Progress 12/01/04 to 11/30/07
Outputs
OUTPUTS: We have introduced the emerging discipline of functional genomics and gene network modeling to the large international avian biology and poultry science communities with invited review articles, book chapters and presentations at international conferences and workshops. Some of the major metabolic and regulatory pathways evoked in the hypothalamus and liver by prolonged fasting and re-feeding have also been introduced to graduate students and undergraduate physiology students in formal courses taught by the PDs at the University of Delaware and University of Maryland. PARTICIPANTS: The majority of the work on this project was completed by Project Director (PD) Larry Cogburn and his post-doctoral research associate, Dr. Nares Trakooljul (a Thai national) at the University of Delaware (UD) and by co-PD Tom Porter and his post-doctoral research associate, Dr. Stacey Higgins, at the University of Maryland (UMd). A considerable about of work in the first year and a half of the project was preformed by Ms. Dianne York, M.Sc., manager of the Microarray Core Facility at the Delaware Biotechnology Institute on the University of Delaware. The two post-doctoral research associates [Nares Trakooljul (UD) and Stacey Higgins (UMd)] received training and professional development in molecular biology, bioinformatics, biostatistics, functional genomics and systems biology. As part of her Master's degree program in Computer and Informational Sciences (CIS) at UD, Arpita Gandhi redesigned the project website and developed very used web-based tools for automated annotation of gene lists generated from analyses of our custom Del-Mar 14K Chicken Integrated Systems microarray (GeneBase) and the Arizona 20.7K chicken long-oligo microarray (Oligo Arrays) used for transcriptional profiling. Dr. Fiona McCarthy (University of Mississippi) kindly re-annotated the genes represented on our Del-Mar 14K chicken microarray. Three undergraduate research assistants at UD (Jessica A. Hall, Danielle Smarsh and Jackie Speier) were Science and Engineering Scholars who contributed to the animal studies and bioinformatics components of the project during summers. Graduate training and professional development opportunities were also provided to Laura E. Ellestad (Ph.D. student) and Jason Saliba (M.S. student) at the University of Maryland. Professional development was provided to the post-doctoral research associates, graduate and undergraduate students, who made presentations on campus and at regional, national and international conferences or workshops. Futhermore, the new knowledge gained from this original functional genomics project on hypothalamic and hepatic gene networks controlling growth and metabolism of the broiler chicken was incorporated into the undergraduate physiology courses taught by the PDs at UD and UMd. TARGET AUDIENCES: This project has served target audiences (students and professionals) in avian genomics, comparative genomics, endocrinology, poultry science, animal science, avian biology, poultry genetics and poultry breeding. The science-based knowledge generated from this functional genomics project was delivered to target audiences with publications (journal articles, book chapters and invited review articles), invited presentations at national and international conferences, workshops on chicken genomics and endocrinology, national and international poultry conferences. PROJECT MODIFICATIONS: A major problem encountered with completion of this research project was that we were unable to print 200 of our custom Del-Mar 14K Chicken Integrated Systems Microarrays (NCBI GEO Platform # GPL1731) developed under a previous USDA-IFAFS project. After printing the first 100-slide batch of the Del-Mar chicken cDNA microarray, we discovered that our original stock plates were depleted of the 17,765 cDNAs used to print microarray slides. Initially, we spent more than six months re-amplifying cDNAs and re-arraying these cDNAs into 384-well plates for printing. Furthermore, the Microarray Core Facility in the Delaware Biotechnology Institute at University of Delaware was closed in the summer of 2006, which made printing of our custom Del-Mar 14K chicken microarray impossible. However, we purchased 200 slides of a 20.7K chicken long oligo array (printed by Dr. Parker Antin at the University of Arizona) and entered this new custom platform called the Arizona Gallus gallus 20.7K Oligo Array v1.0 into NCBI Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgiacc=GPL6049). The use of Arizona 20.7K chicken long oligo array (NCBI GEO Platform # GPL6049) required amplification of our liver and hypothalamic RNA samples before labeling with florescent dyes, which greatly increased the cost of our microarray analysis. Another major problem we encountered was the lack of bioinformatic methods and tools that were required for handling, storing and analyzing large data sets generated from time-course transcriptional scans. Other bioinformatic bottlenecks that delayed interpretation and publication of transcriptional data were the lack of software and methods for analyzing gene networks and biological pathways in the chicken. Likewise, few statisticians had the training and experience to properly analyze and interpret transcriptional scans across multiple tissues or to reconstruct gene interaction networks and biological pathways across multiple physiological systems. However, we hired Dr. Robert J. Tempelman, Michigan State University, as a statistical consultant to analyze the two microarray data sets generated from hybridization of 80 liver RNA samples and provide significant (FDR <0.05) differentially expressed gene list from multiple pair-wise contrast of treatment groups. We also purchased an annual license for Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Inc., Redwood City, CA) to establish gene interaction networks in the liver and hypothalamus of newly-hatched and market age chicken during prolonged fasting and refeeding and to map differentially expressed genes to canonical metabolic and regulatory pathways.
