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Single-cell transcriptomic platforms provide an opportunity to map an organisms response to environmental cues with high resolution. Here, we applied single-nucleus RNA sequencing (snRNA-seq) to establish the tissue and cell type-resolved transcriptome of the adult C. elegans and characterize the inter- and trans-generational transcriptional impact of ethanol. We profiled the transcriptome of 41,749 nuclei resolving into 31 clusters, representing a diverse array of adult cell types including syncytial tissues. Following exposure to human-relevant doses of alcohol, several germline, striated muscle, and neuronal clusters were identified as being the most transcriptionally impacted at the F1 and F3 generations. The effect on germline clusters was confirmed by phenotypic enrichment analysis as well as by functional validation, which revealed a remarkable inter- and trans-generational increase in germline apoptosis, aneuploidy, and embryonic lethality. Together, snRNA-seq represents a valuable approach for the detailed examination of an adult organisms response to environmental exposures.

Toxicoepigenetics examines the health effects of environmental exposure associated with, or mediated by, changes in the epigenome. Despite high expectations, toxicoepigenomic data and methods have yet to become significantly utilized in chemical risk assessment. This article draws on a social science framework to highlight hitherto overlooked structural barriers to the incorporation of toxicoepigenetics in risk assessment and to propose ways forward. The present barriers stem not only from the lack of maturity of the field but also from differences in constraints and standards between the data produced by toxicoepigenetics and the regulatory science data that risk assessment processes require. Criteria and strategies that frame the validation of knowledge used for regulatory purposes limit the application of basic research in toxicoepigenetics toward risk assessment. First, the need in regulatory toxicology for standardized methods that form a consensus between regulatory agencies, basic research, and the industry conflicts with the wealth of heterogeneous data in toxicoepigenetics. Second, molecular epigenetic data do not readily translate into typical toxicological endpoints. Third, toxicoepigenetics investigates new forms of toxicity, in particular low-dose and long-term effects, that do not align well with the traditional framework of regulatory toxicology. We propose that increasing the usefulness of epigenetic data for risk assessment will require deliberate efforts on the part of the toxicoepigenetics community in 4 areas: fostering the understanding of epigenetics among risk assessors, developing knowledge infrastructure to demonstrate applicability, facilitating the normalization and exchange of data, and opening the field to other stakeholders.

Single-cell RNA transcriptomic platforms have significantly contributed to our understanding of tissue heterogeneity as well as of developmental and cellular differentiation trajectories. They also provide an opportunity to map an organism’s response to environmental cues with high resolution and unbiasedly identify the cell types that are the most transcriptionally sensitive to exposures. Here, we applied single nucleus RNA-seq experimental and computational approaches to C. elegans to establish the transcriptome of the adult nematode and comprehensively characterize the transcriptional impact of ethanol as a model environmental exposure on the entire organism at cell type-resolution over several generations. Clustering, tissue and phenotype enrichment, and gene ontology analyses identified 31 clusters representing a diverse number of adult cell types, including those from syncytial and multi-nucleated tissues which are difficult to assess by single cell RNA-seq, such as the mitotic and meiotic germline, hypodermal cells, and the intestine. We applied this method to identify the impact of inter- and trans-generational exposure to two human-relevant doses of alcohol. Cell type proportions were not significantly altered by ethanol. However, Euclidean distance analysis identified several germline, striated muscle, and neuronal clusters as being major transcriptional targets of ethanol at both the F1 and F3 generations although the relative order of clusters changed between generations. The impact on germline clusters was further confirmed by phenotypic enrichment analysis as well as functional validation, namely a remarkable inter- and trans-generational increase in germline apoptosis, aneuploidy, and embryonic lethality. Together, snRNA-seq of the adult C. elegans represents a powerful approach for the detailed examination of an adult organism’s response to environmental cues.

The emergence of the field of transgenerational epigenetics inheritance (TEI) has profoundly reshaped our understanding of the relationships between environment, soma, and germ cells as well as of heredity. TEI refers to the changes in chromatin state, gene expression, and/or phenotypes that are transmitted across several generations without involving changes to the DNA sequences. TEI has direct connections with, and feeds from, the fields of molecular biology, genetics, developmental biology, and reproductive biology, among others. However, the expansion of TEI-related research, has profoundly reshaped boundaries within each field and often led to the erosion of theories and concepts considered as tenets of biology. We first explore how the molecularization of biology has shifted the definition of epigenetics to include the notion of heredity and how epigenetics has refined our understanding of the central dogma of biology. The demonstrated transfer of environmental information from soma to germ cell through extracellular vesicles and subsequent alteration of health outcomes in offspring has put a definite end to the long-held principle of the Weismann barrier. TEI has also simultaneously led to the revival of the inheritance of acquired characteristics while further eroding the concept of an epigenetic "blank slate" in mammals. Using an historical framework, and via the exploration of central studies in the field, in this perspective article, we will draw a compelling argument for the revolutionary aspect of TEI in biology.

