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First published online November 17, 2006
Journal of Experimental Biology 209, 4751-4767 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02555

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Regulation of troponin T expression during muscle development in sea bream Sparus auratus Linnaeus: the potential role of thyroid hormones

M. A. Campinho^1,2, G. E. Sweeney^2,* and D. M. Power¹

¹ CCMAR, FERN, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
² School of Biosciences, University of Wales, Museum Avenue CF11 3US Cardiff, UK

View larger version (90K): [in a new window]   Fig. 1. Clustal X (Thompson et al., 1997) multiple sequence alignment of putative protein sequences of efTnTsb, afTnTsb and LfTnTsb isoforms. Shaded areas indicate identical residues. Accession numbers: efTnTsb, DQ473445; afTnTsb, DQ473443; LfTnTsb, DQ473444; efTnTtn, CR660426; LfTnTtn, CR658326; aTnTtn, CR658422; fTnTmd-2, BJ728074; fTnTmd-1, BJ729852; fTnTss, AAC24595; fTnTgm, AAM21701; fTnTazf, NP571640; fTnTbzf-2, BC065452; fTnTbzf-1, AF425741.

View larger version (71K): [in a new window]   Fig. 2. Northern blot analysis of sea bream fTnT expression. Total RNA (3 µg) of sea bream adult white muscle (lane 1), adult red muscle (lane 2), adult heart (lane 3) and adult liver (lane 4). The probe utilised corresponded to the 3' UTR of sea bream fTnT (top panel). The relative loading of total RNA was determined by staining with ethidium bromide (lower panel).

View larger version (13K): [in a new window]   Fig. 3. Genomic organisation of the putative sea bream, Fugu and Tetraodon fTnT gene predicted after Spidey analysis using the sea bream fTnT isoform cDNA sequence isolated and the Fugu scaffold 1617. The same analysis was carried out using the sea bream fTnT isoforms isolated and Tetraodon scaffold SCAF7217, but including also the Tetraodon full-length cDNA clones CR660426, CR658422 and CR657382. Black boxes represent constitutive protein coding regions whereas the white boxes represent untranslated regions. Broad striped blocks represent alternatively spliced untranslated exons, while narrow striped blocks represent protein coding alternatively spliced exons. Exon II contains the ATG initiation codon (arrowhead) and is composed of part of the 5' UTR and the start of the coding region. The sea bream, Fugu and Tetraodon fTnT locus has 14 exons from which exon I and XIV are untranslated exons. The exon numbers with an asterisk are alternatively spliced exons. Exon V is the larval-specific exon. Flush junction boundaries in exons indicate that they start or end in intact codons; saw tooth boundaries indicate that the upstream exon donates one nt to the codon while the other two are contributed by the downstream exon; concave/convex exon boundaries indicate that codon splitage takes place by the upstream exon donating two nt while the downstream exon contributes one. The efTnTsb isoform results form the incorporation of all exons except exon I. The afTnTsb isoform results from splicing of exons I-IV and VI-XIV whereas in the LfTnTsb isoform exons I-III and VI-XIV are spliced. Both afTnTsb and LfTnTsb have an extra 5' UTR exon, exon I, which is absent from the efTnTsb (Fig. 4). Sequence conservation between this region (exon I) of the presumed adult sea bream fTnT cDNAs and the genomic sequence of Tetraodon was higher than 88%.

View larger version (35K): [in a new window]   Fig. 4. Developmental expression of sea bream fTnT isoforms and 18s ribosomal RNA (18s rRNA) assessed by RT-PCR. (A) Reaction products were fractionated on a 2.5% agarose gel. Embryos from 12, 18 and 36 h.p.f., larvae from 1 to 75 d.p.h. and juveniles of 89 d.p.h. were analysed. Adult white (WM) and red (RM) muscle, heart (H) and liver (L) were also analysed. A no template, negative control was used (C-). Three bands are detected which are products of the sea bream fTnTsb gene. (B) Graph showing the average expression of each fTnT isoform in relation to 18s rRNA of three samples. Values are means ± s.e.m. (C) Graph showing the ratio between the different sea bream fTnT isoforms at various stages and in different tissues.

