Research Article Expression Pattern of Myogenic Regulatory ...Environmental Toxicology Laboratory, Department of Zoology, Centre for Advanced Studies, Visva-Bharati University,...
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Research ArticleExpression Pattern of Myogenic Regulatory Transcription FactormRNAs in the Embryo and Adult Labeo rohita (Hamilton 1822)
1 Molecular Genetics Laboratory Department of Zoology Centre for Advanced Studies Visva-Bharati UniversitySantiniketan West Bengal 731235 India
2 Environmental Toxicology Laboratory Department of Zoology Centre for Advanced Studies Visva-Bharati UniversitySantiniketan West Bengal 731235 India
3 Fish Biology Research Unit Department of Zoology Centre for Advanced Studies Visva-Bharati University SantiniketanWest Bengal 731235 India
Correspondence should be addressed to Ansuman Chattopadhyay chansuman1gmailcom
Received 17 January 2014 Revised 13 March 2014 Accepted 13 March 2014 Published 10 April 2014
Academic Editor Greg Demas
Copyright copy 2014 Archya Sengupta et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Understanding the regulation of skeletal muscle development is important to meet the increasing demand of Indian major carpLabeo rohita Myogenic regulatory factors (MRFs) along with myocyte specific enhancer factor 2 (MEF2) play the pivotal role inthe determination and differentiation of skeletal muscleThemajority of skeletal muscle genes require bothMRFs andMEF2 familymembers to activate their transcription In this study the expression pattern ofMyoD myf-5 myogenin andMEF2Awas observedfrom 6 h after fertilization to 12 months of age using semiquantitative RT-PCR as well as real-time PCR method MyoD and myf-5mRNAs were expressed at high level at the early embryonic stages Myogenin and MEF2A were expressed after MyoD and myf-5and remained active up to adult stage Expression of MyoD was lower than that of Myf-5 after the 5th month Partial sequencing ofMyoD myf-5 and MEF2A was done to draw phylogeny In phylogenetic study LabeoMyoD MEF2A and myf-5 were found to beclosely related to those of common carp The present investigation suggests that the four transcription factors play pivotal role inthe regulation of muscle growth of Labeo rohita in an overlapping and interconnected way
1 Introduction
Rohu Labeo rohita (Hamilton 1822) is one of the most im-portant economic carps in India and other South EastAsian countries With increasing demand more studies arerequired on growth and differentiation of skeletal muscle toimprove the growth rate of this fish The understanding ofthe regulation of embryonic and postnatal skeletal musclegrowth and development is extremely important in thisregard [1] During vertebrate embryogenesis skeletal muscleis derived from somites which is formed by segmentation ofthe paraxial mesoderm lateral to neural tube [2] The trunkmusculature of fish is originated from the segmental platemesoderm flanking the notochord and lying underneath the
presumptive nerve cord Studies in zebrafish (Brachydaniorerio) have revealed that the most medial cells in the segmen-tal plate called adaxial cells commit to become myoblastswith a slow muscle lineage and the fast muscle fibres arederived from the lateral presomitic mesoderm by fusionof several myoblasts to form multinucleated myotubes [3]Development and growth of skeletal muscle are complexdynamic processes involving both the recruitment of newmuscle fibres (hyperplasia) and growth of existing fibres(hypertrophy) [4] Fish have an ability to recruit new skeletalmuscle fibres throughout the larval life and even duringjuvenile and adult life [5] Both hyperplasia and hypertrophyoccur during myogenesis in larval and adult muscle growthof fish which reach a large adult size [6]
Hindawi Publishing CorporationInternational Journal of ZoologyVolume 2014 Article ID 259685 9 pageshttpdxdoiorg1011552014259685
2 International Journal of Zoology
The expression of genes in skeletal cardiac and smoothmuscle cells could be controlled by a shared myogenic regu-latory programme [7] Myogenic regulatory factors (MRFs)family of basic helix-loop-helix (bHLH) transcription factorsplay the pivotal role in the determination and differentiationof skeletal muscle and they have the property of convertinga variety of cells into myoblasts and myotubes [8] Membersof this gene family like MyoD myf-5 and myogenin havebeen identified inmanyfish species and found to be expressedin developing somites and skeletal muscles [9ndash11] though noreport is available in any Indian carp species MyoD andmyf-5 play a common role in establishing myoblast identitywhereas myogenin is involved in terminal differentiationMRF genes are specifically expressed in myoblast cells andregulate expression of different muscle proteins and enzymeslike myosin troponin and creatine kinase [12] MRFs formheterodimers with E-proteins and bind to a consensus DNAsequence known as E box present in the control region ofmany skeletal muscle genesMyocyte specific enhancer factor2 (MEF2) family of transcription factors is another impor-tant regulator of skeletal muscle differentiation The cloningof genes encoding MEF2 factors revealed that these proteinsbelong to the MADS box family of transcription factorsMultiple isoforms of MEF2 have been identified in verte-brates all of which possess a specific DNA binding domaincharacteristic of the MADS box gene family and a highlyconserved MEF2 specific sequence [9] MyoD and MEF2family members function in a combined way to activatemyogenesis Consistent with these observations the majorityof skeletal muscle genes require bothMyoD andMEF2 familymembers to activate their transcription [13] Many studiesrevealed that dynamics of skeletal muscle growth can beaffected by several external factors like photoperiod [14]temperature variation [15] and dietary treatment [4] But noreport is available on age specific expression pattern of thesegenes during growth of Labeo rohita in natural condition
In our study we have assessed the expression of fourimportant regulatory transcription factors in Labeo rohitamRNA expression pattern of MyoD myf-5 myogenin andMEF2A genes from 6 h postfertilization to 12 months of agewas monitored at 16 time points by semiquantitative RT-PCRfollowed by qRT-PCR at 5 time points To the best of ourknowledge this is the first study on the expression pattern ofmyogenic regulatory factors both in embryo and adult Labeorohita This study is important for a better understanding ofthe molecular mechanisms of regulation of skeletal musclegrowth of this important Indian major carp
2 Materials and Methods
21 Chemicals and Reagents TRI reagent for RNA isolationwas procured from Sigma-Aldrich Corp (St Louis MOUSA) Reverse transcriptase and all chemicals of PCR mixwere purchased from Fermentas (USA) All other chemicalsused were of analytical grade and purchased from SiscoResearch Laboratories (Mumbai India) and Merck (Darm-stadt Germany) Custom designed primers were synthesizedfrom Sigma-Aldrich Corp (St Louis MO USA)
22 Maintenance of Fish and Sample Collection Fish weremaintained and cultured in a pond at a local fish farmin Birbhum India Embryos were collected and pooled forRNA isolation Fingerlings of same batch were maintainedin a stocking pond provided with standard diet Fish from1 month of age to 12 months age were collected in the firstweek of every month from the stocking pond Dorsal skeletalmuscle was dissected from each fish and processed for RNAisolation Length and weight of each fish were recordedmonthly for 12 months From each age group 6 individualswere taken randomly for further experiments
23 RNA Extraction Approximately 100mg of pooled wholeembryo or dorsal white muscle fragments (1-monthndash12-month sample) was mechanically homogenized with 1mLof the TRI reagent (Sigma St Louis MO USA) and thetotal RNA was extracted according to the manufacturerrsquosinstructions and stored in DEPC treated nuclease-free waterat minus20∘C RNA was pooled for each age group Samples weresubjected to electrophoresis on 1 agarose gels to confirm theintegrity of the 28S and 18S rRNA bands RNA quality wasassessed as the 260280 nm absorbance ratio and RNA wasquantified from absorbance at 260 nm
24 cDNA Synthesis through RT-PCR Single-strand cDNAwas reverse-transcribed from equal amounts of total RNA(5 120583g) using an oligo-dT
18primer and reverse transcrip-
tase (Fermentas) through RT-PCR The reaction mixturecontained 4 120583L of reverse transcriptase buffer (5 times RTBuffer) 05 120583L (20U) of ribonuclease inhibitor (RiboLockFermentas) 2120583L of dNTP Mix (10mM each) 05 120583g ofoligo-dT
18primer and 1 120583L (200U) of RevertAid H minus
reverse transcriptase (Fermentas) Reaction was carried outfollowing manufacturerrsquos protocol
25 PCR Amplification of Myogenic Regulatory Genes Forpartial amplification ofMyoDmyf-5myogenin andMEF2Agene specific primer pairs were designed using Primer 3software (version 040) based on sequences of carp avail-able inGenBank (httpwwwncbinlmnihgov)120573-Actinwasamplified simultaneously as an internal control and theprimers for 120573-actin were adopted from Li et al 2004 [16]Theprimer sequences are given in Table 1
The PCR was performed following the procedure as perthe manufacturerrsquos instruction for 35 cycles All test sampleswere amplified simultaneously from equal volume of firststrand cDNA with the particular primer pair using a masterPCRmix For each reaction master PCRmix contained PCRbuffer 02mM of dNTPs 25mM MgCl
2 02mM of each
primer template cDNA and 10 unit of TaqDNA polymerase(Fermentas) PCR reactions were run in a programmablethermal cycler (GeneAmp 9700 ABI) with simultaneousNTC (no template control) The PCR products were run in15 agarose gel and visualized in a gel documentation system(Gel Doc EZ Imager Bio-Rad) after staining with ethidiumbromide The densitometric quantification was done usingImageJ (NIH) software
International Journal of Zoology 3
Table1Prim
ersu
sedforp
olym
erasec
hain
reactio
nam
plificatio
nof
LabeoMyoDm
yf-5m
yogenin
MEF
2Aand120573-actin
Gene
Prim
ersequ
ence
Ann
ealin
gtemperature
Source
sequ
ence
forp
rimer
desig
ning
(accessio
nnu
mbersareg
iven)
Positionof
prim
ers
insource
sequ
ence
MyoD
Forw
ard
51015840-C
GAC
TGAG
CAAAG
TCAAC
GA-
31015840590∘
CCtenopharyngodon
idellamRN
AforM
yoD
(GenBa
nkaccession
JQ7938931)
296ndash
315
Reverse
51015840-TTC
CGTC
TTCT
CGAC
TGAC
A-31015840
561ndash542
myf-5
Forw
ard
51015840-G
CCAG
GTC
ACTG
TCTG
CAAT
-31015840
585∘
CCy
prinus
carpiomRN
Aform
yf-5
(GenBa
nkaccession
AB0
128831)
202ndash221
Reverse
51015840-G
CTCA
GAG
CTGCT
TTCC
ATT-31015840
479ndash
460
Myogenin
Forw
ard
51015840-G
GCT
TCGAC
CAAAC
AGGAT
A-31015840
590∘
CCy
prinus
carpiomRN
Aform
yogenin
(GenBa
nkaccession
AB0
128811)
232ndash251
Reverse
51015840-G
CTCC
TGGTG
AGGAG
ACAAG
-31015840
376ndash
357
MEF
2AFo
rward
51015840-ACG
GAT
CATG
GAT
GAG
AGGA-
31015840588∘
CCy
prinus
carpiomRN
AforM
EF2A
(GenBa
nkaccession
AB0
128841)
199ndash
218
Reverse
51015840-TGAC
CGAAAC
AGTC
ATCT
GG-31015840
492ndash473
120573-Actin
Forw
ard
51015840-TGGAAT
CCTG
TGGCA
TCCA
TGAAAC
-31015840
660∘
CLi
etal200
4[16]