Classical mechanisms of volcanic eruptions mostly involve pressure buildup and magma ascent towards the surface1. Such processes produce geophysical and geochemical signals that may be detected and interpreted as eruption precursors1–3. On 22 May 2021, Mount Nyiragongo (Democratic Republic of the Congo), an open-vent volcano with a persistent lava lake perched within its summit crater, shook up this interpretation by producing an approximately six-hour-long flank eruption without apparent precursors, followed—rather than preceded—by lateral magma motion into the crust. Here we show that this reversed sequence was most likely initiated by a rupture of the edifice, producing deadly lava flows and triggering a voluminous 25-km-long dyke intrusion. The dyke propagated southwards at very shallow depth (less than 500 m) underneath the cities of Goma (Democratic Republic of the Congo) and Gisenyi (Rwanda), as well as Lake Kivu. This volcanic crisis raises new questions about the mechanisms controlling such eruptions and the possibility of facing substantially more hazardous events, such as effusions within densely urbanized areas, phreato-magmatism or a limnic eruption from the gas-rich Lake Kivu. It also more generally highlights the challenges faced with open-vent volcanoes for monitoring, early detection and risk management when a significant volume of magma is stored close to the surface., SCOPUS: ar.j, info:eu-repo/semantics/published

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host-microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C. elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host's response to chronic Cu stress.

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host-microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C. elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host's response to chronic Cu stress.

BackgroundAnimal-based studies indicate that bisphenol A (BPA) exposure is detrimental to reproductive health, but its impact on the earliest stages of germ cell development remains poorly defined.ObjectivesUsing a murine in vitro model of early germ cell specification and differentiation, we sought to assess whether exposure to low levels of BPA prior to formation of primordial germ cells (PGCs) alters their differentiation trajectory and unique molecular program.MethodsWe used an established method of in vitro differentiation of mouse embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs) followed by PGC-like cells (PGCLCs), which together recapitulate defined stages of early germ cell development. Cellular consequences were determined using hemocytometer-based cell counting, fixation, and intracellular staining, followed by flow cytometry/fluorescence-activated cell sorting (FACS) of cells exposed to increasing concentrations (range: 1 nM-10 μM) of BPA. To interrogate and characterize gene expression differences resulting from BPA exposure, we also generated RNA-seq libraries from RNA extracted from FACS-purified PGCLCs and performed transcriptome analysis using bioinformatics-based approaches.ResultsExposure of EpiLCs to BPA resulted in higher numbers of cells that were associated with a higher proportion of cells in S-phase as well as a lower proportion undergoing apoptosis; this difference occurred in a concentration-dependent manner. Exposure also resulted in a greater fraction of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively affect PGC specification and resulted in a concentration-dependent effect on PGCLC proliferation in XX but not XY cells. PGCLC transcriptome analysis revealed an aberrant program with significant deregulation of X-linked genes and retrotransposon expression. Differential gene expression analysis also revealed the deregulation of genes associated with lipid metabolism as well as deregulated expre

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host-microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C. elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host's response to chronic Cu stress.

Metabolites control epigenetic mechanisms, and conversly, cell metabolism is regulated at the epigenetic level in response to changes in the cellular environment. In recent years, this metabolo-epigenetic control of gene expression has been implicated in the regulation of multiple stages of embryonic development. The developmental potency of stem cells and their embryonic counterparts is directly determined by metabolic rewiring. Here, we review the current knowledge on the interplay between epigenetics and metabolism in the specific context of early germ cell development. We explore the implications of metabolic rewiring in primordial germ cells in light of their epigenetic remodeling during cell fate determination. Finally, we discuss the relevance of concerted metabolic and epigenetic regulation of primordial germ cells in the context of mammalian transgenerational epigenetic inheritance.

Metabolites control epigenetic mechanisms, and conversly, cell metabolism is regulated at the epigenetic level in response to changes in the cellular environment. In recent years, this metabolo-epigenetic control of gene expression has been implicated in the regulation of multiple stages of embryonic development. The developmental potency of stem cells and their embryonic counterparts is directly determined by metabolic rewiring. Here, we review the current knowledge on the interplay between epigenetics and metabolism in the specific context of early germ cell development. We explore the implications of metabolic rewiring in primordial germ cells in light of their epigenetic remodeling during cell fate determination. Finally, we discuss the relevance of concerted metabolic and epigenetic regulation of primordial germ cells in the context of mammalian transgenerational epigenetic inheritance., info:eu-repo/semantics/published

