H3K27me3 at Pericentromeric Heterochromatin is a Defining Feature of the Early Mouse Blastocyst

Early mouse development is characterized by structural and epigenetic changes at the chromatin level while cells progress towards differentiation. At blastocyst stage, the segregation of the three primordial lineages is accompanied by establishment of differential patterns of DNA methylation and post-translational modifications of histones, such as H3K27me3. In this study, we have analysed the dynamics of H3K27me3 at pericentromeric heterochromatin (PCH) during development of the mouse blastocyst, in comparison with cultured embryonic cells. We show that this histone modification is first enriched at PCH in the whole embryo and evolves into a diffuse distribution in epiblast during its specification and maturation. Concomitantly, the level of transcription from major satellite decreases. Stem cells derived from blastocyst (naïve ESCs and TSCs) do not fully maintain the H3K27me3 enrichment at PCH. Moreover, the dynamic of H3K27me3 at PCH during in vitro conversion from naïve to primed pluripotent state and during ESCs derivation suggests that the mechanisms underlying the control of this histone mark at PCH are different in embryo and in vitro. We also conclude that the non-canonical presence of H3K27me3 at PCH is a defining feature of embryonic cells in the young blastocyst before epiblast segregation.

ECM-integrin signalling instructs cellular position-sensing to pattern the early mouse embryo

Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo give rise to the inner cell mass (ICM) that eventually forms the embryo proper. Yet the molecular basis of how these cells recognise their ‘inside’ position to instruct their fate is unknown. Here we show that provision of extracellular matrix (ECM) to isolated embryonic cells induces ICM specification and alters subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin β1 activity and involves apical to basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for ‘inside’ positional signalling and it is required for proper EPI/PrE patterning. Our findings thus highlight the significance of ECM-integrin adhesion in enabling position-sensing by cells to achieve tissue patterning.

Single cell quantification of ribosome occupancy in early mouse development

Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key regulatory mechanism of genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high coverage measurements enabled the first analysis of allele-specific ribosome engagement in early development and led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes. Finally, by integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle stage oocytes is the predominant determinant of protein abundance in the zygote. Taken together, these findings resolve the long-standing paradox of low correlation between RNA expression and protein abundance in early embryonic development. The novel Ribo-ITP approach will enable numerous applications by providing high coverage and high resolution ribosome occupancy measurements from ultra-low input samples including single cells.

Pak2 reduction induces a failure of early embryonic development in mice

Background The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated. Methods In the present work, Pak2 was reduced using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis. Areas in chromatin with γH2AX were detected by immunofluorescence microscopy and serve as a biomarker of DNA damages. Results We found that Pak2 knockdown significantly reduced blastocyst formation of early embryos. In addition, Pak2 reduction led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. In response to DNA double strand breaks (DSBs), the histone protein H2AX is specifically phosphorylated at serine139 to generate γH2AX, which is used to quantitative DSBs. In this research, Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls. Conclusions Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.

The RNA helicase DDX6 controls early mouse embryogenesis by repressing aberrant inhibition of BMP signaling through miRNA-mediated gene silencing

The evolutionarily conserved RNA helicase DDX6 is a central player of post-transcriptional regulation, but its role during embryogenesis remains elusive. We here demonstrated that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 KO mouse embryos and in vitro epiblast-like cell (EpiLC) induction system. Our study unveiled a great impact of DDX6-mediated RNA regulation on signaling pathways. Deletion of Ddx6 caused the aberrant transcriptional upregulation of the negative regulators of BMP signaling, which accompanied with enhanced Nodal signaling. Ddx6 / pluripotent cells acquired higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal expression in the primitive streak likely promoted endoderm cell fate specification while inhibiting mesoderm development. We further clarified the mechanism how DDX6 regulates cell fate determination of pluripotent cells by genetically dissecting major DDX6 pathways: processing body (P-body) formation, translational repression, mRNA decay, and miRNA-mediated silencing. P-body-related functions were dispensable, but the miRNA pathway was essential for the DDX6 function. DDX6 may prevent aberrant transcriptional upregulation of the negative regulators of BMP signaling by repressing translation of certain transcription factors through the interaction with miRNA-induced silencing complexes (miRISCs). Overall, this delineates how DDX6 affects development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function.

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

Potential genetic robustness of Prnp and Sprn double knockout mouse embryos towards ShRNA-lentiviral inoculation

Shadoo, encoded by Sprn, and PrP, encoded by Prnp, are related proteins whose biological functions are still incompletely understood. Although previous knockdown experiments have suggested the necessity of Shadoo in the absence of PrP during early mouse embryogenesis, little impact of the double-knockout of these two loci was reported. To further investigate this apparent discrepancy, we compared the transcriptome of WT, Prnp0/0 and Prnp0/0, Sprn0/0 E6.5 mouse embryos following inoculation by Sprn-ShRNA or Prnp-ShRNA lentiviral vectors at the one-cell stage. Our results highlighted a significant induction of an apoptotic pathway in Prnp0/0 E6.5 mouse embryos inoculated with Sprn-ShRNA vectors alongside interferon and to a lesser extent inflammatory responses, confirming previous reported experiments. On the contrary, ShRNA vector inoculation in Prnp0/0, Sprn0/0 embryos did not induce apoptosis and resulted in lower interferon responses. Finally, comparisons of the transcriptome of WT and Prnp0/0, Sprn0/0 embryos revealed only slight differences, which may in part explain the genetic robustness observed in the latter genotype.

Advances in live imaging early mouse development: exploring the researcher's interdisciplinary toolkit

ABSTRACT Live imaging is an important part of the developmental biologist's armoury of methods. In the case of the mouse embryo, recent advances in several disciplines including embryo culture, microscopy hardware and computational analysis have all contributed to our ability to probe dynamic events during early development. Together, these advances have provided us with a versatile and powerful ‘toolkit’, enabling us not only to image events during mouse embryogenesis, but also to intervene with them. In this short Spotlight article, we summarise advances and challenges in using live imaging specifically for understanding early mouse embryogenesis.