View larger version (27K): [in a new window]   Fig. 5. Expression of sea bream fTnT gene isoforms afTnTsb (higher molecular mass), LfTnTsb (lower molecular mass) and 18s rRNA assessed by RT-PCR after 7 (64 d.p.h.), 18 (75 d.p.h.) and 31 (89 d.p.h.) days of treatment with T3 and in control animals. Reaction products fractionated on a 2.5% agarose gel (A) and respective graphical representation of the relative expression of isoform afTnTsb (B) and LfTnTsb (C) against 18s; (D) the afTnTsb:LfTnTsb ratio. The efTnTsb isoform is not represented graphically since no expression was detected in any treatment at any sampling point. aSignificant statistical differences between the control and T3-treated groups.

View larger version (28K): [in a new window]   Fig. 6. Expression of sea bream sTnT2, iTnT genes and 18s rRNA determined by RT-PCR after 7 (64 d.p.h.), 18 (75 d.p.h.) and 31 (89 d.p.h.) days of treatment with T3 in control animals. (A) Reaction products fractionated on a 2.5% agarose gel and (B) respective graphs of sTnT2sb expression relative to 18s rRNA. The iTnTsb gene is not represented graphically since no expression was detected in any treatment group at any sampling time. No significant statistical differences were found in sTnT2sb expression between of the T3-treated and the control animals at any sampling time.

View larger version (17K): [in a new window]   Fig. 7. Expression of sea bream sTnT1sb gene isoforms sTnT1sb-1 (lower molecular mass), sTnT1sb-2 (higher molecular mass) and 18s rRNA determined by RT-PCR after 7 (64 d.p.h.), 18 (75 d.p.h.) and 31 (89 d.p.h.) days of treatment with T3 or in control animals. (A) Reaction products fractionated on a 2.5% agarose gel and (B,C) respective graphs of relative expression of isoform sTnT1sb-1 (B) and sTnT1sb-2 (C) against 18s rRNA. (D) The ratio sTnT1sb-1:sTnT1sb-2. aSignificant statistical differences between control and T3-treated animals.

View larger version (9K): [in a new window]   Fig. 8. Sea bream whole body thyroid hormone levels: (A) T4 and (B) T3 levels in control sea bream and in fish treated with T3 (N=5 per sampling time and treatment). Levels were measured after 7 (64 d.p.h.), 18 (75 d.p.h.) and 31 (89 d.p.h.) days of treatment. aSignificant difference (Tukey's HSD, P<0.001) between T3 and the control group.

View larger version (51K): [in a new window]   Fig. 9. (A) Follicle number per slide at the junction of the hypohyal bones (N=3 animals per treatment) in T3 and control sea bream after 7 (64 d.p.h.), 18 (75 d.p.h.) and 31 (89 d.p.h.) days of treatment. (B) Thyrocyte cell height was measured in order to determine thyroid activity. aStatistical significant differences between the T3 treated and the control group. (C) Thyroid follicles in control and T3-treated animals at each sampling point. Scale bar, 100 µm. Black asterisks denote a thyroid follicle.

View larger version (169K): [in a new window]   Fig. A1. Nucleotide sequence and deduced protein sequence of the sea bream fTnT isoforms isolated. Putative isoform efTnTsb (A), isoform afTnTsb (B) and LfTnTsb (C) are shown. The ATG translation initiation codon is shown in bold as well as the TAG STOP codon, which is also indicated by an asterisk. The Kozak consensus sequence is underlined and the double underline indicates the polyadenylation signal. (D) Clustal X (Thompson et al., 1997) multiple nucleotide sequence alignment of sea bream fTnT cDNAs. The position of forward and reverse primers used for RT-PCR expression analysis are indicated by arrows.