Progress 10/01/10 to 09/30/15
Outputs
Target Audience:During this reporting period, presentations of the results of the project were shared with members of the scientific community in several venues including conferences and presentations at the University of Illinois. Our station report was also shared with members of the multi-state committee. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has allowed for professional development in 2 ways. First, the funds provided support the work of graduate and undergraduate students trained in muscle biology and nutritional sciences. Secondly, this multi-state project brings together muscle biology researchers from both within our university and across the country to interact and collaborate. The professional development of young investigators is supported by this group. How have the results been disseminated to communities of interest?Results were disseminated to the scientific community through publications, conferences and the annual meeting of stations. What do you plan to do during the next reporting period to accomplish the goals?Work will continue under the current objectives to identify the interaction of IGF2 and myostatin in the regulation of muscle growth in mice and swine. In 2016, both mouse and swine models will be available for characterization of these effects. Furthermore, now that all muscle biopsies have been obtained and protocols optimized, the analysis of satelite cell activation and growth factor regulation will proceed.
Impacts
What was accomplished under these goals?
Accomplishments: Objective 2: Characterize the Cellular and Molecular Basis of Myogenesis and Muscle Growth Interaction of IGF2 and Myostatin in the Regulation of Muscle Growth and Development 1. Using zinc-finger nuclease technology, we have engineered mutant myostatin pigs lacking one nucleotide in exon 3 of the myostatin gene. This mutation results in a premature stop codon and is predicted to result in pigs lacking functional myostatin. Cloned gilts heterozygous tothis mutation were bred and have successfully farrowed. 2. Pigs heterozygous for this myostatin mutation with either paternal A or paternal G IGF2 alleles are viable, fertile and produce viable offspring. 3. Segregation ratios of the myostatin mutation are consistent with the expected inheritance pattern. 4. Initial evaluations of piglets with homozygous mutations of myostatin suggest that muscle mass, expressed as a percentage of body weight, is increased. 5. To further explore the interaction between IGF2 and myostatin, transcription activator-like effector nucleases (TALEN) with oligodeoxynucleotides templates intended to disrupt the ZBED6 binding site mimicking the G>A substitution that occurs in pigs were used to create mouse models. This resulted in 3 mutant lines with mutations within the ZBED6 binding site. 6. Founders have been bred to produce experimental animals to characterize gene expression, muscle growth and body composition. Satellite Cell Activation and Amino Acid Transporter Expression: 1. We have collected and prepared all human muscle biopsies for immunofluorescence analyses. 2. We have completed preliminary in vitro experiments examining the protein content and gene expression of various amino acid transporters across the lifespan of myoblasts. 3. Preliminary experiments indicate that gene expression of amino acid transporters decreases during myoblast differentiation; however, protein content is increased. Mesenchymal stem cell (MSC) Quantity and Paracrine Factor Secretion: 1. Protocols for isolation of MSCs from fresh human muscle biopsies have been optimized. 2. All human muscle biopsies have been collected from healthy weight, overweight, and obese individuals. 3. Protocols for immunofluorescence detection of MSCs in muscle cross-section have been optimized. 4. Preliminary data indicate that MSCs have higher levels of expression of inflammatory factors (Il-6), growth factors (IGF-1), and extracellular matrix proteins (Collagen 1) than myoblasts.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Clark, D.L., D.I. Clark, J.E. Beever and A.C. Dilger. 2015. Increased prenatal IGF2 expression due to the porcine IGF2 intron3-G3072A mutation may be responsible for increased muscle mass. Journal of Animal Science 93:2546-2558.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Clark, D.L., D.I. Clark, E.K. Hogan, K.A. Kroscher and A.C. Dilger. 2015. Elevated insulin-like growth factor 2 expression may contribute to the hypermuscular phenotype of myostatin null mice. Growth Hormone and IGF Research 25:207-218.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: During this reporting period, presentations of the results of the project were shared with members of the scientific community in several venues including conferences, presentations at the University of Illinois and at the annual multi-state meeting. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has allowed for professional development in 2 ways. First, the funds provided support the work of graduate and undergraduate students trained in muscle biology. Secondly, this multi-state project brings together muscle biology researchers from across the country to interact and collaborate. The professional development of young investigators is supported by this group. How have the results been disseminated to communities of interest? Results were disseminated to the scientific community through publications, conferences and the annual meeting of stations. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, work will continue at the Illinois Station under objectives 1 and 2 of the project. Work is ongoing in the area of IGF2 and myostatin regulation of muscle growth. Signaling mechanism downstream of exogenous growth promotants are being elucidated. The effect of prenatal and postnatal infection on muscle development and growth is being investigated.
