Progress 12/01/06 to 11/30/11
Outputs
OUTPUTS: Seven published manuscripts were directly supported by this project, one additional manuscript is in preparation and eight more publications were indirectly supported. A total of 25 students or postdocs were also trained during the time period this project was active and we made significant progress on achieving our long-term goals. Furthermore, the results obtained from these studies were instrumental in obtaining extramural grant support from the NSF and the USDA and are being used in current pending applications. Briefly, we determined that myostatin's actions are conserved in rainbow trout and that several gene duplication events have produced 4 myostatin paralogs in all salmonids. Current studies are attempting to determine the extent of subfunctionalization among these genes as well as their commercial significance. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our objectives were to (1) isolate and culture primary myosatellite cells from rainbow trout, (2) to determine the effects of IGF-I and myostatin on the proliferation rate and differentiation status of these cells and (3) to begin characterizing the post-receptor signal transduction events of both peptides. We completed all objectives and in addition, generated related and very novel results that better describe the evolution of the myostatin gene family. The combined results are very exciting and could be used to better understand the underlying molecular processes that influence duplicate gene fate and thus, fundamental processes of microevolution. Objectives 1 and 2 additionally revised the field's current understanding of myostatin biology in general and have helped identify markers for assisted selection in rainbow trout.
Publications
- Williams, N., J.Interlichia, M.Jackson, D.Hwang, P.Cohen, and B.D.Rodgers. 2011. Endocrine actions of myostatin; systemic regulation of the insulin-like growth factor (IGF) and IGF binding protein axis.. Endocrinology. 152(1):172-80.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Myostatin is a potent negative regulator of skeletal muscle growth in mammals. Despite high conservation among vertebrate genes, myostatin function in non-mammalian species is poorly defined. Rainbow trout are unique sources of primary myosatellite cells (a.k.a. skeletal muscle stem cells) as, unlike mammals, fish myosatellite cell populations are constantly maintained and significantly contribute to hyperplastic muscle growth even in adults. Compared to mammalian myosatellites, trout cells are easily isolated and can be obtained at far greater numbers. These cells were therefore used to determine the effects of myostatin on proliferation and differentiation. Unlike immortalized mammalian myoblast cell lines, primary trout myosatellites readily differentiate in the presence of serum rather than with serum withdrawal. In fact, differentiation rate was assessed by Myf5, MyoD1, myogenin and MLC expression and was positively correlated to serum concentration. Expression of the different myostatin paralogs increased during differentiation with rtMSTN-1a levels higher than those for -1b and -2a. The rtMSTN-2a transcript, however, was not processed, indicating that autocrine control of myogenesis is regulated by rtMSTN-1 genes and not rtMSTN-2a. Both IGF-I and myostatin temporally stimulated differentiation marker expression, although myostatin's effects were much more pronounced. This differs from studies with mammalian myoblast cell lines where myostatin suppresses rather than stimulates differentiation. Myostatin also suppressed basal and IGF-stimulated proliferation and upregulated rtMSTN-1a expression, which was several fold higher than rtMSTN-1b and -2a. The rtMSTN-2a transcript was always unspliced except in proliferating cells stimulated with IGF-I. Thus, IGF-stimulated differentiation appears to be regulated by the alternative processing of rtMSTN-2a whereas normal differentiation appears to be primarily regulated by the autocrine expression of rtMSTN-1a. These studies are the first to demonstrate myostatin regulation of fish myogenesis. They also suggest that alternative processing of the different myostatin transcripts contributes to gene family subfunctionalization. Furthermore, the general understanding of myostatin action in immortalized mammalian cell lines may not reflect its actions in primary cells as studies with fish and mammalian myosatellites suggest that myostatin activates, rather than inhibits, differentiation. PARTICIPANTS: Dilip Garikipati TARGET AUDIENCES: muscle biologists PROJECT MODIFICATIONS: Not Reported
Impacts
These studies have characterized a novel system for studying skeletal muscle growth and development that can be used by comparative and biomedical researchers alike.
