larvi 2013 Epigenetic regulation of muscle development and growth in Senegalese sole larvae Catarina Campos

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1 lrvi 213 6th fish & shellfish lrviulture symposium Epigeneti regultion of musle development nd growth in Seneglese sole lrve Ctrin Cmpos ghent university, elgium, 2-5 septemer 213

2 EPIGENETIC REGULATION OF MUSCLE DEVELOPMENT AND GROWTH IN SENEGALESE SOLE LARVAE Ctrin Cmpos,,, Luís M.P. Vlente, Luís E.C. Coneição,d, Sofi Engrol nd Jorge M.O. Fernndes CIMAR/CIIMAR & ICBAS Centro Interdisiplinr de Investigção Mrinh e Amientl Univ. Porto, Porto, Portugl CIMAR/CCMAR Centro de Ciênis do Mr do Algrve, Univ. Algrve, Fro, Portugl UiN Fulty of Biosienes nd Aquulture University of Nordlnd, Bodø, Norwy d SPAROS Ld, CRIA - Universidde do Algrve, Cmpus de Gmels, Fro, Portugl. LARVI 213 6th fish & shellfish lrviulture symposium

3 Seneglese sole (Sole seneglensis) is mrine fltfish with high potentil to the quulture industry. However, gret flututions of temperture during prodution re still found, whih n ontriute to vritions on sole s growth nd musle ellulrity. Suh therml plstiity must rise through hnges in multitude of physiologil nd moleulr pthwys, in whih epigeneti gene regultion plys n essentil role epigenetis is ridge etween genotype nd phenotype. LARVI 213 6th fish & shellfish lrviulture symposium

4 DNA methyltion DNA methyltion usully ours on CpG dinuleotides nd is often ssoited with repressed hromtin stte nd inhiition of trnsription. The demethyltion of regultory regions in myogeni genes t the eginning of the differentition progrm is essentil to the ommitment of ells towrds the musle linege. LARVI 213 6th fish & shellfish lrviulture symposium

5 mirnas nt endogenous non-oding RNAs, tht re repressive posttrnsriptionl regultors of gene expression Trnsript degrdtion (requires perfet omplementrity) Cytoplsm Psh Drosh Exportin 5 Trget mrna Trnsltionl repression (requires prtil omplementrity) Pre-miRNA Dier Mture mirna Pri-miRNA Pssenger strnd RISC Argonute Nuleus Some mirnas re strongly expressed in musle nd known to intert with the trnsriptionl networks involved in myogenesis LARVI 213 6th fish & shellfish lrviulture symposium

6 Experimentl pproh Two experiments Inution experiment Lrvl rering experiment Three emryoni tempertures: 15, 18 or 21 ºC Three rering tempertures during pelgi phse: 15, 18 or 21 ºC One rering temperture: 21 ºC until 3 dph Trnsfer to ommon temperture: 2 ºC until 121 dph Musle morphometry, gene expression, DNA methyltion levels nd protein metolism were nlysed. LARVI 213 6th fish & shellfish lrviulture symposium

7 Inution experiment Hours post-fertilistion Dys post-hth ºC ºC ºC ºC Blstul Epioly 2 som Finl som Hthing Mouth Pre-Met opening Met Post-Met 3 dph LARVI 213 6th fish & shellfish lrviulture symposium

8 .5 15ºC 18ºC 21ºC 2 A (mm ) The 18 ºC group hd the highest numer of fst fires, ut no differenes were found etween the 15 nd 21 ºC tretments, whih instigtes the ide tht 18 ºC n e n optiml temperture to inute sole emryos.,, 15 N Fire dimeter (µm) By 3 dph: Weight of 18 nd 21 ºC lrve ws superior to the 15 ºC ones Totl musle re ws similr ross ll tretments, proly due to rering ll lrve t ommon temperture. h t H O M em Pr et M s Po h tm 3 dp LARVI 213 6th fish & shellfish lrviulture symposium

9 Trnsient gene expression during development Genes up-regulted lter in development (lrve) 3 dph Met PreM MO Hth FS 2S 75EP BL myf5, fst, myod2, myl2, myhc nd mrf4 were highest t 18 nd/or 21 ºC t speifi points, whih might hve ontriuted to the generl more pronouned growth of these groups Genes up-regulted in erly development (emryo-hthing) LARVI 213 6th fish & shellfish lrviulture symposium

10 SOLiD sequening of smll RNAs 75Ep 15 ºC 75Ep 21 ºC 2S 15 ºC 2S 21 ºC Hth 15 ºC Hth 21 ºC Met 15 ºC Met 21 ºC 1,4E+6 1,2E+6 # reds 1,E+6 8,E+5 6,E+5 4,E+5 2,E+5,E+ Length (nt) 32 onserved mirnas mongst 149 fmilies Inresing numer nd diversity from Epioly to Metmorphosis # Trimmed Averge # Annotted # Conserved (% Totl) length (nt) mirbse (% Totl) mirnas (42.2) (3.) Ep 21 ºC (24.5) (2.) 231 2S 15 ºC (5.6) (15.1) 265 2S 21 ºC (42.) (11.3) 265 Hth 15 ºC (59.3) (2.2) 285 Hth 21 ºC (52.5) (18.7) 281 Met 15 ºC (69.9) (33.7) 288 Met 21 ºC (67.2) (12.7) 279 Smple # Totl reds 75Ep 15 ºC LARVI 213 6th fish & shellfish lrviulture symposium

