Allard, Patrick (2019) International Worm Meeting "Dissecting the epigenetic mechanisms of environmental memory and transgenerational reproductive dysfunction in C. elegans."

Allard, Patrick

[International Worm Meeting, 2019]

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 several common environmental exposures including to ethanol and the highly prevalent plastic-manufacturing chemical Bisphenol A in C. elegans. We show that both exposures cause a dose-dependent derepression of an epigenomically silenced transgene in the germline although the effect for ethanol is less pronounced than that of BPA. We also show that at the F3 generation, both chemicals lead to an increase in reproductive dysfunctions including an increase in germline apoptosis and embryonic lethality. For BPA, several lines of evidence obtained through 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. 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. Finally, investigation of the reproductive phenotype indicate a pervasive transgenerational disruption of the recombination machinary including an alteration of ZHP-3 and COSA-1 foci number in late pachytene. Together, our results demonstrate the central role of repressive histone modifications in the inheritance of reproductive defects elicited by common environmental chemical exposures.

Allard, Patrick et al. (2009) International Worm Meeting "C. elegans as a model system to understand environmental influence on germline health."

Allard, Patrick, Colaiacovo, Monica

[International Worm Meeting, 2009]

Although it is clear that environmental toxicants can alter reproductive ability, the dissection of the affected molecular pathways has been particularly challenging. We propose to use C. elegans to investigate the genetic mechanisms of meiotic disruption following environmental exposure. As a proof of principle, we concentrated our research on characterizing the biological effects of Bisphenol-A (BPA), a compound commonly used for the production of polycarbonate-containing plastics. Previous work in mice has shown that exposure to BPA in utero leads to aberrations during prophase of meiosis I including incomplete synapsis, end-to-end chromosome fusions, and an increased number of recombination foci corresponding to elevated recombination frequencies and altered exchange distribution. These defects likely result in increased chromosome nondisjunction as highlighted by the greater number of aneuploid eggs and embryos observed. Assessment of the fertility of adult hermaphrodites in C. elegans following exposure to BPA revealed that BPA causes a dramatic increase in embryonic and larval lethality as well as a decrease in the total number of eggs laid compared to control (exposed to vehicle). A two-fold increase in germ cell apoptosis is detected in BPA-exposed worms suggesting the activation of a DNA damage checkpoint that may stem from a defect in DNA double-strand break repair. Analysis of chromosome morphogenesis in control gonads revealed the presence of 6 DAPI-stained bodies, representing the six pairs of attached homologs in oocytes at diakinesis. In contrast, an aberrant DNA morphology is observed at this stage in BPA-exposed worms. Specifically, bivalents were decondensed and chromatin bridges were apparent. Furthermore, disassembly of the synaptonemal complex (SC) is impaired during late prophase as indicated by an incomplete unloading of SYP-1, a structural component of the SC, from chromosomes in late diakinesis. Taken together, these data demonstrate that exposure to BPA disrupts the meiotic machinery in C. elegans. We are currently further characterizing these meiotic defects by examining recombination levels and the expression of other synapsis components in BPA-exposed animals.

Allard, Patrick et al. (2011) International Worm Meeting "High-throughput identification of toxicants that disrupt chromosome segregation during meiosis."

Colaiacovo, Monica P., Allard, Patrick

[International Worm Meeting, 2011]

Abnormal chromosome segregation during meiosis, the process by which haploid sperm and eggs are generated, is a major contributor to aneuploidy and therefore to infertility, miscarriages and birth defects. Although environmental exposure plays a significant role in the etiology of these diseases, there is currently no high-throughput approach for the identification of environmental meiotic disruptors. We propose to use the worm Caenorhabditis elegans in a novel screening strategy for the identification of toxicants altering meiotic chromosome segregation. C. elegans is both a well established meiotic model system that has illuminated our understanding of the genes and pathways governing this process and an emerging animal model used in toxicological studies. Here, we describe a high-throughput method for the identification of environmental aneugens in C. elegans. We have developed a dual luciferase/GFP reporter that is specifically induced in aneuploid embryos. First, worms are exposed to environmental compounds and screened for the presence of aneuploid embryos via a standard luciferase assay. The positive hits are then validated by direct or automated presence of GFP positive embryos in the worms uterus and also by DNA staining for a detailed analysis of the meiotic defects in the germline. In test experiments, we have successfully induced luciferase and GFP expression following exposure to known aneugenic chemicals, such as chemotherapeutic agents, and also detected the expression of the reporters using an automated set-up, indicating that our system is suitable for high-throughput screens. With this novel screening strategy, we address the need for fast and reliable screening of environmental meiotic toxicants and their involvement in the induction of aneuploidies. This work was funded by the Colgate-Palmolive Grant for Alternative Research.

Allard P et al. (2011) Cell Cycle "Mechanistic insights into the action of Bisphenol A on the germline using C. elegans."

Allard P, Colaiacovo MP

[Cell Cycle, 2011]

Feature on: Allard P, et al. Proc Natl Acad Sci USA 2010; 107:20405-10.