Publications
- Williams, N., J.Interlichia, M.Jackson, D.Hwang, P.Cohen, and B.D.Rodgers. 2010. Endocrine actions of myostatin; systemic regulation of the insulin-like growth factor (IGF) and IGF binding protein axis. Endocrinology. 152(1):172-180.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Results from these studies were disseminated at local and national meetings and have been published in peer-reviewed journals as research articles and editorials. They were also presented in guest lectures. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These studies suggest that the biological actions of myostatin in fish may be quite different than in mammals. They specifically suggest that the cytokine may control the growth and development of several tissues in addition to skeletal muscle as well as two key developmental stages: hatching and yolk sak absorption. Thus, industry attempts to enhance muscle growth in commercially important fish species by controlling myostatin production, gene expression and/or bioavailability need to consider these apparently broad actions. Our studies will additionally help in marker assisted selection efforts to develop fish lines with improved growth. The students trained while conducting these studies have already impacted the scientific community as all are currently employed in a related scientific field.
Publications
- Rodgers, B.D., J.P.Interlichia, D.K.Garikipati, R.Mamidi, M.Chandra, O.L.Nelson, C.E.Murry, and L.F.Santana. 2009. Myostatin represses physiological hypertrophy of the heart and excitation-contraction coupling. The Journal of Physiology. 15(587):4873-86.
- Interlichia, J., N.Williams, and B.D.Rodgers. 2009. A rapid, valid and inexpensive bioassay for quantifying growth rate in mice. Growth Hormone and IGF Research.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Results from these studies were disseminated at local and national meetings and have been published in peer-reviewed journals as research articles and editorials. They were also presented in guest lectures. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These studies suggest that the biological actions of myostatin in fish may be quite different than in mammals. They specifically suggest that cytokine may control the growth and development of several tissues in addition to skeletal muscle as well as two key developmental stages: hatching and yolk sak absorption. Thus, industry attempts to enhance muscle growth in commercially important fish species by controlling myostatin production, gene expression and/or bioavailability need to consider these apparently broad actions. Our studies will additionally help in marker assisted selection efforts to develop fish lines with improved growth. The students trained while conducting these studies have already impacted the scientific community as all are currently employed in a related scientific field.
Publications
- Rodgers, B.D., Roalson, E., and Thompson, C. 2008. Phylogenetic analysis of the insulin-like growth factor binding protein (IGFBP) and IGFBP-related protein gene families. General and Comparative Endocrinology. 155:201-207.
- Lin, M., Al-Holy, M.A., al-Qadiri, H.M., Chang, S., Kang, D., Rodgers, B.D., and Rasco, B. 2008. Phylogenetic and spectroscopic analysis of Alicyclobacillus sp. isolates by 16S rDNA sequencing and Fourier Transform Infrared (FT-IR) spectroscopy. Sensing and Instrumentation for Food Safety and Quality. 1Pages x.
- Rodgers, B.D., and Garikipati, D. 2008. Clinical, agricultural and evolutionary biology of myostatin; a comparative review. Endocrine Reviews. 29(5):513-534.
- Davis, L., Rodgers, B.D., and Kelley, K. 2008. Angiotensin II- and glucose-stimulated extracellular matrix production is mediated by the insulin-like growth factor (IGF) axis in a murine mesangial cell line. Endocrine Journal. 33(1):32-39.
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Progress 01/01/07 to 12/31/07
Outputs
Myostatin is an extremely potent negative regulator of vertebrate skeletal muscle development. Our phylogenetic analysis suggests that salmonids should possess four distinct genes, although only MSTN-1 orthologs have been characterized. These studies resulted in the identification of different MSTN-2 orthologs in different fish species including the rainbow trout (rt). Both gene and promoter structures as well as the tissue-specific and developmental expression profiles for both rtMSTN-2a and -2b were determined. Both rainbow trout genes are similarly organized, contain several putative myogenic response elements and are legitimate MSTN-2 orthologs based on Bayesian analyses. However, rtMSTN-2b contains two in-frame stop codons within the first exon and unspliced variants of both transcripts were expressed in a tissue specific manner. Complete splicing of rtMSTN-2a only occurred in brain, where expression is highest, while rtMSTN-2b transcripts were mostly present in
unspliced forms. The presence of stop codons in the rtMSTN-2b open reading frame and the expression of mostly unspliced transcripts indicates that this particular homolog is a pseudogene. These results confirm our previous phylogenetic analysis and suggest that all salmonids likely possess four distinct myostatin genes. The tissue-specific expression and differential processing of both rtMSTN-2 transcripts as well the pseudogenization of rtMSTN-2b may reflect compensatory and adaptive responses to tetraploidization and may help limit rtMSTN-2a's influences primarily to neural tissue.
