Epigenetic inheritance at the agouti locus in the mouse.

Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

The long lasting effects of electrical simulation of the medial preoptic area and medial amygdala on maternal behavior in female rats.

A program of repeated electrical (kindling-like) stimulation of the medial preoptic area (MPOA) or the medial amygdala (MedAmyg) on maternal and other behaviors were investigated. Stimulation was applied daily for 14 days (or until a stage 3 motor seizure was observed) using 2 s trains of biphasic square wave pulses at 60 Hz, 1 ms duration and 300-500 microA. Confirmation of afterdischarge using these parametres was established. In the first experiment, maternally experienced (but not post-partum) MedAmyg stimulated animals became maternal more slowly than did MedAmyg not stimulated animals or than MPOA stimulated animals. In the second experiment, virgin animals were used. MPOA stimulation enhanced the female's preference for pup associated environments in the conditioned place preference (CPP) paradigm. MedAmyg stimulation had no effect on CPP performance, but produced a decreased preference for pup odors in a modified hole board test and increased 'anxiety' in the open field. These results confirm that the MPOA and the MedAmyg are involved in facilitating and attenuating maternal responsiveness and related (precursor?) behaviors, respectively. It appears that chronic (kindling-like) stimulation of these neural substrates enhances their functions.

The effect of kindling of different nuclei in the left and right amygdala on anxiety in the rat.

The effects on rodent anxiety of kindling in the medial or basolateral amygdaloid nuclei in each hemisphere were examined. Anxiety was measured using the hole board and elevated plus maze tests. The animals were kindled in medial or basolateral amygdalas, of either the left or right hemisphere. Controls had electrodes implanted in comparable areas, but were not kindled. Analysis of electrode location showed that some animals were kindled in amygdaloid nuclei other than medial or basolateral amygdala. These animals were labelled outliers. Kindling of the medial/basolateral amygdala in the left hemisphere decreased anxiety for at least 1 week after the last kindled seizure. Right hemisphere medial/basolateral kindling tended to increase anxiety. Outlier-kindled rats were less anxious than their controls regardless of hemisphere 1 week after their last kindled seizure. Clear anxiogenic effects were not likely seen in the right hemisphere in this study because of the electrode locations. The degree of anxiety following kindling was correlated with electrode location in the anterior-posterior plane. More anterior foci in the amygdala were associated with more anxiety. More posterior amygdala foci were associated with less anxiety. These findings point to the importance of kindled focus in the amygdala for behavioral effect. Future research should carefully control the location of kindled foci when investigating effects of amygdala kindling on anxiety and other behaviors.

Somatosensory control of the onset and retention of maternal responsiveness in primiparous Sprague-Dawley rats.

The role of perioral and ventral-trunk somatosensory stimulation from pups mediating the initial expression of maternal behavior and its long-term retention 8 days later, was investigated. Six groups of female rats were permitted to physically interact with four 1-5-day-old foster pups for 1 h, 36 h after Cesarean delivery on gestation day 21. Prior to this maternal experience, dams were subjected to: reduced cutaneous rostral snout sensitivity (anaptia) by injection of lidocaine into the mystacial pads; reduced ventral-trunk sensations by occlusion of the entire ventrum with a full spandex jacket; both manipulations; or control manipulations. Additional groups of females not receiving a maternal experience (inexperienced) also received the somatosensory deprivation or control manipulations. During retention testing, rats in the singly manipulated experienced groups exhibited reduced latencies to become maternal in comparison to their inexperienced counterparts (approximately 3 days vs. 8 days). However, rats previously rendered both anaptic and ventrally-occluded responded like inexperienced rats in showing a long latency to become maternal (8 days). Thus, reduction of either perioral or ventral somatosensory contact from pups did not block the maternal experience effect, but reduction of both of these inputs did.

Preimplantation embryo development in the mouse requires the latency of TRP53 expression, which is induced by a ligand-activated PI3 kinase/AKT/MDM2-mediated signaling pathway.

A universal response to cellular stress is the expression of transformation-related protein 53 (TRP53). This transcription factor reduces cell proliferation and/or survival and is classed as a tumour suppressor protein. Several stresses (including culture) cause increased TRP53 expression in blastocysts and their reduced long-term developmental potential. This study shows that culture from the zygote stage (but not the 2-cell stage) reduced the development of C57BL6 inbred (but not hybrid) strain mouse embryos. Reduced viability was TRP53 dependent, being partially reversed by a TRP53 inhibitor (Pifithrin-alpha). However, the presence of culture did not cause an increase in Trp53 mRNA levels (levels were reduced following culture, P < 0.001). Transformed mouse 3T3 cell double minute 2 (MDM2) causes the ubiquitination and degradation of TRP53. MDM2 activation is accompanied by phosphorylation of Ser-166, and this is commonly catalyzed by the phosphatidylinositol-3 kinase and RAC-alpha serine/threonine-protein kinase (AKT) signaling pathway. Paf is an autocrine embryotrophin that activates the phosphatidylinositol-3 kinase/AKT pathway. High levels of TRP53 expression occurred following the culture of zygotes lacking the Paf receptor (Ptafr(-/-)) and following inhibition of phosphatidylinositol-3 kinase or AKT. Inhibition of MDM2 caused a Trp53-dependent reduction in zygote development. Inbred strain embryos cultured from the zygote stage expressed less phosphorylated MDM2 than similar embryos collected from the uterus. The addition of Paf to the media caused increased phosphorylation of MDM2, and this was blocked by inhibitors of phosphatidylinositol-3 kinase and AKT. The study identifies trophic ligand signaling via the activation of phosphatidylinositol-3 kinase and AKT as a mechanism resulting in the activation of MDM2.