Reverse
51015840-TAAAAC
GCA
GCT
CAGTA
ACAG
TCCG
-31015840
4 International Journal of Zoology
Table2Prim
ersu
sedforq
RT-PCR
ofLa
beoMyoDm
yf-5m
yogenin
MEF
2Aand
GAPD
H
Gene
Prim
ersequ
ence
Ann
ealin
gtemperature
Source
sequ
ence
forp
rimer
desig
ning
(accessio
nnu
mbersareg
iven)
Positionof
prim
ers
insource
sequ
ence
MyoD
Forw
ard
51015840-TCC
AAG
CGCT
GCT
AAG
AAG
T-31015840
590∘
CLa
beorohita
partialm
RNAforM
yoD
(GenBa
nkaccession
KC3445371)
132ndash151
Reverse
51015840-C
ATCA
TGCC
ATCA
GAG
CAGT-31015840
241ndash222
myf-5
Forw
ard
51015840-G
CAGGCT
GAAG
AAG
GTG
AAC
-31015840
590∘
CLa
beorohita
partialm
RNAform
yf-5
(GenBa
nkaccession
KC3445361)
96ndash115
Reverse
51015840-G
GCT
TCCT
CAGGAT
CTCA
AC-31015840
195ndash176
Myogenin
Forw
ard
51015840-G
GCT
TCGAC
CAAAC
AGGAT
A-31015840
590∘
CCy
prinus
carpiomRN
Aform
yogenin
(GenBa
nkaccession
AB0
128811)
232ndash251
Reverse
51015840-G
CTCC
TGGTG
AGGAG
ACAAG
-31015840
376ndash
357
MEF
2AFo
rward
51015840-TGAC
TGTG
AGAT
TGCC
CTGA-
31015840590∘
CLa
beorohita
partialm
RNAforM
EF2A
(GenBa
nkaccession
KC3445351)
94ndash113
Reverse
51015840-C
GTG
GGGTT
CGTT
GTA
TTCT
-31015840
206ndash
187
GAPD
HFo
rward
51015840-AGGGGCT
CAGTA
TGTT
GTG
G-31015840
590∘
CCy
prinus
carpiopartialm
RNAforG
APD
H(G
enBa
nkaccession
AJ8709821)
257ndash276
Reverse
51015840-C
TCTC
TTGGCA
CCAC
CCTT
A-31015840
342ndash323
International Journal of Zoology 5
The PCR products of MyoD myf-5 and MEF2A weresubjected to partial sequence analysis to confirm thegenes Sequencing was done by Xcelris Genomics Pvt LtdNucleotide sequences were translated into amino acidsequences for further analysis Sequence alignments wereobtained using Clustal Omega software (EMBL-EBI httpwwwebiacuk) Phylogenetic and molecular evolutionaryanalyses were conducted using MEGA version 6 [17] Phy-logenetic tree was reconstructed using neighbour-joiningdistance algorithms from translated amino acid sequences inPAM matrix Statistical consistency was evaluated by 1000bootstrap resamplings of the data
26 Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR)was performed using a Bio Rad CFX96 Real-Time PCRSystem (Bio Rad Laboratories Inc Hercules CA USA) andan iQ SYBERGreen superMixKit (Bio Rad Laboratories IncHercules CA USA) which were used in accordance withthe manufacturersrsquo instructions Standard reaction mixtures(15 120583L) were assembled using 75 120583L of iQ SYBERGreen superMix 2x 300 nMof each primer and 100 ng of template cDNAFor amplification of MyoD myf-5 myogenin MEF2A andGAPDH gene specific primer pairs were designed (Table 2)using Primer 3 software (version 040) based on sequencesof carp available in GenBank (httpwwwncbinlmnihgov)
Relative gene expression values were obtained using BioRad CFX manager software (Version 21)
3 Results
31 Fish Growth Mean weight of fish increased at a steadyrate up to 6months of age Duringwinter season (6months to8months of age) the rate ofmonthlyweight gainwas reducedmarginally which again recovered during 9th- to 12th-monthperiodThere was a net increase inmean length in all the timepoints After 12 months the average weight of fish was around700 g and length was approximately 40 cm (Figure 1)
32 mRNA Transcription Pattern of MyogenicRegulatory Factors
321 Reverse Transcriptase PCR (RT-PCR) MyoD mRNAexpression was found to be high in the embryo stagesand decreased gradually after 5 months of age reachingthe minimum at 12 months The highest expression wasobtained at 24 h after fertilization (Figure 2) Expression ofmyf-5 also reached its maximum at 24 h after fertilizationand decreased gradually to reach the basal level (Figure 2)Myogenin expression did not show anymarked alteration andmaintained a steady expression pattern up to 12 months withslightmodulation (Figure 2)MEF2A had a steady expressionpattern with slight elevation during 6 h after fertilization to1 month of age After 1 month it decreased slightly andthen became almost stable throughout the rest part of theexperimental period (Figure 2)
322 Quantitative Real-Time PCR (qRT-PCR) QuantitativePCR was done at 5 time points (6 h 24 h 1 month 6 months
0100200300400500600700800
Wei
ght o
f fish
(g)
Age group of fish (1 month to 12 months)
1 m
onth
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(a)
05
1015202530354045
Leng
th o
f fish
(cm
)
Age group of fish (1 month to 12 months)1
mon
th
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(b)
Figure 1 Monthly growth of Labeo rohita from 1 month to 12months of age (a) Mean weight of fish (b) Mean length of fishValues are expressed as mean plusmn SEM (119873 = 6)
and 12 months) All the four transcription factors showed theexpression pattern similar to that of RT-PCR (Figure 3) andcorroborated with RT-PCR results
33 Construction of Phylogenetic Tree Partial nucleotidecoding sequences of three genes (MyoD myf-5 andMEF2A)were submitted to GenBank (httpwwwncbinlmnihgov)The GenBank accession numbers are KC3445371 for MyoDKC3445361 for myf-5 and KC3445351 for MEF2A Molecu-lar phylogenetic relationship (based on translated amino acidsequences) of Labeo MyoD myf-5 and MEF2A with that ofother closely related species was depicted by reconstructingphylogenetic tree using neighbor-joining method (Figure 4)
4 Discussion
The growth pattern of the experimental fishes followedsimilar annual pattern of growth of Labeo species in situas reported by Jhingran [18] (Figure 1) After fertilizationdifferentiation of skeletal muscle is initiated by MyoD whichbinds directly to the regulatory regions of a wide number ofgenes and regulates their expression during differentiation[19 20] BothMyoD andmyf-5 are necessary for the initiationof myogenesis in vertebrates Disruption of both genes inmice results in the absence of skeletal muscle cells [21]
6 International Journal of Zoology
myf-5
MyoD300
300
(bp)
300
Myogenin
MEF2A200
300
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
120573-Actin
100
bp ru
ler
(a)
002040608
112141618
Rela
tive d
ensit
omet
ric v
alue
(au
)
MyoDmyf-5
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(b)
Relat
ive d
ensit
omet
ric v
alue
002040608
112141618
2
(au
)
MyogeninMEF2A
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(c)
Figure 2 mRNA transcription pattern of myogenic regulatory factors by RT-PCR (a) expression of MyoD myf-5 myogeninand MEF2AmRNA at different time points of growth (6 hours to 12 months) (b) relative densitometric analysis of MyoD and myf-5 expression and (c)relative densitometric analysis of myogenin and MEF2A expression Values are expressed as mean plusmn SEM (119873 = 6)
Myogenic cells undergo active proliferation before cell cyclearrest and fusion into myotubes MyoD and Myf-5 play thecentral role in specifying muscle lineage andMyoD is the keyregulator of maintaining balance between the differentiationand proliferation [22] In the present study the expression ofMyoD andmyf-5 was higher in the embryonic stages of Labeorohita Both the genes followed nearly similar pattern ofexpression as depicted by semiquantitative and quantitativePCR (Figure 3) In mouse myf-5 was reported as the firstexpressedMRF in themyotomal muscle [23] and in commoncarp a high level of mRNA transcripts of myf-5 was detectedat 30 h after fertilization [9] In our study considerableamount of MyoD mRNA transcript was detected at 12 h afterfertilization stage (Figure 2(b)) and myf-5 had the highestlevel of expression at 24 h after fertilization (Figure 2(b))So in this particular fish the MyoD and myf-5 expressionpattern is not similar with the same in other fish speciesreported earlier [9 24]
Postnatal muscle growth involves hypertrophy of musclefibres which require additional nuclei to maintain a relativelyconstant nuclear to cytoplasmic ratio These nuclei are pro-vided by activated myogenic stem cells which also express
myogenic bHLH proteins myf-5 and MyoD expressions inskeletal muscles are followed by upregulation of myogeninand of MEF2 family factors which enhance expression ofmuscle differentiation genes [25] We observed that myo-geninmRNA transcript was present in a considerable amountat all stages with highest value at 24 h (Figure 3) MEF2family of transcription factors specifically bind to an ATrich sequence present inmanymuscle specific promoters andenhancers [26] In zebrafish knockdown ofMEF2A has beenshown to downregulate a large set of genes encoding con-tractile proteins such as troponins myosin heavy and lightchains and 120572-tropomyosin [27] In Labeo rohita MEF2Aexpressed at all the stages showing the high level of expressionat 1 monthThis expression pattern of myogenin andMEF2Ais similar to the pattern in common carp described byKobiyama et al 1998 [9] Muscle growth in fish involvesthe production of new muscle fibres in addition to musclefibre hypertrophy [6]The continued expression of myogeninand MEF2A in Labeo rohita reflects activated myogenic cellswhich help to maintain continuous hypertrophy as well ashyperplasia of skeletal muscle In embryonic stages most ofthe muscle cells remain in the early differentiation stage and
Figure 3 mRNA expression of four myogenic regulatory factors in Labeo rohita at 6 h 24 h 1 month 6 months and 12 months of age (a)MyoD (b) myf-5 (c) myogenin and (d) MEF2A
proliferation stage while most muscle cells of adult are in theterminal differentiation stage [24] Our results clearly showedthat MyoD and myf-5 mRNAs are expressed at high level inthe early embryonic stages whereas myogenin and MEF2Aare expressed afterMyoDandmyf-5 expression and remainedactive in adult stage tomaintain differentiation and growth ofskeletal muscle fibres MyoD is reported to play an importantrole in the arrest of cellular growth Its highest level wasdetected at early G1 phase and the lowest level was at G1 to Sphase transitionMyoD and its cofactors play a critical role inmyoblast cell cycle withdrawal When MyoD is maximal andmyf-5 is down cells exit their cycle into differentiation Theopposite pattern is observed in quiescent nondifferentiatingmyoblasts a high myf-5 and no MyoD [22] In Labeo rohitaboth the weight rate and length rate between the 5th and6th month were significantly greater than those of before 5thmonthThe expression ofMyoDwas lower than that of myf-5after 5thmonth Considering two relationships (expression ofmyf-5MyoD with muscle cells proliferationdifferentiationand muscle cells proliferationdifferentiation with musclehypertrophyhyperplasia) it may be supposed that in thisfish species the muscle development after 5th month was
properly balanced by both hypertrophy and hyperplasiaThe four transcription factors therefore play pivotal rolein the regulation of muscle growth in an overlapping andinterconnected way
Molecular phylogenetic tree showed that Labeo MyoDMEF2A and myf-5 are more closely related to that of com-mon carp (Cyprinus carpio) than any other species Previousreport showed high similarities of MyoD myf-5 andMEF2Asequences between carp and zebrafish but the similaritiesweremore prominent in the bHLH region (MyoD andmyf-5)and MADS box region (MEF2A) than total coding sequence[28]