Camacho, Jessica et al. (2015) International Worm Meeting "Examining environmental effects on the germline epigenome."

Hosohama, Linzi, Camacho, Jessica, Allard, Patrick, Lundby, Zachary, Clark, Amander

[International Worm Meeting, 2015]

The high number of compounds to be tested, the diversity of toxicity endpoints, concentrations, and combinations of chemicals, all provide a challenge in our ability to assess the safety of chemicals. Of particular importance is the potential effect of chemicals on the germline epigenome, which can alter biological processes over several generations. While the cases of DES, vinclozolin and BPA provide evidence of epigenetic effects, there is a great need to explore the influence of environmental compounds on the epigenome and, especially, to develop methods that allow us to quickly and efficiently examine this question. We have established the use of the genetic model system, the worm Caenorhabditis elegans, as a relevant model for epigenetic and reproductive toxicity assessment. By taking advantage of the C. elegans genetic tools, we propose to comprehensively identify chemicals for their ability to disrupt the germline chromatin. To this aim, we are making use of a worm strain where GFP is specifically epigenetically silenced in the germline. We previously showed that valproic acid, a well-known mammalian histone deacetylase inhibitor, disrupts the germline epigenetic state leading to the de-silencing of the transgene in the germline. Our current experiments test this concept further by exposing the worms to environmental compounds. We tested vinclozolin and BPA to analyze a disruption in maintenance and/or establishment of epigenetic marks. Young adult worms were exposed to the compounds for 48 hours, and three subsequent generations were followed and analyzed. Results indicate that de-silencing effects last for at least three generations (up to three-fold compared to our DMSO control). Furthermore, most worms showing de-silencing in the first, second and third generation originate from worms showing de-silencing in the parental exposed generation indicating that the effect is inherited. Thus, we conclude that valproic acid, vinclozolin, and BPA disrupt the germline epigenome over several generations in a heritable fashion. Additionally, in concomitant assays, we are working with the lab of Dr. Amander Clark, using mice primordial germ cell-like cells (PGCLCs) in a mammalian validation approach. Together, we hope to establish a novel in vitro germline differentiation assay to screen for chemicals that affect germline quality.

Barrere-Cain, Rio et al. (2019) International Worm Meeting "Epigenetic Sensitivity to Chemical Exposure in the Aging C. elegans Germline."

Truong, Lisa, Allard, Patrick, Camacho, Jessica, Barrere-Cain, Rio, Chen, Yichang

[International Worm Meeting, 2019]

How an epigenome's sensitivity to chemical exposure changes with age is not fully understood. This is significant for the germline where the genome is kept relatively silent through repressive histone marks such as H3K9me3 (histone 3 lysine 9 trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation). Understanding how epigenetic sensitivity changes with age will inform how age should be accounted for in chemical risk assessments. To investigate the relationship between age and epigenetic sensitivity we used immunofluorescence to quantify changes in H3K9me3 and H3K27me3 levels with age in C. elegans germlines. We performed immunofluorescence for H3K27me3 and H3K9me3 at days 1, 3, 5, and 7 of adulthood, with 5-7 replicates and fluorescence quantified in 3-4 gonads per replicate. Variation in H3K27me3 levels increases with age, suggesting a decrease in epigenetic homeostatic control with germline age. Next, we asked whether maternal age affects transmission of a chemical exposure to future generations. We used a green fluorescent protein (GFP) reporter C. elegans strain, NL2507, where GFP expression indicates levels of chromatin accessibility in C. elegans germlines. Under healthy conditions the GFP reporter is silenced in the germline however, with exposure to bisphenol A (BPA), transcriptional regulation is lost and GFP is expressed (Camacho et al., 2018). We exposed C elegans to 500uM BPA during different 48hr windows in their adult lifespan and analyzed GFP levels in F1 and F3 progeny (30 worms per condition). F3 BPA descendants had GFP expression that was double that of their respective control group, indicating that BPA affects the epigenetics of descendants not directly exposed. Furthermore, the F3 from older mothers had higher levels of GFP expression than the F3 from younger mothers. This suggests that older germlines may be more sensitive to chemical exposures because they are more likely to transmit chemically induced epigenetic perturbations to future generations. The epigenetic sensitivity of older germlines is relevant to humans as we tend to have children later in life. Furthermore, understanding how age impacts epigenetic sensitivity will allow us to more accurately assess the safety of chemicals for both exposed individuals and their descendants.

Kikis EA (2016) Biol Direct "The struggle by Caenorhabditis elegans to maintain proteostasis during aging and disease."

Kikis EA

[Biol Direct, 2016]

The presence of only small amounts of misfolded protein is an indication of a healthy proteome. Maintaining proteome health, or more specifically, "proteostasis," is the purview of the "proteostasis network." This network must respond to constant fluctuations in the amount of destabilized proteins caused by errors in protein synthesis and exposure to acute proteotoxic conditions. Aging is associated with a gradual increase in damaged and misfolded protein, which places additional stress on the machinery of the proteostasis network. In fact, despite the ability of the proteostasis machinery to readjust its stoichiometry in an attempt to maintain homeostasis, the capacity of cells to buffer against misfolding is strikingly limited. Therefore, subtle changes in the folding environment that occur during aging can significantly impact the health of the proteome. This decline and eventual collapse in proteostasis is most pronounced in individuals with neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease that are caused by the misfolding, aggregation, and toxicity of certain proteins. This review discusses how C. elegans models of protein misfolding have contributed to our current understanding of the proteostasis network, its buffering capacity, and its regulation. REVIEWERS: This article was reviewed by Luigi Bubacco, Patrick Lewis and Xavier Roucou.