We live in a complex chemical environment where there are an estimated 350 000 chemical compounds or mixtures commercially produced. A strong body of literature shows that there are time points during early development when an organism's epigenome is particularly sensitive to chemicals in its environment. What is less understood is how gene-environment and epigenetic-environment interactions change with age. This question is bidirectional: (1) how do chemicals in the environment affect the aging process and (2) how does aging affect an organism's response to its chemical environment? The study of gene-environment interactions with age is especially important because, in many parts of the world, older individuals are a large and rapidly growing proportion of the population and because aging is a process universal to most of the animal kingdom. Epigenetics has emerged as a crucial framework for studying aging as epigenetic pathways, often triggered by environmental stimuli, have been shown to be essential regulators of the aging process. In this perspective article, we delineate the connection between aging, epigenetics, and environmental exposures. We discuss why it is essential to consider age when researching how an organism interacts with its environment. We describe recent advances in understanding how the chemical environment affects aging and the gap in research on how age affects an organism's response to the environment. Finally, we highlight how model organisms and network approaches can help fill this crucial gap. Taken together, systemic changes that occur in the epigenome with age indicate that adult organisms cannot be treated as a homogeneous population and that there are discrete mechanisms modulating the aging epigenome that we do not yet understand.

PurposeWe investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications.MethodsFrom 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide.ResultsThe molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality.ConclusionIn this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.

The Weismann barrier has long been regarded as a basic tenet of biology. However, upon close examination of its historical origins and August Weismann's own writings, questions arise as to whether such a status is warranted. As scientific research has advanced, the persistence of the concept of the barrier has left us with the same dichotomies Weismann contended with over 100 years ago: germ or soma, gene or environment, hard or soft inheritance. These dichotomies distract from the more important questions we need to address going forward. In this review, we will examine the theories that have shaped Weismann's thinking, how the concept of the Weismann barrier emerged, and the limitations that it carries. We will contrast the principles underlying the barrier with recent and less recent findings in developmental biology and transgenerational epigenetic inheritance that have profoundly eroded the oppositional view of germline vs. soma. Discarding the barrier allows us to examine the interactive processes and their response to environmental context that generate germ cells in the first place, determine the entirety of what is inherited through them, and set the trajectory for the health status of the progeny they bear.

In a rat model, perinatal nicotine exposure results in an epigenetically driven multi- and trans-generationally transmitted asthmatic phenotype that tends to wane over successive generations. However, the effect of repeat nicotine exposure during the F1 (Filial 1) gestational period on the transmitted phenotype is unknown. Using a well-established rat model, we compared lung function, mesenchymal markers of airway reactivity, and global gonadal DNA methylation changes in F2 offspring in a sex-specific manner following perinatal exposure to nicotine in only the F0 gestation, in both F0 and F1 (F0/F1) gestations, and in neither (control group). Both F0 only and F0/F1 exposure groups showed an asthmatic phenotype, an effect that was more pronounced in the F0/F1 exposure group, especially in males. Testicular global DNA methylation increased, while ovarian global DNA methylation decreased in the F0/F1 exposed group. Since the offspring of smokers are more likely to smoke than the offspring of nonsmokers, this sets the stage for more severe asthma if both mother and grandmother had smoked during their pregnancies. Increased gonadal DNA methylation changes following nicotine reexposure in the F1 generation suggests that epigenetic mechanisms might well underlie the transgenerational inheritance of acquired phenotypic traits in general and nicotine-induced asthma in particular.

In a rat model, perinatal nicotine exposure results in an epigenetically driven multi- and trans-generationally transmitted asthmatic phenotype that tends to wane over successive generations. However, the effect of repeat nicotine exposure during the F1 (Filial 1) gestational period on the transmitted phenotype is unknown. Using a well-established rat model, we compared lung function, mesenchymal markers of airway reactivity, and global gonadal DNA methylation changes in F2 offspring in a sex-specific manner following perinatal exposure to nicotine in only the F0 gestation, in both F0 and F1 (F0/F1) gestations, and in neither (control group). Both F0 only and F0/F1 exposure groups showed an asthmatic phenotype, an effect that was more pronounced in the F0/F1 exposure group, especially in males. Testicular global DNA methylation increased, while ovarian global DNA methylation decreased in the F0/F1 exposed group. Since the offspring of smokers are more likely to smoke than the offspring of nonsmokers, this sets the stage for more severe asthma if both mother and grandmother had smoked during their pregnancies. Increased gonadal DNA methylation changes following nicotine reexposure in the F1 generation suggests that epigenetic mechanisms might well underlie the transgenerational inheritance of acquired phenotypic traits in general and nicotine-induced asthma in particular.

