Larry A. Cogburn

Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg

BackgroundAs uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshells remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the eggs natural defenses.ResultsOur 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hens uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid where eggshell formation takes place.ConclusionsOur original study provides the first detailed description of the chicken uterus transcriptome during formation of the eggshell. We have discovered a cache of about 600 functional genes and identified a large number of encoded proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Some of these uterine genes could prove useful as biological markers for genetic improvement of phenotypic traits (i.e., egg and eggshell quality).

Developmental expression of hepatic growth hormone receptor and insulin-like growth factor-I mRNA in the chicken

We have examined the ontogeny of expression of growth hormone (GH) receptor (GHR) and insulin-like growth factor-I (IGF-I) mRNA in chicken liver from day 13 of incubation until 31 weeks of age. The profiles of GHR and IGF-I mRNA levels were compared to developmental changes in body weight and plasma levels of GH and IGF-I. In the embryo, hepatic GHR mRNA was not detectable until day 15, highest on days 17 and 19, and then declined at hatching (day 21). Following an initial 2-week delay after hatching, there was a progressive increase in hepatic GHR mRNA which continued after the birds reached mature body weight. Plasma GH reached peak levels at 3-4 weeks of age and then fell sharply until maintenance of a low basal level after 10 weeks of age. Thus, there appears to be a strong inverse relationship between expression of the GHR and basal plasma GH levels in the prepubertal chicken. Although IGF-I mRNA was undetectable in embryonic liver by Northern blot analysis, there is a good correlation between expression of hepatic IGF-I mRNA and the plasma IGF-I profile during post-hatching development in the chicken. The highest levels of IGF-I mRNA were reached at 4 weeks of age which was followed by a slow decline to the basal levels maintained after 10 weeks of age. It appears that the decline in plasma IGF-I lags considerably behind the sharp fall in plasma GH levels and expression of hepatic IGF-I mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional genomics in chickens: development of integrated-systems microarrays for transcriptional profiling and discovery of regulatory pathways.

The genetic networks that govern the differentiation and growth of major tissues of economic importance in the chicken are largely unknown. Under a functional genomics project, our consortium has generated 30 609 expressed sequence tags (ESTs) and developed several chicken DNA microarrays, which represent the Chicken Metabolic/Somatic (10 K) and Neuroendocrine/Reproductive (8 K) Systems (http://udgenome.ags.udel.edu/cogburn/). One of the major challenges facing functional genomics is the development of mathematical models to reconstruct functional gene networks and regulatory pathways from vast volumes of microarray data. In initial studies with liver-specific microarrays (3.1 K), we have examined gene expression profiles in liver during the peri-hatch transition and during a strong metabolic perturbation—fasting and re-feeding—in divergently selected broiler chickens (fast vs. slow-growth lines). The expression of many genes controlling metabolic pathways is dramatically altered by these perturbations. Our analysis has revealed a large number of clusters of functionally related genes (mainly metabolic enzymes and transcription factors) that control major metabolic pathways. Currently, we are conducting transcriptional profiling studies of multiple tissues during development of two sets of divergently selected broiler chickens (fast vs. slow growing and fat vs. lean lines). Transcriptional profiling across multiple tissues should permit construction of a detailed genetic blueprint that illustrates the developmental events and hierarchy of genes that govern growth and development of chickens. This review will briefly describe the recent acquisition of chicken genomic resources (ESTs and microarrays) and our consortiums efforts to help launch the new era of functional genomics in the chicken.

Mapping quantitative trait loci affecting fatness and breast muscle weight in meat-type chicken lines divergently selected on abdominal fatness

Quantitative trait loci (QTL) for abdominal fatness and breast muscle weight were investigated in a three-generation design performed by inter-crossing two experimental meat-type chicken lines that were divergently selected on abdominal fatness. A total of 585 F2 male offspring from 5 F1 sires and 38 F1 dams were recorded at 8 weeks of age for live body, abdominal fat and breast muscle weights. One hundred-twenty nine microsatellite markers, evenly located throughout the genome and heterozygous for most of the F1 sires, were used for genotyping the F2 birds. In each sire family, those offspring exhibiting the most extreme values for each trait were genotyped. Multipoint QTL analyses using maximum likelihood methods were performed for abdominal fat and breast muscle weights, which were corrected for the effects of 8-week body weight, dam and hatching group. Isolated markers were assessed by analyses of variance. Two significant QTL were identified on chromosomes 1 and 5 with effects of about one within-family residual standard deviation. One breast muscle QTL was identified on GGA1 with an effect of 2.0 within-family residual standard deviation.