Further extensive studies are warranted to evaluate theregulation of MRF gene expression pattern in fish whichmay provide insight into the signaling pathway controllingmuscle cell differentiation through regulatory transcriptionfactors Information is available on tissue specific embryonicexpression pattern of these transcription factors Contrast-ingly studies on long term age specific pattern of expressionare lacking To the best of our knowledge this is the firstreport on myogenic regulatory transcription factors in any
8 International Journal of Zoology
9499
5199
98
100
100
62
100
01
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
The expression of genes in skeletal cardiac and smoothmuscle cells could be controlled by a shared myogenic regu-latory programme [7] Myogenic regulatory factors (MRFs)family of basic helix-loop-helix (bHLH) transcription factorsplay the pivotal role in the determination and differentiationof skeletal muscle and they have the property of convertinga variety of cells into myoblasts and myotubes [8] Membersof this gene family like MyoD myf-5 and myogenin havebeen identified inmanyfish species and found to be expressedin developing somites and skeletal muscles [9ndash11] though noreport is available in any Indian carp species MyoD andmyf-5 play a common role in establishing myoblast identitywhereas myogenin is involved in terminal differentiationMRF genes are specifically expressed in myoblast cells andregulate expression of different muscle proteins and enzymeslike myosin troponin and creatine kinase [12] MRFs formheterodimers with E-proteins and bind to a consensus DNAsequence known as E box present in the control region ofmany skeletal muscle genesMyocyte specific enhancer factor2 (MEF2) family of transcription factors is another impor-tant regulator of skeletal muscle differentiation The cloningof genes encoding MEF2 factors revealed that these proteinsbelong to the MADS box family of transcription factorsMultiple isoforms of MEF2 have been identified in verte-brates all of which possess a specific DNA binding domaincharacteristic of the MADS box gene family and a highlyconserved MEF2 specific sequence [9] MyoD and MEF2family members function in a combined way to activatemyogenesis Consistent with these observations the majorityof skeletal muscle genes require bothMyoD andMEF2 familymembers to activate their transcription [13] Many studiesrevealed that dynamics of skeletal muscle growth can beaffected by several external factors like photoperiod [14]temperature variation [15] and dietary treatment [4] But noreport is available on age specific expression pattern of thesegenes during growth of Labeo rohita in natural condition
In our study we have assessed the expression of fourimportant regulatory transcription factors in Labeo rohitamRNA expression pattern of MyoD myf-5 myogenin andMEF2A genes from 6 h postfertilization to 12 months of agewas monitored at 16 time points by semiquantitative RT-PCRfollowed by qRT-PCR at 5 time points To the best of ourknowledge this is the first study on the expression pattern ofmyogenic regulatory factors both in embryo and adult Labeorohita This study is important for a better understanding ofthe molecular mechanisms of regulation of skeletal musclegrowth of this important Indian major carp
2 Materials and Methods
21 Chemicals and Reagents TRI reagent for RNA isolationwas procured from Sigma-Aldrich Corp (St Louis MOUSA) Reverse transcriptase and all chemicals of PCR mixwere purchased from Fermentas (USA) All other chemicalsused were of analytical grade and purchased from SiscoResearch Laboratories (Mumbai India) and Merck (Darm-stadt Germany) Custom designed primers were synthesizedfrom Sigma-Aldrich Corp (St Louis MO USA)
22 Maintenance of Fish and Sample Collection Fish weremaintained and cultured in a pond at a local fish farmin Birbhum India Embryos were collected and pooled forRNA isolation Fingerlings of same batch were maintainedin a stocking pond provided with standard diet Fish from1 month of age to 12 months age were collected in the firstweek of every month from the stocking pond Dorsal skeletalmuscle was dissected from each fish and processed for RNAisolation Length and weight of each fish were recordedmonthly for 12 months From each age group 6 individualswere taken randomly for further experiments
23 RNA Extraction Approximately 100mg of pooled wholeembryo or dorsal white muscle fragments (1-monthndash12-month sample) was mechanically homogenized with 1mLof the TRI reagent (Sigma St Louis MO USA) and thetotal RNA was extracted according to the manufacturerrsquosinstructions and stored in DEPC treated nuclease-free waterat minus20∘C RNA was pooled for each age group Samples weresubjected to electrophoresis on 1 agarose gels to confirm theintegrity of the 28S and 18S rRNA bands RNA quality wasassessed as the 260280 nm absorbance ratio and RNA wasquantified from absorbance at 260 nm
24 cDNA Synthesis through RT-PCR Single-strand cDNAwas reverse-transcribed from equal amounts of total RNA(5 120583g) using an oligo-dT
18primer and reverse transcrip-
tase (Fermentas) through RT-PCR The reaction mixturecontained 4 120583L of reverse transcriptase buffer (5 times RTBuffer) 05 120583L (20U) of ribonuclease inhibitor (RiboLockFermentas) 2120583L of dNTP Mix (10mM each) 05 120583g ofoligo-dT
18primer and 1 120583L (200U) of RevertAid H minus
reverse transcriptase (Fermentas) Reaction was carried outfollowing manufacturerrsquos protocol
25 PCR Amplification of Myogenic Regulatory Genes Forpartial amplification ofMyoDmyf-5myogenin andMEF2Agene specific primer pairs were designed using Primer 3software (version 040) based on sequences of carp avail-able inGenBank (httpwwwncbinlmnihgov)120573-Actinwasamplified simultaneously as an internal control and theprimers for 120573-actin were adopted from Li et al 2004 [16]Theprimer sequences are given in Table 1
The PCR was performed following the procedure as perthe manufacturerrsquos instruction for 35 cycles All test sampleswere amplified simultaneously from equal volume of firststrand cDNA with the particular primer pair using a masterPCRmix For each reaction master PCRmix contained PCRbuffer 02mM of dNTPs 25mM MgCl
2 02mM of each
primer template cDNA and 10 unit of TaqDNA polymerase(Fermentas) PCR reactions were run in a programmablethermal cycler (GeneAmp 9700 ABI) with simultaneousNTC (no template control) The PCR products were run in15 agarose gel and visualized in a gel documentation system(Gel Doc EZ Imager Bio-Rad) after staining with ethidiumbromide The densitometric quantification was done usingImageJ (NIH) software
International Journal of Zoology 3
Table1Prim
ersu
sedforp
olym
erasec
hain
reactio
nam
plificatio
nof
LabeoMyoDm
yf-5m
yogenin
MEF
2Aand120573-actin
Gene
Prim
ersequ
ence
Ann
ealin
gtemperature
Source
sequ
ence
forp
rimer
desig
ning
(accessio
nnu
mbersareg
iven)
Positionof
prim
ers
insource
sequ
ence
MyoD
Forw
ard
51015840-C
GAC
TGAG
CAAAG
TCAAC
GA-
31015840590∘
CCtenopharyngodon
idellamRN
AforM
yoD
(GenBa
nkaccession
JQ7938931)
296ndash
315
Reverse
51015840-TTC
CGTC
TTCT
CGAC
TGAC
A-31015840
561ndash542
myf-5
Forw
ard
51015840-G
CCAG
GTC
ACTG
TCTG
CAAT
-31015840
585∘
CCy
prinus
carpiomRN
Aform
yf-5
(GenBa
nkaccession
AB0
128831)
202ndash221
Reverse
51015840-G
CTCA
GAG
CTGCT
TTCC
ATT-31015840
479ndash
460
Myogenin
Forw
ard
51015840-G
GCT
TCGAC
CAAAC
AGGAT
A-31015840
590∘
CCy
prinus
carpiomRN
Aform
yogenin
(GenBa
nkaccession
AB0
128811)
232ndash251
Reverse
51015840-G
CTCC
TGGTG
AGGAG
ACAAG
-31015840
376ndash
357
MEF
2AFo
rward
51015840-ACG
GAT
CATG
GAT
GAG
AGGA-
31015840588∘
CCy
prinus
carpiomRN
AforM
EF2A
(GenBa
nkaccession
AB0
128841)
199ndash
218
Reverse
51015840-TGAC
CGAAAC
AGTC
ATCT
GG-31015840
492ndash473
120573-Actin
Forw
ard
51015840-TGGAAT
CCTG
TGGCA
TCCA
TGAAAC
-31015840
660∘
CLi
etal200
4[16]
Reverse
51015840-TAAAAC
GCA
GCT
CAGTA
ACAG
TCCG
-31015840
4 International Journal of Zoology
Table2Prim
ersu
sedforq
RT-PCR
ofLa
beoMyoDm
yf-5m
yogenin
MEF
2Aand
GAPD
H
Gene
Prim
ersequ
ence
Ann
ealin
gtemperature
Source
sequ
ence
forp
rimer
desig
ning
(accessio
nnu
mbersareg
iven)
Positionof
prim
ers
insource
sequ
ence
MyoD
Forw
ard
51015840-TCC
AAG
CGCT
GCT
AAG
AAG
T-31015840
590∘
CLa
beorohita
partialm
RNAforM
yoD
(GenBa
nkaccession
KC3445371)
132ndash151
Reverse
51015840-C
ATCA
TGCC
ATCA
GAG
CAGT-31015840
241ndash222
myf-5
Forw
ard
51015840-G
CAGGCT
GAAG
AAG
GTG
AAC
-31015840
590∘
CLa
beorohita
partialm
RNAform
yf-5
(GenBa
nkaccession
KC3445361)
96ndash115
Reverse
51015840-G
GCT
TCCT
CAGGAT
CTCA
AC-31015840
195ndash176
Myogenin
Forw
ard
51015840-G
GCT
TCGAC
CAAAC
AGGAT
A-31015840
590∘
CCy
prinus
carpiomRN
Aform
yogenin
(GenBa
nkaccession
AB0
128811)
232ndash251
Reverse
51015840-G
CTCC
TGGTG
AGGAG
ACAAG
-31015840
376ndash
357
MEF
2AFo
rward
51015840-TGAC
TGTG
AGAT
TGCC
CTGA-
31015840590∘
CLa
beorohita
partialm
RNAforM
EF2A
(GenBa
nkaccession
KC3445351)
94ndash113
Reverse
51015840-C
GTG
GGGTT
CGTT
GTA
TTCT
-31015840
206ndash
187
GAPD
HFo
rward
51015840-AGGGGCT
CAGTA
TGTT
GTG
G-31015840
590∘
CCy
prinus
carpiopartialm
RNAforG
APD
H(G
enBa
nkaccession
AJ8709821)
257ndash276
Reverse
51015840-C
TCTC
TTGGCA
CCAC
CCTT
A-31015840
342ndash323
International Journal of Zoology 5
The PCR products of MyoD myf-5 and MEF2A weresubjected to partial sequence analysis to confirm thegenes Sequencing was done by Xcelris Genomics Pvt LtdNucleotide sequences were translated into amino acidsequences for further analysis Sequence alignments wereobtained using Clustal Omega software (EMBL-EBI httpwwwebiacuk) Phylogenetic and molecular evolutionaryanalyses were conducted using MEGA version 6 [17] Phy-logenetic tree was reconstructed using neighbour-joiningdistance algorithms from translated amino acid sequences inPAM matrix Statistical consistency was evaluated by 1000bootstrap resamplings of the data
26 Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR)was performed using a Bio Rad CFX96 Real-Time PCRSystem (Bio Rad Laboratories Inc Hercules CA USA) andan iQ SYBERGreen superMixKit (Bio Rad Laboratories IncHercules CA USA) which were used in accordance withthe manufacturersrsquo instructions Standard reaction mixtures(15 120583L) were assembled using 75 120583L of iQ SYBERGreen superMix 2x 300 nMof each primer and 100 ng of template cDNAFor amplification of MyoD myf-5 myogenin MEF2A andGAPDH gene specific primer pairs were designed (Table 2)using Primer 3 software (version 040) based on sequencesof carp available in GenBank (httpwwwncbinlmnihgov)
Relative gene expression values were obtained using BioRad CFX manager software (Version 21)
3 Results
31 Fish Growth Mean weight of fish increased at a steadyrate up to 6months of age Duringwinter season (6months to8months of age) the rate ofmonthlyweight gainwas reducedmarginally which again recovered during 9th- to 12th-monthperiodThere was a net increase inmean length in all the timepoints After 12 months the average weight of fish was around700 g and length was approximately 40 cm (Figure 1)
32 mRNA Transcription Pattern of MyogenicRegulatory Factors