The Weismann barrier has long been regarded as a basic tenet of biology. However, upon close examination of its historical origins and August Weismann's own writings, questions arise as to whether such a status is warranted. As scientific research has advanced, the persistence of the concept of the barrier has left us with the same dichotomies Weismann contended with over 100 years ago: germ or soma, gene or environment, hard or soft inheritance. These dichotomies distract from the more important questions we need to address going forward. In this review, we will examine the theories that have shaped Weismann's thinking, how the concept of the Weismann barrier emerged, and the limitations that it carries. We will contrast the principles underlying the barrier with recent and less recent findings in developmental biology and transgenerational epigenetic inheritance that have profoundly eroded the oppositional view of germline vs. soma. Discarding the barrier allows us to examine the interactive processes and their response to environmental context that generate germ cells in the first place, determine the entirety of what is inherited through them, and set the trajectory for the health status of the progeny they bear.

We live in a complex chemical environment where there are an estimated 350 000 chemical compounds or mixtures commercially produced. A strong body of literature shows that there are time points during early development when an organism's epigenome is particularly sensitive to chemicals in its environment. What is less understood is how gene-environment and epigenetic-environment interactions change with age. This question is bidirectional: (1) how do chemicals in the environment affect the aging process and (2) how does aging affect an organism's response to its chemical environment? The study of gene-environment interactions with age is especially important because, in many parts of the world, older individuals are a large and rapidly growing proportion of the population and because aging is a process universal to most of the animal kingdom. Epigenetics has emerged as a crucial framework for studying aging as epigenetic pathways, often triggered by environmental stimuli, have been shown to be essential regulators of the aging process. In this perspective article, we delineate the connection between aging, epigenetics, and environmental exposures. We discuss why it is essential to consider age when researching how an organism interacts with its environment. We describe recent advances in understanding how the chemical environment affects aging and the gap in research on how age affects an organism's response to the environment. Finally, we highlight how model organisms and network approaches can help fill this crucial gap. Taken together, systemic changes that occur in the epigenome with age indicate that adult organisms cannot be treated as a homogeneous population and that there are discrete mechanisms modulating the aging epigenome that we do not yet understand.

PurposeWe investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications.MethodsFrom 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide.ResultsThe molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality.ConclusionIn this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.

Recent studies suggested a significant downward trend in population's exposure to bisphenol A (BPA) in the United States. However, the temporal trend of BPA exposure remains unclear in China - a populous country with substantial industrial activities but less efforts made to phase out BPA in consumer products. In addition, it is unclear to what extent a visit from the United States to China could affect human exposure to BPA. In this natural experiment, we measured the concentration of total BPA in 418 urine samples repeatedly collected from 55 Los Angeles residents before, during, and after they spent 10 weeks in Beijing from 2012 to 2017. We found that traveling from Los Angeles to Beijing led to a 2.91-fold (95% CI: 2.43 to 3.50) increase in urinary BPA levels, which fully returned to baseline after study participants came back to Los Angeles. From 2012 to 2017, urinary BPA concentrations decreased in Los Angeles by 25.5% per year (95% CI: -30.8% to -19.8%; p < 0.001) but did not change in Beijing (p = 0.24). Consequently, the concentration ratio of urinary BPA between Beijing and Los Angeles increased from 1.23 (95% CI: 0.82 to 1.85) in 2012 to 4.05 (95% CI: 2.75 to 5.97) in 2017. These results indicate that BPA exposures may increase among international travelers to China. Additional efforts are needed to reduce population's exposure to BPA in China.

PURPOSE: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications. METHODS: From 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide. RESULTS: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality. CONCLUSION: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene(1). The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches(2-5). For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases(6-8). This includes muscle biopsies from patients with undiagnosed rare muscle disorders(6,9), and cultured fibroblasts from patients with mitochondrial disorders(7). However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution., Group Authors: Undiagnosed Dis Network ; Care4Rare Canada Consortium

Despite the high public interest in epigenetics, few scholars have empirically investigated the forms, reasons and consequences of the public circulation of epigenetics. Using an original database focusing on 'lifestyle' or 'everyday' epigenetics, this article aims to promote an open-minded and interdisciplinary dialogue between the public appropriation of epigenetics and the current scientific state of the art. It raises three main questions: Are there any specific modes of circulation of epigenetics in the general public? Why does epigenetics seem so appealing to the public? Within the public repertoire of epigenetics, is it possible to identify some specific knowledge claims and, if so, given the current state of the art, what is their degree of accuracy? The article argues that the social diffusion of epigenetics frequently carries on beliefs and misconceptions about genetics and epigenetics. The social life of epigenetics fuels a collective 'illusion' of control and empowerment on the basis of which new markets expand. More unexpectedly, this article underlines the emergence of a new scientific culture, i.e. the 'scientifization' of the cultural appropriation of epigenetics. Our analysis can inform the scientific community about the current and evolving state of the public representation of epigenetics and help it frame outreach activities.

Objective To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships. Methods We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model. Results The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes.

