A multiscale model via single-cell transcriptomics reveals robust patterning mechanisms during early mammalian embryo development

Vitamin C protects early mouse embryos against juglone toxicity

Mouse pre-implantation embryogenesis is featured by several rounds of sequential cleavages, followed by the first cell lineage formation, specification of the inner cell mass (ICM) and trophectoderm (TE) at blastocyst stage. Establishment of the first cell lineage through asymmetric cell division is a longstanding question in developmental biology. Based on analogy to other lower model organisms or early achievements in mammalian development, three classic models of the first cell lineage specification are proposed to address this question: The prepattern or mosaic model, the inside-outside or positional model, and the polarization model. However, none of these models could well explain the highly regulatory nature of early mammalian embryos. During the past decades, great efforts have been made to elucidate when and how the pre-implantation blastomeres become different and finally segregate from each other. Mounting evidences show that the fate of trophectoderm is regulated by the Hippo/Yap/Tead signaling cascade in mouse early embryogenesis. In addition, single cell profiling and living imaging illustrate the great heterogeneity between blastomeres, providing some explanations for the regulatory nature of mammalian early embryos. Accompanying these progresses, a self-organization model was recently proposed to explain the blastocyst patterning in mammalian early embryos. This new model reconciles the experimental findings that seem to be contradictory to the three classic models and thus is regarded as a reformulation but improvement of classic models. Even so, however, the exact molecular mechanisms underlying this highly dynamic, complex and self-regulative process remain enigmatic. In this review, using mouse as the model system, we firstly summarize the preimplantation development and the classic models of the first cell lineage specification. Then, we highlight recent progresses, especially, the contributions of molecular regulators and heterogeneity of blastomeres in orchestrating the segregation of ICM and TE in mouse early embryos. At last, we summarize the current view on the first cell lineage specification in mammalian early embryogenesis and provide some clues for the future investigations. Altogether, the molecular basis of first cell lineage specification in mammals not only promotes the understanding for the beginning of life, but also contributes to stem cell biology and reproductive medicine.

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

Development of cellular polarity of hamster embryos during compaction

Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker NANOG is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that MED20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.

Chromosome segregation in female germ cells and early embryonic blastomeres is known to be highly prone to errors. The resulting aneuploidy is therefore the most frequent cause of termination of early development and embryo loss in mammals. And in specific cases, when the aneuploidy is actually compatible with embryonic and fetal development, it leads to severe developmental disorders. The main surveillance mechanism, which is essential for the fidelity of chromosome segregation, is the Spindle Assembly Checkpoint (SAC). And although all eukaryotic cells carry genes required for SAC, it is not clear whether this pathway is active in all cell types, including blastomeres of early embryos. In this review, we will summarize and discuss the recent progress in our understanding of the mechanisms controlling chromosome segregation and how they might work in embryos and mammalian embryos in particular. Our conclusion from the current literature is that the early mammalian embryos show limited capabilities to react to chromosome segregation defects, which might, at least partially, explain the widespread problem of aneuploidy during the early development in mammals.

The conversion of lysine to glutamate is needed for signaling in all plants and animals. In mouse embryonic stem (mES) cells, and probably their progenitors, endogenous glutamate production and signaling help maintain cellular pluripotency and proliferation, although the source of glutamate is yet to be determined. If the source of glutamate is lysine, then lysine deprivation caused by maternal low-protein diets could alter early embryo development and, consequently, the health of the offspring in adulthood. For these reasons, we measured three pertinent variables in human embryonic stem (hES) cells as a model for the inner cell masses of human blastocysts. We found that RNA encoding the alpha-aminoadipic semialdehyde synthase enzyme, which regulates glutamate production from lysine, was highly expressed in hES cells. Moreover, the mean amount of lysine consumed by hES cells was 50% greater than the mean amount of glutamate they produced, indicating that lysine is likely converted to glutamate in these cells. Finally, hES cells expressed RNA encoding at least two glutamate receptors. Since this may also be the case for hES progenitor cells in blastocysts, further studies are warranted to verify the presence of this signaling process in hES cells and to determine whether lysine deprivation alters early mammalian embryo development.

Chapter 13 - Differentiation in Early Development

An approach to multiscale modelling with graph grammars.