321 Reverse Transcriptase PCR (RT-PCR) MyoD mRNAexpression was found to be high in the embryo stagesand decreased gradually after 5 months of age reachingthe minimum at 12 months The highest expression wasobtained at 24 h after fertilization (Figure 2) Expression ofmyf-5 also reached its maximum at 24 h after fertilizationand decreased gradually to reach the basal level (Figure 2)Myogenin expression did not show anymarked alteration andmaintained a steady expression pattern up to 12 months withslightmodulation (Figure 2)MEF2A had a steady expressionpattern with slight elevation during 6 h after fertilization to1 month of age After 1 month it decreased slightly andthen became almost stable throughout the rest part of theexperimental period (Figure 2)
322 Quantitative Real-Time PCR (qRT-PCR) QuantitativePCR was done at 5 time points (6 h 24 h 1 month 6 months
0100200300400500600700800
Wei
ght o
f fish
(g)
Age group of fish (1 month to 12 months)
1 m
onth
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(a)
05
1015202530354045
Leng
th o
f fish
(cm
)
Age group of fish (1 month to 12 months)1
mon
th
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(b)
Figure 1 Monthly growth of Labeo rohita from 1 month to 12months of age (a) Mean weight of fish (b) Mean length of fishValues are expressed as mean plusmn SEM (119873 = 6)
and 12 months) All the four transcription factors showed theexpression pattern similar to that of RT-PCR (Figure 3) andcorroborated with RT-PCR results
33 Construction of Phylogenetic Tree Partial nucleotidecoding sequences of three genes (MyoD myf-5 andMEF2A)were submitted to GenBank (httpwwwncbinlmnihgov)The GenBank accession numbers are KC3445371 for MyoDKC3445361 for myf-5 and KC3445351 for MEF2A Molecu-lar phylogenetic relationship (based on translated amino acidsequences) of Labeo MyoD myf-5 and MEF2A with that ofother closely related species was depicted by reconstructingphylogenetic tree using neighbor-joining method (Figure 4)
4 Discussion
The growth pattern of the experimental fishes followedsimilar annual pattern of growth of Labeo species in situas reported by Jhingran [18] (Figure 1) After fertilizationdifferentiation of skeletal muscle is initiated by MyoD whichbinds directly to the regulatory regions of a wide number ofgenes and regulates their expression during differentiation[19 20] BothMyoD andmyf-5 are necessary for the initiationof myogenesis in vertebrates Disruption of both genes inmice results in the absence of skeletal muscle cells [21]
6 International Journal of Zoology
myf-5
MyoD300
300
(bp)
300
Myogenin
MEF2A200
300
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
120573-Actin
100
bp ru
ler
(a)
002040608
112141618
Rela
tive d
ensit
omet
ric v
alue
(au
)
MyoDmyf-5
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(b)
Relat
ive d
ensit
omet
ric v
alue
002040608
112141618
2
(au
)
MyogeninMEF2A
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(c)
Figure 2 mRNA transcription pattern of myogenic regulatory factors by RT-PCR (a) expression of MyoD myf-5 myogeninand MEF2AmRNA at different time points of growth (6 hours to 12 months) (b) relative densitometric analysis of MyoD and myf-5 expression and (c)relative densitometric analysis of myogenin and MEF2A expression Values are expressed as mean plusmn SEM (119873 = 6)
Myogenic cells undergo active proliferation before cell cyclearrest and fusion into myotubes MyoD and Myf-5 play thecentral role in specifying muscle lineage andMyoD is the keyregulator of maintaining balance between the differentiationand proliferation [22] In the present study the expression ofMyoD andmyf-5 was higher in the embryonic stages of Labeorohita Both the genes followed nearly similar pattern ofexpression as depicted by semiquantitative and quantitativePCR (Figure 3) In mouse myf-5 was reported as the firstexpressedMRF in themyotomal muscle [23] and in commoncarp a high level of mRNA transcripts of myf-5 was detectedat 30 h after fertilization [9] In our study considerableamount of MyoD mRNA transcript was detected at 12 h afterfertilization stage (Figure 2(b)) and myf-5 had the highestlevel of expression at 24 h after fertilization (Figure 2(b))So in this particular fish the MyoD and myf-5 expressionpattern is not similar with the same in other fish speciesreported earlier [9 24]
Postnatal muscle growth involves hypertrophy of musclefibres which require additional nuclei to maintain a relativelyconstant nuclear to cytoplasmic ratio These nuclei are pro-vided by activated myogenic stem cells which also express
myogenic bHLH proteins myf-5 and MyoD expressions inskeletal muscles are followed by upregulation of myogeninand of MEF2 family factors which enhance expression ofmuscle differentiation genes [25] We observed that myo-geninmRNA transcript was present in a considerable amountat all stages with highest value at 24 h (Figure 3) MEF2family of transcription factors specifically bind to an ATrich sequence present inmanymuscle specific promoters andenhancers [26] In zebrafish knockdown ofMEF2A has beenshown to downregulate a large set of genes encoding con-tractile proteins such as troponins myosin heavy and lightchains and 120572-tropomyosin [27] In Labeo rohita MEF2Aexpressed at all the stages showing the high level of expressionat 1 monthThis expression pattern of myogenin andMEF2Ais similar to the pattern in common carp described byKobiyama et al 1998 [9] Muscle growth in fish involvesthe production of new muscle fibres in addition to musclefibre hypertrophy [6]The continued expression of myogeninand MEF2A in Labeo rohita reflects activated myogenic cellswhich help to maintain continuous hypertrophy as well ashyperplasia of skeletal muscle In embryonic stages most ofthe muscle cells remain in the early differentiation stage and
Figure 3 mRNA expression of four myogenic regulatory factors in Labeo rohita at 6 h 24 h 1 month 6 months and 12 months of age (a)MyoD (b) myf-5 (c) myogenin and (d) MEF2A
proliferation stage while most muscle cells of adult are in theterminal differentiation stage [24] Our results clearly showedthat MyoD and myf-5 mRNAs are expressed at high level inthe early embryonic stages whereas myogenin and MEF2Aare expressed afterMyoDandmyf-5 expression and remainedactive in adult stage tomaintain differentiation and growth ofskeletal muscle fibres MyoD is reported to play an importantrole in the arrest of cellular growth Its highest level wasdetected at early G1 phase and the lowest level was at G1 to Sphase transitionMyoD and its cofactors play a critical role inmyoblast cell cycle withdrawal When MyoD is maximal andmyf-5 is down cells exit their cycle into differentiation Theopposite pattern is observed in quiescent nondifferentiatingmyoblasts a high myf-5 and no MyoD [22] In Labeo rohitaboth the weight rate and length rate between the 5th and6th month were significantly greater than those of before 5thmonthThe expression ofMyoDwas lower than that of myf-5after 5thmonth Considering two relationships (expression ofmyf-5MyoD with muscle cells proliferationdifferentiationand muscle cells proliferationdifferentiation with musclehypertrophyhyperplasia) it may be supposed that in thisfish species the muscle development after 5th month was
properly balanced by both hypertrophy and hyperplasiaThe four transcription factors therefore play pivotal rolein the regulation of muscle growth in an overlapping andinterconnected way
Molecular phylogenetic tree showed that Labeo MyoDMEF2A and myf-5 are more closely related to that of com-mon carp (Cyprinus carpio) than any other species Previousreport showed high similarities of MyoD myf-5 andMEF2Asequences between carp and zebrafish but the similaritiesweremore prominent in the bHLH region (MyoD andmyf-5)and MADS box region (MEF2A) than total coding sequence[28]
Further extensive studies are warranted to evaluate theregulation of MRF gene expression pattern in fish whichmay provide insight into the signaling pathway controllingmuscle cell differentiation through regulatory transcriptionfactors Information is available on tissue specific embryonicexpression pattern of these transcription factors Contrast-ingly studies on long term age specific pattern of expressionare lacking To the best of our knowledge this is the firstreport on myogenic regulatory transcription factors in any
8 International Journal of Zoology
9499
5199
98
100
100
62
100
01
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
The PCR products of MyoD myf-5 and MEF2A weresubjected to partial sequence analysis to confirm thegenes Sequencing was done by Xcelris Genomics Pvt LtdNucleotide sequences were translated into amino acidsequences for further analysis Sequence alignments wereobtained using Clustal Omega software (EMBL-EBI httpwwwebiacuk) Phylogenetic and molecular evolutionaryanalyses were conducted using MEGA version 6 [17] Phy-logenetic tree was reconstructed using neighbour-joiningdistance algorithms from translated amino acid sequences inPAM matrix Statistical consistency was evaluated by 1000bootstrap resamplings of the data
26 Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR)was performed using a Bio Rad CFX96 Real-Time PCRSystem (Bio Rad Laboratories Inc Hercules CA USA) andan iQ SYBERGreen superMixKit (Bio Rad Laboratories IncHercules CA USA) which were used in accordance withthe manufacturersrsquo instructions Standard reaction mixtures(15 120583L) were assembled using 75 120583L of iQ SYBERGreen superMix 2x 300 nMof each primer and 100 ng of template cDNAFor amplification of MyoD myf-5 myogenin MEF2A andGAPDH gene specific primer pairs were designed (Table 2)using Primer 3 software (version 040) based on sequencesof carp available in GenBank (httpwwwncbinlmnihgov)
Relative gene expression values were obtained using BioRad CFX manager software (Version 21)
3 Results
31 Fish Growth Mean weight of fish increased at a steadyrate up to 6months of age Duringwinter season (6months to8months of age) the rate ofmonthlyweight gainwas reducedmarginally which again recovered during 9th- to 12th-monthperiodThere was a net increase inmean length in all the timepoints After 12 months the average weight of fish was around700 g and length was approximately 40 cm (Figure 1)
32 mRNA Transcription Pattern of MyogenicRegulatory Factors
321 Reverse Transcriptase PCR (RT-PCR) MyoD mRNAexpression was found to be high in the embryo stagesand decreased gradually after 5 months of age reachingthe minimum at 12 months The highest expression wasobtained at 24 h after fertilization (Figure 2) Expression ofmyf-5 also reached its maximum at 24 h after fertilizationand decreased gradually to reach the basal level (Figure 2)Myogenin expression did not show anymarked alteration andmaintained a steady expression pattern up to 12 months withslightmodulation (Figure 2)MEF2A had a steady expressionpattern with slight elevation during 6 h after fertilization to1 month of age After 1 month it decreased slightly andthen became almost stable throughout the rest part of theexperimental period (Figure 2)
322 Quantitative Real-Time PCR (qRT-PCR) QuantitativePCR was done at 5 time points (6 h 24 h 1 month 6 months
0100200300400500600700800
Wei
ght o
f fish
(g)
Age group of fish (1 month to 12 months)
1 m
onth
