod2 CACTACAGCGGGGATTCAGAC CGTTTGCTTCTCCTGGACTC 149bp 83 97 0.99 60 AF478569

Igf1 TGTCTAGCGCTCTTTCCTTTCA AGAGGGTGTGGCTACAGGAGATAC 83bp 79 101 0.99 60 AY996779

Mstn1 GTACGACGTGCTGGGAGACG CGTACGATTCGATTCGTTG 201bp 84.8 99 0.99 60 AF258448.1

Igf2 TGGGATCGTAGAGGAGTGTTGT CTGTAGAGAGGTGGCCGACA 108bp 82 98 0.98 60 AY996778

PCNA TGTTTGAGGCACGTCTGGTT TGGCTAGGTTTCTGTCGC 201bp 87 98 0.99 58 AY550963.1

EF1α CTTCAACGCTCAGGTCATCAT GCACAGCGAAACGACCAAGGGGA 152bp 85.6 97 0.99 60 AF184170

Tm, melting temperature; E, PCR efficiency. Genes are identified as follows: Paired box 7 transcription factor (Pax7), myogenic factor 5 (Myf5), myogenic regulator factor 4 (Mrf4), SRY sex determination factor Y-8 (Sox8), myosing heavy chain (Mhc), myoblast determination factor 2 (Myod2), insulin-like growth factor 1 (Igf1), myostatin 1 (Mstn1), insulin-like growth factor 2 (Igf2), proliferating cell nuclear antigen (PCNA) and elongation factor 1-alpha (EF1α).  

Sparus aurata Pax7

ATGGCGACTCTGCCCGGACCTGTACCGCGGATGATGCGCCCGGCTCTCGGACAGAACTACCCCCGGACTGGATTCCCTCTGGAAGTGTCCACTCCTCTGGGCCAGGGACGGGTCAACCAGCTCGGAGGTGTCTTTATCAACGGCCGGCCGCTGCCCAACCACATCCGACACAAGATAGTGGAGATGGCCCACCACGGGATCCGGCCCTGCGTCATCTCCCGGCAGCTGCGGGTCTCCCACGGCTGCGTCTCCAAGATCCTCTGCCGCTACCAGGAGACCGGCTCCATCCGGCCCGGAGCGATAGGAGGCAGCAAGCCCAGGCAGGTAGCCACGCCGGACGTGGAGAAGCGGATCGAGGAGTACAAGCGGGACAACCCCGGCATGTTCAGCTGGGAGATCCGGGATAAGCTGCTGAAGGACGGCGTGTGTGACAGAAGCACAGTCCCTTCAGTCAGTTCGATCAGCCGTGTACTTCGGGCTCGGTTTGGAAAAAAGGACGATGAGGAGGAGTGTGATAAAAAGGATGAAGACGGAGAGAAGAAAACCAAACACAGCATCGACGGCATCCTCGGCGACAAAGGCAGTCGGATGAACGAAGTGTCAGATGTGGAGTCAGAGCCTGACCTCCCCCTCAAGAGGAAGCAGCGCAGGAGTCGGACCACATTCACAGCAGAGCAGCTGGAGGAGTTGGAGAAGGCTTTTGAAAGAACCCACTACCCCGACATCTACACAAGAGAAGAGCTGGCCCAGGGGACCAAACTCACTGAAGCCAGGGTCCAGGTGTGGTTCAGCAATCGAAGGGCCAGATGGCGGAAACAAGCAGGAGCCAATCAGCTTGCAGCATTTAACCACCTGTTGCCGGGTGGATTTCCTCCCACGGGGATGCCAACACTTCCTACATACCAGCTGCCAGATTCCAGCTACCCAACAAACACTCTGCCCCAAGATGGTAGTGGTACAGTTCACCGTCCCCAGCCGTTACCTCCATCCACCATGCACCAGGGAGGTCTGGGCGCTGACAGCGGCTCCGCTTATGGCCTCTCGTCCAATCGCCACAGCTTCTCCAGCTACTCCGATACTTTCATGAGCCCCTCAGCATCCAGCAACCACATGAACACTGTCGGCAACGGGCTCTCCCCACAGGTGATGAGTATCCTGAGCAACCCCAGCGCCGTACCCTCCCAGCCCCAGCACGACTTTTCCATTTCCCCCCTGCACAGCAGCCTGGAGAGCTCCAACCCCATCTCAGCCAGCTGCAGCCAGCGCTCGGACACCATCAAGAGTGTGGACAGCCTGGCCTCCTCCCAGTCATACTGCCCCCCCACCTACAGCGCCACCAGCTACAGCGTGGACCCCGTCACTGCGGGCTACCAGTACAGCCAGTACGGCCAGACTGCCGTCGACTACCTGGCCAAGAACGTGAGCTTGTCCACCCAGAGGAGGATGAAACTGGGAGACCACTCGGCCGTGCTGGGACTGCTGCAGGTGGAGACAGGACAGGCCTACTGA

Sparus aurata Myf5

ATGGACGTCTTCTCGACATCCAGGTCTACTACGACAGTGCGTGCCCCTCGCCTCCAGACAGCCTGGACTTCGGCCCCGGCATGGAGCTCGACGGCTCCGAGGAGGACGAGCACGTCAGGGTCCCCGGGGCCCCTCACCAGCCGGGACACTGCCTCCAGTGGGCTTGCAAGGCCTGCAAGCGCAAGTCCAACTTTGTGGACCGCAGGCGGGCCGCCACCATGCGCGAGCGCCGGCGACTGAAGAAGGTCAACCACGCTTTCGAGGCTCTGAGGCGCTGCACCTCGGCCAACCCCAGCCAGCGCCTGCCCAAGGTGGAGATCCTGCGCAACGCCATCCAGTACATCGAGAGCCTGCAGGACCTGCTACGAGAGCAGGTGGAGAACTACTACGGCCTTCCTGGAGAGAGCAGCTCGGAGCCCGGGAGCCCGCTGTCCAGCTGCTCCGACGGCATGGTTGACAGCAACAGTCCAGTGTGGCAACATCTGAATACAAACTACAGCAGCAGTTATTCGTATATGAAGAACGACACTTTGGGCGATAAGACAGCCGGAGCCTCCAGTCTCGAGTGTCTCTCCAGCATCGTGGACCGCCTGTCCTCGGTGGAGACCAGCTGCGGACAGCCGCCTCTGAGAGACTCGGCCACCTTCTCCCCCGGCAGCTCCGACTCGCAGCCCTGCACGCCGGAGAGCCCCGGATCCCGGCCCGTCTACCACGTCCTGTGA