Despite the high public interest in epigenetics, few scholars have empirically investigated the forms, reasons and consequences of the public circulation of epigenetics. Using an original database focusing on 'lifestyle' or 'everyday' epigenetics, this article aims to promote an open-minded and interdisciplinary dialogue between the public appropriation of epigenetics and the current scientific state of the art. It raises three main questions: Are there any specific modes of circulation of epigenetics in the general public? Why does epigenetics seem so appealing to the public? Within the public repertoire of epigenetics, is it possible to identify some specific knowledge claims and, if so, given the current state of the art, what is their degree of accuracy? The article argues that the social diffusion of epigenetics frequently carries on beliefs and misconceptions about genetics and epigenetics. The social life of epigenetics fuels a collective 'illusion' of control and empowerment on the basis of which new markets expand. More unexpectedly, this article underlines the emergence of a new scientific culture, i.e. the 'scientifization' of the cultural appropriation of epigenetics. Our analysis can inform the scientific community about the current and evolving state of the public representation of epigenetics and help it frame outreach activities.

We report an effective and environmentally sustainable water treatment approach using enzymes encapsulated in biogenic vault nanoparticles. Manganese peroxidase (MnP), whose stability was remarkably extended by encapsulating into vaults, rapidly catalyzed the biotransformation of endocrine-disrupting compounds, including bisphenol A (BPA), bisphenol F (BPF), and bisphenol AP (BPAP). The vault-encapsulated MnP (vMnP) treatment removed 80-95% of each of the tested bisphenols (BPs) at lower enzyme dosage, while free native MnP (nMnP) only resulted in a 19-36% removal, over a 24-h period. Treatment by vMnP and nMnP resulted in considerable disparities in product species and abundance, which were consistent with the observed changes in the estrogenic activities of BPs. To test if vMnP-catalyzed transformations generated toxic intermediates, we assessed biological hallmarks of BP toxicity, namely, the ability to disrupt reproductive processes. The toxicity of vMnP-treated samples, as measured in the model organism, Caenorhabditis elegans, was dramatically reduced for all three BPs, including the reproductive indicators of BPA exposure such as reduced fertility and increased germ cell death. Collectively, our results indicate that the vMnP system represents an efficient and safe approach for the removal of BPs and promise the development of vault-encapsulated customized enzymes for treating other targeted organic compounds in contaminated waters.

Bisphenol A is an endocrine disruptor associated with hormone synthesis and reproduction alterations. However, the initiating events underpinning these dysfunctions are still unclear. Here, we address the hypothesis that BPA interferes with the highly evolutionary conserved process of mitochondrial cholesterol transport, a crucial step in steroid hormone biosynthesis, by using the model organism C. elegans. We observed that embryonic lethality and germline apoptosis, hallmarks of BPA's reproductive toxicity in C. elegans, are fully rescued by low exogenous cholesterol supplementation. We also observed that increasing BPA concentrations proportionally reduced mitochondrial cholesterol levels. Mutants for strl-1 (ortholog of StAR), but not C41G7.9 (ortholog of TSPO), show reproductive defects similar to BPA's while BPA exposure in a strl-1 background did not worsen these effects. Finally, cholesterol supplementation rescued these defects for all strl-1 genotype/BPA combinations assessed. Together, these results uncover a novel mechanism underlying BPA's germline toxicity through the alteration of cholesterol transport.

Objective To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships. Methods We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model. Results The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes.

How artificial environmental cues are biologically integrated and transgenerationally inherited is still poorly understood. Here, we investigate the mechanisms of inheritance of reproductive outcomes elicited by the model environmental chemical Bisphenol A in C. elegans. We show that Bisphenol A (BPA) exposure causes the derepression of an epigenomically silenced transgene in the germline for 5 generations, regardless of ancestral response. Chromatin immunoprecipitation sequencing (ChIP-seq), histone modification quantitation, and immunofluorescence assays revealed that this effect is associated with a reduction of the repressive marks H3K9me3 and H3K27me3 in whole worms and in germline nuclei in the F3, as well as with reproductive dysfunctions, including germline apoptosis and embryonic lethality. Furthermore, targeting of the Jumonji demethylases JMJD-2 and JMJD-3/UTX-1 restores H3K9me3 and H3K27me3 levels, respectively, and it fully alleviates the BPA-induced transgenerational effects. Together, our results demonstrate the central role of repressive histone modifications in the inheritance of reproductive defects elicited by a common environmental chemical exposure.

How organisms retain a memory of ancestral environmental exposure is a phenomenon that is still poorly understood. Recently published work by our group and others, regarding environmentally induced transgenerational effects, have identified an array of mechanisms, with a particular focus on histone modifications, that shed some light on the underlying epigenetic processes driving long-term generational effects.

