Daniel Nätt

Transgenerational effects of early experience on behavioral, hormonal and gene expression responses to acute stress in the precocial chicken.

Stress during early life can profoundly influence an individuals phenotype. Effects can manifest in the short-term as well as later in life and even in subsequent generations. Transgenerational effects of stress are potentially mediated via modulation of the hypothalamic-pituitary-adrenal axis (HPA) as well as epigenetic mechanisms causing heritable changes in gene expression. To investigate these pathways we subjected domestic chicken (Gallus gallus) to intermittent social isolation for the first three weeks of life. The early life stress resulted in a dampened corticosterone response to restraint stress in affected birds and in their male offspring. Stress-specific genes, such as early growth response 1 (EGR1) and corticotropin releasing hormone receptor 1 (CRHR1), were upregulated immediately after restraint stress, but not under baseline conditions. Treatment differences in gene expression were also correlated across generations which indicate transgenerational epigenetic inheritance. In an associative learning test early stressed birds made more correct choices suggesting a higher coping ability in stressful situations. This study is the first to show transgenerational effects of early life stress in a precocial species by combining behavioral, endocrinological, and transcriptomic measurements.

Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens

BackgroundVariations in gene expression, mediated by epigenetic mechanisms, may cause broad phenotypic effects in animals. However, it has been debated to what extent expression variation and epigenetic modifications, such as patterns of DNA methylation, are transferred across generations, and therefore it is uncertain what role epigenetic variation may play in adaptation.ResultsIn Red Junglefowl, ancestor of domestic chickens, gene expression and methylation profiles in thalamus/hypothalamus differed substantially from that of a domesticated egg laying breed. Expression as well as methylation differences were largely maintained in the offspring, demonstrating reliable inheritance of epigenetic variation. Some of the inherited methylation differences were tissue-specific, and the differential methylation at specific loci were little changed after eight generations of intercrossing between Red Junglefowl and domesticated laying hens. There was an over-representation of differentially expressed and methylated genes in selective sweep regions associated with chicken domestication.ConclusionsOur results show that epigenetic variation is inherited in chickens, and we suggest that selection of favourable epigenomes, either by selection of genotypes affecting epigenetic states, or by selection of methylation states which are inherited independently of sequence differences, may have been an important aspect of chicken domestication.

Transmission of stress-induced learning impairment and associated brain gene expression from parents to offspring in chickens.

Background Stress influences many aspects of animal behaviour and is a major factor driving populations to adapt to changing living conditions, such as during domestication. Stress can affect offspring through non-genetic mechanisms, but recent research indicates that inherited epigenetic modifications of the genome could possibly also be involved. Methodology/Principal Findings Red junglefowl (RJF, ancestors of modern chickens) and domesticated White Leghorn (WL) chickens were raised in a stressful environment (unpredictable light-dark rhythm) and control animals in similar pens, but on a 12/12 h light-dark rhythm. WL in both treatments had poorer spatial learning ability than RJF, and in both populations, stress caused a reduced ability to solve a spatial learning task. Offspring of stressed WL, but not RJF, raised without parental contact, had a reduced spatial learning ability compared to offspring of non-stressed animals in a similar test as that used for their parents. Offspring of stressed WL were also more competitive and grew faster than offspring of non-stressed parents. Using a whole-genome cDNA microarray, we found that in WL, the same changes in hypothalamic gene expression profile caused by stress in the parents were also found in the offspring. In offspring of stressed WL, at least 31 genes were up- or down-regulated in the hypothalamus and pituitary compared to offspring of non-stressed parents. Conclusions/Significance Our results suggest that, in WL the gene expression response to stress, as well as some behavioural stress responses, were transmitted across generations. The ability to transmit epigenetic information and behaviour modifications between generations may therefore have been favoured by domestication. The mechanisms involved remain to be investigated; epigenetic modifications could either have been inherited or acquired de novo in the specific egg environment. In both cases, this would offer a novel explanation to rapid evolutionary adaptation of a population.