Impacts
Poultry producers could benefit from the new knowledge of the negative impact of prolonged feed deprivation on metabolism and subsequent growth of newly hatched chickens during the first two days post-hatching, since feed-deprived chicks never recover from this initial food deficit. Newly hatched broiler chicks are often deprived of feed for one or two days before they are placed in poultry houses; this management practice could reduce the growth rate and feed efficiency. Poultry geneticists and breeders could directly benefit from the dozens of novel candidate genes identified by this project. Some of the functional candidate genes and metabolic loci could serve as molecular markers for breeding broiler chickens with more robust metabolic traits and greater feed efficiency. Furthermore, the large number of Minimal Information about Microarray Experiments (MIAME)-compliant microarray data sets deposited into the National Center for Bioinformatic Information (NCBI) Gene Expression Omnibus (GEO) database could be useful in the near future for meta-analysis of genome-wide transcriptional scans and systems biology modeling.
Publications
- Cogburn,L. A., N. Trakooljul, S.E. Higgins, L. E. Ellestad, R. J. Tempelman and T.E. Porter, 2011. Hepatic transcriptional profiling and gene pathway analysis of newly hatched chicks and market-age broiler chickens during the metabolic perturbation induced by fasting and re-feeding (submitted).
- Higgins, S.E., L.E. Ellestad, N. Trakooljul, L.A. Cogburn, F. McCarthy, J. Saliba and T.E. Porter, 2010. Global gene expression and pathway analysis in the hypothalamus of neonatal chicks during feeding, fasting and delayed feeding. BMC Genomics 11:162.
- Byerly, M. S., J. Simon, E. Lebihan-Duval, M. J. Duclos, L A. Cogburn and T. E. Porter, 2009. Effects of BDNF, T3 and corticosterone on expression of the hypothalamic obesity gene network in vivo and in vitro. Am. J. Physiol.-Regulatory, Integrative and Comparative Physiology 296:R1180-1189.
- Cogburn, L. A., T. E. Porter, M.J. Duclos, J. Simon, S. C.Burgess, J.J. Zhu, H.H. Cheng, J. B. Dodgson and J. Burnside, 2007. Functional genomics of the chicken-a model organism. Poultry Sci. 86:2059-2094.
- Ellestad, L.E., W Carre, M. Muchow , S.A. Jenkin, X. Wang, L.A. Cogburn, and T.E. Porter, 2006. Gene expression profiling during cellular differentiation in the embryonic pituitary gland using cDNA microarrays. Physiol. Genomics 25:414-425.
- W. Carre, X. Wang, T. E. Porter, Y. Nys, J-S Tang, E. Bernberg, R. Morgan, J. Burnside, and Cogburn, L. A., 2006. Chicken functional genomics resource: sequencing and annotation of 35,407 ESTs from single and multiple tissue cDNA libraries and CAP3 assembly of a chicken gene index. Physiol. Genomics 25:514-524.
- Wang, X., W. Carre, L. Rejto and L.A Cogburn, 2005. Transcriptional Profiling in Liver of Hormonally-manipulated Chickens. In: Functional Avian Endocrinology (A Dawson & PJ Sharp, Eds) Narosa Publishing House, New Delhi, India, pp 27-44.
- Burnside, J. L.A. Cogburn, R. Talbot and J.B. Dodgson, 2005. Genomic Tools for Endocrine Research. In: Functional Avian Endocrinology (A Dawson & PJ Sharp, Eds), Narosa Publishing House, New Delhi, India, pp 3-10.
