Progress 06/01/05 to 05/31/08
Outputs
OUTPUTS: We conducted a growth trial of callipyge and normal lambs and collected extensive phenotypic data as well as tissue samples for gene expression and protein analysis. The results of the studies of the callipyge locus were presented at national (Plant and animal Genome Conference (2005-2008) and international meetings (International Society of Animal Genetics (2002 and 2006) and published in peer reviewed journals. This sample collection was leveraged by internal funding and collaboration with the investigators in Australia to conduct a gene profiling experiment using the Affymetrix Bovine Genechip. This greatly expanded the scope of studies beyond the original funded objectives. The combined sample collections and data from Purdue University, Utah State University and CSIRO Livestock Industries has a full range of prenatal development and postnatal growth of skeletal muscles in sheep as well as the callipyge model for postnatal muscle hypertrophy. We have been able to utilize the bovine genome sequence and bovine microarrays to analyze gene expression in sheep. The microarray papers from this work have identified several genes that were completely unknown or not recognized to be involved in muscle growth. This data set will provide a long term resource to investigate muscle growth, production efficiency and meat quality. PARTICIPANTS: Training: Jolena Fleming-Waddell, Ph D. Department of Animal Sciences, Purdue University 2008. Mrs. Waddell's graduate stipend was supported by the grant. She conducted the callipyge lamb growth trial and the quantitative PCR analysis of the genes from the callipyge region (in preparation). She also conducted a microarray analysis of muscle hypertrophy from the samples produced in this study as part of an international research experience in collaboration with R. L. Tellam, CSIRO Livestock Industries. Gayla Olbrigt, Ph D student. Department of Statistics, Purdue University. Mrs. Olbrigt worked on statistical analysis of the microarray data and testing of several algorithms for interpretation of the cross-species hybridization used. Tasia M Taxis, B.S. Department of Animal Sciences, Purdue University 2008. Ms. Taxis worked in the laboratory since her freshman year on several aspects of the project including the callipyge lamb growth trial, RNA isolation and quantitative PCR. She conducted a senior special problems course analyzing gene expression in the liver of callipyge lambs. She has gone on to graduate school at the University of Missouri. Collaborations: Dr. Bruce A. Craig, Department of Statistics, Purdue University Dr. Ross L Tellam, CSIRO Livestock Industries, St. Lucia Queensland Australia. Dr. Jason D. White, School of Veterinary Medicine, University of Melbourne, Victoria, Australia TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project examined the expression of the genes surrounding the callipyge mutation in skeletal muscle, adipose tissue and liver to determine which genes have an overdominant gene expression pattern. Allison Perkins work found that the two genes expressed from the paternally inherited chromosome (DLK1 and PEG11) both show polar overdominance. The DLK1 and PEG11 transcripts abundance are elevated in the muscles of paternal heterozygous (+/C; callipyge) lambs with an intermediate level of expression in the homozygous (C/C) animals. The mutation has a very different effect on the genes expressed from the maternal chromosome (MEG3 and MEG8). There was a equivalent up-regulation of MEG3 and MEG8 in the maternal heterozygous (C/+) and homozygous lambs The most striking difference was the fact the mutation does not effect DLK1 or PEG11 expression in muscle prior to birth but has a major effect after birth which is consistent with the muscle hypertrophy phenotype. The preponderance of evidence indicates DLK1 is the major gene to cause muscle hypertrophy, however, the fact that PEG11 is also up regulated after birth in callipyge lambs indicated it cannot be ruled out as contributing to muscle hypertrophy. Jolena Fleming-Waddell's conducted microarray analysis of gene expression during the onset of muscle hypertrophy. She validated differential gene expression of over thirty genes in callipyge muscle. Genes from several biological processes were identified including transcription factors, genes that regulate signal transduction pathways as well as several genes of completely unknown function. Most of the genes appeared to be responsive to DLK1 expression but one gene appeared to be responsive to PEG11 based on its muscle specific gene expression pattern. This result provided another piece of evidence that PEG11 contributes to muscle hypertrophy in callipyge lambs. Lindsay Wilson and Tasia Taxis analyzed the effect of the mutation on gene expression in adipose tissue and liver and found no direct effect based on genotype. Therefore, the decrease in carcass fat and decrease liver size are likely an indirect effect of muscle hypertrophy such as the repartitioning of nutrient to support muscle growth.