How artificial environmental cues are biologically integrated and transgenerationally inherited is still poorly understood. Here, we investigate the mechanisms of inheritance of reproductive outcomes elicited by the model environmental chemical Bisphenol A in C. elegans. We show that Bisphenol A (BPA) exposure causes the derepression of an epigenomically silenced transgene in the germline for 5 generations, regardless of ancestral response. Chromatin immunoprecipitation sequencing (ChIP-seq), histone modification quantitation, and immunofluorescence assays revealed that this effect is associated with a reduction of the repressive marks H3K9me3 and H3K27me3 in whole worms and in germline nuclei in the F3, as well as with reproductive dysfunctions, including germline apoptosis and embryonic lethality. Furthermore, targeting of the Jumonji demethylases JMJD-2 and JMJD-3/UTX-1 restores H3K9me3 and H3K27me3 levels, respectively, and it fully alleviates the BPA-induced transgenerational effects. Together, our results demonstrate the central role of repressive histone modifications in the inheritance of reproductive defects elicited by a common environmental chemical exposure.

How organisms retain a memory of ancestral environmental exposure is a phenomenon that is still poorly understood. Recently published work by our group and others, regarding environmentally induced transgenerational effects, have identified an array of mechanisms, with a particular focus on histone modifications, that shed some light on the underlying epigenetic processes driving long-term generational effects.

Alternatives analysis (AA) is a method used in regulation and product design to identify, assess, and evaluate the safety and viability of potential substitutes for hazardous chemicals. It requires toxicological data for the existing chemical and potential alternatives. Predictive toxicology uses in silico and in vitro approaches, computational models, and other tools to expedite toxicological data generation in a more cost-effective manner than traditional approaches. The present article briefly reviews the challenges associated with using predictive toxicology in regulatory AA, then presents 4 recommendations for its advancement. It recommends using case studies to advance the integration of predictive toxicology into AA, adopting a stepwise process to employing predictive toxicology in AA beginning with prioritization of chemicals of concern, leveraging existing resources to advance the integration of predictive toxicology into the practice of AA, and supporting transdisciplinary efforts. The further incorporation of predictive toxicology into AA would advance the ability of companies and regulators to select alternatives to harmful ingredients, and potentially increase the use of predictive toxicology in regulation more broadly. Integr Environ Assess Manag 2017;13:915-925. © 2017 SETAC.

BackgroundTen years ago, leaders in the field of toxicology called for a transformation of the discipline and a shift from primarily relying on traditional animal testing to incorporating advances in biotechnology and predictive methodologies into alternative testing strategies (ATS). Governmental agencies and academic and industry partners initiated programs to support such a transformation, but a decade later, the outcomes of these efforts are not well understood.ObjectivesWe aimed to assess the use of ATS and the perceived barriers and drivers to their adoption by toxicologists and by others working in, or closely linked with, the field of toxicology.MethodsWe surveyed 1,381 toxicologists and experts in associated fields regarding the viability and use of ATS and the perceived barriers and drivers of ATS for a range of applications. We performed ranking, hierarchical clustering, and correlation analyses of the survey data.ResultsMany respondents indicated that they were already using ATS, or believed that ATS were already viable approaches, for toxicological assessment of one or more end points in their primary area of interest or concern (26-86%, depending on the specific ATS/application pair). However, the proportions of respondents reporting use of ATS in the previous 12 mo were smaller (4.5-41%). Concern about regulatory acceptance was the most commonly cited factor inhibiting the adoption of ATS, and a variety of technical concerns were also cited as significant barriers to ATS viability. The factors most often cited as playing a significant role (currently or in the future) in driving the adoption of ATS were the need for expedited toxicology information, the need for reduced toxicity testing costs, demand by regulatory agencies, and ethical or moral concerns.ConclusionsOur findings indicate that the transformation of the field of toxicology is partly implemented, but significant barriers to acceptance and adoption remain. https://doi.org/10.1289/EHP1435.

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent potentially unlimited cell sources for clinical applications. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity, as reflected by the reduced expression of major histocompatibility complex class-related molecules. Here we investigated the global immune-related gene expression profiles of human ESCs, hiPSCs and somatic cells and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and three types of human somatic cells: dermal papilla cells, ovarian granulosa cells and foreskin fibroblast cells. We identified the differentially expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1 and KDR, which overlapped with selected immune-related gene lists. In further analyses, mammalian target of rapamycin complex (mTOR) signaling was investigated in the differentiated stem cells following treatment with rapamycin and lentiviral transduction with specific short-hairpin RNAs. We found that the inhibition of mTOR signal pathways significantly downregulated the immunogenicity of differentiated stem cells. We also tested the immune responses induced in differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we observed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together, these data identify candidate immune-related genes that might constitute valuable targets for clinical applications.

