Sex-biased microRNA expression in mammals and birds reveals underlying regulatory mechanisms and a role in dosage compensation

Sexual dimorphism depends on sex-biased gene expression, but the contributions of microRNAs (miRNAs) have not been globally assessed. We therefore produced an extensive small RNA sequencing data set to analyze male and female miRNA expression profiles in mouse, opossum, and chicken. Our analyses uncovered numerous cases of somatic sex-biased miRNA expression, with the largest proportion found in the mouse heart and liver. Sex-biased expression is explained by miRNA-specific regulation, including sex-biased chromatin accessibility at promoters, rather than piggybacking of intronic miRNAs on sex-biased protein-coding genes. In mouse, but not opossum and chicken, sex bias is coordinated across tissues such that autosomal testis-biased miRNAs tend to be somatically male-biased, whereas autosomal ovary-biased miRNAs are female-biased, possibly due to broad hormonal control. In chicken, which has a Z/W sex chromosome system, expression output of genes on the Z Chromosome is expected to be male-biased, since there is no global dosage compensation mechanism that restores expression in ZW females after almost all genes on the W Chromosome decayed. Nevertheless, we found that the dominant liver miRNA, miR-122-5p, is Z-linked but expressed in an unbiased manner, due to the unusual retention of a W-linked copy. Another Z-linked miRNA, the male-biased miR-2954-3p, shows conserved preference for dosage-sensitive genes on the Z Chromosome, based on computational and experimental data from chicken and zebra finch, and acts to equalize male-to-female expression ratios of its targets. Unexpectedly, our findings thus establish miRNA regulation as a novel gene-specific dosage compensation mechanism

Domestication related genotype on ADRA2C - a determinant of fear response in chickens?

White Leghorn (WL) breed is homozygous for an allele on the α2C-AR gene while the Red Junglefowl (RJF) is mostly heterozygous for that. The gene is also hypermethylated in WL. The gene codes for the α2C -adrenergic receptor, which plays various roles including regulation of neurotransmitter release from adrenergic neurons in the central nervous system and sympathetic nerves The aim of this study was to investigate the effects of the variation of α2C-AR gene on the chickens’ behaviour. Various behavioural tests mostly associated with fear and stress was conducted in progeny of an F9 generation of the advanced intercross line (AIL) between RJF and WL, selected for homozygosity of the alternative alleles on the α2C-AR gene. In the emergence test, the stress effect on both poking and total exit latency was significantly higher in WL genotype females in comparison to RJF genotype females (effect on head poking time: WL -70.62 ± 18.603 versus RJF 15.63 ± 29.069; effect on total exit time WL -72.14 ± 20.582 RJF 5.64 ± 30.140). In the aerial predator test RJF genotype birds showed significantly more agitated behaviours after the predator exposure in comparison to WL genotype birds (RJF 27.65 ± 0.700 versus WL 25.09 ± 0.915) Although we found differences in behaviour of individuals with WL genotype, more research is needed to find out how the variation on the ADRA2C gene has played a role in domestication of layer chicken

Domestication Effects on the Stress Response in Chickens : Genetics, Physiology, and Behaviour

Animal domestication, the process where animals become adapted to living in proximity to humans, is associated with the alteration of multiple traits, including decreased fearfulness and stress response. With an estimated population of 50 billion, the domesticated chicken is the most populous avian species in the world. Hundreds of chicken breeds have been developed for meat and egg production, hobby or research purposes. Multidirectional selection and the relaxation of natural selection in captivity have created immense phenotypic diversity amongst domesticates in a relatively short evolutionary time. The extensive phenotypic diversity, existence of the wild ancestor, and feasibility of intercrossing various breeds makes the chicken a suitable model animal for deciphering genetic determinants of complex traits such as stress response. We used chicken domestication as a model to gain insights about the mechanisms that regulate stress response in an avian species. We studied behavioural and physiological stress response in the ancestral Red Junglefowl and one of its domesticated progenies, White Leghorn. An advanced intercross between the aforementioned breeds was later used to map genetic loci underlying modification of stress response. The general pattern of the stress response in chickens was comparable with that reported in mammals, however we identified distinctive differences in the stress modulatory pathways in chickens. We showed that changes in the expression levels of several stress modulatory genes in the brain, the pituitary and the adrenal glands underlie the observed modified stress response in domesticated chickens. Using quantitative trait loci (QTL) mapping, several QTL underlying stress induced corticosterone, aldosterone and baseline dehydroepiandrosterone (DHEA) levels were detected. As a next step, we combined QTL mapping with gene expression (eQTL) mapping and narrowed two QTL down to the putative causal genes, SERPINA10 and PDE1C. Both of these genes were differentially expressed in the adrenal glands of White Leghorn and the Red Junglefowl, had overlapping eQTL with hormonal QTL, and their expression levels in the adrenal glands were correlated with plasma levels of corticosterone and al-dosterone. These two genes thus serve as strong candidates for further functional investigation concerning modification of the stress response during domestication. This dissertation increase the knowledge about genetics and physiology of the stress response in an avian species and its modification during domestication. Our findings expand the basic knowledge about the stress response in chicken, which can potentially be used to improve welfare through appropriate genetic selection

Chicken domestication changes expression of stress-related genes in brain, pituitary and adrenals