Publications
- Fleming, J.N., L.M. Wilson, G. Olbrigt, T. Vuocolo, K. Byrne, B.A. Craig, R.L. Tellam, N.E. Cockett, and C.A. Bidwell. 2006. Analysis of gene expression during the onset of muscle hypertrophy in callipyge lambs. Plant and Animal Genome Conference XIV, San Diego, CA. http://www.intl-pag.org/14/abstracts/PAG14_P724.html
- Vuocolo, T., K. Byrne, S. McWilliam, A. Reverter, N. Cockett and R. Tellam. 2006. Transcriptional profiling of callipyge skeletal muscle. Plant and Animal Genome Conference XIV, San Diego, CA. http://www.intl-pag.org/14/abstracts/PAG14_P753.html.
- Perkins, A. C., L. N. Kramer, D. E. Moody, T. S. Hadfield, N. E. Cockett and C. A. Bidwell (2006) Postnatal changes in gene expression from the callipyge region in sheep skeletal muscle. Anim. Genet. 37:535-542.
- Fleming-Waddell, J. N., L. M. Wilson, G. R. Olbricht, T. Vuocolo, K. Byrne, B. A. Craig, R. L. Tellam, N. E. Cockett and C. A. Bidwell (2007) Analysis of gene expression during the onset of muscle hypertrophy in callipyge lambs. Anim. Genet. 38:28-36.
- Vuocolo, T., K. Byrne, J. White, S. McWilliam, T. Reverter, N. E. Cockett and R. L. Tellam (2007) Identification of a gene network contributing to muscle hypertrophy in callipyge skeletal muscle. Physiological Genomics 28:253-272.
- White, J. D., T. Vuocolog, M. McDonagh, M. D. Grounds, G. Harper, N. E. Cockett and R. Tellam (2008) Cellular analysis of the callipyge phenotype through skeletal muscle development: association of DLK1 with developing satellite cells. Differentiation 76:283-298.
- Vuocolo, T., K. Byrne, J. White, N. Cockett, A. Reverter and R. Tellam. 2006. Changes in skeletal muscle gene expression caused by the callipyge mutation in sheep. Plant and Animal Genome Conference XIV, San Diego, CA. http://www.intl-pag.org/14/abstracts/PAG14_W219.html.
- Bidwell, C.A. and N.E. Cockett. 2006. Genomic regions associated with sheep muscle and carcass traits. J. Anim. Sci. 84 (Suppl. 1), 159.
- Fleming, J.N., L.M. Wilson, G.R. Olbricht, T. Vuocolo, K. Byren, B.A. Craig, R.L. Tellam, N.E. Cockett, C. Bidwell. 2006. Gene expression profiling of two hypertrophied muscles in callipyge lambs. Proceedings of 30th International Conference on Animal Genetics. Porto Seguro, Brazil. August, 2006. http://www.isag.org.uk/pdf/conferences.asp.