Impacts
What was accomplished under these goals?
Objective 1. Characterize the signal transduction pathways that regulate skeletal muscle growth and metabolism including the influence of endogenous growth factors and various production practices. Accomplishments: Insulin-like growth factors (IGF) 1 and 2 regulate prenatal and postnatal skeletal muscle development and growth. While IGF2 is thought to primarily regulate prenatal and not postnatal muscle growth, in swine a single base pair substitution in intron 3 of IGF2 (g3072A) results in increased lean meat yield in pigs. This SNP disrupts the binding of a transcriptional repressor ZBED6, and lack of that repressor binding is thought to result in increased postnatal IGF2 expression, underlying the phenotype of pigs with a paternal A allele, as IGF2 is maternally imprinted. This suggests that IGF2 may be more involved in postnatal muscle growth than previously assumed. Our ongoing work in this area has established the following new information about IGF2 and other regulators of muscle growth in animals: 1. Increased lean meat yield from pigs with IGF2 paternal A alleles is a result of both increased muscle growth and reduced fat deposition. Lean meat yield at market weight (176 d of age, 127 kg live weight) was increased in pigs with paternal A alleles compared with pigs with paternal G alleles resulting from a reduction in back fat depth and an increase in loin eye area at similar hot carcass weights. Body weight was not affected by genotype at any age and muscle weights (longissimus dorsi, semitendinosus and psoas major) were increased by the paternal A allele only in market weight pigs. Increased muscle weights were not observed at younger ages. 2. Increased expression of IGF2 in pigs with paternal A alleles begins as early as late gestation and continues throughout the production phase of pigs. Though expression of IGF2 was similar between genotypes at d 60 of gestation, starting at d 90, IGF2 expression was increased in pigs with the paternal A allele compared with pigs with the paternal G allele. At market weight, IGF2 was increased 4-fold in paternal A compared with paternal G pigs. 3. Muscle fiber number may be increased in pigs with paternal IGF2 A alleles. Despite the increase in postnatal IGF2, muscle fibers of the semitendinosus tended to be smaller in paternal A compared with paternal G pigs. However, there also tended to be more muscle fibers in paternal A pigs. Therefore, it is possible that increased prenatal IGF2 expression noted at d 90 of gestation leads to greater hyperplasia during muscle fiber formation resulting in increased muscle weights observed at market weights. Increased expression of Myf5 and myogenin at d 90 of gestation support this hypothesis. 4. The heavily muscled phenotype of myostatin null mice may be partially attributable to increased IGF2 expression but not to changes in IGF1. Atd 21, 42 and 70 of age, IGF2 expression was increased in myostatin null mice compared with wild type while IGF1 was unchanged. The significance of increased IGF2 expression was confirmed by measuring IGF2 protein by ELISA at d 70; IGF2 protein levels were increased more than 2-fold in myostatin null mice compared with wild type. This increase parallels the 2.9 fold increase in expression of IGF2 at similar ages. Though IGF2 expression was unchanged at birth and 7 d of age, IGF1 expression was actually reduced in myostatin null mice compared with wild type at these ages. Therefore, it is unlikely that IGF1 expression contributes to the hypermuscular phenotype of myostatin null mice. 5. Increased IGF2 expression in myostatin null mice is not variant specific. All three IGF2 variants were increased in concert at both d 21 and 70 of age in myostatin null mice compared with wild type. 6. ZBED6, a repressor of IGF2 expression, is likely not responsible for the reduction in postnatal IGF2 expression compared with fetal expression. In both pig and mouse models, the expression of ZBED6 was not increased with age, which would be expected if ZBED6 was responsible for reductions in IGF2 expression. In fact, ZBED6 expression in pigs was greatest in muscle at d 60 of gestation. 7. Heterozygous mutations of myostatin are viable in pigs with paternal G alleles for IGF2. Using zinc-finger nuclease technology, we have engineered mutant myostatin pigs lacking one nucleotide in exon 3 of the myostatin gene. This mutation results in a premature stop codon and is predicted to result in pigs lacking functional myostatin. This mutation was engineered in a wild-type (paternal G) cell line to prevent interference by the IGF2 paternal A allele. Somatic cell nuclear transfer and embryo transfer resulted in the live birth of 7 cloned gilts, heterozygous for the myostatin mutation. At current, 5 of these gilts are pregnant with offspring sired by IGF2 G/A boars. These pregnancies will result in the F1 generation possessing both heterozygous mutations to allow for further breeding to produce myostatin null pigs of both IGF2 paternal G and IGF2 paternal A genotypes. 