11 A B mir-17-5p mir ºC 21 ºC 5 Reltive expression Reltive expression mir-26 mir Ep 2S C 3dph 75Ep D mir-13 mir S Hth PreMet Met 3dph mir-181-5p mir-181-5p Reltive expression Reltive expression Hth PreMet Met Ep 2S E 3dph 75Ep mir-181-3p Hth PreMet Met 3dph mir-199 mir-199-5p Reltive expression S Hth PreMet Met 3dph G 75Ep 2S Hth PreMet Met 3dph mir-26-3p mir Reltive expression 75Ep qpcr vlidtion of mir-26, mir-181-5p nd mir-26 reveled higher expression t 21 ºC thn t 15 ºC during emryogenesis nd/or t hthing, pointing to higher tivtion of the myogeni proess t higher temperture. 2S F mir-181-3p 35 Reltive expression The very high expression of severl mirnas t pre-metmorphi stge n e ssoited with high growth rte nd preprtion for the metmorphi proess. Hth PreMet Met Ep 2S Hth PreMet Met 3dph LARVI 213 6th fish & shellfish lrviulture symposium

12 Lrvl rering experiment Experimentl setup 8 dph 2 ºC 18 ºC Post-Met 12 dph 9 dph 15 dph 12 dph 15 ºC 83 dph 35 dph 23 dph 39 dph 51 dph 3 15 ºC 3,5 DW (mg) ºC 3, 2,5 2, dph 83 dph 1 dph 121 dph 83 dph 1 dph 121 dph 83 dph 1 dph 121 dph 21 ºC During pelgi phse: High mortlity nd lower growth of the 15 ºC group. 1,5 1, 1 1 dph 2 ºC 35 dph 21 ºC Met 22 dph Pre-Met 16 dph Hthing,5, MO Pre-met Met Post-met By 121 dph: Weight of juveniles from 15 ºC ws similr to 21 ºC, nd oth were lrger thn 18 ºC. LARVI 213 6th fish & shellfish lrviulture symposium

13 25 Fire dimeter (µm) 2 15 During pelgi phse: Fire dimeter ws lrger t 18 nd 21 ºC reltively to 15 ºC. 1 During pelgi phse, the mjority of MRFs s well s myosins, fgf6 nd igf-i were upregulted t 18 ºC nd/or 21 ºC reltively to 15 ºC. 5 Pre-met Met Post-met 83 dph 1 dph Reltive expression 2. Metmorphosis Methyltion nlysis ,,,,.8, mrf4 myog,,.4. myod1 myod2 myf5 px7 myl2 myhc mstn1 fst fgf6 igf-i igf1r igf-ii hsp7 LARVI 213 6th fish & shellfish lrviulture symposium

14 Seneglese sole myogenin promoter: Highly onserved mongst teleost speies. Cn DNA methyltion of myog promoter in sole musle e ffeted y rering temperture? Higher methyltion levels of myog t 15 ºC Totl numer of methylted ytosines Methyltion t CpG sites LARVI 213 6th fish & shellfish lrviulture symposium

15 By 1 dph: Inresed hypertrophy of musle fires in the18 ºC group, whih might indue lower growth pity. 15 < 1 µm (%) 1 dph 1 5 Pre-met Met Post-met 83 dph 1 dph ºC Musle - 1 dph 2. Reltive expression Fire dimeter (µm) 18 ºC 21 ºC ,,.8.4. myod1 myod2 myf5 mrf4 myog px7 myl2 myhc mstn1 fst fgf6 igf-i igf1r igf-ii hsp7 LARVI 213 6th fish & shellfish lrviulture symposium

16 Artemi rdiolelling feeding trils: metmorphosis nd enthi post-lrve. LARVI 213 6th fish & shellfish lrviulture symposium

17 Protein digestiility nd retention is ffeted y rering temperture during metmorphosis lowest t 15 ºC. Evution Asorption Digestiility (% of totl fed) 6% 4% 2% % Retention nd tolism (% Asorption) 1% 1% 8% Ctolism Retention,, 15 ºC 18 ºC 21 ºC 8% 6% 4% 2% % 15 ºC 18 ºC 21 ºC LARVI 213 6th fish & shellfish lrviulture symposium

18 After trnsfer to ommon temperture, post-lrve from 15 ºC showed the highest protein digestiility. Evution 24 HAF Digestiility (% totl fed) 8% 6% 4% 2% % Retention 1% Retention nd Ctolism (% Asorption) Digestiility 1% Ctolism 24 HAF 8% 6% 4% 2% % LARVI 213 6th fish & shellfish lrviulture symposium

19 Conlusions Seneglese sole pelgi lrve more suseptile to 15 ºC thn emryos. Inution t 18 ºC + lrvl rering t 21 ºC promoted lrvl musle hyperplsti growth, whih n hve positive implitions on musle growth potentil. Therml plstiity of myogeni genes nd mirnas my e responsile for the oserved growth effets during the inution experiment. Rering pelgi lrve t 15 ºC gretly deresed growth nd survivl, deresed protein sorption nd retention nd inresed DNA methyltion levels of myog promoter in skeletl musle however, mehnism of ompenstory growth ws lter oserved in the 15 ºC juveniles. Rering pelgi lrve t 21 ºC promoted good growth nd survivl nd therefore is pproprite for Seneglese sole pelgi lrve. LARVI 213 6th fish & shellfish lrviulture symposium

20 Tke home messge Wter temperture during erly ontogeny hs mjor effets on Seneglese sole growth potentil, oth in short nd longterm, with implitions on musle ellulrity, gene expression, gene regultion nd protein utilistion. LARVI 213 6th fish & shellfish lrviulture symposium

21 Aknowledgements COST Ation FA81 SFRH / BD / / 28 João Sendão Mri Filip Cstnheir Pulo Gvi Helen Teixeir Filip Roh Ver Rodrigues Els Crit André Sntos Alessi Ginnetto Arvind Sundrm Teshome Bizuyehu LARVI 213 6th fish & shellfish lrviulture symposium