Preimplantation embryo development in the mouse requires the latency of TRP53 expression, which is induced by a ligand-activated PI3 kinase/AKT/MDM2-mediated signaling pathway.

A universal response to cellular stress is the expression of transformation-related protein 53 (TRP53). This transcription factor reduces cell proliferation and/or survival and is classed as a tumour suppressor protein. Several stresses (including culture) cause increased TRP53 expression in blastocysts and their reduced long-term developmental potential. This study shows that culture from the zygote stage (but not the 2-cell stage) reduced the development of C57BL6 inbred (but not hybrid) strain mouse embryos. Reduced viability was TRP53 dependent, being partially reversed by a TRP53 inhibitor (Pifithrin-alpha). However, the presence of culture did not cause an increase in Trp53 mRNA levels (levels were reduced following culture, P < 0.001). Transformed mouse 3T3 cell double minute 2 (MDM2) causes the ubiquitination and degradation of TRP53. MDM2 activation is accompanied by phosphorylation of Ser-166, and this is commonly catalyzed by the phosphatidylinositol-3 kinase and RAC-alpha serine/threonine-protein kinase (AKT) signaling pathway. Paf is an autocrine embryotrophin that activates the phosphatidylinositol-3 kinase/AKT pathway. High levels of TRP53 expression occurred following the culture of zygotes lacking the Paf receptor (Ptafr(-/-)) and following inhibition of phosphatidylinositol-3 kinase or AKT. Inhibition of MDM2 caused a Trp53-dependent reduction in zygote development. Inbred strain embryos cultured from the zygote stage expressed less phosphorylated MDM2 than similar embryos collected from the uterus. The addition of Paf to the media caused increased phosphorylation of MDM2, and this was blocked by inhibitors of phosphatidylinositol-3 kinase and AKT. The study identifies trophic ligand signaling via the activation of phosphatidylinositol-3 kinase and AKT as a mechanism resulting in the activation of MDM2.

Aggressive behaviour of male mice (Mus musculus) towards familiar and unfamiliar opponents.

Two types of colonies of laboratory mice were employed; hierarchically organized ones formed by placing five unfamiliar 8-week-old mice in a cage together and amicably organized colonies in which four male litter mates were kept together throughout the whole period of the experiment. During a 21-day pre-experimental period intra-colony aggressive behaviour was recorded. A dominant animal and ranked subordinates occurred in every hierarchical colony, whilst no aggression was recorded in any of the amicable colonies. During a 25-day period unfamiliar adult male or female mice were introduced daily into the amicable or hierarchical colonies for 10 min. In a third experiment juvenile mice 17, 24, 31 or 38 days old were introduced into hierarchically organized colonies during a 20-day period. In all hierarchical colonies the stranger was attacked irrespective of sex and age; the majority of attacks were carried out by the dominant mouse. Aggression by the dominant declined exponentially throughout the experimental period and regression analyses compared the different data. Unfamiliar adult females were the recipients of fewer attacks than unfamiliar adult males and the age of juvenile strangers was found to be an important factor. Amicably organized mice initially did not attack strangers, but over a period of 25 days the number of attacks on unfamiliar males gradually increased.

The marks, mechanisms and memory of epigenetic states in mammals.

It is well recognized that there is a surprising degree of phenotypic variation among genetically identical individuals, even when the environmental influences, in the strict sense of the word, are identical. Genetic textbooks acknowledge this fact and use different terms, such as 'intangible variation' or 'developmental noise', to describe it. We believe that this intangible variation results from the stochastic establishment of epigenetic modifications to the DNA nucleotide sequence. These modifications, which may involve cytosine methylation and chromatin remodelling, result in alterations in gene expression which, in turn, affects the phenotype of the organism. Recent evidence, from our work and that of others in mice, suggests that these epigenetic modifications, which in the past were thought to be cleared and reset on passage through the germline, may sometimes be inherited to the next generation. This is termed epigenetic inheritance, and while this process has been well recognized in plants, the recent findings in mice force us to consider the implications of this type of inheritance in mammals. At this stage we do not know how extensive this phenomenon is in humans, but it may well turn out to be the explanation for some diseases which appear to be sporadic or show only weak genetic linkage.

Reactivation of heritably silenced gene expression in mice.

Epigenetic modifications that suppress gene activity in mammals are generally considered to be cleared in the germline, restoring totipotency of the genome. Here we report the germline inheritance of transcriptional silencing in mice, and reversion to activity after as many as three generations in the silent state. In a series of lines made with a LacZ transgene, one line exhibits variable expressivity: genotypically identical littermates have proportions of beta-Gal-positive erythrocytes that vary over at least four orders of magnitude, and in some offspring expression is completely silenced. The silent state of the transgene is inherited for multiple generations in the founder strain irrespective of the sex of the parent, implying maintenance of the epigenetic state through meiosis. Crosses of silenced mice with C57BL/6 mice result in reactivation of the transgene in approximately a third of F(1) littermates. The silencing involves a stochastic, all-or-none mechanism. Furthermore, silencing is transcriptional and correlates with methylation of the transgene as well as an inaccessible chromatin structure; these changes are reversed when expression is reactivated. This work supports the notion that silent genetic information in mammals can be inherited and later reactivated, and implies a mode of phenotypic inheritance that is less stable than Mendelian inheritance.