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(a)
05
1015202530354045
Leng
th o
f fish
(cm
)
Age group of fish (1 month to 12 months)1
mon
th
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(b)
Figure 1 Monthly growth of Labeo rohita from 1 month to 12months of age (a) Mean weight of fish (b) Mean length of fishValues are expressed as mean plusmn SEM (119873 = 6)
and 12 months) All the four transcription factors showed theexpression pattern similar to that of RT-PCR (Figure 3) andcorroborated with RT-PCR results
33 Construction of Phylogenetic Tree Partial nucleotidecoding sequences of three genes (MyoD myf-5 andMEF2A)were submitted to GenBank (httpwwwncbinlmnihgov)The GenBank accession numbers are KC3445371 for MyoDKC3445361 for myf-5 and KC3445351 for MEF2A Molecu-lar phylogenetic relationship (based on translated amino acidsequences) of Labeo MyoD myf-5 and MEF2A with that ofother closely related species was depicted by reconstructingphylogenetic tree using neighbor-joining method (Figure 4)
4 Discussion
The growth pattern of the experimental fishes followedsimilar annual pattern of growth of Labeo species in situas reported by Jhingran [18] (Figure 1) After fertilizationdifferentiation of skeletal muscle is initiated by MyoD whichbinds directly to the regulatory regions of a wide number ofgenes and regulates their expression during differentiation[19 20] BothMyoD andmyf-5 are necessary for the initiationof myogenesis in vertebrates Disruption of both genes inmice results in the absence of skeletal muscle cells [21]
6 International Journal of Zoology
myf-5
MyoD300
300
(bp)
300
Myogenin
MEF2A200
300
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
120573-Actin
100
bp ru
ler
(a)
002040608
112141618
Rela
tive d
ensit
omet
ric v
alue
(au
)
MyoDmyf-5
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(b)
Relat
ive d
ensit
omet
ric v
alue
002040608
112141618
2
(au
)
MyogeninMEF2A
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(c)
Figure 2 mRNA transcription pattern of myogenic regulatory factors by RT-PCR (a) expression of MyoD myf-5 myogeninand MEF2AmRNA at different time points of growth (6 hours to 12 months) (b) relative densitometric analysis of MyoD and myf-5 expression and (c)relative densitometric analysis of myogenin and MEF2A expression Values are expressed as mean plusmn SEM (119873 = 6)
Myogenic cells undergo active proliferation before cell cyclearrest and fusion into myotubes MyoD and Myf-5 play thecentral role in specifying muscle lineage andMyoD is the keyregulator of maintaining balance between the differentiationand proliferation [22] In the present study the expression ofMyoD andmyf-5 was higher in the embryonic stages of Labeorohita Both the genes followed nearly similar pattern ofexpression as depicted by semiquantitative and quantitativePCR (Figure 3) In mouse myf-5 was reported as the firstexpressedMRF in themyotomal muscle [23] and in commoncarp a high level of mRNA transcripts of myf-5 was detectedat 30 h after fertilization [9] In our study considerableamount of MyoD mRNA transcript was detected at 12 h afterfertilization stage (Figure 2(b)) and myf-5 had the highestlevel of expression at 24 h after fertilization (Figure 2(b))So in this particular fish the MyoD and myf-5 expressionpattern is not similar with the same in other fish speciesreported earlier [9 24]
Postnatal muscle growth involves hypertrophy of musclefibres which require additional nuclei to maintain a relativelyconstant nuclear to cytoplasmic ratio These nuclei are pro-vided by activated myogenic stem cells which also express
myogenic bHLH proteins myf-5 and MyoD expressions inskeletal muscles are followed by upregulation of myogeninand of MEF2 family factors which enhance expression ofmuscle differentiation genes [25] We observed that myo-geninmRNA transcript was present in a considerable amountat all stages with highest value at 24 h (Figure 3) MEF2family of transcription factors specifically bind to an ATrich sequence present inmanymuscle specific promoters andenhancers [26] In zebrafish knockdown ofMEF2A has beenshown to downregulate a large set of genes encoding con-tractile proteins such as troponins myosin heavy and lightchains and 120572-tropomyosin [27] In Labeo rohita MEF2Aexpressed at all the stages showing the high level of expressionat 1 monthThis expression pattern of myogenin andMEF2Ais similar to the pattern in common carp described byKobiyama et al 1998 [9] Muscle growth in fish involvesthe production of new muscle fibres in addition to musclefibre hypertrophy [6]The continued expression of myogeninand MEF2A in Labeo rohita reflects activated myogenic cellswhich help to maintain continuous hypertrophy as well ashyperplasia of skeletal muscle In embryonic stages most ofthe muscle cells remain in the early differentiation stage and
Figure 3 mRNA expression of four myogenic regulatory factors in Labeo rohita at 6 h 24 h 1 month 6 months and 12 months of age (a)MyoD (b) myf-5 (c) myogenin and (d) MEF2A
proliferation stage while most muscle cells of adult are in theterminal differentiation stage [24] Our results clearly showedthat MyoD and myf-5 mRNAs are expressed at high level inthe early embryonic stages whereas myogenin and MEF2Aare expressed afterMyoDandmyf-5 expression and remainedactive in adult stage tomaintain differentiation and growth ofskeletal muscle fibres MyoD is reported to play an importantrole in the arrest of cellular growth Its highest level wasdetected at early G1 phase and the lowest level was at G1 to Sphase transitionMyoD and its cofactors play a critical role inmyoblast cell cycle withdrawal When MyoD is maximal andmyf-5 is down cells exit their cycle into differentiation Theopposite pattern is observed in quiescent nondifferentiatingmyoblasts a high myf-5 and no MyoD [22] In Labeo rohitaboth the weight rate and length rate between the 5th and6th month were significantly greater than those of before 5thmonthThe expression ofMyoDwas lower than that of myf-5after 5thmonth Considering two relationships (expression ofmyf-5MyoD with muscle cells proliferationdifferentiationand muscle cells proliferationdifferentiation with musclehypertrophyhyperplasia) it may be supposed that in thisfish species the muscle development after 5th month was
properly balanced by both hypertrophy and hyperplasiaThe four transcription factors therefore play pivotal rolein the regulation of muscle growth in an overlapping andinterconnected way
Molecular phylogenetic tree showed that Labeo MyoDMEF2A and myf-5 are more closely related to that of com-mon carp (Cyprinus carpio) than any other species Previousreport showed high similarities of MyoD myf-5 andMEF2Asequences between carp and zebrafish but the similaritiesweremore prominent in the bHLH region (MyoD andmyf-5)and MADS box region (MEF2A) than total coding sequence[28]
Further extensive studies are warranted to evaluate theregulation of MRF gene expression pattern in fish whichmay provide insight into the signaling pathway controllingmuscle cell differentiation through regulatory transcriptionfactors Information is available on tissue specific embryonicexpression pattern of these transcription factors Contrast-ingly studies on long term age specific pattern of expressionare lacking To the best of our knowledge this is the firstreport on myogenic regulatory transcription factors in any
8 International Journal of Zoology
9499
5199
98
100
100
62
100
01
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
The PCR products of MyoD myf-5 and MEF2A weresubjected to partial sequence analysis to confirm thegenes Sequencing was done by Xcelris Genomics Pvt LtdNucleotide sequences were translated into amino acidsequences for further analysis Sequence alignments wereobtained using Clustal Omega software (EMBL-EBI httpwwwebiacuk) Phylogenetic and molecular evolutionaryanalyses were conducted using MEGA version 6 [17] Phy-logenetic tree was reconstructed using neighbour-joiningdistance algorithms from translated amino acid sequences inPAM matrix Statistical consistency was evaluated by 1000bootstrap resamplings of the data
26 Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR)was performed using a Bio Rad CFX96 Real-Time PCRSystem (Bio Rad Laboratories Inc Hercules CA USA) andan iQ SYBERGreen superMixKit (Bio Rad Laboratories IncHercules CA USA) which were used in accordance withthe manufacturersrsquo instructions Standard reaction mixtures(15 120583L) were assembled using 75 120583L of iQ SYBERGreen superMix 2x 300 nMof each primer and 100 ng of template cDNAFor amplification of MyoD myf-5 myogenin MEF2A andGAPDH gene specific primer pairs were designed (Table 2)using Primer 3 software (version 040) based on sequencesof carp available in GenBank (httpwwwncbinlmnihgov)
Relative gene expression values were obtained using BioRad CFX manager software (Version 21)
3 Results
31 Fish Growth Mean weight of fish increased at a steadyrate up to 6months of age Duringwinter season (6months to8months of age) the rate ofmonthlyweight gainwas reducedmarginally which again recovered during 9th- to 12th-monthperiodThere was a net increase inmean length in all the timepoints After 12 months the average weight of fish was around700 g and length was approximately 40 cm (Figure 1)
32 mRNA Transcription Pattern of MyogenicRegulatory Factors
321 Reverse Transcriptase PCR (RT-PCR) MyoD mRNAexpression was found to be high in the embryo stagesand decreased gradually after 5 months of age reachingthe minimum at 12 months The highest expression wasobtained at 24 h after fertilization (Figure 2) Expression ofmyf-5 also reached its maximum at 24 h after fertilizationand decreased gradually to reach the basal level (Figure 2)Myogenin expression did not show anymarked alteration andmaintained a steady expression pattern up to 12 months withslightmodulation (Figure 2)MEF2A had a steady expressionpattern with slight elevation during 6 h after fertilization to1 month of age After 1 month it decreased slightly andthen became almost stable throughout the rest part of theexperimental period (Figure 2)
322 Quantitative Real-Time PCR (qRT-PCR) QuantitativePCR was done at 5 time points (6 h 24 h 1 month 6 months
0100200300400500600700800
Wei
ght o
f fish
(g)
Age group of fish (1 month to 12 months)
1 m
onth
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(a)
05
1015202530354045
Leng
th o
f fish
(cm
)
Age group of fish (1 month to 12 months)1
mon
th
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(b)
Figure 1 Monthly growth of Labeo rohita from 1 month to 12months of age (a) Mean weight of fish (b) Mean length of fishValues are expressed as mean plusmn SEM (119873 = 6)
and 12 months) All the four transcription factors showed theexpression pattern similar to that of RT-PCR (Figure 3) andcorroborated with RT-PCR results
33 Construction of Phylogenetic Tree Partial nucleotidecoding sequences of three genes (MyoD myf-5 andMEF2A)were submitted to GenBank (httpwwwncbinlmnihgov)The GenBank accession numbers are KC3445371 for MyoDKC3445361 for myf-5 and KC3445351 for MEF2A Molecu-lar phylogenetic relationship (based on translated amino acidsequences) of Labeo MyoD myf-5 and MEF2A with that ofother closely related species was depicted by reconstructingphylogenetic tree using neighbor-joining method (Figure 4)
