Epigenetic regulation of muscle development and growth in ... 3 - Tuesday/17.00-17.20... ·...

larvi 2013

ghent university, belgium, 2-5 september 2013

6th fish & shellfish larviculture symposium

Epigenetic regulation of muscledevelopment and growthin Senegalese sole larvae

Catarina Campos

a CIMAR/CIIMAR & ICBASCentro Interdisciplinar de InvestigaçãoMarinha e Ambiental

Univ. Porto,Porto, Portugal

b CIMAR/CCMARCentro de Ciências do Mar do Algarve,

Univ. Algarve,Faro, Portugal

c UiNFaculty of Biosciences and Aquaculture

University of Nordland,Bodø, Norway

d SPAROS Lda,CRIA - Universidade do Algarve, Campus de

Gambelas, 8005-139 Faro,Portugal.

EPIGENETIC REGULATION OF MUSCLE DEVELOPMENTAND GROWTH IN SENEGALESE SOLE LARVAE

Catarina Campos a,b,c, Luísa M.P. Valente a, Luís E.C. Conceição b,d,Sofia Engrola b and Jorge M.O. Fernandes c

LARVI 2013 – 6th fish & shellfish larviculture symposium


Senegalese sole (Solea senegalensis) is a marine flatfish with highpotential to the aquaculture industry.

However, great fluctuations of temperature during production arestill found, which can contribute to variations on sole’s growth and

muscle cellularity.

Such thermal plasticity must arise through changes in a multitude ofphysiological and molecular pathways, in which epigenetic gene

regulation plays an essential role → epigenetics is a bridgebetween genotype and phenotype.

DNA methylation


DNA methylation usuallyoccurs on CpG

dinucleotides and is oftenassociated with a

repressed chromatin stateand inhibition of

transcription.

The demethylation of regulatory regions in myogenic genes at thebeginning of the differentiation program is essential to the commitment of

cells towards the muscle lineage.

miRNAs


18–24 nt endogenous non-coding RNAs, that are repressive post-transcriptional regulators of gene expression

Some miRNAs are strongly expressed in muscle and known to interactwith the transcriptional networks involved in myogenesis

Exportin 5Exportin 5Exportin 5

Cytoplasm

Nucleus

Target mRNAPasha

Drosha Pre-miRNA

DicerDicerDicer

RISCMature miRNA

Argonaute

Transcript degradation(requires perfect complementarity)

Translational repression(requires partial complementarity)

Passenger strandPri-miRNA


Experimental approach

Two experiments

Incubation experiment Larval rearing experiment

Three embryonic temperatures:15, 18 or 21 ºC

One rearing temperature:21 ºC until 30 dph

Three rearing temperatures duringpelagic phase: 15, 18 or 21 ºC

Transfer to common temperature:20 ºC until 121 dph

Muscle morphometry, gene expression, DNA methylation levels andprotein metabolism were analysed.

Blastula Epiboly 20 som Finalsom

Hatching Mouthopening

Met 30 dph

15 ºC

21 ºC

18 ºC

9

9.5

10

12

14

22

20

24

36

24

28

40

34

42

62

Hours post-fertilisation Days post-hatch

21 ºC

Pre-Met

2

2

3

8

8

9

14

14

15

30

30

30

Post-Met

22

22

23


Incubation experiment

By 30 dph:• Weight of 18 and 21 ºC larvae wassuperior to the 15 ºC ones.

• The 18 ºC group had the highest numberof fast fibres, but no differences werefound between the 15 and 21 ºCtreatments, which instigates the idea that18 ºC can be an optimal temperature toincubate sole embryos.

• Total muscle area was similar across alltreatments, probably due to rearing alllarvae at a common temperature.

A (m

m2 )

0.0

0.1

0.2

0.3

0.4

0.5

a

bb

ab b

a b b

ab b

aa,b b

aa,b

b

N

0

500

1000

1500

2000

Hatch MOPreM Met

PostM30 dph

Fibr

e di

amet

er (µ

m)

0

5

10

15

20

15ºC18ºC21ºC

ac

b

ab c

ab c

a b b a b b

ab b


Transient gene expression during development

Genes up-regulated in early development (embryo-hatching)

Genes up-regulated later in development (larvae)