Sparus aurata MRF4

ATGATGGACCTTTTTGAGACCAACACTTATCTTTTTAATGATTTGCGCTATTTGGAAGAAGGAGATCATGGACAACTGCAGCACCTGGACATGTCGGGGGTTTCCCCTCTGTACAACGGCACCGACAGCCCGCTGTCACCGGGCCAGGATGATAATGTTCCGTCCGAGACCGGCGGGGAGAGCAGCGGGGAGGAGCACGTCCTTGCGCCCCCGGGTCTCCGTGCGCACTGCGAGGGCCAGTGCCTCATGTGGGCCTGCAAGATCTGCAAGAGGAAGTCGGCGCCCACGGACCGACGCAAGGCCGCCACGCTCAGGGAGAGGAGGAGGCTCAAGAAGATCAACGAGGCCTTCGACGCGCTGAAGAGGAAGACCGTGCCCAACCCCAACCAGCGGCTACCCAAGGTGGAGATTTTACGCAGCGCCATCAGCTACATCGAGCGGCTGCAGGAGCTGCTGCAGACGCTGGACGAGCAGGACAAAAACGGATCATCCCACAGCTTTAAAGGCAAAGAGCACACTGTGGCCAGTCATGAGTATCACTGGAAAAAGGCCTCGGAGAGCTGGTCGACCTCTGCTGATCATATCACTGCAGCAATGACAAACCAGAGAGACGGAACAAGTGAATCTTCGGCGTCCTCCAGCCTCCTCCGTCTCTCCTAACTCGAGGTACCGTGCAGG

Sparus aurata Sox8

ATGTTAAAATCGACCGAGGAGCATGACAAGTGTGTCGGCGACCAGCCCTGCAGTCCATCCGGCACCAACAGCTCCATGTCCCAGGACGAGTCCGACTCGGACGCTCCGTCCTCGCCGACGGGCTCGGACGGCCATGGGTCCCTGCTCACCGGTCTGGGCAAGAAGCTGGACTCCGAGGACGACGACAGGTTCCCGGCTTGCATACGGGACGCCGTCTCTCAGGTCCTCAAGGGATACGACTGGTCCTTGGTGCCCATGCCCGTGAGGGGGAA

Suppl. 1

CGGATCCCTAAAGAATAAACCGCACGTCAAGAGACCCATGAATGCGTTCATGGTTTGGGCGCAGGCTGCCCGCAGAAAGCTCGCGGATCAGTATCCACACTTGCACAACGCCGAGCTGAGCAAGACTCTGGGGAAGCTGTGGCGTTTGCTCTCAGAAAATGAGAAGAGGCCGTTTGTGGAGGAAGCAGAGAGGCTTCGGGTCCAGCACAAGAAAGATCACCCGGACTACAAGTACCAGCCCCGGCGACGGAAGAATGTGAAACCAGGCCAGAGCGACTCCGACTCGGGAGCAGAGCTGGCTCATCACATGTACAAAGCCGAGCCGGGGATGGGAGGACTGGCGGGGATCAGCGACGGACACCACCACCCTGAACATGCAGGGCAGCCTCATGGTCCCCCCACACCACCCACCACTCCCAAAACAGACCTGCACCACGGGGTGACGCAGGATCTGAAGCACGAGGGCCGCCGTCTTGTTGACGGTAGCGGGCAAAACATCGACTTCAGCAACGTCGACATCTCTGAGCTCAGCACTGATGTCATCAGCAACATGGAGACCTTTCGACGTGCACGAGTTCGACCAGTACCTCCCGCTCAACGGCCACGCCTCGGCCTCCTCCGCCCTGCCCTCAGACCACAGCCATGGGCAGCCTCCGGCGCCTGCCGGATCTTACGCTTCCTCTTACGGCCACGTGGGTGCCAACGGGCGGCGTGGAGCCGCAAGGGCAACATGTCCTCCACCTCTCCCTCAGCCGGCGAGGTGGGCCAGCACCGGCTCCACATCAAGACGGAGCAGCTGAGCCCGAGCCACTACAGCGAGCACTCCCACAGGTCCCCCTCACACTCCGATTACAGCTCCTACAGCAGCCAGGCTTGCGTCACCTCGGCCTCGTCGGCTGCCTCAGCCGCAGCGTCTTTCTCCAGCTCCCAGTGTGACTATACTGACATCCAGAGCTCCAACTATTACAACCCTTACTCCGGCTACCCCTCCAGCCTCTACCAGTACCCTTACTTCCACTCCTCCAGGCGGCCTTACAGCAGCCCCATCCTCAACAGTCTGTCCATGGCTCCTGCCCACAGCCCCACCGCCTCCAGCTGGGACCAGCCCGTCTACACCACGCTGTCTCGGCCATAAAAAGAGGACAAGGAAAAACGGCGTCAGTTCGCTCCCAGAGACTCACTCAGATCGATGCACTACTAATGAAGGAAGTTGAGGAGCAGGTGGAGAGGTGTGTTGTGTGTGTGAGGCCACCTGCGACCATCTGAACGTGAAACAAAAAAAAAAAAAAAAAAAAA

Sparus aurata Myf5

MELDGSEEDEHVRVPGAPHQPGHCLQWACKACKRKSNFVDRRRAATMRERRRLKKVNHAFEALRRCTSANPSQRLPKVEILRNAIQYIESLQDLLREQVENYYGLPGESSSEPGSPLSSCSDGMVDSNSPVWQHLNTNYSSSYSYMKNDTLGDKTAGASSLECLSSIVDRLSSVETSCGQPPLRDSATFSPGSSDSQPCTPESPGSRPVYHVL*

Sparus aurata MRF4

MMDLFETNTYLFNDLRYLEEGDHGQLQHLDMSGVSPLYNGTDSPLSPGQDDNVPSETGGE SSGEEHVLAPPGLRAHCEGQCLMWACKICKRKSAPTDRRKAATLRERRRLKKINEAFDAL KRKTVPNPNQRLPKVEILRSAISYIERLQELLQTLDEQDKNGSSHSFKGKEHTVASHEYH WKKASESWSTSADHITAAMTNQRDGTSESSASSSLLRLS*

Sparus aurata Pax7

MATLPGPVPRMMRPALGQNYPRTGFPLEVSTPLGQGRVNQLGGVFINGRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPRQVATPDVEKRIEEYKRDNPGMFSWEIRDKLLKDGVCDRSTVPSVSSISRVLRARFGKKDDEEECDKKDEDGEKKTKHSIDGILGDKGSRMNEVSDVESEPDLPLKRKQRRSRTTFTAEQLEELEKAFERTHYPDIYTREELAQGTKLTEARVQVWFSNRRARWRKQAGANQLAAFNHLLPGGFPPTGMPTLPTYQLPDSSYPTNTLPQDGSGTVHRPQPLPPSTMHQGGLGADSGSAYGLSSNRHSFSSYSDTFMSPSASSNHMNTVGNGLSPQVMSILSNPSAVPSQPQHDFSISPLHSSLESSNPISASCSQRSDTIKSVDSLASSQSYCPPTYSATSYSVDPVTAGYQYSQYGQTAVDYLAKNVSLSTQRRMKLGDHSAVLGLLQVETGQAY*

