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Troponin T isoform expression is modulated during Atlantic halibut metamorphosis.

Campinho MA, Silva N, Nowell MA, Llewellyn L, Sweeney GE, Power DM - BMC Dev. Biol. (2007)

Bottom Line: In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment.Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations.We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: CCMAR, FERN, Universidade do Algarve, Campus de Gambelas, Faro, Portugal. macampin@ualg.pt <macampin@ualg.pt>

ABSTRACT

Background: Flatfish metamorphosis is a thyroid hormone (TH) driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT), a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied.

Results: In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT) gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT) expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature.

Conclusion: Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

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Schematic representation of muscle ontogeny in halibut during metamorphosis; Schematic representation of the muscle ontogeny in halibut from larvae (stg 5) to juvenile (stg 10) and the overall expression pattern of halibut TnT genes. There is a general increase in myotome volume during metamorphosis. Note that throughout muscle ontogeny, prior to and after metamorphosis, symmetry is maintained both in the sagital and longitudinal planes. In pre-metamorphic halibut (stg 5 and 6) white muscle hyperplasic small diameter fibers are located in the most apical and lateral sides of the myotome regions (insert) which predominantly express efTnThh (black dots). As white muscle fibers increase in size the expression of fTnThh isoforms disappears. The expression of the other fTnThh isoforms (light grey dots) co-localizes with efTnThh expression up until stg 7. When the animals reach the beginning of metamorphosis (stg 7) all mature white muscle fibers express fTnThh-1 and -2 isoforms (light grey shading) and efTnThh is only found in smaller diameter hyperplasic fibers in the periphery of the myotomes around the myosepta (black dots). As the animals enter the climax of metamorphosis expression of the low molecular weight fTnThh isoforms is located in the entire myotome (light grey shading) and efTnThh is confined to scattered presumptive satellite cells (black dots). At climax (stg 9) and in juvenile (stg 10) white muscle fTnThh-1 and -2 isoforms are expressed in all the myotome with varying intensity and in fibers close to the myosepta expression is higher. In pre-metamorphic halibut red muscle (dark grey) all cells express sTnT2hh and AfTnThh. The expression of the red muscle specific Troponin T genes is constant and restricted to the outer red muscle throughout halibut metamorphosis and in juvenile animals.
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Figure 8: Schematic representation of muscle ontogeny in halibut during metamorphosis; Schematic representation of the muscle ontogeny in halibut from larvae (stg 5) to juvenile (stg 10) and the overall expression pattern of halibut TnT genes. There is a general increase in myotome volume during metamorphosis. Note that throughout muscle ontogeny, prior to and after metamorphosis, symmetry is maintained both in the sagital and longitudinal planes. In pre-metamorphic halibut (stg 5 and 6) white muscle hyperplasic small diameter fibers are located in the most apical and lateral sides of the myotome regions (insert) which predominantly express efTnThh (black dots). As white muscle fibers increase in size the expression of fTnThh isoforms disappears. The expression of the other fTnThh isoforms (light grey dots) co-localizes with efTnThh expression up until stg 7. When the animals reach the beginning of metamorphosis (stg 7) all mature white muscle fibers express fTnThh-1 and -2 isoforms (light grey shading) and efTnThh is only found in smaller diameter hyperplasic fibers in the periphery of the myotomes around the myosepta (black dots). As the animals enter the climax of metamorphosis expression of the low molecular weight fTnThh isoforms is located in the entire myotome (light grey shading) and efTnThh is confined to scattered presumptive satellite cells (black dots). At climax (stg 9) and in juvenile (stg 10) white muscle fTnThh-1 and -2 isoforms are expressed in all the myotome with varying intensity and in fibers close to the myosepta expression is higher. In pre-metamorphic halibut red muscle (dark grey) all cells express sTnT2hh and AfTnThh. The expression of the red muscle specific Troponin T genes is constant and restricted to the outer red muscle throughout halibut metamorphosis and in juvenile animals.