Alternatives analysis (AA) is a method used in regulation and product design to identify, assess, and evaluate the safety and viability of potential substitutes for hazardous chemicals. It requires toxicological data for the existing chemical and potential alternatives. Predictive toxicology uses in silico and in vitro approaches, computational models, and other tools to expedite toxicological data generation in a more cost-effective manner than traditional approaches. The present article briefly reviews the challenges associated with using predictive toxicology in regulatory AA, then presents 4 recommendations for its advancement. It recommends using case studies to advance the integration of predictive toxicology into AA, adopting a stepwise process to employing predictive toxicology in AA beginning with prioritization of chemicals of concern, leveraging existing resources to advance the integration of predictive toxicology into the practice of AA, and supporting transdisciplinary efforts. The further incorporation of predictive toxicology into AA would advance the ability of companies and regulators to select alternatives to harmful ingredients, and potentially increase the use of predictive toxicology in regulation more broadly. Integr Environ Assess Manag 2017;13:915-925. © 2017 SETAC.

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent potentially unlimited cell sources for clinical applications. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity, as reflected by the reduced expression of major histocompatibility complex class-related molecules. Here we investigated the global immune-related gene expression profiles of human ESCs, hiPSCs and somatic cells and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and three types of human somatic cells: dermal papilla cells, ovarian granulosa cells and foreskin fibroblast cells. We identified the differentially expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1 and KDR, which overlapped with selected immune-related gene lists. In further analyses, mammalian target of rapamycin complex (mTOR) signaling was investigated in the differentiated stem cells following treatment with rapamycin and lentiviral transduction with specific short-hairpin RNAs. We found that the inhibition of mTOR signal pathways significantly downregulated the immunogenicity of differentiated stem cells. We also tested the immune responses induced in differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we observed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together, these data identify candidate immune-related genes that might constitute valuable targets for clinical applications.

BackgroundTen years ago, leaders in the field of toxicology called for a transformation of the discipline and a shift from primarily relying on traditional animal testing to incorporating advances in biotechnology and predictive methodologies into alternative testing strategies (ATS). Governmental agencies and academic and industry partners initiated programs to support such a transformation, but a decade later, the outcomes of these efforts are not well understood.ObjectivesWe aimed to assess the use of ATS and the perceived barriers and drivers to their adoption by toxicologists and by others working in, or closely linked with, the field of toxicology.MethodsWe surveyed 1,381 toxicologists and experts in associated fields regarding the viability and use of ATS and the perceived barriers and drivers of ATS for a range of applications. We performed ranking, hierarchical clustering, and correlation analyses of the survey data.ResultsMany respondents indicated that they were already using ATS, or believed that ATS were already viable approaches, for toxicological assessment of one or more end points in their primary area of interest or concern (26-86%, depending on the specific ATS/application pair). However, the proportions of respondents reporting use of ATS in the previous 12 mo were smaller (4.5-41%). Concern about regulatory acceptance was the most commonly cited factor inhibiting the adoption of ATS, and a variety of technical concerns were also cited as significant barriers to ATS viability. The factors most often cited as playing a significant role (currently or in the future) in driving the adoption of ATS were the need for expedited toxicology information, the need for reduced toxicity testing costs, demand by regulatory agencies, and ethical or moral concerns.ConclusionsOur findings indicate that the transformation of the field of toxicology is partly implemented, but significant barriers to acceptance and adoption remain. https://doi.org/10.1289/EHP1435.

BackgroundDespite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals.ObjectivesWe developed an assay in Caenorhabditis elegans that fluorescently marks aneuploid embryos after chemical exposure.MethodsWe qualified the predictive value of the assay against chemotherapeutic agents as well as environmental compounds from the ToxCast Phase I library by comparing results from the C. elegans assay with the comprehensive mammalian in vivo end point data from the ToxRef database.ResultsThe assay was highly predictive of mammalian reproductive toxicities, with a 69% maximum balanced accuracy. We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression.ConclusionsThis C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function.

BackgroundDespite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals.ObjectivesWe developed an assay in Caenorhabditis elegans that fluorescently marks aneuploid embryos after chemical exposure.MethodsWe qualified the predictive value of the assay against chemotherapeutic agents as well as environmental compounds from the ToxCast Phase I library by comparing results from the C. elegans assay with the comprehensive mammalian in vivo end point data from the ToxRef database.ResultsThe assay was highly predictive of mammalian reproductive toxicities, with a 69% maximum balanced accuracy. We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression.ConclusionsThis C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function.

Concerns about the safety of Bisphenol A, a chemical found in plastics, receipts, food packaging and more, have led to its replacement with substitutes now found in a multitude of consumer products. However, several popular BPA-free alternatives, such as Bisphenol S, share a high degree of structural similarity with BPA, suggesting that these substitutes may disrupt similar developmental and reproductive pathways. We compared the effects of BPA and BPS on germline and reproductive functions using the genetic model system Caenorhabditis elegans. We found that, similarly to BPA, BPS caused severe reproductive defects including germline apoptosis and embryonic lethality. However, meiotic recombination, targeted gene expression, whole transcriptome and ontology analyses as well as ToxCast data mining all indicate that these effects are partly achieved via mechanisms distinct from BPAs. These findings therefore raise new concerns about the safety of BPA alternatives and the risk associated with human exposure to mixtures.

