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Changes in pituitary gene expression between embryonic day 17 and post-hatch day 3.

ABSTRACT: A major research focus in our laboratory is the development of the chicken pituitary gland. As this tissue does not yield sufficient RNA to analyze using cDNA microarrays, mRNA must be amplified by reverse transcription with an oligo-dT primer containing the T7 RNA polymerase promoter site followed by in vitro transcription using T7 RNA polymerase. Therefore, this preliminary study investigating differential gene expression between embryonic day 17, when all the major cell types in the chick anterior pituitary gland have differentiated, and post-hatch day 3 was conducted to verify that amplified antisense RNA could be used in conjunction with our neuroendocrine microarrays containing 5,128 unique cDNAs to detect changes in gene expression. Of the 662 cDNAs that were expressed at significantly different levels between the two ages (P < 0.05, n=3 samples/age), 159 exhibited substantial differences where the higher mean was at least 1.6-fold greater than the lower mean. The majority of the genes (117) increased between e17 and d3. Several genes which may play a role in neuroendocrine development were identified, including many transcription factors, signaling molecules, and morphogeneic factors. Keywords: time course during development (2 ages)

ORGANISM(S): Gallus gallus

PROVIDER: GSE3226 | GEO | 2005/09/02

SECONDARY ACCESSION(S): PRJNA92751

REPOSITORIES: GEO

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Project description:Chickens divergently selected for either high abdominal fat content (fat genotype) or low abdominal fat content (lean genotype) at SRA-INRA, France were used to profile pituitary gland gene expression during juvenile development (1 to 7 weeks of age) and to identify differentially expressed genes associated with genotype and age. The fat line (FL) and lean line (LL) chickens are different in various phenotypic and metabolic measurements, including abdominal fatness, plasma glycemia, and T3. The FL and LL chickens represent unique models for characterizing biomedical and agricultural traits. The Del-Mar 14K Chicken Integrated Systems microarrays were used for a transcriptional scan in the pituitary gland during juvenile development using a reference design for the hybridization of total RNA from the pituitary gland. A reference RNA pool was made from an equal amount of high-quality total RNA extracted from all of the samples. Each of the microarrays was co-hybridized with Cy3-labeled cDNA targets from one of the pituitary samples and Cy5-labled cDNA targets from the reference pituitary RNA pool. Log2-transformed fluorescence intensities were analyzed with a mixed model. Two-way ANOVA (SAS) of log2 ratios was used to detect significant (p<0.05) differences by line, age, and the line-by-age interaction. There were 1150 significantly different genes between the two lines, and 339 of these genes exhibited greater than 0.68-fold differences in their log2 ratios (highest group mean at least 160% of the lowest group mean). One thousand four hundred twenty nine genes were significantly different by age and of these, 583 exhibited fold changes greater than 0.68 in the log2 ratio. There were 145 genes that significantly differed in their line-by-age interaction, and 62 of these exhibited greater than 0.68-fold differences. There were 386 genes with significant differences by line or for line-by-age interaction with at least a 0.68-fold change in log2 ratio and n ≥ 2 for each experimental group. Four biological replicates were analyzed, each consisting of a complete set of individual samples from pituitary glands of Fat and Lean line chickens at week 1, week 3, week 5, or week 7 of age. Amplified antisense RNA from each sample was analyzed using Del-Mar 14 K array in a reference design. Each experimental sample (Fat and Lean Weeks 1, 3, 5, and 7) was labeled with Cy3 and hybridized with an aliquot of the Cy5 reference pool, which was generated from all 32 RNA samples.

Project description:Several processes governing growth and metabolism are regulated by the neuroendocrine system, particularly the trophic hormones produced in the anterior pituitary gland. However, many of the differences that exist in pituitary gene expression and functional gene networks associated with divergent growth characteristics are still largely unknown. Using the Del-Mar 14K Chicken Integrated Systems microarray, this experiment evaluated global gene expression in the anterior pituitary gland during the first 7 weeks of post-hatch growth in two lines of chickens genetically selected for high growth (HG) or low growth (LG) at SRA-INRA, France. The HG and LG chickens differ in body weight, abdominal fat content, and plasma hormone and metabolite levels, making them a valuable model for both biomedical and agricultural traits. Amplified total RNA extracted from individual anterior pituitary glands collected from 4 birds from each line at each age (n=4) was produced. Cy-3 labeled cDNA from these samples was hybridized with an aliquot of Cy-5 labeled cDNA made from a pool of total RNA from all 32 samples. Data were analyzed as log2-(Cy3intensity (norm)/Cy5intensity (raw)) by two-way ANOVA using a linear mixed model procedure (PROC MIXED; SAS institute Inc., Cary, NC), and a total of 2,310 spots were significantly different (P<0.05) by line (1,100), age (1,058), or the line-by-age interaction (353). Of these, we identified 291 spots representing 263 genes as differentially expressed between HG and LG birds, where there was a >160% difference between the highest expression in one line and the lowest expression in the other and a significant line-by-age (110) or line (181) effect. Four anterior pituitary hormones were contained in this set of differentially expressed genes: thyroid-stimulating hormone ?-subunit, luteinizing hormone ?-subunit, and follicle-stimulating hormone ?-subunit mRNA levels were higher in the HG line; and growth hormone mRNA was elevated in the LG line. Pathway analysis revealed differences in expression between HG and LG lines associated with molecular transport, endocrine system development and function, organ and tissue morphology, and cellular compromise. Potential alteration in pituitary function between the two lines was highlighted in a network containing genes associated with hormone expression and secretion, as well as those potentially involved in pituitary gland morphology and vascularization.

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Changes in pituitary gene expression between embryonic day 17 and post-hatch day 3.

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