Measurement of developmental changes in plasma insulin-like growth factor-I levels of broiler chickens by radioreceptor assay and radioimmunoassay

The main purpose of this study was to examine the relationship between insulin-like growth factor-I (IGF-I) and growth hormone (GH) during embryonic and posthatching development of broiler chickens. Two heterologous assays were validated for measurement of IGF-I in chicken and turkey plasma. A radioreceptor assay (RRA), utilizing microsomal membranes prepared from human placenta, was modified and validated for measurement of IGF peptide (mainly IGF-I). A double-antibody radioimmunoassay (RIA) was validated for measurement of immunoreactive IGF-I levels in chicken and turkey plasma. In both assay systems, recombinant-derived human IGF-I was used for standards and trace hormone. Hypophysectomy in turkey poults reduced plasma levels of IGF (RRA) by 35% and IGF-I (RIA) by 59% as compared to intact control turkeys. In Experiment 1, 14 chicken embryos were bled at 15, 17, 19, and 21 days of incubation and at 1 week of age to determine plasma levels of IGF-I and GH. Plasma IGF levels (RRA) remained constant during late incubation, but increased significantly (P less than 0.05) at 1 week of age. Plasma IGF-I levels (RIA) declined 2 days before hatching; however, plasma levels of IGF-I were sharply elevated (P less than 0.05) at 1 week of age. Plasma GH concentrations were low in embryos and were greatly elevated (P less than 0.05) at hatching (21 days of incubation) and at 1 week of age. In Experiment 2, 12 different broiler cockerels were weighed and then bled by cardiac puncture each week from hatching (1 day of age) to 7 weeks of age. The plasma profiles of IGF, IGF-I, GH, triiodothyronine (T3), and thyroxine (T4) were each compared to relative growth rate by analysis of covariance. Plasma IGF and IGF-I levels increased progressively from 0 to 3 weeks of age and were maintained in a plateau from 3 to 7 weeks of age. Plasma GH levels reached a peak at 4 weeks of age, but declined sharply thereafter, while IGF and IGF-I levels remained elevated. Plasma T3 concentrations were progressively increased and reached peak concentrations at 3 weeks of age, while plasma T4 levels increased only at 6 and 7 weeks of age. There was a high correlation (P less than 0.01) between relative growth rate and age-related changes in plasma levels of IGF (r = 0.96), IGF-I (r = 0.97), and T3 (r = 0.94); however, there was no correlation between relative growth rate and changes in plasma GH or T4.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of pinealectomy on plasma and extrapineal melatonin rhythms in young chickens (Gallus domesticus)

A specific radioimmunoassay was validated for the quantitative measurement of melatonin (MT) levels in plasma and homogenates of the pineal gland, Harderian gland, or retinae of young chickens. Single-comb White Leghorn (SCWL) cockerels were raised under a 12L:12D light/dark cycle for two experiments. In Experiment 1, 12 birds were bled and immediately killed for their pineal glands at 4-hr intervals during a single light/dark cycle at 25 days of age (25 da) for simultaneous determination of changes in MT levels in the plasma and pineal gland. Plasma MT levels were low during photophase (100 pg/ml) and reached a distinct peak (390 pg/ml) at mid-scotophase. A parallel MT rhythm was found in pineal homogenates where the average MT content during scotophase (7.4 ng/gland) was 10 times higher than the average MT content of pineal glands obtained during photophase. In Experiment 2, SCWL cockerels were either pinealectomized or sham-operated (PN) at 8 to 10 da. At 25 da, six birds from each surgical treatment group, including unoperated controls (C), were bled at 4-hr intervals, corresponding to those in Experiment 1, during a single light/dark cycle. Immediately after being bled, each bird was killed and the eyes and Harderian glands were removed for measurement of their MT contents. Pinealectomy completely abolished the plasma MT rhythm which in intact chicks (PN and C) reached a sharp peak (298 pg/ml) at mid-scotophase. Although not affected by surgical treatment, retinal MT levels showed a higher amplitude rhythm with a prominent peak (4 ng/retina) at mid-scotophase that was 15 times higher than the average retinal MT content during photophase. A modest nocturnal MT rhythm was found in the Harderian gland where the average MT level for all surgical treatment groups during scotophase (89 pg/100 mg wet wt) was only 51% higher than that observed for photophase. These data indicate that the plasma MT rhythm in chickens is derived solely from MT secreted into blood by the pineal gland and that the extrapineal MT produced rhythmically in both the retina and Harderian gland does not contribute to the plasma MT rhythm.