Sparus aurata Sox8

Suppl. 2

MLKSTEEHDKCVGDQPCSPSGTNSSMSQDESDSDAPSSPTGSDGHGSLLTGLGKKLDSEDDDRFPACIRDAVSQVLKGYDWSLVPMPVRGNGSLKNKPHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSENEKRPFVEEAERLRVQHKKDHPDYKYQPRRRKNVKPGQSDSDSGAELAHHMYKAEPGMGGLAGISDGHHHPEHAGQPHGPPTPPTTPKTDLHHGVTQDLKHEGRRLVDGSGQNIDFSNVDISELSTDVISNMETFRRARVRPVPPAQRPRLGLLRPALRPQPWAASGACRILRFLLRPRGCQRAAWSRKGNMSSTSPSAGEVGQHRLHIKTEQLSPSHYSEHSHRSPSHSDYSSYSSQACVTSASSAASAAASFSSSQCDYTDIQSSNYYNPYSGYPSSLYQYPYFHSSRRPYSSPILNSLSMAPAHSPTASSWDQPVYTTLSRP*

MRF4

Sparus Takifugu Salmo Sternopygus Danio Xenopus Mus

Sparus 100

Takifugu 84 100

Salmo 79 78.5 100

Sternopygus 75.9 73.8 82.9 100

Danio 78.2 77.5 83.3 83 100

Xenopus 61.3 62.4 66.5 64.4 68.2 100

Mus 63.5 66.3 68.7 68.3 66.5 78.3 100

MRF4 Sparus_aurata -MMDLFETNTYLFNDLRYLEEGDHGQLQHLDMSGVSPLYNGTDSPLSPGQDDNVPSETGG Takifugu_rubrip -MMDLFETNTYLFNDLGYLEEGDHGPLQHLDMSGVSPLYNGNDRPLSPGQ-DNVPSETGG Salmo_salar -MMDLFETHTYFFNDLRYL-EGDHGPLQHLDMAGVSPLYHGNDSPLSPGG-D--PSETGC Sternopygus_mac -MMDLFETNTYFFNDLRYI-EGDHGTLQHLDIPEVSPLYQGNDSPPSPGQ-DQAPTETGC Danio_rerio -MMDLFETNAYFFNDLRYL-EGDHGT---LDMPGVSPLYEGNDSPLSPGQ-DPVPSETGC Xenopus_laevis -MMDLFETNSYFF----YL-DGDNGAFQQLGVADGSPVYPGSEGTLSPCR-DQLPVDAGS Mus_musculus MMMDLFETGSYFF----YL-DGENVTLQPLEVAEGSPLYPGSDGTLSPCQ-DQMPQEAGS Gallus_gallus MMMDLFETGSYFF----YL-DGENGALQQLEMAEGSPLYPGSDGTLSPCQ-DQLPPEAGS

******* :*:* *: :*:: * :. **:* *.: . ** * * ::*

Sparus_aurata ESSGEEHVLAPPGLR-AHCEGQCLMWACKICKRKSAPTDRRKAATLRERRRLKKINEAFD Takifugu_rubrip ESSGDEHVLAPPGLR-SHCEGQCLMWACKICKRKSAPTDRRKAATLRERRRLKKINEAFD Salmo_salar DSSGEEHVLAPPGLQ-PHCEGQCLIWACKVCKRKSAPTDRRKAATLRERRRLKRINEAFD Sternopygus_mac DSSGEEHVLAPPGLQ-AHCEGQCLIWACKICKRKSAPTDRRKAATLRERRRLKKINEAFD Danio_rerio ESSGEEHVLAPPGLQ-AHCEGQCLMWACKICKRKSAPTDRRKAATLRERRRLKKINEAFD Xenopus_laevis DRSEEEHVLAPPGLQ-PHCPGQCLIWACKTCKRKSAPTDRRKAATLRERRRLKKINEAFE Mus_musculus DSSGEEHVLAPPGLQPPHCPGQCLIWACKTCKRKSAPTDRRKAATLRERRRLKKINEAFE Gallus_gallus DSSGEEHVLAPPGLQPPHCPGQCLIWACKTCKRKSAPTDRRKAATLRERRRLKKINEAFE

: * :*********: .** ****:**** ***********************:*****:

Sparus_aurata ALKRKTVPNPNQRLPKVEILRSAISYIERLQELLQTLDEQDK--N--GSSHSFKGKEHTV Takifugu_rubrip ALKRKTVANPNQRLPKVEILRSAISYIERLQDLLQTLDEQERSQS--GASDTRNDKEQNR Salmo_salar ALKKKTVPNPNQRLPKVEILRSAINYIEQLQDLLHTLDEQENPPQ-----NGYNVKEHHA Sternopygus_mac ALKKKTVPNPNQRLPKVEILRSAINYIEKLQDLLHTLDEQAKLPD--NNTYNCSAKDHSV Danio_rerio ALKKKTVPNPNQRLPKVEILRSAINYIEKLQDLLHSLDEQEQSND--TDPYTYNLKENHV Xenopus_laevis ALKRRTVANPNQRLPKVEILRSAINYIERLQDLLHSLDQQDKPQKADEEPFSYNSKEAPV Mus_musculus ALKRRTVANPNQRLPKVEILRSAISYIERLQDLLHRLDQQEKMQELGVDPYSYKPKQEIL Gallus_gallus ALKRRTVANPNQRLPKVEILRSAISYIERLQDLLHRLDQQDKMQEVAADPFSFSPKQGNV

***::**.****************.***:**:**: **:* . . . *:

Sparus_aurata ASH-EYHWKKASE-SWSTSADHITAA-MTNQRD-GT-SESSASSSLLRLSSIVNXINMTK Takifugu_rubrip PSGVDYRWKKASN-TWPTSADHSA---IINQRD-GN-CESSATSSLLCLSSIVSSISDDK Salmo_salar SNK-EYHWKKNCQ-NWQTSADHSNAP-MTNQRE-GF-TESSASTSLLRLSSIVDSISSEE