8. Changes in myostatin expression in adipose tissue may underlie metabolic changes observed in cats based on photoperiod. Myostatin expression in adipose was increased in short-day housed cats compared with long-day housed cats. This accompanied changes in other genes involved in cell division and development and protein turnover. In contrast, short-day housed cats had decreased expression of genes related to immune function. The interaction of adipose tissue and whole body metabolism in determining the body composition of cats and other animals requires more investigation. 9. Differences in the reductions of growth-regulating genes observed with age do not explain muscle growth differences between sexes or genotypes. A set of 7 genes (Ezh2, Gpc3, Mdk, Mest, Mycn, Peg3, and Plagl1) had been previously implicated in regulating the growth rates of organs as their expression declines with increasing age and growth. However, we have determined that while the expression of these genes does decline with age in the muscles of mice, the rate of that decline did not differ between wild type and more heavily muscle myostatin null mice, nor did it differ between male and female mice.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jones, J.C., Kroscher, K.A. and Dilger, A.C. Reductions in expression of growth regulating genes in skeletal muscles and organs with age. BMC Physiology 14:3.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Clark, D.I., Clark, D.L., Rochell, S.J., Dilger, R.N., Johnson, R.W. and Dilger, A.C. Effects of maternal and post natal infection with porcine reproductive and respiratory syndrome virus on muscle growth and development in piglets. Experimental Biology. San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Clark, D.L., Clark, D.I., Hogan, E.K., Kroscher, K.A. and Dilger, A.C. Expression of insulin-like growth factor family members in myostatin null mice. Experimental Biology. 26-30 April 2014, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Kroscher, K.A., Clark, D.L. and Dilger, A.C. Expression of growth regulatory genes in skeletal muscle of myostatin null mice. Experimental Biology. 26-30 April 2014, San Diego, CA.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Mori, A., Kappen, K.L., Dilger, A.C. and Swanson, K.S. Effect of photoperiod on the feline transcriptome as assessed by RNA sequencing. BMC Veterinary Research 10:146.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Hogan, E.K., Clark, D.L. and Dilger, A.C. Temporal postmortem RNA degradation in subcutaneous adipose tissue in swine. Midwest Section of Animal Sciences Meeting. 17-19 March 2014, Des Moines, IA.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Clark, D.L., Bohrer, B.M., Tav�rez, M.A., Boler, D.D., Beever, J.E. and Dilger, A.C. Effects of the porcine IGF2 intron3-G3072A mutation on carcass cutability, meat quality and bacon processing. Journal of Animal Science 92:5778-5788.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Clark, D.L., Clark, D.I., Beever, J.E. and Dilger, A.C. Effect of the porcine IGF2 intron3 G3072A substitution on development and growth of skeletal muscle. Experimental Biology. 26-30 April 2014, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Clark, D.L., Beever, J.E. and Dilger, A.C. Effect of the porcine IGF2 intron3-G3072A substitution on carcass cutability and meat quality. Midwest Section of Animal Sciences Meeting. 17-19 March 2014, Des Moines, IA.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The main target audience during this reporting period was the scientific community, specifically those working in animal sciences and muscle biology. Outreach to the local community (K-12, public) was also conducted informally as was interaction with the meat and animal pharmaceutical industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project allowed for the training of 2 graduate students and several undergraduate students in our labortory. It also allowed us to leverage other federally appropriated money to extend the research of other investigators into the area of muscle biology and growth. Two students, supported by this project, will graduate in 2014 with an MS and a PhD degree. How have the results been disseminated to communities of interest? These results were dissemenated to the scientific community in the form of abstracts and presentations. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, we plan to complete our work regarding the influence of IGF2 SNP on muscle growth and development. We will present these results as abstracts at 2 upcoming meetings. Furthermore, we will complete the phenotypic evalation of piglets from dams exposed to PRRS prenatally and present those results as well. Publication of these results isplanned for 2014.