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Progress 06/01/06 to 05/31/07
Outputs
OUTPUTS: Sheep with the callipyge trait have increased muscling predominantly in the loin and pelvic limbs. The callipyge mutation is a single nucleotide polymorphism between the DLK1 and MEG3 genes. The phenotype is inherited in a non-Mendelian manner called polar overdominance. Callipyge lambs inherit the wild type allele from the dam and the callipyge allele from the sire (+/C). Animals that are homozygous for the mutation (C/C) do not have muscle hypertrophy. We have previously reported that the presence of the mutation increases the expression of the DLK1 gene a parent-of-origin specific effect. Furthermore, there are some differences in the effect of the maternal allele specific genes relative to the paternal allele specific genes. We examined the expression of the DLK1 protein to determine if post-transcriptional mechanisms may be involved in polar overdominance for objective 2. Fetal antigen 1 (FA1) is a secreted proteolytic product of the DLK1 gene. We examined the
expression of the DLK1 protein in frozen sections of the semimembranosus (SM) muscle in collaboration with Dr. Jason D. White, Univ. Melbourne using an anti-bovine FA1 antibody provided by Dr. Ross L. Tellam CSIRO Livestock Industries in Australia. The DLK1 protein was detected in the myofiber basement membrane of callipyge (+/C) and homozygous (C/C) lambs but not in normal (+/+) or maternal heterozygous lambs (C/+). There were substantially fewer DLK1 positive myofibers in C/C lambs than in callipyge lambs (+/C). Western blot analysis of total membrane proteins from the SM also showed significantly more DLK1 in callipyge lambs that in the other three genotypes (P<.05). The affected muscles of callipyge lambs grow by hypertrophy. There is an increase in the size and proportion of fast twitch glycolytic myofibers. Therefore, we examined the effect of the callipyge mutation on four skeletal muscle myosin isoforms as downstream targets of elevated DLK1 expression in the longissimus dorsi
(LD) and SM that undergo hypertrophy and the supraspinatus (SS) that does not undergo hypertrophy. The transcript abundance of MYH7 (Type I, slow twitch, oxidative) in the LD was significantly lower (P=.0423) in callipyge lambs than in normal lambs. There was a trend (P=.0749) for lower expression of MYH7 in the SM of callipyge lambs. There was also a significant effect of genotype on the expression of MYH2 (Type IIa, fast twitch, oxidative) in the LD (P=.0007) and SM (P<.0001). Callipyge lambs had a 6-8 fold reduction in MYH2 transcript abundance relative to normal lambs. There was no effect of genotype on the expression of MYH1 (fast twitch, glycolytic) but there was a highly significant effect of genotype on MYH4 (fast twitch, glycolytic) in both the LD (P<.0001) and SM (P<.0001). During the initial onset of muscle hypertrophy (20 to 30 days), there was a 10-13 fold increase in MYH4 in the LD and a 20-48 fold increase of MYH4 in the SM. These large differences between genotypes
declined with age but remained 3-5 fold higher in callipyge lambs at 60 days of age. There was no effect of genotype for any of the four myosin isoforms tested in the SS.
PARTICIPANTS: Jolena Fleming-Waddell, Department of Animal Sciences, Purdue University is a Ph. D candidate with stipend support from this grant. She has done a substantial amount of the lab work described in this report. Tasia M Taxis, Department of Animal Sciences, Purdue University is an undergraduate student who has received research experience and training in animal handling and sampling, genotyping, RNA isolation and quantitaitve PCR by working on this project with hourly wage support. Dr. Ross L. Tellam, CSIRO Livestock Industries, St. Lucia, Queensland Australia is a collaborator and provided the anti-bovine FA1 antibodies. Dr. Jason D. White, School of Veterinary Medicine, University of Melbourne, Melbourne, Victoria, Australia collaborated on the histological analysis of DLK1 expression in the skeletal muscles of callipyge lambs.
TARGET AUDIENCES: Muscle biologists
Impacts
These results further support the hypothesis that up-regulation of DLK1 causes muscle hypertrophy in callipyge lambs. There was a substantial increase in DLK1 transcript abundance that is accompanied by an increase in the DLK1 protein in the myofibers of hypertrophy responsive muscles. There is a smaller increase in DLK1 transcript abundance homozygous (C/C) animals. In general, it appears that polar overdominance is due to a transcriptional mechanism and not a post-transcriptional mechanism. Increased DLK1 protein was detected in the myofibers of C/C animals however far fewer fibers are affected. The muscle specific changes in myosin isoform expression correspond well to the changes in DLK1 expression. Changes in DLK1 and myosin isoform expression were seen only in the hypertrophy responsive longissimus dorsi and semimembranosus and not the supraspinatus which does not hypertrophy. There was some decrease in the expression of two myosin isoforms. The very large
increase in the expression of the glycolytic MYH4 gene was likely responsible for the myofiber hypertrophy and increase protein accretion in callipyge lambs. The inheritance of the mutation and the parent of origin specific up-regulation of DLK1 initiate a biochemical pathway causing muscle growth in callipyge lambs. The MYH4 gene is the likely end point of that pathway. Understanding how the DLK1 gene causes such a large increase in MYH4 gene expression will identify mechanisms that control muscle growth.