Impacts
These studies have clarified many nomenclature discrepancies in the field and identified additional targets for QTL analysis. They also identified alternative mechanisms for growth regulation in a commercially important fish species, the rainbow trout.
Publications
- Garikipati, D., E. Roalson, S.A. Ghar, and B.D. Rodgers. 2007. Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a and -2b): genomic organization, differential expression and pseudogenization. Endocrinology 148(5):2106-2115.
- Rodgers, B.D., E. Roalson, G.M. Weber, S.B. Roberts, and F.W. Goetz. 2007. A proposed nomenclature consensus for the myostatin gene family. American Journal of Physiology: Endocrinology and Metabolism. 292:E371-E372.
- Helterline, D.L., D. Garikipati, D.L. Stenkamp, and B.D. Rodgers. 2007. Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish. General and Comparative Endocrinology 151(1):90-97.
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Progress 01/01/06 to 12/31/06
Outputs
Understanding of myostatin gene phylogenies and expression patterns in fish has been significantly revised thanks to the efforts of my laboratory personnel. To summarize, we have identified and cloned four novel genes in the rainbow trout (rtMSTN-1a, -1b, -2a & -2b), we have generated gene-specific assays for quantifying their expression and have used these assays to define their developmental and tissue-specific expression patterns. We have also performed similar assays using a model system, zebrafish, and have started to investigate myostatin's actions directly on myosatellite cells isolated from rainbow trout. These studies have together resulted in the production of nine manuscripts (7 published/in press & 2 in review - published articles below) as well as seven published abstracts (not shown) of presentations at various annual and regional meetings including those of the Endocrine Society, the World Aquaculture Society and the Society of Developmental Biology.
Three MS students (Tovah Kerr, Deri Helterline & Sally Wang-Jun Lin) have also completed their degrees working on these studies and a PhD student, Dilip Garikipati, will complete his dissertation studies in Spring 2007.
Impacts
These studies suggest that the biological actions of myostatin in fish may be quite different than in mammals. They specifically suggest that the cytokine may control the growth and development of several tissues in addition to skeletal muscle as well as two key developmental stages: hatching and yolk sak absorption. Thus, industry attempts to enhance muscle growth in commercially important fish species by controlling myostatin production, gene expression and/or bioavailability need to consider these apparently broad actions. Our studies will additionally help in marker assisted selection efforts to develop fish lines with improved growth. The students trained while conducting these studies have already impacted the scientific community as all are currently employed in a related scientific field.
Publications
- Rodgers, B.D. 2005. Insulin-like growth factor-I downregulates embryonic myosin heavy chain (eMyHC) protein in myoblast nuclei. GH IGF Res 15(6):377-83.
- Oufattole, M., S.W.J. Lin, B. Liu, D. Mascarenhas, P. Cohen and B.D. Rodgers. 2006. RNA polymerase II subunit 3 (Rpb3), a potential nuclear target of IGFBP-3. Endocrinology 147(5):2138-46.
- Garikipati, D., S.A. Ghar and B.D. Rodgers. 2006. Identification, characterization and quantitative expression analysis of rainbow trout myostatin-1a and -1b genes. J Endocrinol 190(3):879-88.
- Rodgers, B.D., E.H. Roalson, G.M. Weber, S.B. Roberts and F.W. Goetz. A proposed nomenclature consensus for the myostatin gene family. Am J Physiol; Endocrinol Metab Sep. 26, 2006 (Epub ahead of print). http://ajpendo.physiology.org/
- Kerr, T., E.H. Roalson and B.D. Rodgers. 2005. Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in Zebrafish. Evo Devo 7(5):391-401.