4 Discussion
The growth pattern of the experimental fishes followedsimilar annual pattern of growth of Labeo species in situas reported by Jhingran [18] (Figure 1) After fertilizationdifferentiation of skeletal muscle is initiated by MyoD whichbinds directly to the regulatory regions of a wide number ofgenes and regulates their expression during differentiation[19 20] BothMyoD andmyf-5 are necessary for the initiationof myogenesis in vertebrates Disruption of both genes inmice results in the absence of skeletal muscle cells [21]
6 International Journal of Zoology
myf-5
MyoD300
300
(bp)
300
Myogenin
MEF2A200
300
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
120573-Actin
100
bp ru
ler
(a)
002040608
112141618
Rela
tive d
ensit
omet
ric v
alue
(au
)
MyoDmyf-5
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(b)
Relat
ive d
ensit
omet
ric v
alue
002040608
112141618
2
(au
)
MyogeninMEF2A
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(c)
Figure 2 mRNA transcription pattern of myogenic regulatory factors by RT-PCR (a) expression of MyoD myf-5 myogeninand MEF2AmRNA at different time points of growth (6 hours to 12 months) (b) relative densitometric analysis of MyoD and myf-5 expression and (c)relative densitometric analysis of myogenin and MEF2A expression Values are expressed as mean plusmn SEM (119873 = 6)
Myogenic cells undergo active proliferation before cell cyclearrest and fusion into myotubes MyoD and Myf-5 play thecentral role in specifying muscle lineage andMyoD is the keyregulator of maintaining balance between the differentiationand proliferation [22] In the present study the expression ofMyoD andmyf-5 was higher in the embryonic stages of Labeorohita Both the genes followed nearly similar pattern ofexpression as depicted by semiquantitative and quantitativePCR (Figure 3) In mouse myf-5 was reported as the firstexpressedMRF in themyotomal muscle [23] and in commoncarp a high level of mRNA transcripts of myf-5 was detectedat 30 h after fertilization [9] In our study considerableamount of MyoD mRNA transcript was detected at 12 h afterfertilization stage (Figure 2(b)) and myf-5 had the highestlevel of expression at 24 h after fertilization (Figure 2(b))So in this particular fish the MyoD and myf-5 expressionpattern is not similar with the same in other fish speciesreported earlier [9 24]
Postnatal muscle growth involves hypertrophy of musclefibres which require additional nuclei to maintain a relativelyconstant nuclear to cytoplasmic ratio These nuclei are pro-vided by activated myogenic stem cells which also express
myogenic bHLH proteins myf-5 and MyoD expressions inskeletal muscles are followed by upregulation of myogeninand of MEF2 family factors which enhance expression ofmuscle differentiation genes [25] We observed that myo-geninmRNA transcript was present in a considerable amountat all stages with highest value at 24 h (Figure 3) MEF2family of transcription factors specifically bind to an ATrich sequence present inmanymuscle specific promoters andenhancers [26] In zebrafish knockdown ofMEF2A has beenshown to downregulate a large set of genes encoding con-tractile proteins such as troponins myosin heavy and lightchains and 120572-tropomyosin [27] In Labeo rohita MEF2Aexpressed at all the stages showing the high level of expressionat 1 monthThis expression pattern of myogenin andMEF2Ais similar to the pattern in common carp described byKobiyama et al 1998 [9] Muscle growth in fish involvesthe production of new muscle fibres in addition to musclefibre hypertrophy [6]The continued expression of myogeninand MEF2A in Labeo rohita reflects activated myogenic cellswhich help to maintain continuous hypertrophy as well ashyperplasia of skeletal muscle In embryonic stages most ofthe muscle cells remain in the early differentiation stage and
Figure 3 mRNA expression of four myogenic regulatory factors in Labeo rohita at 6 h 24 h 1 month 6 months and 12 months of age (a)MyoD (b) myf-5 (c) myogenin and (d) MEF2A
proliferation stage while most muscle cells of adult are in theterminal differentiation stage [24] Our results clearly showedthat MyoD and myf-5 mRNAs are expressed at high level inthe early embryonic stages whereas myogenin and MEF2Aare expressed afterMyoDandmyf-5 expression and remainedactive in adult stage tomaintain differentiation and growth ofskeletal muscle fibres MyoD is reported to play an importantrole in the arrest of cellular growth Its highest level wasdetected at early G1 phase and the lowest level was at G1 to Sphase transitionMyoD and its cofactors play a critical role inmyoblast cell cycle withdrawal When MyoD is maximal andmyf-5 is down cells exit their cycle into differentiation Theopposite pattern is observed in quiescent nondifferentiatingmyoblasts a high myf-5 and no MyoD [22] In Labeo rohitaboth the weight rate and length rate between the 5th and6th month were significantly greater than those of before 5thmonthThe expression ofMyoDwas lower than that of myf-5after 5thmonth Considering two relationships (expression ofmyf-5MyoD with muscle cells proliferationdifferentiationand muscle cells proliferationdifferentiation with musclehypertrophyhyperplasia) it may be supposed that in thisfish species the muscle development after 5th month was
properly balanced by both hypertrophy and hyperplasiaThe four transcription factors therefore play pivotal rolein the regulation of muscle growth in an overlapping andinterconnected way
Molecular phylogenetic tree showed that Labeo MyoDMEF2A and myf-5 are more closely related to that of com-mon carp (Cyprinus carpio) than any other species Previousreport showed high similarities of MyoD myf-5 andMEF2Asequences between carp and zebrafish but the similaritiesweremore prominent in the bHLH region (MyoD andmyf-5)and MADS box region (MEF2A) than total coding sequence[28]
Further extensive studies are warranted to evaluate theregulation of MRF gene expression pattern in fish whichmay provide insight into the signaling pathway controllingmuscle cell differentiation through regulatory transcriptionfactors Information is available on tissue specific embryonicexpression pattern of these transcription factors Contrast-ingly studies on long term age specific pattern of expressionare lacking To the best of our knowledge this is the firstreport on myogenic regulatory transcription factors in any
8 International Journal of Zoology
9499
5199
98
100
100
62
100
01
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
The PCR products of MyoD myf-5 and MEF2A weresubjected to partial sequence analysis to confirm thegenes Sequencing was done by Xcelris Genomics Pvt LtdNucleotide sequences were translated into amino acidsequences for further analysis Sequence alignments wereobtained using Clustal Omega software (EMBL-EBI httpwwwebiacuk) Phylogenetic and molecular evolutionaryanalyses were conducted using MEGA version 6 [17] Phy-logenetic tree was reconstructed using neighbour-joiningdistance algorithms from translated amino acid sequences inPAM matrix Statistical consistency was evaluated by 1000bootstrap resamplings of the data
26 Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR)was performed using a Bio Rad CFX96 Real-Time PCRSystem (Bio Rad Laboratories Inc Hercules CA USA) andan iQ SYBERGreen superMixKit (Bio Rad Laboratories IncHercules CA USA) which were used in accordance withthe manufacturersrsquo instructions Standard reaction mixtures(15 120583L) were assembled using 75 120583L of iQ SYBERGreen superMix 2x 300 nMof each primer and 100 ng of template cDNAFor amplification of MyoD myf-5 myogenin MEF2A andGAPDH gene specific primer pairs were designed (Table 2)using Primer 3 software (version 040) based on sequencesof carp available in GenBank (httpwwwncbinlmnihgov)
Relative gene expression values were obtained using BioRad CFX manager software (Version 21)
3 Results
31 Fish Growth Mean weight of fish increased at a steadyrate up to 6months of age Duringwinter season (6months to8months of age) the rate ofmonthlyweight gainwas reducedmarginally which again recovered during 9th- to 12th-monthperiodThere was a net increase inmean length in all the timepoints After 12 months the average weight of fish was around700 g and length was approximately 40 cm (Figure 1)
32 mRNA Transcription Pattern of MyogenicRegulatory Factors
321 Reverse Transcriptase PCR (RT-PCR) MyoD mRNAexpression was found to be high in the embryo stagesand decreased gradually after 5 months of age reachingthe minimum at 12 months The highest expression wasobtained at 24 h after fertilization (Figure 2) Expression ofmyf-5 also reached its maximum at 24 h after fertilizationand decreased gradually to reach the basal level (Figure 2)Myogenin expression did not show anymarked alteration andmaintained a steady expression pattern up to 12 months withslightmodulation (Figure 2)MEF2A had a steady expressionpattern with slight elevation during 6 h after fertilization to1 month of age After 1 month it decreased slightly andthen became almost stable throughout the rest part of theexperimental period (Figure 2)
322 Quantitative Real-Time PCR (qRT-PCR) QuantitativePCR was done at 5 time points (6 h 24 h 1 month 6 months
0100200300400500600700800
Wei
ght o
f fish
(g)
Age group of fish (1 month to 12 months)
1 m
onth
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(a)
05
1015202530354045
Leng
th o
f fish
(cm
)
Age group of fish (1 month to 12 months)1
mon
th
2m
onth
s
3m
onth
s
4m
onth
s
5m
onth
s
6m
onth
s
7m
onth
s
8m
onth
s
9m
onth
s
10m
onth
s
11m
onth
s
12m
onth
s
(b)
Figure 1 Monthly growth of Labeo rohita from 1 month to 12months of age (a) Mean weight of fish (b) Mean length of fishValues are expressed as mean plusmn SEM (119873 = 6)
and 12 months) All the four transcription factors showed theexpression pattern similar to that of RT-PCR (Figure 3) andcorroborated with RT-PCR results
33 Construction of Phylogenetic Tree Partial nucleotidecoding sequences of three genes (MyoD myf-5 andMEF2A)were submitted to GenBank (httpwwwncbinlmnihgov)The GenBank accession numbers are KC3445371 for MyoDKC3445361 for myf-5 and KC3445351 for MEF2A Molecu-lar phylogenetic relationship (based on translated amino acidsequences) of Labeo MyoD myf-5 and MEF2A with that ofother closely related species was depicted by reconstructingphylogenetic tree using neighbor-joining method (Figure 4)
4 Discussion
The growth pattern of the experimental fishes followedsimilar annual pattern of growth of Labeo species in situas reported by Jhingran [18] (Figure 1) After fertilizationdifferentiation of skeletal muscle is initiated by MyoD whichbinds directly to the regulatory regions of a wide number ofgenes and regulates their expression during differentiation[19 20] BothMyoD andmyf-5 are necessary for the initiationof myogenesis in vertebrates Disruption of both genes inmice results in the absence of skeletal muscle cells [21]
6 International Journal of Zoology
myf-5
MyoD300
300
(bp)
300
Myogenin
MEF2A200
300