Publications
- Perkins, A.C., L.N. Kramer, T.S. Hadfield, D.E. Moody, N.E. Cockett and C.A. Bidwell. 2006. Postnatal changes in the expression of genes located in the callipyge region in sheep skeletal muscle. Animal Genetics 37, 535-542
- Fleming-Waddell, J.N., L.M. Wilson, G.R. Olbricht, T. Vuocolo, K. Byrne, B.A. Craig, R. L. Tellam, N.E. Cockett, and C.A. Bidwell. 2007. Analysis of gene expression during the onset of muscle hypertrophy in callipyge lambs. Animal Genetics 38, 28-36.
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Progress 06/01/05 to 06/01/06
Outputs
Sheep with the callipyge trait have altered carcass composition with increased muscling and decreased fat. The callipyge mutation lies in an imprinted gene cluster containing at several genes and is a single nucleotide polymorphism between the DLK1 and MEG3 genes. We are reporting progress on objective 1 by examining expression of genes from the callipyge region in muscle and adipose tissue. The expression of five genes surrounding the callipyge mutation was analyzed in skeletal muscles from lambs at prenatal and postnatal ages that coincide with the development of muscle hypertrophy. Genotype-specific changes in transcript abundance were detected for paternal allele-specific DLK1 and PEG11 and the maternal allele-specific MEG3, PEG11AS and MEG8 when the mutation was inherited on the same chromosome (a cis effect). There were differences in the temporal and muscle-specific effects on expression between the maternal allele-specific genes and paternal allele-specific
genes. Maternal inheritance of the callipyge allele had a significant effect on the expression of MEG3 and MEG8 at prenatal and postnatal ages, whereas paternal inheritance of DLK1 and PEG11 affected only postnatal expression. Maternal inheritance of the mutation caused similar changes in MEG3 and MEG8 expression in muscles that undergo hypertrophy and muscles that do not hypertrophy. Paternal inheritance of the mutation caused changes in PEG11 expression in both muscles although the magnitude of expression in semimembranosus was more than 100 fold greater than in supraspinatus. DLK1 expression was up-regulated in callipyge animals at both postnatal ages in the semimembranosus but there was no effect of genotype on DLK1 expression in the supraspinatus at any age. The DLK1 gene is also well know to be involved in adipocyte differentiation so we hypothesized that a cis effect of the callipyge mutation on DLK1 expression in adipose tissue could be responsible for the decreased fat
composition . Subcutaneous and visceral adipose samples were collected from 10 days of age to market size. Several genes were measured including those in the callipyge locus: DLK1, MEG3, and MEG8, as well as leptin and PPAR gamma as indicators of adipocyte function. No effect of callipyge genotype on DLK1 or MEG3 expression was detected for either fat depot. Transcript abundance of DLK1 and MEG3 was shown to decrease in SQ and visceral tissues as an effect of age. In addition, data revealed no differences between genotypes in the marker for adiposity, leptin. However at 84d, an increase in transcript abundance of leptin indicated adipose tissues in all animals behave as expected. Overall, neither DLK1 nor MEG3 expression was altered in subcutaneous or visceral adipose depots, suggesting that the callipyge mutation does not perturb adipocyte function directly. Therefore, the decrease in body fat accretion in callipyge animals is likely to be an indirect effect of repartitioning energy
into muscle hypertrophy
Impacts
Our analysis of gene expression in skeletal muscle and fat indicate the callipyge mutation has a complex effect on gene expression in skeletal muscle but no detectable effect in adipose tissue. The effect of the mutation on the expression of the two genes expressed from the paternally inherited chromosome (DLK1 and PEG11) is very different from the genes expressed from the maternal chromosome MEG3 and MEG8. The most striking difference was the fact the mutation does not effect DLK1 or PEG11 expression in muscle prior to birth but has a major effect after birth which is consistent with the muscle hypertrophy phenotype. Although DLK1 is the major candidate gene to cause muscle hypertrophy, the fact that PEG11 is also up regulated after birth in callipyge lambs indicated it cannot be ruled out as contributing to muscle hypertrophy. Therefore the unique polar overdominant inheritance may be due to the effect of both genes. Furthermore, it does not appear that the
callipyge mutation has any direct effect adipose tissue and the reduced fat in the carcass is an indirect effect of the muscle growth.