Gene imprinting is a widely observed epigenetic phenomenon in maize endosperm; however, whether it also occurs in the maize embryo remains controversial. Here, we used high-throughput RNA sequencing on laser capture microdissected and manually dissected maize embryos from reciprocal crosses between inbred lines B73 and Mo17 at six time points (3-13days after pollination, DAP) to analyze allelic gene expression patterns. Co-expression analysis revealed sequential gene activation during maize embryo development. Gene imprinting was observed in maize embryos, and a greater number of imprinted genes were identified at early embryo stages. Sixty-four strongly imprinted genes were identified (at the threshold of 9:1) on manually dissected embryos 5-13 DAP (more imprinted genes at 5 DAP). Forty-one strongly imprinted genes were identified from laser capture microdissected embryos at 3 and 5 DAP (more imprinted genes at 3 DAP). Furthermore, of the 56 genes that were completely imprinted (at the threshold of 99:1), 36 were not previously identified as imprinted genes in endosperm or embryos. In situ hybridization demonstrated that most of the imprinted genes were expressed abundantly in maize embryonic tissue. Our results shed lights on early maize embryo development and provide evidence to support that gene imprinting occurs in maize embryos.

Corticosteroids are increasingly being used during the peri-implantation period to treat women with repeated IVF failure and recurrent miscarriage. However, the direct effects of prednisolone (PRDL), one of the commonly used corticosteroids on early embryo development is not understood. To mimic the possible clinical scenario and to understand the embryonic response to direct PRDL exposure, this pilot study was conducted in a mouse model. Cleavage stage embryos exposed to 3 and 30 µM PRDL in vitro were assessed for peri-implantation developmental potential, genetic integrity, inner cell mass (ICM) proliferation and pluripotency markers in the proliferated ICM cells. Exposure to 30 µM PRDL delayed the embryonic progression beyond compaction (P < 0.05) in comparison to vehicle control and, had reduced total cell number (P < 0.001) than all other groups. In addition, 30 µM PRDL exposure resulted in poor hatching potential (P < 0.05) and increased apoptosis in blastocysts (P < 0.05) compared to 3 µM PRDL. On the other hand, completely formed ICM outgrowths were significantly higher (P < 0.05) in 3 µM PRDL compared to control. However, no significant differences were observed in the expression of pluripotency genes. In conclusion, the trend observed in embryos exposed to PRDL in vitro provides important information concerning the use of this drug when treating patients at the peri-implantation phase of IVF cycles. However, the clinical value of this observation on human embryo development needs further research.

Chemical vapor deposition (CVD) is a crucial technique in the preparation of high-quality thin films and coatings, and is widely used in various industries including semiconductor, optics, and nuclear fuel, due to its operation simplicity and high growth rate. The complexity of the CVD process arises from numerous parameters, such as precursor chemistry, temperature, pressure, gas flow dynamics, and substrate characteristics. These multiscale parameters make the optimization of the CVD process a challenging task. Numerical simulations are widely used to model and analyze the CVD complex systems, and can be divided into nanoscale, mesoscale, and macroscale methods. Numerical simulation is aimed at optimizing the CVD process, but the inter-scale parameters still need to be extracted in modeling processes. However, multiscale coupling modeling becomes a powerful method to solve these challenges by providing a comprehensive framework that integrates phenomena occurring at different scales. This review presents an overview of the CVD process, the common critical parameters, and an in-depth analysis of CVD models in different scales. Then various multiscale models are discussed. This review highlights the models in different scales, integrates these models into multiscale frameworks, discusses typical multiscale coupling CVD models applied in practice, and summarizes the parameters that can transfer information between different scales. Finally, the schemes of multiscale coupling are given as a prospective view. By offering a comprehensive view of the current state of multiscale CVD models, this review aims to bridge the gap between theory and practice, and provide insights that could lead to a more efficient and precise control of the CVD process.

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

High-throughput single-cell sequencing technologies hold tremendous potential for defining cell types in an unbiased fashion using gene expression and epigenomic state. A key challenge in realizing this potential is integrating single-cell datasets from multiple protocols, biological contexts, and data modalities into a joint definition of cellular identity. We previously developed an approach, called linked inference of genomic experimental relationships (LIGER), that uses integrative nonnegative matrix factorization to address this challenge. Here, we provide a step-by-step protocol for using LIGER to jointly define cell types from multiple single-cell datasets. The main stages of the protocol are data preprocessing and normalization, joint factorization, quantile normalization and joint clustering, and visualization. We describe how to jointly define cell types from single-cell RNA-seq (scRNA-seq) and single-nucleus ATAC-seq (snATAC-seq) data, but similar steps apply across a wide range of other settings and data types, including cross-species analysis, single-nucleus DNA methylation, and spatial transcriptomics. Our protocol contains examples of expected results, describes common pitfalls, and relies only on our freely available, open-source R implementation of LIGER. We also provide R Markdown tutorials showing the outputs from each individual code segment. The analysis process can be performed in 1-4 h, depending on dataset size, and assumes no specialized bioinformatics training.

Shifting early embryology paradigms: Applications of stem cell-based embryo models in bioengineering