Suppl. 3

Sternopygus_mac GNG-EYHWKKTCQ-NWQGNAEHSKLPQLPYQRE-GSAAESLASSSLRRLSSIVDSISTEE Danio_rerio TPS-EYHWKKTCQ-SWQENPDHSSSQ-MAGHRE-GAVLESSESSSLRRLSSIVDSISTEE Xenopus_laevis QSE-DF--LSTCHPEWHHIPDHSRMP-NLNIKEEGS-LQENSSSSLQCLSSIVDSISSDE Mus_musculus EGA-DF--LRTCSPQWPSVSDHSRGL-VITAKEGGANVDASASSSLQRLSSIVDSISSEE Gallus_gallus PGS-DF--LSTCGSDWHSASDHSRAL-GGSPKAGGSMVESSASSSLRCLSSIVDSISSDE

:: . * .:* : * : ::** *****. *. :

Sparus_aurata KSTF---GGIAYRKLKL Takifugu_rubrip TNLR---QGVQEN---- Salmo_salar KPT--CNEEVSEK---- Sternopygus_mac TKAH-RPEESTE----- Danio_rerio PKAR-CPSQISEK---- Xenopus_laevis PRHPCTIQELVEN---- Mus_musculus RKLP-SVEEVVEK---- Gallus_gallus PKLP-GAEEAVEK----

Pax7

Sparus Salmo Salvenius Sternopygus Danio Xenopus Gallus Mus Oreochromis

Sparus 100

Salmo 91 100

Salvenius 93.2 95.6 100

Sternopygus 92.7 92 94 100

Danio 93.8 93 95 96.8 100

Xenopus 85.4 84.3 86.5 85.3 86.3 100

Gallus 84.5 82 84 84.3 84.5 86 100

Mus 88.2 85.6 87.4 88.2 88.3 88.7 91.5 100

Oreochromis 89.4 90.9 88.5 90.4 90.4 84.6 85.6 84.6 100

PAX7 Sparus_aurata MATLPGPVPRMMRPALGQNYPRTGFPLE-------------VSTPLGQGRVNQLGGVFIN

Salmo_salar MATLPGTVPRMMRPAPGQNYPRTGFPLEGQYAGPEYVYCGTVSTPLGQGRVNQLGGVFIN Salvenius_alpin MATLPGTVPRMMRTAPGQNYPRTGFPLE-------------VSTPLGQGRVNQLGGVFIN Sternopygus_mac MATLPGTVPRMVRPAPGQNYPRTGFPLE-------------VSTPLGQGRVNQLGGVFIN Danio_rerio MATLPGTVPRMMRPAPGQNYPRTGFPLE-------------VSTPLGQGRVNQLGGVFIN Xenopus_laevis MAALPGTIPRMMRPAPGQNYPRTGFPLE-------------VSTPLGQGRVNQLGGVFIN Gallus_gallus MAALPGTVPRMMRPAPGQNYPRTGFPLE-------------VSTPLGQGRVNQLGGVFIN Mus_musculus MAALPGAVPRMMRPGPGQNYPRTGFPLE-------------VSTPLGQGRVNQLGGVFIN Takifugu_rubrip MSSLPGTIPRMMRPAPGQNYPRTGFPLEAHTLGPQSQHLNIVSTPLGQGRVNQLGGVFIN Oryzias_latipes MSSLQGPEPRMMRAAPGHNYPRAGFPLE-------------VSTPLGQGRVNQLGGVFIN

*::* *. ***:*.. *:****:***** *******************

Sparus_aurata GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Salmo_salar GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Salvenius_alpin GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Sternopygus_mac GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Danio_rerio GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Xenopus_laevis GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Gallus_gallus GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Mus_musculus GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Takifugu_rubrip GRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR Oryzias_latipes GRPLPNHIRHKIVEMAHHGVRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPR

*******************:****************************************

Sparus_aurata QVATPDVEKRIEEYKRDNPGMFSWEIRDKLLKDGVCDRSTVP--------SVSSISRVLR Salmo_salar QVATPDVEKRIEEYKRENPGMFSWEIRDKLLKDGVCDRSTVPSGE---ASSVSSISRVLR Salvenius_alpin QVSTPDVEKRIEEYKRENPGMFSWEIRDKLLKDGVCDRSTVPSGE---A-SVSSISRVLR Sternopygus_mac QVATPDVEKRIEEYKRENPGMFSWEIRDKLLKDGVCDRSTVPSGE---ASSVSSISRVLR Danio_rerio QVATPDVEKRIEEYKRENPGMFSWEIRDKLLKDGVCDRGTVPSGE---ASSVSSISRVLR Xenopus_laevis -VATPDVEKKIEEYKRENPGMFSWEIRDRLLKDGHCDRSSVPSG------LVSSISRVLR Gallus_gallus QVATPDVEKKIEEYKRENPGMFSWEIRDRLLKDGHCDRSTVP--------SVSSISRVLR Mus_musculus QVATPDVEKKIEEYKRENPGMFSWEIRDRLLKDGHCDRSTVP--------SVSSISRVLR Takifugu_rubrip QVATPDVEKRIEEYKRDNPGMFSWEIRDKLLKDGVCDRSTVPSGE---ASSVSSISRVLR Oryzias_latipes QVASLDVEKRIEEYRRDNPGMFSWEIRDRLLKDGVCERSTAPSGALMPASELSSISRVLR

*:: ****:****:*:***********:***** *:*.:.* :********

Sparus_aurata ARFGKKDDEEECDKKDEDGEKKTKHSIDGILGDKGSRMNEVSDVESEP-DLPLKRKQRRS Salmo_salar ARFGKKDDDDDSDKKDEDGEKKTKHSIDGILGDKGNRTDEGSDVESEP-DLPLKRKQRRS Salvenius_alpin ARFGKKDDDDDSDKKDEDGEKKTKHSIDGILGDK----DEGSDVESEP-DLPLKRKQRRS Sternopygus_mac ARFGKKDDDDECDKKDEDGEKKTKHSIDGILGDKGHRTDEGSDVESEP-DLPLKRKQRRS Danio_rerio ARFGKKDDDDECDKKDEDGEKKTKHSIDGILGDKGNRTDEGSDVESEP-DLPLKRKQRRS Xenopus_laevis IKFGKKEEDDDCDKKEEDGEKKAKHSIDGILGDKGNRIDEGSDVESEP-DLPLKRKQRRS Gallus_gallus IKFGKKEEEEDCDKKEEDGEKKAKHSIDGILGDKGNRLDEGSDVESEP-DLPLKRKQRRS Mus_musculus IKFGKKEDDEEGDKKEEDGEKKAKHSIDGILGDKGNRLDEGSDVESEP-DLPLKRKQRRS Takifugu_rubrip ARFGKKDDEEECDKKDEDGEKKTKHSIDGILGDKA----PGSSADGGPSDLVIKERMRGR Oryzias_latipes ARFGSKFEESDFD-----------HSIDGILAD---------------------------

