Project description:The contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes. In many organisms dosage compensation has thus evolved to equalize sex-linked gene expression in males and females1,2, in mammals achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. Another form of dosage compensation ensures that expression levels on the X chromosome and on autosomes are balanced3,4. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma5,6. Here we use a microarray approach to show that male day 18 chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4-1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci2. Intriguingly, without global dosage compensation, female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds. The pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Experiment Overall Design: Sample collection Experiment Overall Design: Fertilized eggs from White Leghorn fowl were purchased from OVA Production (MorgongM-CM-%va, Sweden). The eggs were incubated at 37.5C and 60% relative humidity, and were turned every 3 hours. After 18 days of incubation (ed18), the embryos were euthanized by decapitation. A piece from the apical part of the heart was collected and the left gonad and the brain were excised. The cerebellum, the optic lobes and the cerebral hemispheres were removed from the brain and, consequently, the brain sample included the intact diencephalon and remaining parts from other regions. The samples were immediately frozen in liquid nitrogen and then stored at -70 M-BM-0C. The embryos were sexed by ocular inspection of the gonads and MM-CM-<llerian ducts. Experiment Overall Design: RNA preparation and microarray hybridization Experiment Overall Design: Heart, left gonads and diencephalon from each of four male and four female embryos were homogenized by syringe and needle followed by use of the Homogenizer system (Invitrogen, Carlsbad, CA, USA). RNA was extracted from heart and brain homogenates with the PureLink Micro-to-Midi Total RNA Purification System (Invitrogen), and RNA was then DNase treated with a DNA-free kit (Ambion, Austin, TX, USA). Due to limited amounts of available tissue, gonad RNA extraction was performed with an RNeasy Micro Kit (Qiagen, Hilden, Germany) with integrated DNase treatment. RNA concentration was measured with ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and RNA quality was evaluated using the Agilent 2100 Bioanalyzer system (Agilent Technologies Inc, Palo Alto, CA). Two micrograms of total RNA from each sample were used to prepare biotinylated fragmented cRNA according to the GeneChipM-BM-. Expression Analysis Technical Manual (Rev. 5, Affymetrix Inc., Santa Clara, CA). Affymetrix GeneChipM-BM-. expression arrays (Human Genome U133 Plus 2.0 Array) were hybridized for 16 hours in a 45M-BM-0C incubator, rotated at 60 rpm. According to the GeneChipM-BM-. Expression Analysis Technical Manual (Rev. 5, Affymetrix Inc., Santa Clara, CA), the arrays were then washed and stained using the Fluidics Station 450 and finally scanned using the GeneChipM-BM-. Scanner 3000 7G. In total, 24 hybridizations were made (4 individuals x 3 tissues x 2 sexes), however, three samples failed to meet Affymetrix quality control criteria and were removed from further analysis (heart and brain from one male, and gonads from one female). Experiment Overall Design: Microarray data analysis. All pre-processing and statistical analysis of microarray data was performed in R version 2.4.1 using Bioconductor packages release 1.9 (ref. 29). The CEL files were processed using GCRMA30, a background adjustment method taking into account the GC content of probes when assessing non-specific binding, followed by quantile normalization and median-polish summarization of probe intensities into probe set intensities. A linear model was fitted to the log2 of the expression levels based on all probe sets and considering sex and tissue as a combined factor using the Limma package. After pre-processing and linear model fitting the probe sets were filtered on expression; an expression threshold was set on both average expression level and absent/present calls from the R implementation of the Affymetrix MAS 5.0 algorithm. Only probe sets with average expression over a defined threshold and present in more than half of the samples within at least one tissue-sex combination were considered as significantly expressed. This resulted in 15,398 probe sets for heart, 16,846 for brain and 17,438 for gonads, and these probe sets composed the reference for analysis of differently expressed genes between the sexes. Experiment Overall Design: Annotation of probe sets Experiment Overall Design: Annotations for the probe sets were extracted from Ensembl (www.ensembl.org) via biomRt in R. The Ensembl mapping of probe sets is based on alignments of individual probes to the chicken genome version 2.1 (WASHUC2 May 2006) and covers 21,885 of the 37,693 chicken-specific probe sets, which is close to the total number of protein-coding genes in the chicken genome identified by Ensembl. Several transcripts are represented by more than one probe set; the 21,885 probe sets with annotation corresponds to 14,414 unique transcripts. The genomic location for probe sets was taken from Ensembl. Experiment Overall Design: Statistical analysis Experiment Overall Design: Fold-change was calculated as the average male expression over ave Experiment Overall Design: rage female expression, and a Bayesian moderated t-statistic for differential expression between males and females was then generated for each tissue. To take multiple testing into account p-values (corrected p-values) were adjusted using the Benjamini and Hochberg false discovery rate (FDR) method32. Probe sets with an absolute fold-change value larger than various threshold levels were considered sex-biased. Statistical testing of differences in levels of hybridization intensities between sex and/or chromosome categories was done by bootstrapping. The FisherM-bM-^@M-^Ys exact test was used to test the distribution of gene ontology terms and the distribution of sex-biased genes across chromosomes. Spearman rank correlation coefficients were calculated for the continuous fold-change values and several genomic parameters including gene density, microsatellite and CR1 retrotransposons repeat density, and GC content, all taken from Ensembl. These parameters were estimated based on averaging over a 100 kb window surrounding each gene.

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