Identification of QTL controlling meat quality traits in an F2 cross between two chicken lines selected for either low or high growth rate

BackgroundMeat technological traits (i.e. meat pH, water retention and color) are important considerations for improving further processing of chicken meat. These quality traits were originally characterized in experimental lines selected for high (HG) and low (LG) growth. Presently, quantitative trait loci (QTL) for these traits were analyzed in an F2 population issued from the HG × LG cross. A total of 698 animals in 50 full-sib families were genotyped for 108 microsatellite markers covering 21 linkage groups.ResultsThe HG and LG birds exhibit large differences in body weight and abdominal fat content. Several meat quality traits [pH at 15 min post-slaughter (pH15) and ultimate pH (pHu), breast color-redness (BCo-R) and breast color-yellowness (BCo-Y)] were lower in HG chickens. In contrast, meat color-lightness (BCo-L) was higher in HG chickens, whereas meat drip loss (DL) was similar in both lines. HG birds were more active on the shackle line. Association analyses were performed using maximum-likelihood interval mapping in QTLMAP. Five genome-wide significant QTLs were revealed: two for pH15 on GGA1 and GGA2, one for DL on GGA1, one for BCo-R and one for BCo-Y both on GGA11. In addition, four suggestive QTLs were identified by QTLMAP for BCo-Y, pHu, pH15 and DL on GGA1, GGA4, GGA12 and GGA14, respectively. The QTL effects, averaged on heterozygous families, ranged from 12 to 31% of the phenotypic variance. Further analyses with QTLExpress confirmed the two genome-wide QTLs for meat color on GGA11, failed to identify the genome-wide QTL for pH15 on GGA2, and revealed only suggestive QTLs for pH15 and DL on GGA1. However, QTLExpress qualified the QTL for pHu on GGA4 as genome-wide.ConclusionThe present study identified genome-wide significant QTLs for all meat technological traits presently assessed in these chickens, except for meat lightness. This study highlights the effects of divergent selection for growth rate on some behavioral traits, muscle biochemistry and ultimately meat quality traits. Several QTL regions were identified that are worthy of further characterization. Some QTLs may in fact co-localize, suggesting pleiotropic effects for some chromosomal regions.

Discovery of the Elusive Leptin in Birds: Identification of Several ‘Missing Links’ in the Evolution of Leptin and Its Receptor

Leptin is a pleiotropic protein best known for regulation of appetite and fat storage in mammals. While many leptin orthologs have been identified among vertebrates, an authentic leptin in birds has remained elusive and controversial. Here we identify leptin sequence from the Peregrine falcon, Falco peregrinus (pfleptin), and identify sequences from two other birds (mallard and zebra finch), and ‘missing’ vertebrates (elephant shark, alligator, Indian python, Chinese soft-shelled turtle, and coelacanth). The pattern of genes surrounding leptin (snd1, rbm28) is syntenic between the falcon and mammalian genomes. Phylogenetic analysis of all known leptin protein sequences improves our understanding of leptin’s evolution. Structural modeling of leptin orthologs highlights a highly conserved hydrophobic core in the four-helix cytokine packing domain. A docked model of leptin with the leptin receptor for Peregrine falcon reveals several conserved amino acids important for the interaction and possible coevolution of leptin with its receptor. We also show for the first time, an authentic avian leptin sequence that activates the JAK-STAT signaling pathway. These newly identified sequences, structures, and tools for avian leptin and its receptor will allow elucidation of the function of these proteins in feral and domestic birds.