:**.* ::.: * *******.*

Sparus_aurata RTTFTAEQLEELEKAFERTHYPDIYTREELAQGTKLTEARVQVWFSNRRARWRKQAGANQ Salmo_salar RTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQ Salvenius_alpin RTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQ Sternopygus_mac RTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKRAGANQ

Danio_rerio RTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQ Xenopus_laevis RTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQ Gallus_gallus RTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQ Mus_musculus RTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQ Takifugu_rubrip RVS--------------------------------------------------------- Oryzias_latipes ------------------------------------------------------------

Sparus_aurata LAAFNHLLPGGFPPTGMPTLPTYQLPDSSYPTNTLPQDGSGTVHRPQPLPPSTMHQGGL- Salmo_salar LAAFNHLLPGGFPPTGMPSLPTYQLPESSYPGTTLSQDGSSTLHRPQPLPPSSMHQGGL- Salvenius_alpin LAAFNHLLPGGFPPTGMPSLPTYQLPECSYPGTTLSQDGSSTLHRPQPLPPSSMHQGGL- Sternopygus_mac LAAFNHLLPGGFPPTGMPTLPTYQLPETTYPSTTLSQDGSSTLHRPQPLPPSSMHQGGL- Danio_rerio LAAFNHLLPGGFPPTGMPTLPTYQLPESTYPSTTLSQDGSSTLHRPQPLPPSSMHQGGL- Xenopus_laevis LAAFNHLLPGGFPPTGMPALPHYQLPDSSYSAASLGQDVGSTVHRPQPLPPSSMHQGGL- Gallus_gallus LAAFNHLLPGGFPPTGMPTLPPYQLPDSTYPTTTISQDGGSTVHRPQPLPPSTMHQGGL- Mus_musculus LAAFNHLLPGGFPPTGMPTLPPYQLPDSTYPTTTISQDGGSTVHRPQPLPPSTMHQGGLA Takifugu_rubrip ------------------------------------------------------------ Oryzias_latipes ------------------------------------------------------------

Sparus_aurata ----GADSGSAYGLSSNRHSFSSYSDTFMSPSASSNHMNTVGNGLSP------------- Salmo_salar -----SDGSSAYGLSSNRHSFSSYSDSFMSQSAASNHMNPVSNGLSP------------- Salvenius_alpin -----SDGSSPYGLSSNRHSFSSYSDTFMSQSAASNHMNPVSNGLSP------------- Sternopygus_mac ----SADTSSAYGLSSNRHTFSSYSDTFMSPSASSNHMNAVSNGLSP------------- Danio_rerio ----SADSGSAYGLSSNRHTFSSYSETFMSPTASSNHMNPVSNGLSP------------- Xenopus_laevis ---SAADTGSAYG---ARHSFSSYSDTFINPGGPSNHMNPVNNGLSP------------- Gallus_gallus AAAAAADSSSAYG---ARHSFSSYSDSFMNAAAPANHMNPVSNGLSPQKQGAQNKMQCSR Mus_musculus AAAAAADTSSAYG---ARHSFSSYSDSFMNPGAPSNHMNPVSNGLSP------------- Takifugu_rubrip ------------------------------------------------------------ Oryzias_latipes ------------------------------------------------------------

Sparus_aurata ---------QVMSILSNPSAVPSQPQHDFSISPLHSSLESSNPISASCSQRSDTIKSVDS Salmo_salar ---------QVMSILSNPNAVPSQSQHDFSISPLHGSLESSNPISASCSQRSDSIKGVDS Salvenius_alpin ---------QVMSILSNPNAVPSQSQHDFSISPLHGSLESSNPISASCSQRSDSIKGVDS Sternopygus_mac ---------QVMSILSNPSAVPSQPQHDFSISPLHGGLDTSNPISASCSQRSDPIKSADS Danio_rerio ---------QVMSILSNPSAVPSQPQHDFSISPLHGGLEASNPISASCSQRSDPIKSVDS Xenopus_laevis ---------QVMSILNSPGGVGPQSQSDFSISPLHGGLETSNSLSASCSQRSDSIKSVDS Gallus_gallus WNLTIALNNQVMSILSNPSGVPPQPQADFSISPLHGGLDTTNSISASCSQRSDSIKSVDS Mus_musculus ---------QVMSILSNPSAVPPQPQADFSISPLHGGLDSASSISASCSQRADSIKPGDS Takifugu_rubrip ------------------------------------------------------------ Oryzias_latipes ------------------------------------------------------------

Sparus_aurata LASSQSYCPPTYSATSYSVDPVTAGYQYSQYGQTAVDYLAKNVSLSTQRRMKLGD-HSAV Salmo_salar LASSQSYCPPTYSATSYSVDPVTAGYQYSQYGQTAVDYLAKNVSLSTQRRMKLGDTGSAV Salvenius_alpin LASSQSYCPPTYSATSYSVDPVTAGYQYSQYGQTAVDYLTKNVSLSTQRRMKLGD-HSAV Sternopygus_mac LASSQSYCPPTYSATSYSVDPVAAGYQYSQYGQSAVDYLAKNVSLSTQRRMKLGE-PSAV Danio_rerio LASTQSYCPPTYSATSYSVDPVTAGYQYSQYGQTAVDYLAKNVSLSTQRRMKLGD-HSAV Xenopus_laevis LSTSQSYCAPTYSSSGYSMDPV-ASYQYGQYGQTAVDYLAKNVSLSTQRRMKLGD-HSAV Gallus_gallus LPTSQSYCPPTYSTTSYSVDPV-AGYQYGQYGQTAVDYLTKNVSLSTQRRMKLGE-HSAV

Mus_musculus LPTSQSYCPPTYSTTGYSVDPV-AGYQYSQYGQTAVDYLAKNVSLSTQRRMKLGE-HSAV Takifugu_rubrip ------------------------------------------------------------ Oryzias_latipes ------------------------------------------------------------

Sparus_aurata LGLLQVETGQAY Salmo_salar LGLLQVETGQAY Salvenius_alpin LGLLQVETGQAY Sternopygus_mac LGLLQVETGQAY Danio_rerio LGLLQVETGQAY Xenopus_laevis LGLLPVETGQAY Gallus_gallus LGLLPVETGQAY Mus_musculus LGLLPVETGQAY Takifugu_rubrip ---------GAR Oryzias_latipes ------------

