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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
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ms. has 47 pages, 7 figures, 2 tables, 5 suppl. files 25
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*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
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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
224
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
239
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
297
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
7 0
8 0
9 0
1 0 0
a b a a b c
b c b c c a b c a b c
d a y o f c u l t u r e
% D
esm
in-p
ositi
ve c
ells
10
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
100
Akt
-p/A
KT
rela
tive
expr
essi
on
Fed Fasted
*
A
c-met
0
50
100
150
c-m
et/a
ctin
rela
tive
expr
essi
on
Fed Fasted
*
C
mTOR-p
0
20
40
60
80
100
mTO
R-p
/mTO
R re
lativ
e ex
pres
sion
Fed Fasted
*
B
AKT-p
AKT
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
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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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0
50000
100000
150000
200000
250000
300000
Sple
en
Live
r
Hea
rt
Kidn
ey
Brai
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Adip
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Red
Mus
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Whi
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Myf
5 ex
pres
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(a.u
.)
Spl
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Live
r
Hea
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Kid
ney
Bra
in
Adi
pose
0
5
10
15
20
25
30
Myf
5 ex
pres
sion
( a.u
)
0
5000
10000
15000
20000
25000
30000
35000
Sple
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Live
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Hear
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Kidn
ey
Brai
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Adip
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Red
mus
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Whi
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MR
F4 e
xpre
ssio
n (a
.u.)
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
Spl
een
Live
r
Hea
rt
Kid
ney
Bra
in
Adi
pose
MR
F4
exp
ress
ion
(a.u
)
Suppl. 4
0
10000
20000
30000
40000
50000
60000
70000
Sple
en
Live
r
Hea
rt
Kidn
ey
Brai
n
Adip
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Red
Mus
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Whi
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7 ex
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Sple
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Live
r
Hea
rt
Kidn
ey
Brai
n
Adip
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Red
Mus
cle
Whi
te M
uscl
e 0
50
100
150
200
250
300
Sox
8 ex
pres
sion
(a.u
.)
Spl
een
Live
r
Hea
rt
Kid
ney
Adi
pose
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
10,00
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
10,00
Pax
7 ex
pres
sion
(a.u
)