The Role of p66Shc in Mouse Blastocyst Development

The earliest cell fate specification events during mammalian development occur in the blastocyst-stage preimplantation embryo, during which a pluripotent cell population is established. These cells form the basis of the developing fetus and must be correctly specified in order for successful development to occur. Cell signalling in response to environmental cues has a critical role in cell differentiation. The signalling adaptor protein p66Shc is expressed in mammalian embryos and promotes apoptosis and permanent embryo arrest in response to stress-inducing conditions. However, loss-of-function studies suggest that p66Shc may be important for embryonic development to the blastocyst stage. In this thesis, I aimed to determine the role of p66Shc in mouse blastocyst development and mouse embryonic stem cell function. Through a combination of environmental modulation of p66Shc expression, experimental knockdown, and genetic knockout of p66Shc in mouse preimplantation embryos and mouse embryonic stem cells, I demonstrated that p66Shc is required for normal embryo physiology, and correct cell lineage-associated marker expression in the blastocyst inner cell mass and mouse embryonic stem cells. First, I observed that p66Shc is normally upregulated at the blastocyst stage in vivo, and oxygen-induced increases in p66Shc expression are associated with altered embryo metabolism in vitro. Secondly, I demonstrated that knockdown of p66Shc transcript abundance significantly alters the timing and proportion of cells expressing lineage-associated transcription factors in the blastocyst inner cell mass. Lastly, I observed that knockout of p66Shc in mouse embryonic stem cells alters the expression of the core pluripotency marker NANOG and causes an upregulation of mesoderm-associated markers during stem cell differentiation. Collectively, my work provides insight into a novel role for p66Shc during preimplantation embryo development, expanding the diversity of cellular functions attributed to p66Shc in mammalian development

Knockdown of p66Shc Alters Lineage-Associated Transcription Factor Expression in Mouse Blastocysts

The p66Shc adaptor protein regulates apoptosis and senescence during early mammalian development. However, p66Shc expression during mouse preimplantation development is upregulated at the blastocyst stage. Our objective was to determine the biological function of p66Shc during mouse blastocyst development. In this study, we demonstrate that a reduced p66Shc transcript abundance following its short interfering RNA (siRNA)-mediated knockdown alters the spatiotemporal expression of cell lineage-associated transcription factors in the inner cell mass (ICM) of the mouse blastocyst. P66Shc knockdown blastocysts restrict OCT3/4 earlier to the inner cells of the early blastocyst and have ICMs containing significantly higher OCT3/4 levels, more GATA4-positive cells, and fewer NANOG-positive cells. P66Shc knockdown blastocysts also show a significantly reduced ability to form ICM-derived outgrowths when explanted in vitro. The increase in cells expressing primitive endoderm markers may be due to increased ERK1/2 activity, as it is reversed by ERK1/2 inhibition. These results suggest that p66Shc may regulate the relative abundance and timing of lineage-associated transcription factor expression in the blastocyst ICM

P66Shc, a key regulator of metabolism and mitochondrial ROS production, is dysregulated by mouse embryo culture.