Publications
- Perkins, A.C., L.N. Kramer, J.M. Day, T.S. Hadfield, D.E. Moody, N.E. Cockett and C.A. Bidwell. 2006. Postnatal changes in the expression of genes located in the callipyge region in sheep skeletal muscle. Animal Genetics doi: 10.1111/j.1365-2052.2006.01518.x
- Skipwith, A., N.E. Cockett and C. A. Bidwell. 2006. Regulatory activity of DNA sequences surrounding the callipyge mutation on ovine chromosome 18. Plant and Animal Genome Conference XIV, San Diego, CA. http://www.intl-pag.org/14/abstracts/PAG14_P786.html.
- Wilson, L.M., J.N. Waddell, M. Neary, N.E. Cockett, C. Bidwell. 2006. The callipyge mutation does not alter DLK1 expression in visceral or subcutaneous adipose tissue in sheep. Proceedings of 30th International Conference on Animal Genetics. Porto Seguro, Brazil. August, 2006. http://www.isag.org.uk/pdf/conferences.asp.
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Progress 10/01/04 to 09/30/05
Outputs
The phenotype of callipyge lambs consists of pronounced hypertrophy of the muscles of loin and pelvic limbs and a lesser degree of hypertrophy in the muscles of the thoracic limbs. Callipyge lambs also have reduced carcass fat as well as lighter livers and pelts. This resulted in callipyge lambs having a higher dressing percentage and altered carcass composition. The callipyge mutation has been identified as a single nucleotide polymorphism in an intergenic region between DLK1 and GTL2 in an imprinted gene cluster. We have previously reported that the callipyge mutation has altered the expression of at least 5 of the genes in the region in skeletal muscle and DLK1 and/or PEG11 were candidate genes for causing muscle hypertrophy. We hypothesized that if the change in liver size was a direct effect of the callipyge mutation, the liver would exhibit differential expression of genes that surround the mutation. In order to investigate this, liver samples were collected
from 15 lambs (callipyge phenotype, +/C, n = 4; maternal heterozygotes, C/+, n= 4; homozygotes C/C, n= 3; and normal, +/+, n=4) at 8 weeks of age. The effect of inheritance of the callipyge mutation on gene expression in the liver was determined by quantitative PCR (measured as the log of transcript abundance per 100 ng total RNA). The genes that were quantified from the callipyge locus included DLK1, GTL2, PEG11AS, PEG11 and MEG8. G3PD was also quantified for sample standardization. There was no significant effect of genotype on G3PD, GTL2, MEG8, PEG11AS, or PEG11 transcript abundance. A significant effect of genotype was seen for DLK1 expression (P = 0.01) with the callipyge lambs having the lowest mean transcript abundance. Orthogonal contrasts were used to analyze the genetic model which indicated additive (p = 0.010) and reciprocal heterozygote (p = 0.043) models to be significant. In addition, the polar overdominance contrast was also significant (p = 0.003) for DLK1 transcript
abundance. The decrease in liver DLK1 expression found only in callipyge animals suggests a direct effect of the callipyge mutation on postnatal liver growth.
Impacts
The size of internal organs makes a direct contribution to the dressing percentage of meat animal carcasses and the liver is one of the largest internal organs. Callipyge lambs have smaller livers than normal lambs and we have experimental data showing that DLK1 expression is reduced in the livers of callipyge lambs. This suggests that liver size in callipyge lambs is a direct effect of gene expression from the callipyge locus and not a indirect effect of altered carcass composition due to muscle hypertrophy.
Publications
- Fleming, J.N., J. M. Smith, T. S. Hadfield, S. L. Eng, D. E. Moody, N. E. Cockett, and C. A. Bidwell. 2005. Decreased expression of DLK1 in the livers of 8 week old callipyge lambs. Journal of Animal Sciences 83 (1), 280.
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