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
120573-Actin
100
bp ru
ler
(a)
002040608
112141618
Rela
tive d
ensit
omet
ric v
alue
(au
)
MyoDmyf-5
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(b)
Relat
ive d
ensit
omet
ric v
alue
002040608
112141618
2
(au
)
MyogeninMEF2A
Age group of fish (6h to 12 months)
6h
12h
24h
48
h1
mon
th2
mon
ths
3m
onth
s4
mon
ths
5m
onth
s6
mon
ths
7m
onth
s8
mon
ths
9m
onth
s10
mon
ths
11m
onth
s12
mon
ths
(c)
Figure 2 mRNA transcription pattern of myogenic regulatory factors by RT-PCR (a) expression of MyoD myf-5 myogeninand MEF2AmRNA at different time points of growth (6 hours to 12 months) (b) relative densitometric analysis of MyoD and myf-5 expression and (c)relative densitometric analysis of myogenin and MEF2A expression Values are expressed as mean plusmn SEM (119873 = 6)
Myogenic cells undergo active proliferation before cell cyclearrest and fusion into myotubes MyoD and Myf-5 play thecentral role in specifying muscle lineage andMyoD is the keyregulator of maintaining balance between the differentiationand proliferation [22] In the present study the expression ofMyoD andmyf-5 was higher in the embryonic stages of Labeorohita Both the genes followed nearly similar pattern ofexpression as depicted by semiquantitative and quantitativePCR (Figure 3) In mouse myf-5 was reported as the firstexpressedMRF in themyotomal muscle [23] and in commoncarp a high level of mRNA transcripts of myf-5 was detectedat 30 h after fertilization [9] In our study considerableamount of MyoD mRNA transcript was detected at 12 h afterfertilization stage (Figure 2(b)) and myf-5 had the highestlevel of expression at 24 h after fertilization (Figure 2(b))So in this particular fish the MyoD and myf-5 expressionpattern is not similar with the same in other fish speciesreported earlier [9 24]
Postnatal muscle growth involves hypertrophy of musclefibres which require additional nuclei to maintain a relativelyconstant nuclear to cytoplasmic ratio These nuclei are pro-vided by activated myogenic stem cells which also express
myogenic bHLH proteins myf-5 and MyoD expressions inskeletal muscles are followed by upregulation of myogeninand of MEF2 family factors which enhance expression ofmuscle differentiation genes [25] We observed that myo-geninmRNA transcript was present in a considerable amountat all stages with highest value at 24 h (Figure 3) MEF2family of transcription factors specifically bind to an ATrich sequence present inmanymuscle specific promoters andenhancers [26] In zebrafish knockdown ofMEF2A has beenshown to downregulate a large set of genes encoding con-tractile proteins such as troponins myosin heavy and lightchains and 120572-tropomyosin [27] In Labeo rohita MEF2Aexpressed at all the stages showing the high level of expressionat 1 monthThis expression pattern of myogenin andMEF2Ais similar to the pattern in common carp described byKobiyama et al 1998 [9] Muscle growth in fish involvesthe production of new muscle fibres in addition to musclefibre hypertrophy [6]The continued expression of myogeninand MEF2A in Labeo rohita reflects activated myogenic cellswhich help to maintain continuous hypertrophy as well ashyperplasia of skeletal muscle In embryonic stages most ofthe muscle cells remain in the early differentiation stage and
Figure 3 mRNA expression of four myogenic regulatory factors in Labeo rohita at 6 h 24 h 1 month 6 months and 12 months of age (a)MyoD (b) myf-5 (c) myogenin and (d) MEF2A
proliferation stage while most muscle cells of adult are in theterminal differentiation stage [24] Our results clearly showedthat MyoD and myf-5 mRNAs are expressed at high level inthe early embryonic stages whereas myogenin and MEF2Aare expressed afterMyoDandmyf-5 expression and remainedactive in adult stage tomaintain differentiation and growth ofskeletal muscle fibres MyoD is reported to play an importantrole in the arrest of cellular growth Its highest level wasdetected at early G1 phase and the lowest level was at G1 to Sphase transitionMyoD and its cofactors play a critical role inmyoblast cell cycle withdrawal When MyoD is maximal andmyf-5 is down cells exit their cycle into differentiation Theopposite pattern is observed in quiescent nondifferentiatingmyoblasts a high myf-5 and no MyoD [22] In Labeo rohitaboth the weight rate and length rate between the 5th and6th month were significantly greater than those of before 5thmonthThe expression ofMyoDwas lower than that of myf-5after 5thmonth Considering two relationships (expression ofmyf-5MyoD with muscle cells proliferationdifferentiationand muscle cells proliferationdifferentiation with musclehypertrophyhyperplasia) it may be supposed that in thisfish species the muscle development after 5th month was
properly balanced by both hypertrophy and hyperplasiaThe four transcription factors therefore play pivotal rolein the regulation of muscle growth in an overlapping andinterconnected way
Molecular phylogenetic tree showed that Labeo MyoDMEF2A and myf-5 are more closely related to that of com-mon carp (Cyprinus carpio) than any other species Previousreport showed high similarities of MyoD myf-5 andMEF2Asequences between carp and zebrafish but the similaritiesweremore prominent in the bHLH region (MyoD andmyf-5)and MADS box region (MEF2A) than total coding sequence[28]
Further extensive studies are warranted to evaluate theregulation of MRF gene expression pattern in fish whichmay provide insight into the signaling pathway controllingmuscle cell differentiation through regulatory transcriptionfactors Information is available on tissue specific embryonicexpression pattern of these transcription factors Contrast-ingly studies on long term age specific pattern of expressionare lacking To the best of our knowledge this is the firstreport on myogenic regulatory transcription factors in any
8 International Journal of Zoology
9499
5199
98
100
100
62
100
01
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
Figure 2 mRNA transcription pattern of myogenic regulatory factors by RT-PCR (a) expression of MyoD myf-5 myogeninand MEF2AmRNA at different time points of growth (6 hours to 12 months) (b) relative densitometric analysis of MyoD and myf-5 expression and (c)relative densitometric analysis of myogenin and MEF2A expression Values are expressed as mean plusmn SEM (119873 = 6)
Myogenic cells undergo active proliferation before cell cyclearrest and fusion into myotubes MyoD and Myf-5 play thecentral role in specifying muscle lineage andMyoD is the keyregulator of maintaining balance between the differentiationand proliferation [22] In the present study the expression ofMyoD andmyf-5 was higher in the embryonic stages of Labeorohita Both the genes followed nearly similar pattern ofexpression as depicted by semiquantitative and quantitativePCR (Figure 3) In mouse myf-5 was reported as the firstexpressedMRF in themyotomal muscle [23] and in commoncarp a high level of mRNA transcripts of myf-5 was detectedat 30 h after fertilization [9] In our study considerableamount of MyoD mRNA transcript was detected at 12 h afterfertilization stage (Figure 2(b)) and myf-5 had the highestlevel of expression at 24 h after fertilization (Figure 2(b))So in this particular fish the MyoD and myf-5 expressionpattern is not similar with the same in other fish speciesreported earlier [9 24]
Postnatal muscle growth involves hypertrophy of musclefibres which require additional nuclei to maintain a relativelyconstant nuclear to cytoplasmic ratio These nuclei are pro-vided by activated myogenic stem cells which also express
myogenic bHLH proteins myf-5 and MyoD expressions inskeletal muscles are followed by upregulation of myogeninand of MEF2 family factors which enhance expression ofmuscle differentiation genes [25] We observed that myo-geninmRNA transcript was present in a considerable amountat all stages with highest value at 24 h (Figure 3) MEF2family of transcription factors specifically bind to an ATrich sequence present inmanymuscle specific promoters andenhancers [26] In zebrafish knockdown ofMEF2A has beenshown to downregulate a large set of genes encoding con-tractile proteins such as troponins myosin heavy and lightchains and 120572-tropomyosin [27] In Labeo rohita MEF2Aexpressed at all the stages showing the high level of expressionat 1 monthThis expression pattern of myogenin andMEF2Ais similar to the pattern in common carp described byKobiyama et al 1998 [9] Muscle growth in fish involvesthe production of new muscle fibres in addition to musclefibre hypertrophy [6]The continued expression of myogeninand MEF2A in Labeo rohita reflects activated myogenic cellswhich help to maintain continuous hypertrophy as well ashyperplasia of skeletal muscle In embryonic stages most ofthe muscle cells remain in the early differentiation stage and
Figure 3 mRNA expression of four myogenic regulatory factors in Labeo rohita at 6 h 24 h 1 month 6 months and 12 months of age (a)MyoD (b) myf-5 (c) myogenin and (d) MEF2A
proliferation stage while most muscle cells of adult are in theterminal differentiation stage [24] Our results clearly showedthat MyoD and myf-5 mRNAs are expressed at high level inthe early embryonic stages whereas myogenin and MEF2Aare expressed afterMyoDandmyf-5 expression and remainedactive in adult stage tomaintain differentiation and growth ofskeletal muscle fibres MyoD is reported to play an importantrole in the arrest of cellular growth Its highest level wasdetected at early G1 phase and the lowest level was at G1 to Sphase transitionMyoD and its cofactors play a critical role inmyoblast cell cycle withdrawal When MyoD is maximal andmyf-5 is down cells exit their cycle into differentiation Theopposite pattern is observed in quiescent nondifferentiatingmyoblasts a high myf-5 and no MyoD [22] In Labeo rohitaboth the weight rate and length rate between the 5th and6th month were significantly greater than those of before 5thmonthThe expression ofMyoDwas lower than that of myf-5after 5thmonth Considering two relationships (expression ofmyf-5MyoD with muscle cells proliferationdifferentiationand muscle cells proliferationdifferentiation with musclehypertrophyhyperplasia) it may be supposed that in thisfish species the muscle development after 5th month was
properly balanced by both hypertrophy and hyperplasiaThe four transcription factors therefore play pivotal rolein the regulation of muscle growth in an overlapping andinterconnected way
Molecular phylogenetic tree showed that Labeo MyoDMEF2A and myf-5 are more closely related to that of com-mon carp (Cyprinus carpio) than any other species Previousreport showed high similarities of MyoD myf-5 andMEF2Asequences between carp and zebrafish but the similaritiesweremore prominent in the bHLH region (MyoD andmyf-5)and MADS box region (MEF2A) than total coding sequence[28]
Further extensive studies are warranted to evaluate theregulation of MRF gene expression pattern in fish whichmay provide insight into the signaling pathway controllingmuscle cell differentiation through regulatory transcriptionfactors Information is available on tissue specific embryonicexpression pattern of these transcription factors Contrast-ingly studies on long term age specific pattern of expressionare lacking To the best of our knowledge this is the firstreport on myogenic regulatory transcription factors in any