- Porter T.E., Ellestad L.E., 2005. Gene expression profiling in the developing neuroendocrine system of the chick. In: Functional Avian Endocrinology, Dawson A, Sharp PJ (Eds.), Narosa Publishing House, New Dehli, pp. 45-56.
- Porter TE, Ellestad LE, 2005. Characterization of gene expression profiles in the hypothalamus and pituitary during chick embryonic development using cDNA microarrays. Plant and Animal Genome XI Conference, San Diego, CA.
- Byerly MS, Simon J, Duclos M, Porter TE, 2005. Hypothalamic gene expression patterns during the development of adiposity. Program Annual Meeting of the North American Association for the Study of Obesity.
- Cogburn, L.A., T.E. Porter, S.E. Aggrey, E. Le Bihan-Duval, M. Duclos and J. Simon, 2011. Meta-analysis of the chicken transcriptome across metabolic tissues, divergent genotypes and juvenile ages. BARD Workshop: Affecting early life poultry development to improve domestic fowl livestock, Jerusalem, Israel, March 6-11.
- Higgins, S.E., L.E. Ellestad, N. Trakooljul, J. Saliba, F. McCarthy, L.A. Cogburn, T.E. Porter, 2008. Differential gene expression in the hypothalamus of hatchling chicks during the fasting and re-feeding response. Poultry Science Association Annual Meeting, Niagara Falls, Ontario, Canada, July 20-23.
- Hall, J. A., R. J. Tempelman, and L. A. Cogburn, 2006. Analysis of hepatic gene expression in chickens with hormonally-induced lean and fat phenotypes. FASEB J. 20: A523-a.
- Byerly M.S., M.J.Duclos, L.A.Cogburn, J. Simon, T.E. Porter, 2006. In vivo and in vitro analyses of a potential hypothalamic pathway for regulation of metabolic rate and body fat. Program 36th Annual Meeting of the Society for Neuroscience.
- Porter TE, Ellestad LE, Muchow M, Jenkins SA, Byerly MS, 2006. Analysis of global gene expression in the avian neuroendocrine system. Annual Meeting Poultry Science Association, Edmonton, Alberta, Canada, July 16-19.
- Cogburn, L.A., T. E. Porter, S.E. Aggrey, E. Lebihan-Duval, M. Duclos and J. Simon, 2010. Meta-analysis of the chicken transcriptome across multiple tissues genotypes and ages during juvenile development. XIIIth European Poultry Congress (EPC 2010), Tours, France, Aug. 23-27.
- Cogburn L.A., Trakooljul N., Hall J.A., Smarsh D. N., Wang X., Carre W., and Simon J., 2008. Functional genomics of the avian somatic endocrine system. Nineth International Symposium on Avian Endocrinology, Leuven, Belgium, July 11-15.
- Cogburn, L.A., 2008. Gene expression patterns in somatic tissue of divergently selected broiler chickens. XXIII World Poultry Congress, Brisbane, Australia, June 29-July 4.
- Cogburn, L.A., 2008. Functional genomics of the chicken. Annual National Poultry Breeders Roundtable, St Louis, MO. May 8-9.
- Cogburn, LA, 2006. Functional mapping of gene networks controlling growth and metabolism. Functional Genomics: Building the Bridge between the Genome and Phenome, Ancillary Scientists Symposium, Poultry Science Association Annual Meeting,Edmonton, Alberta, Canada, July 16.