Impacts
What was accomplished under these goals?
In the past year, the Illinois Station completed work under two of the three objectives of the overall project. This work includes: Objective 1. Characterize the signal transduction pathways that regulate skeletal muscle growth and metabolism including the influence of endogenous growth factors and various production practices. In swine, a QTL that explained 15-30% of variation in muscle mass and 10-20% of variation in back fat was first reported in Large White and Peitran breeds. The mutation was later identified as a single base pair substitution in intron 3 of IGF2 (g3072A). The substitution altered a conserved CpG island that is hypomethylated in skeletal muscle and is thought to be a binding site for ZBED6, a repressor. Interestingly, IGF2 is one of many maternally imprinted genes. The mutation (inherited paternal A) resulted in an increase in IGF2 expression in postnatal muscle though it did not alter IGF2 expression in fetal muscle. Increased postnatal IGF2 expression may be responsible for increased differentiation in satellite cells resulting in increased muscle hypertrophy. However, the phenotype of IGF2 intron 3 g3072A animals has not been fully characterized; therefore, the mechanism by which this mutation alters muscle growth remains unclear despite the A allele being largely fixed in commercial swine populations. To more fully characterize the phenotype of this mutation and determine the mechanism of increased muscle growth in pigs carrying a paternal A allele, IGF2 mutant and wild-type animals were produced with a common genetic background and analyzed from day 60 of gestation through market weight. The mutation did not alter body weight at any time point measured. However, the mutation did alter carcass composition. Pigs with a mutant A allele had decreased backfat thickness (P <0.05) and increased loin muscle area (P <0.05) and longissimus weight (P <0.01), thereby resulting in a 2.4-percentage unit increase in lean carcass cutting yield (P <0.0001). In contrast, the mutation only resulted in minimal changes in fresh meat quality parameters. The A genotype did increase post-natal IGF2 expression, up to a 4-fold increase in expression at a market weight time point (P <0.0001), without significantly altering IGF1 expression. However, IGF type 1 and type 2 receptors, myostatin, and ZBED6 expression do not appear to be altered by the mutation. Objective 2. Characterize the cellular and molecular basis of myogenesis. Alterations in muscle development due to prenatal insults can have life-long implications for both animal production and human health. We have recently began a series of projects to determine the fetal programming that results from nutritional alterations or from maternal infection. Choline, an essential nutrient, plays a key role in regulating growth and brain development. It is unclear, however, whether prenatal or postnatal deficiency in choline can alter skeletal muscle growth. Treatments were arranged in a 2x2 factorial with factors of choline status (deficient vs sufficient) and timing of treatment (prenatal vs postnatal). Sows were fed diets either deficient or sufficient in choline, and piglets were raised artificially on milk replacer deficient or sufficient in choline. Gene expression was analyzed in the longissimus dorsi muscle while cell size was measured in the semitendinosus muscle. Expression of IGF1 , IGF2, and myogenin were unaffected by treatment, while postnatal choline deficiency increased the expression of MyoD. Both prenatal and postnatal choline deficiency resulted in an increase in myostatin expression. Furthermore, MHC1 gene expression was increased and MHC2b expression was decreased in postnatal choline deficient animals. Expression of MHC2a and MHC2x were unaltered by treatment. Additionally, choline deficiency did not alter average muscle cell size. To determine the influence of maternal infection on prenatal muscle development, sows were infected with porcine respiratory and reproductive syndrome (PRRS) virus at day 76 of gestation, and muscle samples were collected at birth and 28 days of age. As expected, maternal PRRS virus infection decreased crown to rump length, birth weight and the weight of the longissimus dorsi and psoas major muscles. Semitendinosus muscle has been collected to determine muscle cell size and number, while the longissimus dorsi and psoas major was collected for muscle gene expression. To contrast prenatal exposure to PRRS with a postnatal infection, we also collected tissue samples from pigs inoculated with PRRS virus at 5 weeks of age and sacrificed at 7 weeks of age. Inoculation with PRRS virus resulted in decreased body weight and crown to rump length and decreased longissimus dorsi and semitendinosus weights. However, PRRS virus did not alter the weight of the psoas major.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Marron, B.M., Clark, D.L., Dilger, A.C. and Beever, J.E. Gene-Editing of Porcine Myostatin using Zinc-Finger Nucleases. Plant and Animal Genome XXI. 12-16 January 2013, San Diego, CA.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: An experiment investigating the influence of genes important in the proliferation of organ cells on the growth of skeletal muscle was completed. These results are contained in the thesis of Jennifer Jones whose work was supported by the project. She recently completed her MS degree. Publication of this work is currently under review. This project also informs my lectures as I teach Animal Growth and Development and Muscle Biology to undergraduate and graduate students. PARTICIPANTS: Two graduate fellows, Jennifer Jones and Daniel Clark, are supported by this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This year, results of research related to muscle growth have resulted in a change in knowledge for myself and my students. The input from collaborators from other stations has improved my own experiments and provided opportunities for further training of my students. In the future, collaborations within this project will extend my skill set and allow new techniques to be introduced into my laboratory furthering my work.