Inheritance of Acquired Behaviour Adaptations and Brain Gene Expression in Chickens

Background Environmental challenges may affect both the exposed individuals and their offspring. We investigated possible adaptive aspects of such cross-generation transmissions, and hypothesized that chronic unpredictable food access would cause chickens to show a more conservative feeding strategy and to be more dominant, and that these adaptations would be transmitted to the offspring. Methodology/Principal Findings Parents were raised in an unpredictable (UL) or in predictable diurnal light rhythm (PL, 12∶12 h light∶dark). In a foraging test, UL birds pecked more at freely available, rather than at hidden and more attractive food, compared to birds from the PL group. Female offspring of UL birds, raised in predictable light conditions without parental contact, showed a similar foraging behavior, differing from offspring of PL birds. Furthermore, adult offspring of UL birds performed more food pecks in a dominance test, showed a higher preference for high energy food, survived better, and were heavier than offspring of PL parents. Using cDNA microarrays, we found that the differential brain gene expression caused by the challenge was mirrored in the offspring. In particular, several immunoglobulin genes seemed to be affected similarly in both UL parents and their offspring. Estradiol levels were significantly higher in egg yolk from UL birds, suggesting one possible mechanism for these effects. Conclusions/Significance Our findings suggest that unpredictable food access caused seemingly adaptive responses in feeding behavior, which may have been transmitted to the offspring by means of epigenetic mechanisms, including regulation of immune genes. This may have prepared the offspring for coping with an unpredictable environment.

Large sex differences in chicken behavior and brain gene expression coincide with few differences in promoter DNA-methylation.

While behavioral sex differences have repeatedly been reported across taxa, the underlying epigenetic mechanisms in the brain are mostly lacking. Birds have previously shown to have only limited dosage compensation, leading to high sex bias of Z-chromosome gene expression. In chickens, a male hyper-methylated region (MHM) on the Z-chromosome has been associated with a local type of dosage compensation, but a more detailed characterization of the avian methylome is limiting our interpretations. Here we report an analysis of genome wide sex differences in promoter DNA-methylation and gene expression in the brain of three weeks old chickens, and associated sex differences in behavior of Red Junglefowl (ancestor of domestic chickens). Combining DNA-methylation tiling arrays with gene expression microarrays we show that a specific locus of the MHM region, together with the promoter for the zinc finger RNA binding protein (ZFR) gene on chromosome 1, is strongly associated with sex dimorphism in gene expression. Except for this, we found few differences in promoter DNA-methylation, even though hundreds of genes were robustly differentially expressed across distantly related breeds. Several of the differentially expressed genes are known to affect behavior, and as suggested from their functional annotation, we found that female Red Junglefowl are more explorative and fearful in a range of tests performed throughout their lives. This paper identifies new sites and, with increased resolution, confirms known sites where DNA-methylation seems to affect sexually dimorphic gene expression, but the general lack of this association is noticeable and strengthens the view that birds do not have dosage compensation.

Brain gene expression in relation to fearfulness in female red junglefowl (Gallus gallus).

The biology of fear is central to animal welfare and has been a major target for selection during domestication. Fear responses were studied in female red junglefowl (RJF), the ancestor of domesticated chickens. A total of 31 females were tested in a ground predator test, an aerial predator test and a tonic immobility (TI) test, in order to assess their level of fearfulness across different situations. Two to six variables from each test were entered into a principal component (PC) analysis, which showed one major fearfulness component (explaining 27% of the variance). Based on the PC scores, four high‐ and four low‐fearful birds were then selected for gene expression analysis. From each of these birds, the midbrain region (including thalamus, hypothalamus, pituitary, mesencephalon, pons, nucleus tractus solitarii and medulla oblongata), was collected and global gene expression compared between groups using a 14k chicken cDNA microarray. There were 13 significantly differentially expressed (DE) genes (based on M > 1 and B > 0; FDR‐adjusted P < 0.05) between the fearful and non‐fearful females. Among the DE genes, we identified the neuroprotein Axin1, two potential DNA/RNA regulating proteins and a retrotransposon transcript situated in a well‐studied quantitative trait loci (QTL) region on chromosome 1, known to affect several domestication‐related traits. The differentially expressed genes may be part of a possible molecular mechanism controlling fear responses in fowl.

High cortisol in 5-year-old children causes loss of DNA methylation in SINE retrotransposons: a possible role for ZNF263 in stress-related diseases

BackgroundChildhood stress leads to increased risk of many adult diseases, such as major depression and cardiovascular disease. Studies show that adults with experienced childhood stress have specific epigenetic changes, but to understand the pathways that lead to disease, we also need to study the epigenetic link prospectively in children.ResultsHere, we studied a homogenous group of 48 5-year-old children. By combining hair cortisol measurements (a well-documented biomarker for chronic stress), with whole-genome DNA-methylation sequencing, we show that high cortisol associates with a genome-wide decrease in DNA methylation and targets short interspersed nuclear elements (SINEs; a type of retrotransposon) and genes important for calcium transport: phenomena commonly affected in stress-related diseases and in biological aging. More importantly, we identify a zinc-finger transcription factor, ZNF263, whose binding sites where highly overrepresented in regions experiencing methylation loss. This type of zinc-finger protein has previously shown to be involved in the defense against retrotransposons.ConclusionsOur results show that stress in preschool children leads to changes in DNA methylation similar to those seen in biological aging. We suggest that this may affect future disease susceptibility by alterations in the epigenetic mechanisms that keep retrotransposons dormant. Future treatments for stress- and age-related diseases may therefore seek to target zinc-finger proteins that epigenetically control retrotransposon reactivation, such as ZNF263.