Sox8

Sparus Takifugu Oryzias Salmo Xenopus Gallus Mus

Sparus 100

Takifugu 84.2 100

Oryzias 83.5 87.8 100

Salmon 80.8 84.9 82.4 100

Xenopus 71.6 77.4 77.7 79.3 100

Gallus 74.5 80.1 79.6 80.3 87.4 100

Mus 69.9 75.8 75 74.7 81.2 82.2 100

SOX8 Gasterostreus_a MLKMTEEHDKCGSDQPCSPSGTNSSMSQ-DESDSDAPSSPTGSDGQGSL---------LA

Tetraodont_nigr MLKMTEEHDKCVSDQPCSPSGTNSSMSQ-DESDSDAPSSPTGSDGQGSL---------LT Takifugu_rubrip MLKMTEEHDKCVNDQPCSPSGTNSSMSQ-DESDSDAPSSPTGSDGQGSL---------LT Oryzias_latipes MLKMTEEHDKCVSEQPCSPSGTNSSMSQ-DESDSDAPSSPTGSDGHGSL---------LA Sparus_aurata MLKSTEEHDKCVGDQPCSPSGTNSSMSQ-DESDSDAPSSPTGSDGHGSL---------LT Salmo_salar MLKMTAEHDKSVSDPPCSPAGTTSSMSQ-DDSDSDAPSSPTGSDGHGSL---------LA Xenopus_laevis MLNMSSDQ-----EPPCSPTGTASSMSHVSDSDSDSPLSPAGSEGRGSH---------RP Gallus_gallus MLNMTEEHDKAL-EAPCSPAGTTSSMSH-VDSDSDSPLSPAGSEGLGCAPAPAPRPPGAA Mus_musculus MLDMSEAR----AQPPCSPSGTASSMSHVEDSDSDAPPSPAGSEGLGRA-----------

**. : : : ****:** ****: :****:* **:**:* *

Gasterostreus_a GLGKKLDSED--DDRFPACIRDAVSQVLKGYDWSLVPMPVR--GSGSLKNKPHVKRPMNA Tetraodont_nigr SLGRKVDSED--DERFPACIRDAVSQVLKGYDWSLVPMPVR--GNGSLKNKPHVKRPMNA Takifugu_rubrip SLGRKVDSED--DERFPACIRDAVSQVLKGYDWSLVPMPVR--GNGSLKNKPHVKRPMNA Oryzias_latipes GLSKKLDSED--DERFPACIRDAVSQVLKGYDWSLVPMPVR--GNGSLKSKPHVKRPMNA Sparus_aurata GLGKKLDSED--DDRFPACIRDAVSQVLKGYDWSLVPMPVR--GNGSLKNKPHVKRPMNA Salmo_salar GIGKKLDGED--DDRFPACIRDAVSQVLKGYDWSLVPMPVR--GNGSLKNKPHVKRPMNA Xenopus_laevis PGISKRDGEEPMDERFPACIRDAVSQVLKGYDWSLVPMPVR--GSGGLKAKPHVKRPMNA Gallus_gallus PLGAKVDAAE-VDERFPACIRDAVSQVLKGYDWSLVPMPVR--GNGSLKAKPHVKRPMNA Mus_musculus GGGGRGDTAEAADERFPACIRDAVSQVLKGYDWSLVPMPVRGGGGGTLKAKPHVKRPMNA

: * : *:*************************** *.* ** **********

Gasterostreus_a FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSEGEKRPFVDEAERLRVQHKKDHPD Tetraodont_nigr FMVWAQAARRKLADQYPHLHNAELSKALGKLWRLLSESEKRPFVDEAERLRIQHKKDHPD Takifugu_rubrip FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSESEKRPFVDEAERLRIQHKKDHPD Oryzias_latipes FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSESEKRPFVDEAERLRVQHKKDHPD Sparus_aurata FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSENEKRPFVEEAERLRVQHKKDHPD Salmo_salar FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSENEKRPFVEEAERLRVQHKKDHPD Xenopus_laevis FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSENEKRPFVEEAERLRVQHKKDHPD Gallus_gallus FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSENEKRPFVEEAERLRVQHKKDHPD Mus_musculus FMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLSESEKRPFVEEAERLRVQHKKDHPD

**************************:**********.******:******:********

Gasterostreus_a YKYQPRRRKNMKPGQSDSDSGAELAHH----MYKAEPGMGGLAGMTDGHHHPE-HTGQQH Tetraodont_nigr YKYQPRRRKNVKPGQSDSDSGAELAHH----MYKAEPGMGGMGGITDAHHHAE-HAGQPH Takifugu_rubrip YKYQPRRRKNAKPGQSDSDSGAELANH----MYKAEPGMGGLAGLTDAHHHAE-HAGQPH Oryzias_latipes YKYQPRRRKNVKPGQSDSESGAEMAHH----MYKSEPGMGGMAGMTEGHHHPD-HTGQPH Sparus_aurata YKYQPRRRKNVKPGQSDSDSGAELAHH----MYKAEPGMGGLAGISDGHHHPE-HAGQPH Salmo_salar YKYQPRRRKSVKPGQSDSDSGAELGNH----MYKAEPGL---------------LAGQPH Xenopus_laevis YKYQPRRRKSVKAGQSDSDSGAELGHHPGSQMYKSDSGMGSMG---ENHLHSE-HAGQNH Gallus_gallus YKYQPRRRKSVKAGQSDSDSGAELSHHAGTQIYKADSGLGGMA---DGHHHGE-HAGQPH Mus_musculus YKYQPRRRKSVKTGRSDSDSGTELGHHPGGPMYKADAVLG------EAHHHSDHHTGQTH

*********. *.*:***:**:*:.:* :**::. : :** *

Gasterostreus_a GPPTPPTTPKTDLH---HGLKQDLKHEGRRLVDSNRR----------------------- Tetraodont_nigr GPPTPPTTPKTDLH---HGAKQDLKHEGRRLIDSSRQNIDFSNVDISELSTDVISSMETF Takifugu_rubrip GPPTPPTTPKTDLH---HGMRQDLKHEGRRLVDSGRQNIDFSNVDISELSTDVISNMETF Oryzias_latipes GPPTPPTTPKTDLH---HGVKHDLKHEGRRLIDSSRQNIDFSNVDISELSTDVISNIETF Sparus_aurata GPPTPPTTPKTDLH---HGVTQDLKHEGRRLVDGSGQNIDFSNVDISELSTDVISNMETF Salmo_salar GPPTPPTTPKTDLH---HGGKQDMKHEGRRLLDSGRQNIDFSNVDISELSTDVISNMEAF Xenopus_laevis GPPTPPTTPKTDLH---HGGKQELKHEGRRMMDNGRQNIDFSNVDINELSSEVISNIEAF Gallus_gallus GPPTPPTTPKTDLH---HGSKQELKHEGRRLVESGRQNIDFSNVDISELSSEVINNMETF

Mus_musculus GPPTPPTTPKTDLHQASNGSKQELRLEGRRLVDSGRQNIDFSNVDISELSSEVISNMDTF ************** :* :::: ****:::.. :

