METTL3-mediated mRNA N6-methyladenosine is required for oocyte and follicle development in mice.

Proper follicle development is very important for the production of mature oocytes, which is essential for the maintenance of female fertility. This complex biological process requires precise gene regulation. The most abundant modification of mRNA, N6-methyladenosine (m6A), is involved in many RNA metabolism processes, including RNA splicing, translation, stability, and degradation. Here, we report that m6A plays essential roles during oocyte and follicle development. Oocyte-specific inactivation of the key m6A methyltransferase Mettl3 with Gdf9-Cre caused DNA damage accumulation in oocytes, defective follicle development, and abnormal ovulation. Mechanistically, combined RNA-seq and m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq) data from oocytes revealed, that we found METTL3 targets Itsn2 for m6A modification and then enhances its stability to influence the oocytes meiosis. Taken together, our findings highlight the crucial roles of mRNA m6A modification in follicle development and coordination of RNA stabilization during oocyte growth.

Implantation initiation of self-assembled embryo-like structures generated using three types of mouse blastocyst-derived stem cells.

Spatially ordered embryo-like structures self-assembled from blastocyst-derived stem cells can be generated to mimic embryogenesis in vitro. However, the assembly system and developmental potential of such structures needs to be further studied. Here, we devise a nonadherent-suspension-shaking system to generate self-assembled embryo-like structures (ETX-embryoids) using mouse embryonic, trophoblast and extra-embryonic endoderm stem cells. When cultured together, the three cell types aggregate and sort into lineage-specific compartments. Signaling among these compartments results in molecular and morphogenic events that closely mimic those observed in wild-type embryos. These ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues. After transplantation into the pseudopregnant mouse uterus, ETX-embryoids efficiently initiate implantation and trigger the formation of decidual tissues. The ability of the three cell types to self-assemble into an embryo-like structure in vitro provides a powerful model system for studying embryogenesis.

Long non-coding RNAs involved in the regulatory network during porcine pre-implantation embryonic development and iPSC induction.

Long non-coding RNAs (lncRNA) play a key role in the orchestration of transcriptional regulation during development and many other cellular processes. The importance of the regulatory co-expression network was highlighted in the identification of the mechanism of these processes in humans and mice. However, elucidation of the properties of porcine lncRNAs involved in the regulatory network during pre-implantation embryonic development and fibroblast reprogramming to induced pluripotent stem cell (iPSC) has been limited to date. Using a weighted gene co-expression network analysis, we constructed the regulatory network and determined that the novel lncRNAs were functionally involved in key events of embryonic development during the pre-implantation period; moreover, reprogramming could be delineated by a small number of potentially functional modules of co-expressed genes. These findings indicate that lncRNAs may be involved in the transcriptional regulation of zygotic genome activation, first lineage segregation and somatic reprogramming to pluripotency. Furthermore, we performed a conservation and synteny analysis with the significant lncRNAs involved in these vital events and validated the results via experimental assays. In summary, the current findings provide a valuable resource to dissect the protein coding gene and lncRNA regulatory networks that underlie the progressive development of embryos and somatic reprogramming.

Lineage specification revealed by single-cell gene expression analysis in porcine preimplantation embryos.

After zygotic genome activation and lineage specification, zygotes develop into late blastocysts comprising three distinct cell types. The molecular mechanisms underlying this progress are largely unknown in pigs. Here, we intended to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the porcine blastocysts. Using a quantitative microfluidics approach in single cells, we detected mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency simultaneously in 480 individual cells derived from porcine preimplantation embryos. The developmental transitions can be distinguished based on distinctive gene expression profiles, and we identified paired box 6 (PAX6) and aquaporin 3 (AQP3) expressed in early and late developmental stages, respectively. Two lineages can be segregated in porcine early and late blastocysts by the expression patterns of lineage-specific genes such as DAB2, clathrin adaptor protein (DAB2) for trophectoderm (TE), platelet derived growth factor receptor alpha (PDGFRA), Nanog homeobox (NANOG), fibronectin 1 (FN1), hepatocyte nuclear factor 4 alpha (HNF4A), goosecoid homeobox (GSC), nuclear receptor subfamily 5 group A member 2 (NR5A2), and lysine acetyltransferase 6A (KAT6A; previously known as MYST3) for inner cell mass (ICM). However, the epiblast and primitive endoderm cannot be identified in late blastocysts, and those TE or ICM lineage-specific genes were low expressed in blastomeres from the morula. Our results shed light on early cell fate determination in porcine preimplantation embryos and offer theoretical support for deriving porcine embryonic stem cells.

Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos.

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.

Bovine lineage specification revealed by single-cell gene expression analysis from zygote to blastocyst.

Preimplantation embryos undergo zygotic genome activation and lineage specification resulting in three distinct cell types in the late blastocyst. The molecular mechanisms underlying this progress are largely unknown in bovines. Here, we sought to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the bovine blastocyst. Using a quantitative microfluidics approach in single cells, we analyzed mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency in parallel in 384 individual cells from bovine preimplantation embryos. The developmental transitions can be distinguished by distinctive gene expression profiles and we identified NOTCH1, expressed in early developmental stages, while T-box 3 (TBX3) and fibroblast growth factor receptor 4 (FGFR4), expressed in late developmental stages. Three lineages can be segregated in bovine expanded blastocysts based on the expression patterns of lineage-specific genes such as disabled homolog 2 (DAB2), caudal type homeobox 2 (CDX2), ATPase H+/K+ transporting non-gastric alpha2 subunit (ATP12A), keratin 8 (KRT8), and transcription factor AP-2 alpha (TFAP2A) for trophectoderm; GATA binding protein 6 (GATA6) and goosecoid homeobox (GSC) for primitive endoderm; and Nanog homeobox (NANOG), teratocarcinoma-derived growth factor 1 (TDGF1), and PR/SET domain 14 (PRDM14) for epiblast. Moreover, some lineage-specific genes were coexpressed in blastomeres from the morula. The commitment to trophectoderm and inner cell mass lineages in bovines occurs later than in the mouse, and KRT8 might be an earlier marker for bovine trophectoderm cells. We determined that TDGF1 and PRDM14 might play pivotal roles in the primitive endoderm and epiblast specification of bovine blastocysts. Our results shed light on early cell fate determination in bovine preimplantation embryos and offer theoretical support for deriving bovine embryonic stem cells.

Improvement in Mouse iPSC Induction by Rab32 Reveals the Importance of Lipid Metabolism during Reprogramming.

Induced pluripotent stem cells (iPSCs) have variable expression levels of a series of genes that affect their pluripotent potential, but the regulatory mechanisms controlling reprogramming remain unclear. By testing the efficiency of iPSC generation using Oct4, Sox2, Klf4 (termed OSK) plus one additional gene, we found that Rab32 improved reprogramming efficiency. We established a system for detecting the number and the size of lipid droplets and autophagosomes per cell for tracking their morphological changes during reprogramming. Our results showed that Rab32 increased lipid storage during the early and middle stages, and also increased autophagy during the middle stage of reprogramming. These findings were further confirmed by the up-regulation of lipid biosynthesis and autophagosome formation related genes, of which their expression could improve iPSC induction. The inhibition of lipid biosynthesis and autophagosome formation significantly reduced reprogramming efficiency, and the inhibition of lipid synthesis phenotype could be rescued by the overexpression of Rab32. In addition, the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, was up-regulated by Rab32. These results demonstrated that Rab32 could improve the induction of iPSCs through the enhancement of lipid biosynthesis, highlighting the importance of lipid metabolism during reprogramming.