STUDY QUESTION: Do high oxygen tension and high glucose concentrations dysregulate p66Shc (Src homologous-collagen homologue adaptor protein) expression during mouse preimplantation embryo culture? SUMMARY ANSWER: Compared with mouse blastocysts in vivo, P66Shc mRNA and protein levels in blastocysts maintained in vitro increased under high oxygen tension (21%), but not high glucose concentration. WHAT IS KNOWN ALREADY: Growth in culture adversely impacts preimplantation embryo development and alters the expression levels of the oxidative stress adaptor protein p66Shc, but it is not known if p66Shc expression is linked to metabolic changes observed in cultured embryos. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We used a standard wild-type CD1 mouse model of preimplantation embryo development and embryo culture with different atmospheric oxygen tension and glucose media concentrations. Changes to p66Shc expression in mouse blastocysts were measured using quantitative RT-PCR, immunoblotting and immunofluorescence followed by confocal microscopy. Changes to oxidative phosphorylation metabolism were measured by total ATP content and superoxide production. Statistical analyses were performed on a minimum of three experimental replicates using Students\u27 t-test or one-way ANOVA. MAIN RESULTS AND THE ROLE OF CHANCE: P66Shc is basally expressed during in vivo mouse preimplantation development. Within in vivo blastocysts, p66Shc is primarily localized to the cell periphery of the trophectoderm. Blastocysts cultured under atmospheric oxygen levels have significantly increased p66Shc mRNA transcript and protein abundances compared to in vivo controls (P \u3c 0.05). However, the ratio of phosphorylated serine 36 (S36) p66Shc to total p66Shc decreased in culture regardless of O2 atmosphere used, supporting a shift in the mitochondrial fraction of p66Shc. Total p66Shc localized to the cell periphery of the blastocyst trophectoderm and phosphorylated S36 p66Shc displayed nuclear and cytoplasmic immunoreactivity, suggesting distinct compartmentalization of phosphorylated S36 p66Shc and the remaining p66Shc fraction. Glucose concentration in the culture medium did not significantly change p66Shc mRNA or protein abundance or its localization. Blastocysts cultured under low or high oxygen conditions exhibited significantly decreased cellular ATP and increased superoxide production compared to in vivo derived embryos (P \u3c 0.05). LIMITATIONS/REASONS FOR CAUTION: This study associates embryonic p66Shc expression levels with metabolic abnormalities but does not directly implicate p66Shc in metabolic changes. Additionally, we used one formulation of embryo culture medium that differs from that used in other mouse model studies and from clinical media used to support human blastocyst development. Our findings may, therefore, be limited to this media, or may be a species-specific phenomenon. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to show distinct immunolocalization of p66Shc to the trophectoderm of mouse blastocysts and that its levels are abnormally increased in embryos exposed to culture conditions. Changes in p66Shc expression and/or localization could possibly serve as a molecular marker of embryo viability for clinical applications. The outcomes provide insight into the potential metabolic role of p66Shc. Metabolic anomalies are induced even under the current optimal culture conditions, which could negatively impact trophectoderm and placental development. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Canadian Institutes of Health Research (CIHR) operating funds, Ontario Graduate Scholarship (OGS). There are no competing interests

Treatment with AICAR inhibits blastocyst development, trophectodermdifferentiation and tight junction formation and function in mice

STUDY QUESTION: What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? SUMMARY ANSWER: AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. WHAT IS KNOWN ALREADY: AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. STUDY DESIGN, SIZE, DURATION: Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 μM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 μM AICAR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. MAIN RESULTS AND THE ROLE OF CHANCE: Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. WIDER IMPLICATIONS OF THE FINDINGS: Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. STUDY FUNDING AND COMPETING INTEREST(S): Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests

Four Futures For Occupational Safety and Health

Rapid changes to the nature of work have challenged the capacity of existing occupational safety and health (OSH) systems to ensure safe and productive workplaces. An effective response will require an expanded focus that includes new tools for anticipating and preparing for an uncertain future. Researchers at the U.S. National Institute for Occupational Safety and Health (NIOSH) have adopted the practice of strategic foresight to structure inquiry into how the future will impact OSH. Rooted in futures studies and strategic management, foresight creates well-researched and informed future scenarios that help organizations better prepare for potential challenges and take advantage of new opportunities. This paper summarizes the inaugural NIOSH strategic foresight project, which sought to promote institutional capacity in applied foresight while exploring the future of OSH research and practice activities. With multidisciplinary teams of subject matter experts at NIOSH, we undertook extensive exploration and information synthesis to inform the development of four alternative future scenarios for OSH. We describe the methods we developed to craft these futures and discuss their implications for OSH, including strategic responses that can serve as the basis for an action-oriented roadmap toward a preferred future

Leveraging Strategic Foresight to advance Worker Safety, Health, and Well-Being

Attending to the ever-expanding list of factors impacting work, the workplace, and the workforce will require innovative methods and approaches for occupational safety and health (OSH) research and practice. This paper explores strategic foresight as a tool that can enhance OSH capacity to anticipate, and even shape, the future as it pertains to work. Equal parts science and art, strategic foresight includes the development and analysis of plausible alternative futures as inputs to strategic plans and actions. Here, we review several published foresight approaches and examples of work-related futures scenarios. We also present a working foresight framework tailored for OSH and offer recommendations for next steps to incorporate strategic foresight into research and practice in order to advance worker safety, health, and well-being