Soltesz, Z. et al. (2011) International Worm Meeting "Carbon dioxide avoidance is mediated by a diverse set of sensory neurons in C. elegans."

Soltesz, Z., de Bono, M., Bretscher, A. J.

[International Worm Meeting, 2011]

Monitoring carbon dioxide levels has a twofold importance for many living organisms: CO2 can act as a sensory cue for food or other animals, while regulating internal CO2 levels is an important part of homeostasis. C. elegans relies on diffusion for gas exchange, and avoids CO2 levels as low as 1%. We are interested in the neural and molecular mechanisms underlying the C. elegans CO2 avoidance behaviour. Mutants defective in the tax-4 or tax-2 genes, which encode the a and b subunits, respectively, of a cGMP-gated ion channel, showed reduced CO2 avoidance in behavioural assays1,2. By expressing tax-2 cDNA from neuron-specific promoters in tax-2 mutants to rescue the avoidance behaviour, and by imaging neurons using the genetically encoded calcium indicator YC3.60, we have shown that sensory neurons previously implicated in oxygen, temperature, and salt-sensing, including BAG, AFD and ASE, are CO2 sensors as well3. We have observed both persistent and transient cell-intrinsic calcium-responses in several sensory neurons, suggesting that CO2 stimuli could modulate neural activity in C. elegans in a complex manner. We are therefore investigating how CO2 stimuli can affect neural processing in downstream neurons. 1 Andrew Jonathan Bretscher, Karl Emanuel Busch, and Mario de Bono, A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. PNAS 105(23):8044-8049 2 Elissa A. Hallem and Paul W. Sternberg, Acute carbon dioxide avoidance in Caenorhabditis elegans. PNAS 105(23):8038-8043 3 Andrew Jonathan Bretscher, Eiji Kodama-Namba, Karl Emanuel Busch, Robin Joseph Murphy, Zoltan Soltesz, Patrick Laurent and Mario de Bono, Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior. Neuron 69(6):1099-1113.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Voegtlin, Thomas et al. (2011) International Worm Meeting "An Open-Source Neuromechanical Model of C. Elegans Locomotion."

Cohen, Netta, Voegtlin, Thomas

[International Worm Meeting, 2011]

A neuromechanical model of locomotion in C. elegans was recently proposed by Jordan H. Boyle [1]. One of the main results is that both swimming and crawling can be generated by a single neural circuit, reflexively modulated by the environment. This supports the known experimental results showing that different forms of C. elegans forward locomotion (e.g., swimming and crawling) can be described by a modulation of a single biomechanical gait [2]. The modelling result illustrates the importance and the potential of neuromechanical simulations for the analysis of the worm's behaviour. In order to continue this work, and to make it usable by a broader audience, we have developed a similar neuromechanical model of the worm using CLONES. CLONES (Closed Loop Neural Simulation) is an open source framework for neuromechanical simulations. CLONES implements a communication interface between a neural simulator, called BRIAN [3], and a physics engine for biomedical applications, called SOFA [4]. BRIAN and SOFA are open-source simulators that are easy to use and provide high performance. Our implementation of the worm's locomotion reproduces the neural model described in [1]. However, there are two key differences between the original physical model and our implementation. Firstly, Boyle's model considers that the body of the worm has zero mass (a low Reynolds number approximation). In contrast, the SOFA simulator allows us to integrate equations with mass and inertia. Secondly, the original model uses rigid rods of fixed length orthogonal to the body axis (approximating the incompressibility of the body due to high internal pressure). In SOFA rigid rods are modeled as springs of very high stiffness. The physical system simulated in SOFA is described using a XML syntax. The neural network model interpreted by BRIAN is written in Python, using MATLAB-like syntax. Thus, the model is completely interpreted, and it is possible to visualize/interact with the simulation during runtime. Physical environments containing obstacles or chemical concentration gradients can be defined easily. References 1. Boyle JH: C. elegans locomotion: an integrated approach. PhD thesis, university of Leeds, 2009 2. Berri S, Boyle JH, Tassieri M, Hope IA and Cohen N, Forward locomotion of the nematode C. elegans is achieved through modulation of a single gait HFSP J 3:186, 2009; 3. Goodman DF, Brette R: Brian: a simulator for spiking neural networks in Python. Front Neuroinform 2:5, 2008 4. Allard J, Cotin S, Faure F, Bensoussan PJ, Poyer F, Duriez C, Delingette H, Grisoni L: SOFA - an Open Source Framework for Medical Simulation. Medicine Meets Virtual Reality (MMVR'15), pp. 13-18, 2007.