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Progress 12/01/04 to 11/30/05
Outputs
OUTPUTS: This research project had three major objectives: (1) analyze transcriptional profiles in the liver and hypothalamus of newly hatched chicks during a strong metabolic perturbation, the fasting and refeeding response, (2) analyze transcriptional profiles in the liver and hypothalamus of market age chickens during a strong metabolic perturbation, the fasting and refeeding response, and (3) verify gene expression patterns by qRT-PCR analysis and develop models of major gene networks that control feed intake and energy metabolism in the chicken. The animal experiments were completed in the first year of the project. Total RNA was isolated from the liver and hypothalamic tissue and plasma metabolite profiles determined in the second year of the project. During the third year, two microarray hybridization experiments have been completed on liver samples collected from two fasting-refeeding perturbation studies conducted in the newly hatched chick (50 samples) and the market-age
(six-week-old) broiler chicken (30 samples). The chicken RNA samples were amplified, labeled with Alexa 555 (green) fluorescent dye, and hybridized along with (pooled liver) amplified RNA (aRNA) labeled with Alexa647 (red) fluorescent dye in a reference RNA hybridization design. A chicken microarray platform containing 20,460 70mer oligos and 216 control spots (20.7K) was created and submitted to NCBI Gene Expression Omnibus (GEO) website (Platform GPL6049). Minimum information about a microarray experiment (MIAME)-compliant microarray data files for the newly hatched chick (GEO Series # GSE9500) and market age chickens (not assigned) have been deposited into the NCBI GEO database (www.ncbi.nlm.nih.gov/geo). In a third hybridization experiment, hypothalamic aRNA samples from the newly-hatched chick fasting-refeeding study were hybridized to 40 chicken 20.7K oligo arrays in a similar reference RNA hybridization design. The final hybridization of hypothalamic aRNA samples from the
market age chickens is currently in progress. Independent qRT-PCR analysis was used to verify gene expression patterns revealed by microarray analysis of liver and hypothalamus aRNA samples from the newly hatched chick experiment and the liver aRNA samples from the market age chicken experiment.
PARTICIPANTS: Nares Trakooljul is a post-doctoral research associate at the University of Delaware who has worked on this project since November 1, 2005. Dr. Trakooljul was responsible for isolation and amplification of RNA from the liver samples collected from two experiments
Impacts
One important gene we found differentially regulated in the hypothalamus in response to fasting and refeeding is the leptin receptor, which was upregulated in the hypothalamus of chicks fasted at 24h post-hatch, and again on d4 in refed chicks. Leptin is a hormone produced primarily by adipocytes and associated with regulation of feed intake in mammals. In mammals, increased levels of leptin function to decrease feed intake. However, the role of leptin in the chicken is not completely characterized. We also observed increased thyrotropin releasing hormone (TRH) expression in the hypothalamus of chicks following fasting for two days, but not one day. Additionally, TRH levels remained increased after 4h of refeeding following 48h of fasting. As TRH stimulates secretion of thyroid stimulating hormone (TSH) by the thyrotrophs of the pituitary, analysis of gene expression levels in the pituitary during the proposed research would contribute to understanding the interactions
between the two organs during this metabolic challenge. Expression of mRNA encoding the FK506 binding protein 51 (FKBP51) was also differentially regulated in this model. This protein functions as an immunophilin cochaperone, and is associated with steroid receptor complexes along with Hsp90. We observed levels of FKBP51 that were increased at hatch, d1, and in birds fasted for 24h. Levels of FKBP51 were relatively lower in chicks fed for 48h, and they remained at this level on days 3 and 4. As glucocorticoid receptors are frequently associated with this protein and glucocorticoids are known to increase synthesis of growth hormone, the difference in the expression levels of this transcript between fed chicks on day 2 and fasted chicks on day 2 may provide key information relating to the gene networks activated in this model due to restriction of feed. Pro-opiomelanocortin (POMC) was also differentially regulated in the hypothalamus. POMC is a precursor to several hormones, including
a-melanocyte-stimulating hormone (aMSH), adrenocorticotropic hormone (ACTH), beta-endorphin, and beta-lipotropin. We observed relatively lower expression of POMC in chicks fasted for 2 days, and also in chicks that had been fasted for 2 days and refed for 4h. The differential regulation of this gene may result in the secretion of multiple hormones, including aMSH which is known regulate metabolic rate through the autonomic nervous system. Analysis of gene expression downstream of the hypothalamus in the pituitary is necessary to better understand the effects of fasting on regulation of the POMC gene. These data from the hypothalamus confirm that the model of fasting of neonatal chicks, and refeeding for 4, 24 or 48h indeed caused differential expression of genes associated with metabolism and feed intake. The analyses of these data are underway currently to determine which gene networks are involved in regulation of feed intake and metabolism in the chicken.
Publications
- Cogburn, L. A., T. E. Porter, M.J. Duclos, J. Simon, S. Burgess, J. B. Dodgson, H.H. Cheng and J. Burnside, 2007. Functional genomics of the chicken:A model organism. Poultry Sci. 86:2059-2094
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