Majorana Electroformed Copper Mechanical Analysis

The MAJORANA DEMONSTRATOR is a large array of ultra-low background high-purity germanium detectors, enriched in 76Ge, designed to search for zero-neutrino double-beta decay. The DEMONSTRATOR will utilize ultra high purity electroformed copper for a variety of detector components and shielding. A preliminary mechanical evaluation was performed on the Majorana prototype electroformed copper material. Several samples were removed from a variety of positions on the mandrel. Tensile testing, optical metallography, scanning electron microscopy, and hardness testing were conducted to evaluate mechanical response. Analyses carried out on the Majorana prototype copper to this point show consistent mechanical response from a variety of test locations. Evaluation shows the copper meets or exceeds the design specifications

Draft genomes of 12 Bifidobacterium isolates from human IBD fecal samples

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GLP-1 action in the mouse bed nucleus of the stria terminalis

Glucagon-like peptide-1 (GLP-1) injected into the brain reduces food intake. Similarly, activation of preproglucagon (PPG) cells in the hindbrain which synthesize GLP-1, reduces food intake. However, it is far from clear whether this happens because of satiety, nausea, reduced reward, or even stress. Here we explore the role of the bed nucleus of the stria terminalis (BNST), an area involved in feeding control as well as stress responses, in GLP-1 responses. Using cre-expressing mice we visualized projections of NTS PPG neurons and GLP-1R-expressing BNST cells with AAV-driven Channelrhodopsin-YFP expression. The BNST displayed many varicose YFP+ PPG axons in the ventral and less in the dorsal regions. Mice which express RFP in GLP-1R neurons had RFP+ cells throughout the BNST with the highest density in the dorsal part, suggesting that PPG neuron-derived GLP-1 acts in the BNST. Indeed, injection of GLP-1 into the BNST reduced chow intake during the dark phase, whereas injection of the GLP-1 receptor antagonist Ex9 increased feeding. BNST-specific GLP-1-induced food suppression was less effective in mice on high fat (HF, 60%) diet, and Ex9 had no effect. Restraint stress-induced hypophagia was attenuated by BNST Ex9 treatment, further supporting a role for endogenous brain GLP-1. Finally, whole-cell patch clamp recordings of RFP+ BNST neurons demonstrated that GLP-1 elicited either a depolarizing or hyperpolarizing reversible response that was of opposite polarity to that under dopamine. Our data support a physiological role for BNST GLP-1R in feeding, and suggest complex cellular responses to GLP-1 in this nucleus

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Naert, T., Tulkens, D., Edwards, N. A., Carron, M., Shaidani, N. I., Wlizla, M., Boel, A., Demuynck, S., Horb, M. E., Coucke, P., Willaert, A., Zorn, A. M., & Vleminckx, K. Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos. Scientific Reports, 10(1), (2020): 14662, doi:10.1038/s41598-020-71412-0.CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.Research in the Vleminckx laboratory is supported by the Research Foundation—Flanders (FWO-Vlaanderen) (Grants G0A1515N and G029413N), by the Belgian Science Policy (Interuniversity Attraction Poles—IAP7/07) and by the Concerted Research Actions from Ghent University (BOF15/GOA/011). Further support was obtained by the Hercules Foundation, Flanders (Grant AUGE/11/14) and the Desmoid Tumor Research Foundation and the Desmoid Tumour Foundation Canada. T.N. is funded by “Kom op tegen Kanker” (Stand up to Cancer), the Flemish cancer society and previously held PhD fellowship with VLAIO-HERMES during the course of this work. D.T. and M. C. hold a PhD fellowship from the Research Foundation-Flanders (FWO-Vlaanderen). The Zorn Lab is supported by Funding from NIH National Institute of Child Health and Human Development (NICHD) P01 HD093363. A.W. and A.B. are supported by the Ghent University (Universiteit Gent) Methusalem grant BOFMET2015000401 to Anne De Paepe. The National Xenopus Resource and Horb lab is supported by funding from the National Institutes of Health (P40 OD010997 and R01 HD084409)