Gasterostreus_a -------------HASGSSAVPSDHG-HGQAPVPGGSYASSY--GHAG----TNGSVWSR Tetraodont_nigr DVHEFDQYLPLNGHTSSSSSLPSDQP-----PAPVSSYASSY--GHAG----VNGPAWSR Takifugu_rubrip DVHEFDQYLPLNGHTSSSSGLPSDQP-----PAPVGSYASSY--GHAG----INGSAWSR Oryzias_latipes DVHEFDQYLPLNGHMSGSAALSSDHS-HGPVPVPGSSYSTSY--SHTG----SNGTSWSR Sparus_aurata RRARVRPVPPAQRPRL--GLLRPALR-PQPWAASGACRILRFLLRPRG----CQRAAWSR Salmo_salar DVHEFDQYLPLNGHASTAGAGVDHHGHHGLNPASGAGSYTSY--SHAT----ANGAVWSR Xenopus_laevis DVHEFDQYLPLNGH----GAIPADHG---QNTTAA-PYGPSY--PHAA--GATPAPVWSH Gallus_gallus DVHEFDQYLPLNGH----TAMPADHG-----PGAG-FYSTSY--SHSAAGAGGAGQVWTH Mus_musculus DVHEFDQYLPLNGH----SALPTEPS---QATASGSYGGASY--SHSGATGIGASPVWAH

. . : *::

Gasterostreus_a KGAMSSSAPSAGEL---GQHRLQIKTEQLSPSHYS--EHSHGSPPHSDYG-SYSSQACVT Tetraodont_nigr KGAMPSSSPSPGEV---GQHRLHIKTEQLSPSHYS--EHSHRSPPHSDYG-SYSSPACVT Takifugu_rubrip KGAMSSSSPSSGEV---GQHRLQIKTEQLSPSHYS--EHSHRSPPHSDYG-SYSSPACVT Oryzias_latipes KSTMSSSSPSTSEV---G--RLHIKTEQLSPSHYS--EHSHGSPSHSDYG-SYSSQACVT Sparus_aurata KGNMSSNSPSAGRG---GAARLHIKTEQLN------------------------------ Salmo_salar KSAAMS-ASSSTSSEAVPQHRAHIKTEQLSPSHYSGDSHSHSSPSHSDYSHSYTAQTCVT Xenopus_laevis KSSSTS-SSSSIES---GQQRPHIKTEQLSPSHYN--DQSQGSPTHSDYN-TYSAQACAT Gallus_gallus KSPASA-SPSSADS---GQQRPHIKTEQLSPSHYS--DQSHGSPAHSDYG-SYSTQACAT Mus_musculus KGAPSA-SASPTEA---GPLRPQIKTEQLSPSHYN--DQSHGSPGRADYG-SYSAQASVT

*. : :.*. * :******.

Gasterostreus_a SATSA--AAASFSSSQCDYTDLQSSNYYNPYSGYPSSLYQYPYFHSSRRPY-SSPILNSL Tetraodont_nigr SATSA--ASVPFSGSQCDYSDIQSSNYYNPYSSYSSSLYQYPYFHSSRRPY-GSPILNSL Takifugu_rubrip SATSA--ASVPFSGSQCDYSDIQSTNYYNPYSSYSSGLYQYPYFHSSRRPY-GSPILNSL Oryzias_latipes SATSAPSAPASFTSSQCDYTDLQSSSYYNRYTGYPSSIYQYPYLHST-RPY-SSTILNSL Sparus_aurata ------------------------------------------------------------ Salmo_salar SS----TAAASFSSSQCDYTDLQSSNYYNPYSGYPSSLYQYPYFHSSRRAYHGSPILNSL Xenopus_laevis TVSSA-TVPTAFPSSQCDYTDLPSSNYYNPYSGYPSSLYQYPYFHSSRRPY-ATPILNSL Gallus_gallus TASTA-TAAASFSSSQCDYTDLQSSNYYNPYPGYPSSIYQYPYFHSSRRPY-ATPILNGL Mus_musculus TAASA-TAASSFASAQCDYTDLQASNYYSPYPGYPPSLYQYPYFHSSRRPY-ASPLLNGL

Gasterostreus_a SMAPTHSPNASSWD----QPVYTTLSRP Tetraodont_nigr SMAPAHSPTGSGWD----QPVYTTLSRP Takifugu_rubrip SMAPAHSPTGSGWD----QPVYTTLSRP Oryzias_latipes SMAPTHSPTSTNWD----QPVYTTLSRP Sparus_aurata ---------------------------- Salmo_salar SIPPTHSPPTSKRGTSRCTPPYP--DRR Xenopus_laevis SIPPSHSPTS-NWD----QPVYTTLTRP Gallus_gallus SIPPAHSPTA-NWD----QPVYTTLTRP Mus_musculus SMPPAHSPSS-NWD----QPVYTTLTRP

Myf5

Sparus Micropterus Lateolabrax Epinephelus Takifugu Salmo Oncorhynchus Danio Gallus Xenopus Mus

Sparus 100

Micropterus 91.6 100

Lateolabrax 90.8 92.6 100

Epinephelus 86.4 89 85.9 100

Takifugu 85.9 88.2 86.4 85.5 100

Salmo 79 79 78.6 78.1 74.2 100

Oncorhynchus 82.5 82.5 82.1 81.2 77.2 96.5 100

Danio 74.6 75.9 76.4 75.5 71.6 78.6 80.8 100

Gallus 58.7 60 59.2 58.3 57.9 57.3 60 59.5 100

Xenopus 62.3 63.3 62.8 61.4 58.1 59.7 60.3 63.3 67.4 100

Mus 59.1 58.7 57.9 60.4 59.3 55.6 58.5 59.7 69.3 74.2 100

MYF5 Sparus_aurata MDV-----FSTSQVYYDSACPSPPDS---LDFGPG----------MGLDGSEEDEHVRVP Micropterus_sal MDV-----FSPSQVYYDRACASSPDS---LEFGPG----------VELDGSEEDEHVRVP Laterolabrax_ja MDV-----FSTSQVYYDSACASSPDS---LEFGPG----------MELAGSDEDEHVRVP Orzya_latipes MDV-----FSPSQAFYDRACALSPDD---LEFGSS----------LELTGSEEDEHVRVP Takifugu_rubrip MDV-----FSPSQVYYDTMCASSPDS---SEFGPG----------VELAGSGEDEHIRVP Salmo_salar MDV-----FSQSQIFYDSACASSPED---LDFGPG-----------ELDGSEEDEHVRVP Onchorhycchus_m MDV-----FSQSQVFYDSACASSPED---LDFGPR-----------ELDGSEEDEHVRVP Danio_rerio MDV-----FSTSQIFYDSTCASSPED---LEFGAS----------GELTGSEEDEHVRAP Gallus_gallus MEVMDSCQFSPSELFYDSSCLSSPEGEFPEDFEPRELPPFGAPAPTEPACPEEEEHVRAP Xenopus_laevis M------------------------------------SLYGAHK-KDLQESDEDEHVRAP Mus_musculus MDMTDGCQFSPSEYFYEGSCIPSPEDEFGDQFEPR-VAAFGAHK-AELQGSDDEEHVRAP

