Research Article Expression Pattern of Myogenic Regulatory ...Environmental Toxicology Laboratory, Department of Zoology, Centre for Advanced Studies, Visva-Bharati University, Santiniketan,WestBengal,

Research ArticleExpression Pattern of Myogenic Regulatory Transcription FactormRNAs in the Embryo and Adult Labeo rohita (Hamilton 1822)

Archya Sengupta1 Sandip Mukherjee2 Shelley Bhattacharya2

Samar Kumar Saha3 and Ansuman Chattopadhyay1

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


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


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]


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


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


0

02

04

06

08

1

12Re

lativ

e fol

d of

expr

essio

n

MyoD relative gene expression1 month 6 months 12 months6h 24h

(a)

0

02

04

06

08

1

12

14

1 month 6 months 12 months

Relat

ive f

old

of ex

pres

sion

myf-5 relative gene expression6h 24h

(b)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion

Myogenin relative gene expression1 month 6 months 12 months6h 24h

(c)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion

MEF2A relative gene expression1 month 6 months 12 months6h 24h

(d)

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


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


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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


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


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


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





3 Results





0100200300400500600700800

Wei

ght o

f fish

(g)


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

)


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)




4 Discussion



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


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


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)






0

02

04

06

08

1

12Re

lativ

e fol

d of

expr

essio

n


(a)

0

02

04

06

08

1

12

14


Relat

ive f

old

of ex

pres

sion


(b)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(c)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(d)







9499

5199

98

100

100

62

100

01








(a)

54

100

10099

100

01





(b)

100

9372

100

01





(c)



5 Conclusion




Acknowledgments




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


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


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





3 Results





0100200300400500600700800

Wei

ght o

f fish

(g)


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

)


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)




4 Discussion



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


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


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)






0

02

04

06

08

1

12Re

lativ

e fol

d of

expr

essio

n


(a)

0

02

04

06

08

1

12

14


Relat

ive f

old

of ex

pres

sion


(b)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(c)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(d)







9499

5199

98

100

100

62

100

01








(a)

54

100

10099

100

01





(b)

100

9372

100

01





(c)



5 Conclusion




Acknowledgments




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


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





3 Results





0100200300400500600700800

Wei

ght o

f fish

(g)


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

)


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)




4 Discussion



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


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


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)






0

02

04

06

08

1

12Re

lativ

e fol

d of

expr

essio

n


(a)

0

02

04

06

08

1

12

14


Relat

ive f

old

of ex

pres

sion


(b)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(c)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(d)







9499

5199

98

100

100

62

100

01








(a)

54

100

10099

100

01





(b)

100

9372

100

01





(c)



5 Conclusion




Acknowledgments




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results





0100200300400500600700800

Wei

ght o

f fish

(g)


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

)


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)




4 Discussion



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


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


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)






0

02

04

06

08

1

12Re

lativ

e fol

d of

expr

essio

n


(a)

0

02

04

06

08

1

12

14


Relat

ive f

old

of ex

pres

sion


(b)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(c)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(d)







9499

5199

98

100

100

62

100

01








(a)

54

100

10099

100

01





(b)

100

9372

100

01





(c)



5 Conclusion




Acknowledgments




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


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


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


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)






0

02

04

06

08

1

12Re

lativ

e fol

d of

expr

essio

n


(a)

0

02

04

06

08

1

12

14


Relat

ive f

old

of ex

pres

sion


(b)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(c)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(d)







9499

5199

98

100

100

62

100

01








(a)

54

100

10099

100

01





(b)

100

9372

100

01





(c)



5 Conclusion




Acknowledgments




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

02

04

06

08

1

12Re

lativ

e fol

d of

expr

essio

n


(a)

0

02

04

06

08

1

12

14


Relat

ive f

old

of ex

pres

sion


(b)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(c)

0

02

04

06

08

1

12

Relat

ive f

old

of ex

pres

sion


(d)







9499

5199

98

100

100

62

100

01








(a)

54

100

10099

100

01





(b)

100

9372

100

01





(c)



5 Conclusion




Acknowledgments




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


9499

5199

98

100

100

62

100

01








(a)

54

100

10099

100

01





(b)

100

9372

100

01





(c)



5 Conclusion




Acknowledgments




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Expression Pattern of Myogenic Regulatory ...Environmental Toxicology Laboratory, Department of Zoology, Centre for Advanced Studies, Visva-Bharati University, Santiniketan,WestBengal,