Publications
- Jones, J.C. 2012. Reductions in expression of growth regulating genes are observed in skeletal muscle and organs with age. M.S. Thesis, University of Illinois, Urbana-Champaign.
- Baer, A.A, Jones, J.C., Jones, K.A. and Dilger, A.C. 2012. Synergistic effects of Beta-adrenergic agonists and zinc supplementation. ASAS Midwest Section Meeting, Abstract 243P.
- Jones, J.C. and Dilger, A.C. 2012. Similar reductions in expression of proliferative genes are observed in skeletal muscle and organs with age. The FASEB Journal, 26:1086.6.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: In this, my first year as part of the NC-1184 Multi-state project, I have completed several activities related to beginning my research program in the molecular regulation of muscle growth. I planned an experiment investigating the influence of genes important in the proliferation of organ cells on the growth of skeletal muscle. I am mentoring a graduate fellow who is completing the work involved in this project and the results will form the basis of her Masters of Science thesis. Information from this Multi-State project also informs my teaching of Animal Growth and Development, a course in the curriculum at the University of Illinois. In connection with work and expertise of this project, I also completed consulting this year with a small start-up company interested in the manipulation of muscle growth of chicken embryos. Finally, I submitted an AFRI grant this year whose results will continue the work of this Multi-State project. PARTICIPANTS: Individuals: Two graduate students are being supported by this project including Jennifer Jones and Arica Baer. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This year, results of research related to muscle growth have resulted in a change in knowledge for myself and my students. Due to research in the area of swine growth rate and meat quality, we now understand that the rate of growth in swine may be inversely correlated with meat quality though it has positive impact on the yield of meat. In the future, collaborations within this project will extend my skill set and allow new techniques to be introduced into my laboratory furthering my work.
Publications
- Boler, D.D., Killefer, J., Meeuwse, D.M., King, V.L., McKeith, F.K. and Dilger, A.C. 2011. Effects of harvest time post-second injection on carcass cutting yields and bacon characteristics of immunologically castrated male pigs. Journal of Animal Science (In Press).
- Clark, D.L., Boler, D.D., Kutzler, L.W., Jones, K.A., McKeith, F.K., Killefer, J., Carr, T.R. and Dilger, A.C. 2011. Muscle gene expression associated with increased marbling in beef cattle. Anim Biotechnology, 22:51-63.
- Tavarez, M.A., Boler, D.D., Bess, K.N., Zhao, J., Yan, F., Dilger, A.C., McKeith, F.K. and Killefer, J. 2011. Effect of antioxidant inclusion and oil quality on broiler performance, meat quality, and lipid oxidation. Poultry Science, 90:922-930.
- Boler, D.D., Holmer, S.F., Duncan, D.A., Carr, S.M., Ritter, M.J., Petry, D.B., Hinson, R.B., Allee, G.L., McKeith, F.K. and Killfer, J. 2010. Fresh meat and further processing characteristics of ham muscles from finishing pigs fed Ractopamine hydrochloride (Paylean). Journal of Animal Science, 89: 210-220.
- Lowe, B.K., Clark, D.L., Boler, D.D., Dilger, A.C., McKeith, F.K., Eggert, J.M., Newcom, D.W. and Killefer, J. 2011. Characterization of loin shape from Duroc and Duroc composite finishing gilts. Meat Science, 87:146-150.
- Dilger, A.C., Spurlock, M.E., Grant, A.L. and Gerrard, D.E. 2010. Myostatin null mice respond differently to dietary-induced and genetic obesity. Anim Sci Journal, 81:586-593.
- Clark, D.L. 2010. Transcription profiles of differentially marbled beef cattle. MS Thesis.
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