Hugh Morgan

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 ( Avy/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.

Transgenerational inheritance of epigenetic states at the murine AxinFu allele occurs after maternal and paternal transmission

Phenotypic variation that cannot be explained by genetic or environmental heterogeneity has intrigued geneticists for decades. The molecular basis of this phenomenon, however, is largely a mystery. Axin-fused (AxinFu), first identified in 1937, is a classic example of a mammalian allele displaying extremely variable expression states. Here we demonstrate that the presence or absence of its characteristic phenotype, a kinked tail, correlates with differential DNA methylation at a retrotransposon within AxinFu and identify mutant transcripts arising adjacent to the retrotransposon LTR that are likely to be causative of the phenotype. Furthermore, the epigenetic state at AxinFu can be inherited transgenerationally after both maternal and paternal transmission. This is in contrast to epigenetic inheritance at the murine agouti-viable yellow (Avy) allele, which occurs through the female only. Unlike the egg, the sperm contributes very little (if any) cytoplasm to the zygote, and therefore paternal inheritance at AxinFu argues against the possibility that the effects are due to cytoplasmic or metabolic influences. Consistent with the idea of transgenerational inheritance of epigenetic marks, we find that the methylation state of AxinFu in mature sperm reflects the methylation state of the allele in the somatic tissue of the animal, suggesting that it does not undergo epigenetic reprogramming during gametogenesis. Finally, we show that epigenetic inheritance is influenced by strain background. These findings enable us to propose a model for transgenerational epigenetic inheritance in mammals.

Reactivation of heritably silenced gene expression in mice.

Abstract. 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 β-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 F1 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.

A mouse informatics platform for phenotypic and translational discovery

The International Mouse Phenotyping Consortium (IMPC) is providing the world’s first functional catalogue of a mammalian genome by characterising a knockout mouse strain for every gene. A robust and highly structured informatics platform has been developed to systematically collate, analyse and disseminate the data produced by the IMPC. As the first phase of the project, in which 5000 new knockout strains are being broadly phenotyped, nears completion, the informatics platform is extending and adapting to support the increasing volume and complexity of the data produced as well as addressing a large volume of users and emerging user groups. An intuitive interface helps researchers explore IMPC data by giving overviews and the ability to find and visualise data that support a phenotype assertion. Dedicated disease pages allow researchers to find new mouse models of human diseases, and novel viewers provide high-resolution images of embryonic and adult dysmorphologies. With each monthly release, the informatics platform will continue to evolve to support the increased data volume and to maintain its position as the primary route of access to IMPC data and as an invaluable resource for clinical and non-clinical researchers.

Is real reform of the Medicare Benefits Schedule for psychiatrists in Australia economically, socially or professionally desirable?

OBJECTIVE To propose alternative Medicare Benefits Schedule-based funding models for outpatient psychiatric services in Australia. METHOD Development of alternative funding schedules for a variety of under-serviced populations. CONCLUSIONS Consideration of alternative systems is necessary to address the restrictive work practices, inequity and poor distribution of the psychiatric workforce.

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 (Reprinted with Correction)

Abstract 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.

Accessing and mining data from large-scale mouse phenotyping projects.

Comprehensive phenotyping through the International Mouse Phenotyping Consortium (IMPC)-www.mousephenotype.org-will reveal the pleiotropic functions of every gene in the mouse genome and uncover the wider role of genetic loci within diverse biological systems. The informatics challenge will be to develop an infrastructure to acquire the diverse and complex data sets generated from broad-based phenotyping and disseminate these data in an integrated manner to the scientific community. We describe here the current methodologies implemented to capture and disseminate these data, and plans within the Knockout Mouse Phenotyping Project (KOMP2) (http://commonfund.nih.gov/KOMP2/)-funded informatics consortium to scale these implementations to manage the surge in data from the IMPC.

Reproducibility and replicability of rodent phenotyping in preclinical studies

The scientific community is increasingly concerned with the proportion of published “discoveries” that are not replicated in subsequent studies. The field of rodent behavioral phenotyping was one of the first to raise this concern, and to relate it to other methodological issues: the complex interaction between genotype and environment; the definitions of behavioral constructs; and the use of laboratory mice and rats as model species for investigating human health and disease mechanisms. In January 2015, researchers from various disciplines gathered at Tel Aviv University to discuss these issues. The general consensus was that the issue is prevalent and of concern, and should be addressed at the statistical, methodological and policy levels, but is not so severe as to call into question the validity and the usefulness of model organisms as a whole. Well-organized community efforts, coupled with improved data and metadata sharing, have a key role in identifying specific problems and promoting effective solutions. Replicability is closely related to validity, may affect generalizability and translation of findings, and has important ethical implications.

A bioimage informatics platform for high-throughput embryo phenotyping

Abstract High-throughput phenotyping is a cornerstone of numerous functional genomics projects. In recent years, imaging screens have become increasingly important in understanding gene–phenotype relationships in studies of cells, tissues and whole organisms. Three-dimensional (3D) imaging has risen to prominence in the field of developmental biology for its ability to capture whole embryo morphology and gene expression, as exemplified by the International Mouse Phenotyping Consortium (IMPC). Large volumes of image data are being acquired by multiple institutions around the world that encompass a range of modalities, proprietary software and metadata. To facilitate robust downstream analysis, images and metadata must be standardized to account for these differences. As an open scientific enterprise, making the data readily accessible is essential so that members of biomedical and clinical research communities can study the images for themselves without the need for highly specialized software or technical expertise. In this article, we present a platform of software tools that facilitate the upload, analysis and dissemination of 3D images for the IMPC. Over 750 reconstructions from 80 embryonic lethal and subviable lines have been captured to date, all of which are openly accessible at mousephenotype.org. Although designed for the IMPC, all software is available under an open-source licence for others to use and develop further. Ongoing developments aim to increase throughput and improve the analysis and dissemination of image data. Furthermore, we aim to ensure that images are searchable so that users can locate relevant images associated with genes, phenotypes or human diseases of interest.

Comparative visualization of genotype-phenotype relationships

A defining feature of Phenoview is its ability to comparatively visualize data for multiple genotype-phenotype combinations simultaneously. This mix-and-match option is suited to investigating the knockout effects of a genotype on multiple traits, and also to the investigation of multiple genotypes that affect the same trait. Selecting the comparative visualization mode displays multiple graphs that reflect selections made by the user (Fig. 1b). These genotype-phenotype combinations are selectable on the basis of user-defined P-value thresholds configurable on the heat-map interface. Phenoview enables real-time interaction with the displayed data, allowing users to interactively filter out data points by gender and zygosity and to dynamically configure the statistics displayed. It also allows the generated graphs to be saved as image files. Phenoview is context sensitive and automatically chooses the appropriate display type to match the data. It can also display digital media (e.g., X-ray or LacZ expression), with the flexibility to adjust brightness, contrast, zooming and panning (Fig. 1c). To permit further analysis of the data using alternative tools, Phenoview also allows downloading of the raw data (Supplementary Note). By collating data from multiple centers, Phenoview provides a single, convenient data access point for researchers, increasing accessibility to the phenotype data. Through these tools, the IMPC aims to advance our understanding of genotype-phenotype relationships6 by enabling systematic analysis of large-scale phenotype data.