Mentions: In contrast to fTnThh, expression of halibut AfTnThh (Fig. 6A) and sTnT2hh (Fig. 7A) detected by RT-PCR does not change during metamorphosis (Fig. 7). However, 4 alternatively spliced isoforms of AfTnThh are detected (Fig. 8A and 8D). Sequence analysis reveals that the AfTnThh isoforms are a result of alternative splicing of exons IV, V and VII (Fig. 2) and are designated AfTnThh-1 to -4. The largest product, AfTnThh-1 (439 bp) is identical to the AfTnThh cDNA isolated by library screening and includes all alternatively spliced exons (Fig. 6A and 6D); AfTnThh-2 (412 bp; DQ680177) lacks exons IV and VII but exon V is maintained (Fig. 6A and 6D); AfTnThh-3 (301 bp; DQ680178) is composed of exon IV and VII but exon V is spliced out (Fig. 6A and 6D); and in AfTnThh-4 (283 bp; DQ680179) only exon IV is spliced in and all other alternatively spliced exons (exons V and VII) are spliced out (Fig. 6A and 6D). All the halibut AfTnThh isoforms detected contain exon VI (Fig. 6D) and neither the isoform expression pattern nor the ratio between these AfTnThh isoforms is altered during metamorphosis (Fig. 6A–C). A small but statistically significant (HSD, p ≤ 0.005; Fig. 6A and 6B) decrease in expression of all the isoforms is observed in stg 10 juveniles; AfTnThh-1 is the predominant isoform throughout the halibut's life. In turn, AfTnThh-2 is the second most abundant isoform and its expression increases after climax (HSD, p ≤ 0.006; Fig. 6A and 6B). However, in adult red muscle the ratio of the two isoforms is identical to pre-metamorphic stg 5 animals (Fig. 6A and 6C). The AfTnThh-3 and -4 isoforms have identical (HSD, p > 0.05), very low expression (Fig. 6A–C) and their expression and ratio in relation to other isoforms does not change during metamorphosis (HSD, p > 0.05; Fig. 6A–C). Moreover, in adult red muscle these low molecular weight isoforms are not expressed (Fig. 6A and 6B).


Troponin T isoform expression is modulated during Atlantic halibut metamorphosis.

Campinho MA, Silva N, Nowell MA, Llewellyn L, Sweeney GE, Power DM - BMC Dev. Biol. (2007)

