Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

Single-cell perturbation dissects transcription factor control of progression speed and trajectory choice in early T-cell development

In early T-cell development, single cells dynamically shift expression of multiple transcription factors (TFs) during transition from multipotentiality to T-lineage commitment, but the functional roles of many TFs have been obscure. Here, synchronized in vitro differentiation systems, scRNA-seq with batch indexing, and controlled gene-disruption strategies have unraveled single-cell impacts of perturbing individual TFs at two stages in early T-cell development. Single-cell CRISPR perturbation revealed that early-acting TFs Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) each play individualized roles promoting or retarding T-lineage progression and suppressing alternative trajectories, collectively determining population dynamics and path topologies. Later, during T-lineage commitment, cells prevented from expressing TF Bcl11b "realized" this abnormality not with a developmental block, but by shifting into a divergent path via bZIP and Sox TF activation as well as E protein antagonism, finally exiting the T-lineage trajectory. These TFs thus exert a network of impacts to control progression kinetics, trajectories, and differentiation outcomes of early pro-T cells.

Improved development of mouse SCNT embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†

In mammalian cloning by somatic cell nuclear transfer (SCNT), treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives—such as trichostatin A—characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit Class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1 to 7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2 to 7.3%). Thus, inhibition of Class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8–10 h because longer inhibition with Class I inhibitors causes a 2-cell developmental block. Therefore, we used Ky-29, with higher selectivity for Class IIa than Class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the 2-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the 1-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies.

PSIV-B-38 Late-Breaking: Effect of Glutamate (Glu) on Developmental Block and the MZT Relative Genes Expression in Mouse embryo during in Vitro Culture

The risk of developmental block in mammal’s embryos is high during in vitro as compare to in vivo environment because the in vitro embryo-culture systems are suboptimal. During in vitro-culture the balance between ROS production and elimination is disturbed and may lead to 2-cell block in mouse embryos [1]. In the current study, we investigated the effects of Glu as anti-developmental block during IVC on ZGA and MZT on mouse embryos. The mouse embryos were divided into control and different level of Glu treated group. The cleavage rate was determined, the ROS and GSH level was investigated using DCHF-DA and CMF2HC respectively. The mRNA expression level of ZGA marker gene such as Eif-1α, Muerv l, Zscan4d and Hsp70.1 was analyzed among the groups using RT-PCR. The transition rate from 2-cell to 4-cell was significantly higher in 6mmol/L Glu treated group as compare to control and others treated groups. No significant difference was recorded in the level of ROS and GSH during MZT stage among the different groups. The mRNA expression level of ZGA marker gene was significantly increased at middle and late stage in 6mmol/L Glu treated group as compare to control and others treated groups. In conclusion, this study shows that the concentration of 6mmol/L Glu could maintain the dynamic balance of GSH and ROS, increase the expression of ZGA marker gene and maintain its high expression pattern of time series, directly participate in the ZGA activated process; ultimately reduce the risk of developmental block to ensure the successful completion of MZT. Reference [1] Lee MT, Bonneau AR, Giraldez AJ.Zygotic Genome Activation during the Maternal-to-Zygotic Transition. Annual Rev Cell Dev Biol [J], 2014, 30:581–613.

Psychological Model of Development of Professional Axiological Orientations in Prospective Teachers at the Stage of Higher Education

The article defines the relevance of developing professional axiological orientations in prospective teachers and reviews the main research ideas on the topic. The aim of the article was to create a psychological model of development of professional axiological orientations of prospective teachers at the stage of higher education. The main methods of the study included theoretical analysis of the research literature on the topic of teachers’ axiological orientations, as well as the method modelling that included the creation of the model of development of professional axiological orientations of prospective teachers at the stage of higher education. The authors presented the model that included the following inter-connected blocks: developmental, processing and resulting blocks. The developmental block includes the content, psychological-pedagogic conditions and mechanisms of the prospective teachers’ professional axiological orientations development. The processing block defines the stages of the development. The resulting block includes the criteria and levels of the development. The content of the article reveals an opportunity for setting new goals in conducting professional axiological development of prospective teachers. Reaching these goals by integrating the international and national experience would help to efficiently organize the educational and mentoring process at the stage of higher education, which defines the future perspective of the study.