Concerns about the safety of Bisphenol A, a chemical found in plastics, receipts, food packaging and more, have led to its replacement with substitutes now found in a multitude of consumer products. However, several popular BPA-free alternatives, such as Bisphenol S, share a high degree of structural similarity with BPA, suggesting that these substitutes may disrupt similar developmental and reproductive pathways. We compared the effects of BPA and BPS on germline and reproductive functions using the genetic model system Caenorhabditis elegans. We found that, similarly to BPA, BPS caused severe reproductive defects including germline apoptosis and embryonic lethality. However, meiotic recombination, targeted gene expression, whole transcriptome and ontology analyses as well as ToxCast data mining all indicate that these effects are partly achieved via mechanisms distinct from BPAs. These findings therefore raise new concerns about the safety of BPA alternatives and the risk associated with human exposure to mixtures.

The comprehensive identification and mechanistic analysis of reproductive toxicants constitutes one of the major hurdles in the toxicological assessment of chemicals originating from the large number of chemicals to be tested and the difficulty in examining germ cells at various stages of their development. We previously described the development of an assay in the roundworm Caenorhabditis elegans that allows the detection of chemicals bearing aneugenic activity and that could be used for the detection of germline toxicity. We present here new evidence for the reproductive toxicity of three pesticides identified in our germline toxicity assay: Maneb, Diazinon and Fenarimol. We show that all three pesticides cause an acute germline nuclear loss in exposed nematodes in a dose-dependent fashion. The loss of germline nuclei coincides with the meiotic stage of pachytene during Prophase I and is dependent on the germline apoptotic machinery suggesting activation of a meiotic checkpoint. Further investigation revealed a profound dysregulation of the meiotic program revealed by (1) an alteration of the kinetics of double strand repair, (2) the disruption of the process of chromosome morphogenesis at the end of Prophase I and (3) the reorganization of the meiotic differentiation gradient inherent to the C. elegans germline following exposure to Maneb and Diazinon. These defects correlate with a significant increase in embryonic lethality and a corresponding decrease in the number of progeny. These results therefore provide strong evidence for the reproductive toxicity of Maneb, Diazinon and Fenarimol rooted in the alteration of early steps of germ cell differentiation.

Germ cells are unique in their ability to transfer genetic information and traits from generation to generation. As such, the proper development of germ cells and the integrity of their genome are paramount to the health of organisms and the survival of species. Germ cells are also exquisitely sensitive to environmental influences although the testing of germ cell toxicity, especially in females, has proven particularly challenging. In this review, we first describe the remarkable odyssey of germ cells in mammals, with an emphasis on the female germline, from their initial specification early during embryogenesis to the generation of mature gametes in adults. We also describe the current methods used in germ cell toxicity testing and their limitations in examining the complex features of mammalian germ cell development. To bypass these challenges, we propose the use of alternative model systems such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and in vitro germ cell methods that have distinct advantages over traditional toxicity models. We discuss the benefits and limitations of each approach, their application to germ cell toxicity studies, and the need for computational approaches to maximize the usefulness of these models. Together, the inclusion of these alternative germ cell toxicity models will be invaluable for the examination of stages not easily accessible in mammals as well as the large scale, high-throughput investigation of germ cell toxicity.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals. We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

The comprehensive identification and mechanistic analysis of reproductive toxicants constitutes one of the major hurdles in the toxicological assessment of chemicals originating from the large number of chemicals to be tested and the difficulty in examining germ cells at various stages of their development. We previously described the development of an assay in the roundworm Caenorhabditis elegans that allows the detection of chemicals bearing aneugenic activity and that could be used for the detection of germline toxicity. We present here new evidence for the reproductive toxicity of three pesticides identified in our germline toxicity assay: Maneb, Diazinon and Fenarimol. We show that all three pesticides cause an acute germline nuclear loss in exposed nematodes in a dose-dependent fashion. The loss of germline nuclei coincides with the meiotic stage of pachytene during Prophase I and is dependent on the germline apoptotic machinery suggesting activation of a meiotic checkpoint. Further investigation revealed a profound dysregulation of the meiotic program revealed by (1) an alteration of the kinetics of double strand repair, (2) the disruption of the process of chromosome morphogenesis at the end of Prophase I and (3) the reorganization of the meiotic differentiation gradient inherent to the C. elegans germline following exposure to Maneb and Diazinon. These defects correlate with a significant increase in embryonic lethality and a corresponding decrease in the number of progeny. These results therefore provide strong evidence for the reproductive toxicity of Maneb, Diazinon and Fenarimol rooted in the alteration of early steps of germ cell differentiation.