Schematic representation of muscle ontogeny in halibut during metamorphosis; Schematic representation of the muscle ontogeny in halibut from larvae (stg 5) to juvenile (stg 10) and the overall expression pattern of halibut TnT genes. There is a general increase in myotome volume during metamorphosis. Note that throughout muscle ontogeny, prior to and after metamorphosis, symmetry is maintained both in the sagital and longitudinal planes. In pre-metamorphic halibut (stg 5 and 6) white muscle hyperplasic small diameter fibers are located in the most apical and lateral sides of the myotome regions (insert) which predominantly express efTnThh (black dots). As white muscle fibers increase in size the expression of fTnThh isoforms disappears. The expression of the other fTnThh isoforms (light grey dots) co-localizes with efTnThh expression up until stg 7. When the animals reach the beginning of metamorphosis (stg 7) all mature white muscle fibers express fTnThh-1 and -2 isoforms (light grey shading) and efTnThh is only found in smaller diameter hyperplasic fibers in the periphery of the myotomes around the myosepta (black dots). As the animals enter the climax of metamorphosis expression of the low molecular weight fTnThh isoforms is located in the entire myotome (light grey shading) and efTnThh is confined to scattered presumptive satellite cells (black dots). At climax (stg 9) and in juvenile (stg 10) white muscle fTnThh-1 and -2 isoforms are expressed in all the myotome with varying intensity and in fibers close to the myosepta expression is higher. In pre-metamorphic halibut red muscle (dark grey) all cells express sTnT2hh and AfTnThh. The expression of the red muscle specific Troponin T genes is constant and restricted to the outer red muscle throughout halibut metamorphosis and in juvenile animals.
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Figure 8: Schematic representation of muscle ontogeny in halibut during metamorphosis; Schematic representation of the muscle ontogeny in halibut from larvae (stg 5) to juvenile (stg 10) and the overall expression pattern of halibut TnT genes. There is a general increase in myotome volume during metamorphosis. Note that throughout muscle ontogeny, prior to and after metamorphosis, symmetry is maintained both in the sagital and longitudinal planes. In pre-metamorphic halibut (stg 5 and 6) white muscle hyperplasic small diameter fibers are located in the most apical and lateral sides of the myotome regions (insert) which predominantly express efTnThh (black dots). As white muscle fibers increase in size the expression of fTnThh isoforms disappears. The expression of the other fTnThh isoforms (light grey dots) co-localizes with efTnThh expression up until stg 7. When the animals reach the beginning of metamorphosis (stg 7) all mature white muscle fibers express fTnThh-1 and -2 isoforms (light grey shading) and efTnThh is only found in smaller diameter hyperplasic fibers in the periphery of the myotomes around the myosepta (black dots). As the animals enter the climax of metamorphosis expression of the low molecular weight fTnThh isoforms is located in the entire myotome (light grey shading) and efTnThh is confined to scattered presumptive satellite cells (black dots). At climax (stg 9) and in juvenile (stg 10) white muscle fTnThh-1 and -2 isoforms are expressed in all the myotome with varying intensity and in fibers close to the myosepta expression is higher. In pre-metamorphic halibut red muscle (dark grey) all cells express sTnT2hh and AfTnThh. The expression of the red muscle specific Troponin T genes is constant and restricted to the outer red muscle throughout halibut metamorphosis and in juvenile animals.
Mentions: In contrast to fTnThh, expression of halibut AfTnThh (Fig. 6A) and sTnT2hh (Fig. 7A) detected by RT-PCR does not change during metamorphosis (Fig. 7). However, 4 alternatively spliced isoforms of AfTnThh are detected (Fig. 8A and 8D). Sequence analysis reveals that the AfTnThh isoforms are a result of alternative splicing of exons IV, V and VII (Fig. 2) and are designated AfTnThh-1 to -4. The largest product, AfTnThh-1 (439 bp) is identical to the AfTnThh cDNA isolated by library screening and includes all alternatively spliced exons (Fig. 6A and 6D); AfTnThh-2 (412 bp; DQ680177) lacks exons IV and VII but exon V is maintained (Fig. 6A and 6D); AfTnThh-3 (301 bp; DQ680178) is composed of exon IV and VII but exon V is spliced out (Fig. 6A and 6D); and in AfTnThh-4 (283 bp; DQ680179) only exon IV is spliced in and all other alternatively spliced exons (exons V and VII) are spliced out (Fig. 6A and 6D). All the halibut AfTnThh isoforms detected contain exon VI (Fig. 6D) and neither the isoform expression pattern nor the ratio between these AfTnThh isoforms is altered during metamorphosis (Fig. 6A–C). A small but statistically significant (HSD, p ≤ 0.005; Fig. 6A and 6B) decrease in expression of all the isoforms is observed in stg 10 juveniles; AfTnThh-1 is the predominant isoform throughout the halibut's life. In turn, AfTnThh-2 is the second most abundant isoform and its expression increases after climax (HSD, p ≤ 0.006; Fig. 6A and 6B). However, in adult red muscle the ratio of the two isoforms is identical to pre-metamorphic stg 5 animals (Fig. 6A and 6C). The AfTnThh-3 and -4 isoforms have identical (HSD, p > 0.05), very low expression (Fig. 6A–C) and their expression and ratio in relation to other isoforms does not change during metamorphosis (HSD, p > 0.05; Fig. 6A–C). Moreover, in adult red muscle these low molecular weight isoforms are not expressed (Fig. 6A and 6B).

Bottom Line: In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment.Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations.We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: CCMAR, FERN, Universidade do Algarve, Campus de Gambelas, Faro, Portugal. macampin@ualg.pt <macampin@ualg.pt>

ABSTRACT

Background: Flatfish metamorphosis is a thyroid hormone (TH) driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT), a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied.

Results: In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT) gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT) expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature.

Conclusion: Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

Show MeSH
Related in: MedlinePlus