Domesticated species have an attenuated behavioral and physiological stress response compared to their wild counterparts, but the genetic mechanisms underlying this change are not fully understood. We investigated gene expression of a panel of stress response-related genes in five tissues known for their involvement in the stress response: hippocampus, hypothalamus, pituitary, adrenal glands and liver of domesticated White Leghorn chickens and compared it with the wild ancestor of all domesticated breeds, the Red Junglefowl. Gene expression was measured both at baseline and after 45 min of restraint stress. Most of the changes in gene expression related to stress were similar to mammals, with an upregulation of genes such as FKBP5, C-FOS and EGR1 in hippocampus and hypothalamus and StAR, MC2R and TH in adrenal glands. We also found a decrease in the expression of CRHR1 in the pituitary of chickens after stress, which could be involved in negative feedback regulation of the stress response. Furthermore, we observed a downregulation of EGR1 and C-FOS in the pituitary following stress, which could be a potential link between stress and its effects on reproduction and growth in chickens. We also found changes in the expression of important genes between breeds such as GR in the hypothalamus, POMC and PC1 in the pituitary and CYP11A1 and HSD3B2 in the adrenal glands. These results suggest that the domesticated White Leghorn may have a higher capacity for negative feedback of the HPA axis, a lower capacity for synthesis of ACTH in the pituitary and a reduced synthesis rate of corticosterone in the adrenal glands compared to Red Junglefowl. All of these findings could explain the attenuated stress response in the domesticated birds

The Strong Selective Sweep Candidate Gene ADRA2C Does Not Explain Domestication Related Changes In The Stress Response Of Chickens

Analysis of selective sweeps to pinpoint causative genomic regions involved in chicken domestication has revealed a strongselective sweep on chromosome 4 in layer chickens. The autoregulatory a-adrenergic receptor 2C (ADRA2C) gene is theclosest to the selective sweep and was proposed as an important gene in the domestication of layer chickens. The ADRA2Cpromoter region was also hypermethylated in comparison to the non-selected ancestor of all domesticated chicken breeds,the Red Junglefowl, further supporting its relevance. In mice the receptor is involved in the fight-or-flight response as itmodulates epinephrine release from the adrenals. To investigate the involvement of ADRA2C in chicken domestication, wemeasured gene expression in the adrenals and radiolabeled receptor ligand in three brain regions comparing the domesticWhite Leghorn strain with the wild ancestor Red Junglefowl. In adrenals ADRA2C was twofold greater expressed than therelated receptor gene ADRA2A, indicating that ADRA2C is the predominant modulator of epinephrine release but no straindifferences were measured. In hypothalamus and amygdala, regions associated with the stress response, and in striatum,receptor binding pIC50 values ranged between 8.1–8.4, and the level was not influenced by the genotyped allele. Becausechicken strains differ in morphology, physiology and behavior, differences attributed to a single gene may be lost in thenoise caused by the heterogeneous genetic background. Therefore an F10 advanced intercross strain between WhiteLeghorn and Red Junglefowl was used to investigate effects of ADRA2C alleles on fear related behaviors and fecundity. Wedid not find compelling genotype effects in open field, tonic immobility, aerial predator, associative learning or fecundity.Therefore we conclude that ADRA2C is probably not involved in the domestication of the stress response in chicken, and thestrong selective sweep is probably caused by selection of some unknown genetic element in the vicinity of the gene

Domestication Effects on Stress Induced Steroid Secretion and Adrenal Gene Expression in Chickens

Understanding the genetic basis of phenotypic diversity is a challenge in contemporary biology. Domestication provides a model for unravelling aspects of the genetic basis of stress sensitivity. The ancestral Red Junglefowl (RJF) exhibits greater fear-related behaviour and a more pronounced HPA-axis reactivity than its domesticated counterpart, the White Leghorn (WL). By comparing hormones (plasmatic) and adrenal global gene transcription profiles between WL and RJF in response to an acute stress event, we investigated the molecular basis for the altered physiological stress responsiveness in domesticated chickens. Basal levels of pregnenolone and dehydroepiandrosterone as well as corticosterone response were lower in WL. Microarray analysis of gene expression in adrenal glands showed a significant breed effect in a large number of transcripts with over-representation of genes in the channel activity pathway. The expression of the best-known steroidogenesis genes were similar across the breeds used. Transcription levels of acute stress response genes such as StAR, CH25 and POMC were upregulated in response to acute stress. Dampened HPA reactivity in domesticated chickens was associated with changes in the expression of several genes that presents potentially minor regulatory effects rather than by means of change in expression of critical steroidogenic genes in the adrenal.Funding agencies: Swedish Research Council (VR) [621-2011-4731]; Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) [221-2011-1088]; SRC [621-2011-5523]; ERC [322206]; Swedish Centre of Excellence in Animal Welfare</p

Adrenergic receptor gene expression in adrenal glands.

<p>A) <i>ADRA2C</i> and B) <i>ADRA2A</i> where the line indicates average value relative to housekeeping genes and the dots are the individual data points.</p

Behavioural variables from Tonic Immobility (TI) and Aerial Predator tests.

<p>A) TI induction attempts and B) TI duration (s). C) and D) Relaxed behavior pre and post exposure to the aerial predator in males (C) and in females (D). Data shown as mean and standard deviation. Star indicates a significant difference between genotypes (p<0.05).</p