Transcriptional analysis of abdominal fat in genetically fat and lean chickens reveals adipokines, lipogenic genes and a link between hemostasis and leanness

BackgroundThis descriptive study of the abdominal fat transcriptome takes advantage of two experimental lines of meat-type chickens (Gallus domesticus), which were selected over seven generations for a large difference in abdominal (visceral) fatness. At the age of selection (9 wk), the fat line (FL) and lean line (LL) chickens exhibit a 2.5-fold difference in abdominal fat weight, while their feed intake and body weight are similar. These unique avian models were originally created to unravel genetic and endocrine regulation of adiposity and lipogenesis in meat-type chickens. The Del-Mar 14K Chicken Integrated Systems microarray was used for a time-course analysis of gene expression in abdominal fat of FL and LL chickens during juvenile development (1–11 weeks of age).ResultsMicroarray analysis of abdominal fat in FL and LL chickens revealed 131 differentially expressed (DE) genes (FDR≤0.05) as the main effect of genotype, 254 DE genes as an interaction of age and genotype and 3,195 DE genes (FDR≤0.01) as the main effect of age. The most notable discoveries in the abdominal fat transcriptome were higher expression of many genes involved in blood coagulation in the LL and up-regulation of numerous adipogenic and lipogenic genes in FL chickens. Many of these DE genes belong to pathways controlling the synthesis, metabolism and transport of lipids or endocrine signaling pathways activated by adipokines, retinoid and thyroid hormones.ConclusionsThe present study provides a dynamic view of differential gene transcription in abdominal fat of chickens genetically selected for fatness (FL) or leanness (LL). Remarkably, the LL chickens over-express a large number of hemostatic genes that could be involved in proteolytic processing of adipokines and endocrine factors, which contribute to their higher lipolysis and export of stored lipids. Some of these changes are already present at 1 week of age before the divergence in fatness. In contrast, the FL chickens have enhanced expression of numerous lipogenic genes mainly after onset of divergence, presumably directed by multiple transcription factors. This transcriptional analysis shows that abdominal fat of the chicken serves a dual function as both an endocrine organ and an active metabolic tissue, which could play a more significant role in lipogenesis than previously thought.

Effects of BDNF, T3, and corticosterone on expression of the hypothalamic obesity gene network in vivo and in vitro

Hypothalamic neuropeptides, neurotrophins, and systemic hormones modulate food intake and body composition. Although advances toward elucidating these interactions have been made, many aspects of the underlying mechanisms remain vague. Hypothalami from fat and lean chicken lines were assessed for differential expression of anabolic/orexigenic and catabolic/anorexigenic genes. Effects of triiodothyronine (T(3)), corticosterone (Cort), and brain-derived neurotrophic factor (BDNF) on expression of anabolic/orexigenic and catabolic/anorexigenic genes were tested in cultures of hypothalamic neurons. From this, we found that BDNF increased and T(3) decreased gene expression for BDNF, leptin receptor (LEPR), pro-opiomelanocortin (POMC), thyrotropin releasing hormone (TRH), and agouti-related protein (AGRP). Thyroid hormone levels were manipulated during development to show that T(3) inhibited BDNF, TRH, and BDNF receptor gene expression. Delivery of T(3), Cort, T(3) plus Cort, or vehicle in vivo continuously for 72 h indicated that Cort and T(3) have overlapping roles in regulating TRH, LEPR, and POMC gene expression and that Cort and T(3) regulate BDNF, neuropeptide Y, and AGRP in opposite directions. Collectively, these findings suggest that interactions between the neuropeptide BDNF and the hormones T(3) and/or Cort may constitute a homeostatic mechanism that links hypothalamic energy regulation controlling body composition.