15°C

18°C

21°C

15°C

18°C

21°C

15°C

18°C

21°C

15°C

18°C

21°C

15°C

18°C

21°C

15°C

18°C

21°C

15°C

18°C

21°C

15°C

18°C

21°C

15°C

18°C

21°C

BL 75EP FS20S 30 dphMO PreM MetHatch

myf5, fst, myod2,mylc2, myHC and

mrf4 were highest at18 and/or 21 ºC at

specific points,which might havecontributed to the

general morepronounced growth

of these groups

0,0E+00

2,0E+05

4,0E+05

6,0E+05

8,0E+05

1,0E+06

1,2E+06

1,4E+06

# re

ads

Length (nt)

75Ep 15 ºC75Ep 21 ºC20S 15 ºC20S 21 ºCHatch 15 ºCHatch 21 ºCMet 15 ºCMet 21 ºC

SOLiD sequencing of small RNAs

Sample # Total reads# Trimmed

(% Total)

Average

length (nt)

# Annotated

miRBase (% Total)

# Conserved

miRNAs75Ep 15 ºC 6 642 089 2 802 986 (42.2) 23.5 200 224 (3.0) 232

75Ep 21 ºC 8 842 550 2 169 069 (24.5) 21.1 174 027 (2.0) 231

20S 15 ºC 6 566 636 3 320 266 (50.6) 22.4 993 981 (15.1) 265

20S 21 ºC 6 101 835 2 564 327 (42.0) 22.7 689 954 (11.3) 265

Hatch 15 ºC 7 268 950 4 312 425 (59.3) 23.0 1 467 039 (20.2) 285

Hatch 21 ºC 6 200 277 3 256 433 (52.5) 22.4 1 162 239 (18.7) 281

Met 15 ºC 7 105 697 4 965 849 (69.9) 23.0 2 391 674 (33.7) 288

Met 21 ºC 6 325 932 4 247 722 (67.2) 25.2 804 587 (12.7) 279

Increasingnumber and

diversity fromEpiboly to

Metamorphosis• 320 conserved miRNAs amongst 149 families


• The very high expression of several miRNAs ata pre-metamorphic stage can be associated with

a high growth rate and preparation for themetamorphic process.

• qPCR validation of miR-26a, miR-181a-5p andmiR-206 revealed higher expression at 21 ºCthan at 15 ºC during embryogenesis and/or athatching, pointing to a higher activation of the

myogenic process at a higher temperature.


miR-130c

75Ep 20S Hatch PreMet Met 30dph

Rel

ativ

e ex

pres

sion

0

20

40

60

80

100

*

*

*

miR-26a


Rel

ativ

e ex

pres

sion

0

1

2

3

4

5

6

**

miR-181a-5p


Rel

ativ

e ex

pres

sion

0

10

20

30

40

50

60

miR-181a-3p


Rel

ativ

e ex

pres

sion

0

5

10

15

20

25

30

35

*

*

miR-17a-5p


Rel

ativ

e ex

pres

sion

0

20

40

60

80

100

120

140 15 ºC21 ºC

miR-199a-5p


Rel

ativ

e ex

pres

sion

0

20

40

60

80

100

120

140

160

180

*

miR-206-3p


Rel

ativ

e ex

pres

sion

0

5

10

15

20

25

30

35

*

*

A B

C D

E F

G

*

*

*

miR-17a miR-26a

miR-130c miR-181a-5p

miR-181a-3p miR-199

miR-206

Larval rearing experiment

21 ºC

18 ºC

15 ºC

20 ºC

Experimental setupMet

16 d

ph

22 d

ph

35 d

ph

8 dph 35 dph

39 dph

51 dph

20 ºC

Hatching

12 dph

9 dph

12 dph

15 dph

23 dph

83 dph

83 dph

83 dph

100 dph

100 dph

100 dph

Pre-Met Post-Met 100 dph83 dph 121 dph

121 dph

121 dph

121 dph


0

50

100

150

200

250

300

DW

(mg)

15 ºC18 ºC21 ºC

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

MO Pre-met Met Post-met

a

cb

a

cb

a

ba

a

cb

a

bb

a

b

a

During pelagic phase:

• High mortality andlower growth of the

15 ºC group.

By 121 dph:• Weight of juveniles

from 15 ºC was similarto 21 ºC, and both were

larger than 18 ºC.