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Progress 01/01/05 to 12/31/05
Outputs
Myostatin is a member of the transforming Growth Factor (TGF) beta super-family and a potent negative regulator of skeletal muscle development. Although myostatin cDNA clones have been characterized in several diverse species, the genomic organization and bioactivity of myostatin homologues in non-mammalian vertebrates has not. A recent phylogenetic analysis of the myostatin subfamily indicates that an early gene duplication event produced MSTN-1 and MSTN-2 sister clades in the boney fishes while a second isolated event within the Salmonidae family produced additional orthologues in each clade. Thus, all salmonids should possess four distinct myostatin genes, MSTN-1a, -1b, 2a and -2b, of which only MSTN-1a and -1b cDNA clones have been characterized. We therefore isolated complete genomic clones for all four rainbow trout genes using gene-specific and degenerate primers in genome walking and PCR assays. Each gene is similarly organized into three exons, a pattern
conserved among all vertebrates, although intron sequences and sizes are only conserved between orthologues. MSTN-2b contains a 51 bp deletion within the second intron and two in-frame stop condons within the first exon, suggesting pseudogenization. In silico subsequence analysis of the promoter regions revealed the presence of several E-boxes and other putative myogenic response elements. However, a MyoD binding site, which is an important regulator of mammalian myostatin gene expression during myogenesis, was only identified in the MSTN-2a promoter. A quantitative analysis of the embryonic expression profile for both MSTN-1 genes indicates that MSTN-1a expression is consistently greater than that of MSTN-1b and that neither gene is significantly expressed throughout gastrulation. Expression increases during somitogenesis and peaks as this developmental period ends. After eyeing, however, MSTN-1a and -1b mRNA levels rise considerably and steadily until adulthood. A similar analysis
of adult tissue expression indicates that all four genes are expressed in most tissues although mRNA levels are highest in brain, muscle and spleen. Furthermore, non-spliced variants of both MSTN-2 transcripts were present in all tissues sampled except brain, which is the first report of tissue-dependent processing of myostatin pre-mRNA for any species. These data together suggest that the genomic organization of all myostatin genes is extremely well conserved in vertebrates and that the expression of MSTN-1a, -1b and -2a are often differentially regulated in adult tissues and throughout embryonic development. The likely pseudogenization of MSTN-2b and the tissue-specific differential processing of MSTN-2 transcripts may reflect adaptive changes to tetraploidization and the potential negative effects of too much myostatin.
Impacts
Manipulating myostatin production and/or bioactivity has the potential to revolutionize the aquaculture industry as the dramatic increase in muscle mass associated with myostatin-null animals has never been attained through the administration of growth promoters. However, blindly targeting all myostatin genes in rainbow trout will likely have very negative effects. Our work suggests that the cytokines may regulate many critical processes including brain and immune cell development. Any attempt to manipulate myostatin must therefore target a specific gene and tissue. Our work is setting the stage for just such advances.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The myostatin-null phenotype in mammals is characterized by extreme gains in skeletal muscle mass or double muscling as the cytokine negatively regulates skeletal muscle growth. Recent attempts, however, to reproduce a comparable phenotype in zebrafish have failed. Several aspects of myostatin biology in the fishes differ significantly from those in mammals and at least two distinct paralogues have been identified in some species, which possibly suggests functional divergence between the different vertebrate classes or between fish paralogues. We therefore conducted a phylogenetic analysis of the entire myostatin gene sub-family. Maximum likelihood, Bayesian inference and bootstrap analyses indicated a monophyletic distribution of all myostatin genes with two distinct fish clades: MSTN-1 and -2. These analyses further indicated that all Salmonid genes described are actually MSTN-1 orthologues (1a & 1b) and that additional MSTN-2 paralogues likely exist in most fish
species. An additional zebrafish homologue was identified by BLAST searches of the zebrafish HTGS database and was subsequently cloned. Comparative sequence analysis of both genes (zfMSTN-1 & -2) revealed many differences, primarily within the latency associated peptide regions, but also within the bioactive domains. The 2 kb promoter region of zfMSTN-2 contained many putative cis regulatory elements that are active during myogenesis, but are lacking in the zfMSTN-1 promoter. In fact, zfMSTN-2 expression was limited to the early stages of somitogenesis, while zfMSTN-1 was expressed throughout embryogenesis. These data suggest that zfMSTN-2 may be more closely associated with skeletal muscle growth and development. We have additionally isolated and characterized the rainbow trout MSTN-1a and -1b genomic clones. Each gene is segregated into three exons separated by introns of different sizes. However, the respective exons in each gene are identical in size, which independently supports
the orthologous relationship suggested by the phylogenetic analysis. Future studies will determine the cis regulatory elements responsible for tissue specific expression of each gene as the 2 kb region promoter region of each has also been cloned.
Impacts
These studies resolve the previous ambiguity in classification of fish myostatin genes, specifically among the Salmonids, and propose a revised nomenclature that is based upon the true evolutionary relationships between each gene. The characterization of the genomic clones will add to the understanding of myostatin evolution and will eventually aid in determining the tissue-specific function of each gene.
Publications
- No publications reported this period
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