8 International Journal of Zoology
9499
5199
98
100
100
62
100
01
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
Figure 3 mRNA expression of four myogenic regulatory factors in Labeo rohita at 6 h 24 h 1 month 6 months and 12 months of age (a)MyoD (b) myf-5 (c) myogenin and (d) MEF2A
proliferation stage while most muscle cells of adult are in theterminal differentiation stage [24] Our results clearly showedthat MyoD and myf-5 mRNAs are expressed at high level inthe early embryonic stages whereas myogenin and MEF2Aare expressed afterMyoDandmyf-5 expression and remainedactive in adult stage tomaintain differentiation and growth ofskeletal muscle fibres MyoD is reported to play an importantrole in the arrest of cellular growth Its highest level wasdetected at early G1 phase and the lowest level was at G1 to Sphase transitionMyoD and its cofactors play a critical role inmyoblast cell cycle withdrawal When MyoD is maximal andmyf-5 is down cells exit their cycle into differentiation Theopposite pattern is observed in quiescent nondifferentiatingmyoblasts a high myf-5 and no MyoD [22] In Labeo rohitaboth the weight rate and length rate between the 5th and6th month were significantly greater than those of before 5thmonthThe expression ofMyoDwas lower than that of myf-5after 5thmonth Considering two relationships (expression ofmyf-5MyoD with muscle cells proliferationdifferentiationand muscle cells proliferationdifferentiation with musclehypertrophyhyperplasia) it may be supposed that in thisfish species the muscle development after 5th month was
properly balanced by both hypertrophy and hyperplasiaThe four transcription factors therefore play pivotal rolein the regulation of muscle growth in an overlapping andinterconnected way
Molecular phylogenetic tree showed that Labeo MyoDMEF2A and myf-5 are more closely related to that of com-mon carp (Cyprinus carpio) than any other species Previousreport showed high similarities of MyoD myf-5 andMEF2Asequences between carp and zebrafish but the similaritiesweremore prominent in the bHLH region (MyoD andmyf-5)and MADS box region (MEF2A) than total coding sequence[28]
Further extensive studies are warranted to evaluate theregulation of MRF gene expression pattern in fish whichmay provide insight into the signaling pathway controllingmuscle cell differentiation through regulatory transcriptionfactors Information is available on tissue specific embryonicexpression pattern of these transcription factors Contrast-ingly studies on long term age specific pattern of expressionare lacking To the best of our knowledge this is the firstreport on myogenic regulatory transcription factors in any
8 International Journal of Zoology
9499
5199
98
100
100
62
100
01
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
C idella MyoD (JQ 7938931)S dolichonema MyoD (KC1841221)
S prenanti MyoD (JQ7938941)
C carpio MyoD (AB0128821)L rohita MyoD (KC3445371)
I punctatus MyoD (AY5343281)O mykiss MyoD (X757981)
S salar MyoD (AJ8518271)H molitrix MyoD (GU2184631)
D rerio MyoD (AF 3185032)
H nobilis MyoD (GU2184641)G morhua MyoD (AF 3299032)
(a)
54
100
10099
100
01
O mykiss Myf-5 (AY 7512831)D rerio Myf-5 (AF 2534701)
C molitorella Myf-5 (GU 2895081)S Salar Myf-5 (NM 0011236441)
S prenanti Myf -5 (JQ 7938951)C idella Myf-5 (GU 2902271)
C carpio Myf -5 (AB 0128831)L rohita Myf-5 (KC 3445361)
(b)
100
9372
100
01
L rohita MEF2A (KC 3445351)C carpio MEF2A (AB 0128841)
I punctatus MEF2A (JT 4193411)C batrachus MEF2A (KF 0072291)
D rerio MEF2A (U 665681)O niloticus MEF2A (XM 0054707861)
G morhua MEF2A (GQ 3061511)
(c)
Figure 4 Molecular phylogenetic tree of (a) MyoD (b) myf-5 and (c) MEF2A This dendrogram is based on the translated amino acidsequences GenBank accession numbers are given in the brackets and sequences acquired from this study are underlined accordingly
Indian carp describing monthly expression pattern of thesetranscription factors in juvenile and adult Labeo rohita
5 Conclusion
This study demonstrated the mRNA transcription pattern ofMyoD myf-5 myogenin and MEF2A in embryo and adultLabeo rohita and depicted probable phylogenetic relationshipof this fish with other related species with respect to MyoDmyf-5 and MEF2A gene MyoD was expressed first in theembryo along with myf-5 to initiate myogenesis MEF2Aandmyogenin were expressed throughout the developmentalperiod to help in differentiation of muscle cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to the Department of Science andTechnology Government of West Bengal for the projectGrant [917(Sanc)STPSampT2G-12009] to Ansuman Chat-topadhyay and Junior Research Fellowship to Archya Sen-gupta Archya Sengupta is also thankful to UGC for non-NET fellowship Sandip Mukherjee gratefully acknowledges
International Journal of Zoology 9
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999
UGC for BSR fellowship and Shelley Bhattacharya gratefullyacknowledges the NASI Senior Scientist Platinum JubileeFellowship The authors gratefully acknowledge the labora-tory and academic support of Professor Samir Bhattacharyaand Dr Jolly Basak and the academic help of Dr AparajitaChatterjee and Dr Atish Ray
References
[1] J M Reecy S A Miller andMWebster ldquoRecent advances thatimpact skeletalmuscle growth and development researchrdquo Jour-nal of Animal Science vol 81 supplement 1 pp E1ndashE8 2003
[2] F Wachtler and B Christ ldquoThe basic embryology of skeletalmuscle formation in vertebrates the avian modelrdquo Seminars inDevelopmental Biology vol 3 pp 217ndash227 1992
[3] S H Devoto E Melancon J S Eisen and M WesterfieldldquoIdentification of separate slow and fast muscle precursor cellsin vivo prior to somite formationrdquoDevelopment vol 122 no 11pp 3371ndash3380 1996
[4] H Alami-Durante C Wrutniak-Cabello S J Kaushik and FMedale ldquoSkeletal muscle cellularity and expression ofmyogenicregulatory factors and myosin heavy chains in rainbow trout(Oncorhynchus mykiss) effects of changes in dietary plant pro-tein sources and amino acid profilesrdquoComparative Biochemistryand Physiology Part A vol 156 no 4 pp 561ndash568 2010
[5] A Rowlerson and A Veggetti ldquoCellular mechanisms of post-embryonic muscle growth in aquaculture speciesrdquo Fish Physiol-ogy vol 18 pp 103ndash140 2001
[6] I A Johnston N J Cole M Abercromby and V L A VieiraldquoEmbryonic temperaturemodulatesmuscle growth characteris-tics in larval and juvenile herringrdquoThe Journal of ExperimentalBiology vol 201 no 5 pp 623ndash646 1998
[7] E N Olson M Perry and R A Schulz ldquoRegulation of muscledifferentiation by the MEF2 family of MADS box transcriptionfactorsrdquo Developmental Biology vol 172 no 1 pp 2ndash14 1995
[8] HWeintraub ldquoTheMyoD family andmyogenesis redundancynetworks and thresholdsrdquo Cell vol 75 no 7 pp 1241ndash12441993
[9] A Kobiyama Y Nihei Y Hirayama et al ldquoMolecular cloningand developmental expression patterns of theMyoD andMEF2families of muscle transcription factors in the carprdquoThe Journalof Experimental Biology vol 201 no 20 pp 2801ndash2813 1998
[10] Y-H Chen W-C Lee C-H Cheng and H-J Tsai ldquoMus-cle regulatory factor gene Zebrafish (Danio rerio) myogenincDNArdquo Comparative Biochemistry and Physiology Part B vol127 no 1 pp 97ndash103 2000
[11] X Tan L Hoang and S J Du ldquoCharacterization of muscle-reg-ulatory genes Myf5 and Myogenin from striped bass and pro-moter analysis of muscle-specific expressionrdquo Marine Biotech-nology vol 4 no 6 pp 537ndash545 2002
[12] S J Du J Gao and V Anyangwe ldquoMuscle-specific expressionof myogenin in zebrafish embryos is controlled by multipleregulatory elements in the promoterrdquoComparative Biochemistryand Physiology Part B vol 134 no 1 pp 123ndash134 2003
[13] E Dodou S-M Xu and B L Black ldquomef2c is activated directlyby myogenic basic helix-loop-helix proteins during skeletalmuscle development in vivordquoMechanisms of Development vol120 no 9 pp 1021ndash1032 2003
[14] I A Johnston S Manthri A Smart P Campbell D Nickelland R Alderson ldquoPlasticity of muscle fibre number in seawater
stages of Atlantic salmon in response to photoperiod manipu-lationrdquoThe Journal of Experimental Biology vol 206 no 19 pp3425ndash3435 2003
[15] H Alami-Durante N Olive and M Rouel ldquoEarly thermal his-tory significantly affects the seasonal hyperplastic process oc-curring in the myotomal white muscle of Dicentrarchus labraxjuvenilesrdquo Cell and Tissue Research vol 327 no 3 pp 553ndash5702007
[16] N Li J Alam M I Venkatesan et al ldquoNrf2 is a key transcrip-tion factor that regulates antioxidant defense in macrophagesand epithelial cells protecting against the proinflammatoryand oxidizing effects of diesel exhaust chemicalsrdquo Journal ofImmunology vol 173 no 5 pp 3467ndash3481 2004
[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 pp 2725ndash2729 2013
[18] V G Jhingran Fish and Fisheries of India Hindustan Publish-ing Delhi India 3rd edition 1991
[19] DA Bergstrom BH PennA Strand R L S PerryMA Rud-nicki and S J Tapscott ldquoPromoter-specific regulation ofMyoDbinding and signal transduction cooperate to pattern geneexpressionrdquoMolecular Cell vol 9 no 3 pp 587ndash600 2002
[20] C A Berkes D A Bergstrom B H Penn K J Seaver P SKnoepfler and S J Tapscott ldquoPbx marks genes for activationbyMyoD indicating a role for a homeodomain protein in estab-lishing myogenic potentialrdquo Molecular Cell vol 14 no 4 pp465ndash477 2004
[21] M A Rudnicki P N J Schnegelsberg R H Stead T Braun HH Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993
[22] M Kitzmann and A Fernandez ldquoCrosstalk between cell cycleregulators and themyogenic factorMyoD in skeletalmyoblastsrdquoCellular and Molecular Life Sciences vol 58 no 4 pp 571ndash5792001
[23] M-O Ott E Bober G Lyons H Arnold andM BuckinghamldquoEarly expression of the myogenic regulatory gene myf-5in precursor cells of skeletal muscle in the mouse embryordquoDevelopment vol 111 no 4 pp 1097ndash1107 1991
[24] Y Zhang X Tan P Xu W Sun Y Xu and P Zhang ldquoQuan-titative comparison of the expression of myogenic regulatoryfactors in flounder (Paralichthys olivaceus) embryos and adulttissuesrdquo Chinese Journal of Oceanology and Limnology vol 28no 2 pp 248ndash253 2010
[25] K Yun and B Wold ldquoSkeletal muscle determination and dif-ferentiation story of a core regulatory network and its contextrdquoCurrent Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996
[26] P-Y Rescan J Montfort C Ralliere A Le Cam D Esquerreand K Hugot ldquoDynamic gene expression in fish muscle duringrecovery growth induced by a fasting-refeeding schedulerdquo BMCGenomics vol 8 article 438 2007
[27] Y Wang L Qian Y Dong et al ldquoMyocyte-specific enhancerfactor 2A is essential for zebrafish posterior somite develop-mentrdquoMechanisms of Development vol 123 no 10 pp 783ndash7912006
[28] S Watabe ldquoMyogenic regulatory factors and muscle differenti-ation during ontogeny in fishrdquo Journal of Fish Biology vol 55pp 1ndash18 1999