Dependence-induced increase of alcohol self-administration and compulsive drinking mediated by the histone methyltransferase PRDM2

Epigenetic processes have been implicated in the pathophysiology of alcohol dependence, but the specific molecular mechanisms mediating dependence-induced neuroadaptations remain largely unknown. Here, we found that a history of alcohol dependence persistently decreased the expression of Prdm2, a histone methyltransferase that monomethylates histone 3 at the lysine 9 residue (H3K9me1), in the rat dorsomedial prefrontal cortex (dmPFC). Downregulation of Prdm2 was associated with decreased H3K9me1, supporting that changes in Prdm2 mRNA levels affected its activity. Chromatin immunoprecipitation followed by massively parallel DNA sequencing showed that genes involved in synaptic communication are epigenetically regulated by H3K9me1 in dependent rats. In non-dependent rats, viral-vector-mediated knockdown of Prdm2 in the dmPFC resulted in expression changes similar to those observed following a history of alcohol dependence. Prdm2 knockdown resulted in increased alcohol self-administration, increased aversion-resistant alcohol intake and enhanced stress-induced relapse to alcohol seeking, a phenocopy of postdependent rats. Collectively, these results identify a novel epigenetic mechanism that contributes to the development of alcohol-seeking behavior following a history of dependence.

Early Stress Causes Sex-Specific, Life-Long Changes in Behaviour, Levels of Gonadal Hormones, and Gene Expression in Chickens

Early stress can have long-lasting phenotypic effects. Previous research shows that male and female chickens differ in many behavioural aspects, and respond differently to chronic stress. The present experiment aimed to broadly characterize long-term sex differences in responses to brief events of stress experienced during the first weeks of life. Chicks from a commercial egg-laying hybrid were exposed to stress by inducing periods of social isolation during their first three weeks of life, followed by a broad behavioural, physiological and genomic characterization throughout life. Early stressed males, but not females, where more anxious in an open field-test, stayed shorter in tonic immobility and tended to have delayed sexual maturity, as shown by a tendency for lower levels of testosterone compared to controls. While early stressed females did not differ from non-stressed in fear and sexual maturation, they were more socially dominant than controls. The differential gene expression profile in hypothalamus was significantly correlated from 28 to 213 days of age in males, but not in females. In conclusion, early stress had a more pronounced long-term effect on male than on female chickens, as evidenced by behavioral, endocrine and genomic responses. This may either be attributed to inherent sex differences due to evolutionary causes, or possibly to different stress related selection pressures on the two sexes during commercial chicken breeding.

A molecular mechanism for choosing alcohol over an alternative reward

Finding the vulnerable minority “Only” about 10 to 15% of people exposed to alcohol develop alcohol-related problems. The behavioral repertoire of people confronted with opportunities to consume alcohol involves numerous choices between this drug reward and healthy alternatives. Augier et al. established a choice procedure that begins to address alcohol addiction in rats (see the Perspective by Spanagel). They found that a minority of outbred rats continued to self-administer alcohol even when a high-value alternative (such as sugar) was available. That minority displayed a remarkable constellation of behavioral traits resembling the human clinical condition, including a high motivation to obtain alcohol and continued use despite adverse consequences. The cause was impaired GABA (γ-aminobutyric acid) clearance in the central amygdala. Postmortem tissue analysis supported the possibility of a similar pathology in human alcoholism. Science, this issue p. 1321; see also p. 1298 Impaired GABA clearance within the central amygdala provides a molecular mechanism behind preferentially choosing alcohol. Alcohol addiction leads to increased choice of alcohol over healthy rewards. We established an exclusive choice procedure in which ~15% of outbred rats chose alcohol over a high-value reward. These animals displayed addiction-like traits, including high motivation to obtain alcohol and pursuit of this drug despite adverse consequences. Expression of the γ-aminobutyric acid (GABA) transporter GAT-3 was selectively decreased within the amygdala of alcohol-choosing rats, whereas a knockdown of this transcript reversed choice preference of rats that originally chose a sweet solution over alcohol. GAT-3 expression was selectively decreased in the central amygdala of alcohol-dependent people compared to those who died of unrelated causes. Impaired GABA clearance within the amygdala contributes to alcohol addiction, appears to translate between species, and may offer targets for new pharmacotherapies for treating this disorder.