Pre-met Met Post-met 83 dph 100 dph

Fibr

e di

amet

er (µ

m)

0

5

10

15

20

25

abc

abc

a

bc

ab

c

ab c

Metamorphosis

myod1 myod2 myf5 mrf4 myog pax7 mylc2 myHC mstn1 fst fgf6 igf-I igf1r igf-II hsp70

Rel

ativ

e ex

pres

sion

0.0

0.4

0.8

1.2

1.6

2.0

b

a

a,b

b

aa,b

b

aa,b

c

a

bb

a

a,bb

a

a,b

b

a

a,bba,b

a


During pelagic phase:

• Fibre diameter was larger at 18and 21 ºC relatively to 15 ºC.

• During pelagic phase, themajority of MRFs as well as

myosins, fgf6 and igf-I were up-regulated at 18 ºC and/or 21 ºC

relatively to 15 ºC.

Methylation analysis


• Higher methylation levels of myog at 15 ºC

Total number of methylated cytosines Methylation at CpG sites

Senegalese sole myogenin promoter:• Highly conserved amongst teleost species.

• Can DNA methylation of myog promoter insole muscle be affected by rearing

temperature?

Muscle - 100 dph

myod1 myod2 myf5 mrf4 myog pax7 mylc2 myHC mstn1 fst fgf6 igf-I igf1r igf-II hsp70

Rel

ativ

e ex

pres

sion

0.0

0.4

0.8

1.2

1.6

2.0

a

a,b

b

aa

b

a

a,b

b

Pre-met Met Post-met 83 dph 100 dph

Fibr

e di

amet

er (µ

m)

0

5

10

15

20

25

abc

abc

a

bc

ab

c

ab c


By 100 dph:• Increased hypertrophy of muscle fibres inthe18 ºC group, which might induce a lower

growth capacity.

15 ºC 18 ºC 21 ºC

< 10

µm

(%) 1

00 d

ph

0

3

6

9

12

15

b

a a


Artemia radiolabelling feeding trials:metamorphosis and benthic post-larvae.


• Protein digestibility and retention is affected by rearingtemperature during metamorphosis – lowest at 15 ºC.

0%

20%

40%

60%

80%

100%

15 ºC 18 ºC 21 ºC

Ret

entio

n an

d ca

tabo

lism

(% A

bsor

ptio

n)

CatabolismRetention

0%

20%

40%

60%

80%

100%

15 ºC 18 ºC 21 ºC

Dig

estib

ility

(% o

f tot

al fe

d)

EvacuationAbsorption

ab c

a bc

a b a,b

a,bba


• After transfer to a common temperature, post-larvae from 15 ºCshowed the highest protein digestibility.

0%

20%

40%

60%

80%

100%

15°C 18°C 21°C

Dig

estib

ility

(% to

tal f

ed)

24 HAF Evacuation

Digestibility

0%

20%

40%

60%

80%

100%

15°C 18°C 21°C

Ret

entio

n an

d C

atab

olis

m(%

Abs

orpt

ion)

24 HAF Catabolism

Retention

a

a

b

b

b

b

Conclusions

• Senegalese sole pelagic larvae → more susceptible to 15 ºC than embryos.

• Incubation at 18 ºC + larval rearing at 21 ºC → promoted larval musclehyperplastic growth, which can have positive implications on muscle growthpotential.

• Thermal plasticity of myogenic genes and miRNAs may be responsible forthe observed growth effects during the incubation experiment.

• Rearing pelagic larvae at 15 ºC → greatly decreased growth and survival,decreased protein absorption and retention and increased DNA methylationlevels of myog promoter in skeletal muscle → however, a mechanism ofcompensatory growth was later observed in the 15 ºC juveniles.

• Rearing pelagic larvae at 21 ºC → promoted a good growth and survival andtherefore is appropriate for Senegalese sole pelagic larvae.


Take home message

• Water temperature during early ontogeny has major effectson Senegalese sole growth potential, both in short and long-

term, with implications on muscle cellularity, gene expression,gene regulation and protein utilisation.


SFRH / BD / 43633 / 2008

João SendãoMaria Filipa CastanheiraPaulo GavaiaHelena TeixeiraFilipa RochaVera RodriguesElsa CabritaAndré Santos

Alessia GiannettoArvind SundaramTeshome Bizuayehu

COST Action FA0801

Acknowledgements


Epigenetic regulation of muscle development and growth in ... 3 - Tuesday/17.00-17.20... ·...

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