Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells.

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

Metabolomics to Explore Imidacloprid-Induced Toxicity in the Central Nervous System of the Freshwater Snail Lymnaea stagnalis.

Modern toxicology is seeking new testing methods to better understand toxicological effects. One of the most concerning chemicals is the neonicotinoid pesticide imidacloprid. Although imidacloprid is designed to target insects, recent studies have shown adverse effects on nontarget species. Metabolomics was applied to investigate imidacloprid-induced sublethal toxicity in the central nervous system of the freshwater snail Lymnaea stagnalis. The snails (n = 10 snails) were exposed for 10 days to increasing imidacloprid concentrations (0.1, 1, 10, and 100 µg/L). The comparison between control and exposure groups highlighted the involvement and perturbation of many biological pathways. The levels of several metabolites belonging to different metabolite classes were significantly changed by imidacloprid exposure. A change in the amino acids and nucleotide metabolites like tryptophan, proline, phenylalanine, uridine, and guanosine was found. Many fatty acids were down-regulated, and the levels of the polyamines, spermidine and putrescine, were found to be increased which is an indication of neuron cell injury. A turnover increase between choline and acetylcholine led us to hypothesize an increase in cholinergic gene expression to overcome imidacloprid binding to the nicotinic acetylcholine receptors. Metabolomics revealed imidacloprid induced metabolic changes at low and environmentally relevant concentration in a nontarget species and generated a novel mechanistic hypothesis.

In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.

The Role of Epigenetics in the Latent Effects of Early Life Exposure to Obesogenic Endocrine Disrupting Chemicals.

Recent research supports a role for exposure to endocrine-disrupting chemicals (EDCs) in the global obesity epidemic. Obesogenic EDCs have the potential to inappropriately stimulate adipogenesis and fat storage, influence metabolism and energy balance and increase susceptibility to obesity. Developmental exposure to obesogenic EDCs is proposed to interfere with epigenetic programming of gene regulation, partly by activation of nuclear receptors, thereby influencing the risk of obesity later in life. The goal of this minireview is to briefly describe the epigenetic mechanisms underlying developmental plasticity and to evaluate the evidence of a mechanistic link between altered epigenetic gene regulation by early life EDC exposure and latent onset of obesity. We summarize the results of recent in vitro, in vivo, and transgenerational studies, which clearly show that the obesogenic effects of EDCs such as tributyltin, brominated diphenyl ether 47, and polycyclic aromatic hydrocarbons are mediated by the activation and associated altered methylation of peroxisome proliferator-activated receptor-γ, the master regulator of adipogenesis, or its target genes. Importantly, studies are emerging that assess the effects of EDCs on the interplay between DNA methylation and histone modifications in altered chromatin structure. These types of studies coupled with genome-wide rather than gene-specific analyses are needed to improve mechanistic understanding of epigenetic changes by EDC exposure. Current advances in the field of epigenomics have led to the first potential epigenetic markers for obesity that can be detected at birth, providing an important basis to determine the effects of developmental exposure to obesogenic EDCs in humans.

Acute and chronic toxicity of short chained perfluoroalkyl substances to Daphnia magna.

The aim of this study was to evaluate the aquatic toxicity of a C4-C6 chemistry based fluoroalkylated polymer and the perfluoroalkyl carboxylic acids, PFBA, PFHxA and PFOA to Daphnia magna. The acute toxicity decreased with decreasing carbon chain length, but the polymer did not show a dose related effect. In a chronic toxicity test performed with PFHxA, mortality was observed at similar concentrations as in the acute toxicity test, indicating that toxicity did not increase with increasing exposure time. Effects on mortality, reproduction and population growth rate occurred at similar concentrations, indicating no specific effect of PFHxA on sublethal endpoints. C4-C6 chemistry is thus less hazardous to daphnids than C7-C8 chemistry. Yet, these compounds are persistent, hard to remove from the environment and production volumes are increasing.