* . ::**:*.*

Sparus_aurata GAPHQPGHCLQWACKACKRKSNFVDRRRAATMRERRRLKKVNHAFEALRRCTSANPSQRL Micropterus_sal GAPHQPGHCLQWACKACKRKSNFVDRRRAATMRERRRLKKVNHAFEALRRCTSANPSQRL Laterolabrax_ja GAPHQPGHCLQWACKACKRKSNFVDRRRAATMRERRRLKKVNHAFEALRRCTSANPSQRL Orzya_latipes GAPHQPGHCLQWACKACKRKSSFVDRRRAATMRERRRLKKVNHAFEALRRCTSANPSQRL Takifugu_rubrip GAPHQPGHCLPWACKACKRKSNFVDRRRAATMRERRRLKKVNHAFDALRRCTSANSSQRL

Salmo_salar GTPHQAGHCLQWACKACKRKSSTVDRRRAATMRERRRLRKVNHGFEALRRCTSANHSQRL Onchorhycchus_m GTPHQAGHCLQWACKACKRKSSTVDRRRAATMRERRRLKKVNHGFEALRRCTSANPSQRL Danio_rerio GAPHQPGHCLQWACKACKRKASTVDRRRAATMRERRRLKKVNHAFEALRRCTSANPSQRL Gallus_gallus SGHHQAGHCLMWACKACKRKSTTMDRRKAATMRERRRLKKVNQAFETLKRCTTANPNQRL Xenopus_laevis IGHHQAGNCLMWACKACKRKSSTMDRRKAATMRERRRLKKVNQAFETLKRCTTTNPNQRL Mus_musculus TGHHQAGHCLMWACKACKRKSTTMDRRKAATMRERRRLKKVNQAFETLKRCTTTNPNQRL

**.*:** *********:. :***:**********:***:.*::*:***::* .***

Sparus_aurata PKVEILRNAIQYIESLQDLLREQVENYYGLPGESSSEPGSPLSSCSDGMVDSNSP-VWQH Micropterus_sal PKVEILRNAIHYIESLQDLLREQVENYYCLPGESSSEPGSPLSSCSDGMADSNSP-VWQH Laterolabrax_ja PKVEILRNAIQYIESLQDLLREQVENYYCLPGESSSEPGSPLSSCSDGMTDSNSS-LWQQ Orzya_latipes PKVEILRNAIQYIESLQELLREQVENFYGLPGESSSEPGSPLSSCSDGMAGGSSP-VWQQ Takifugu_rubrip PKVEILRNAIQYIESLQELLREQVESYYGLPGESGSEPGSPLSNCSDGPADSNSP-VWQQ Salmo_salar PKVEILRNAIQYIESLQELLHEHVENYYGLPGESSSEPGSPSSSRSDSMVDCNIPVVWPQ Onchorhycchus_m PKVEILRNAIQYIESLQELLHEHVENYYGLPGESSSEPGSPSSSCSDSMVDCNSPVVWPQ Danio_rerio PKVEILRNAIQYIESLQELLREQVENYYSLPMESSSEPASPSSSCSESMVDCNSP-VWPQ Gallus_gallus PKVEILRNAIRYIESLQELLREQVENYYHLPGQSCSEPTSPSSSCSDVMADSRSP-VWPA Xenopus_laevis PKVEILRNAIKYIESLQDLLQEQVENYYSLPGQSCTEPGSPTSSCSDGMTDCSSP-QWSG Mus_musculus PKVEILRNAIRYIESLQELLREQVENYYSLPGQSCSEPTSPTSNCSDGMPECNSP-VWSR

**********:******:**:*:**.:* ** :* :** ** *. *: . *

Sparus_aurata LNTNYSSSYSY-MKNDTLGDKTA-GASSLECLSSIVDRLSSVETSCGQPPLRDSATFSPG Micropterus_sal LNANYSNRYSY-AKNESVGDKTA-GASSLECLSSIVDRLSSVESSCGPVALRDMATFSPG Laterolabrax_ja LNANYGNSYSY-AKNDGLGDKAP-GASSLECLSSIVDRLSSVESSCGPAALRDMATFSPG Orzya_latipes LNANYSSSYSY-TKNESLDNKTA-GASSLECLSSIVDRLSSVESSGGPAALRDAATFSPS Takifugu_rubrip MNAVYSSGYLY-AKNEILTDKTA-GASSLECLSSIVDRLSSVESSCGPAALRDAATFSPG Salmo_salar MNTSYGNNYSY-TKNVSSGERGA-GASSLARLSNIVDRLSSVDAS-APAGLRDMLTFSPS Onchorhycchus_m MNTSYGNNYSY-TKNVSSGERGA-GASSLACLSSIVDRLSSVDAS-APAGLRDMLTFSPS Danio_rerio MNQNYGNNYNFDVQNASTMERTP-GVSSLQCLSSIVDRLSSVD----PAGMRNMVVLSPT Gallus_gallus RGSSFEAGYCR-EMPHGYATEQSGALSSLDCLSSIVDRLSPAEEP--GLPLRHAGSLSPG Xenopus_laevis RNSSFDNVYCS-DLQTSFSSTKL-TLSSLDCLSSIVDRISSSEQC--SLPIPDSLSLSPT Mus_musculus KNSSFDSIYCP-DVSNACAADKS-SVSSLDCLSSIVDRITSTEPS--ELALQDTASLSPA

. : * *** **.****::. : : . :**

Sparus_aurata -SSDSQPCTPESPGSRPVYHVL Micropterus_sal -SSDSQPCTPESPGCRPVYHVL Laterolabrax_ja -SSDSQPCTPESPGTRPVYHVL Orzya_latipes -SSDSQPCTPENSGSRPVYHVL Takifugu_rubrip -SAESQPCTPESPGSRPVYHVL Salmo_salar -STDSQPCTTESPGTRPVYHVL Onchorhycchus_m -STDSQPCTPESPGTRPVYHVL Danio_rerio -GSDSQSSSPDSPNNRPVYHVL Gallus_gallus ASIDSGPGTPGSPPPRRTYQAL Xenopus_laevis SSTDSFPRSPDMPDCRPIYHVL Mus_musculus TSANSQPATPGPSSSRLIYHVL

. :* . :. . * *:.*

 

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