Early Mammalian Embryonic Development Research Articles

Crosstalk between Signaling Pathways and Energy Metabolism in Pluripotency.

The sequential change from totipotency to multipotency occurs during early mammalian embryo development. However, due to the lack of cellular models to recapitulate the distinct potency of stem cells at each stage, their molecular and cellular characteristics remain ambiguous. The establishment of isogenic naïve and primed pluripotent stem cells to represent the pluripotency in the inner cell mass of the pre-implantation blastocyst and in the epiblast from the post-implantation embryo allows the understanding of the distinctive characteristics of two different states of pluripotent stem cells. This review discusses the prominent disparities between naïve and primed pluripotency, including signaling pathways, metabolism, and epigenetic status, ultimately facilitating a comprehensive understanding of their significance during early mammalian embryonic development.

Lipidomic remodeling during mammalian preimplantation embryonic development

Abstract The dynamic changes in lipids during early embryonic development in mammals have not yet been comprehensively investigated. In a recent paper published in Nature Cell Biology, Jin Zhang et al. reported the dynamic lipid landscapes during preimplantation embryonic development in mice and humans. They highlight the crucial role of lipid unsaturation in regulating embryogenesis.

Differential alternative splicing landscape identifies potentially functional RNA binding proteins in early embryonic development in mammals

Alternative splicing (AS) as one of the important post-transcriptional regulatory mechanisms has been poorly studied during embryogenesis. In this study, we comprehensively collected and analyzed the transcriptome data of early embryos from human and mouse. We found that AS plays an important role in this process and predicted candidate RNA binding protein (RBP) regulators that are associated with reproductive development. The predicted RBPs such as EIF4A3, MAK16, SRSF2, and UTP23 were found to be associated with reproductive disorders. By Smart-seq2 sequencing analysis, we identified 5445 aberrant alternative splicing events in Eif4a3-knockdown embryos. These events were preferentially associated with RNA processing. In conclusion, our work on the landscape and potential function of alternative splicing events will boost further investigation of detailed mechanisms and key factors regulating mammalian early embryo development and promote the inspiration of pharmaceutical approaches for disorders in this crucial biology process.

Low-input lipidomics reveals lipid metabolism remodelling during early mammalian embryo development.

Lipids are indispensable for energy storage, membrane structure and cell signalling. However, dynamic changes in various categories of endogenous lipids in mammalian early embryonic development have not been systematically characterized. Here we comprehensively investigated the dynamic lipid landscape during mouse and human early embryo development. Lipid signatures of different developmental stages are distinct, particularly for the phospholipid classes. We highlight that the high degree of phospholipid unsaturation is a conserved feature as embryos develop to the blastocyst stage. Moreover, we show that lipid desaturases such as SCD1 are required for in vitro blastocyst development and blastocyst implantation. One of the mechanisms is through the regulation of unsaturated fatty-acid-mediated fluidity of the plasma membrane and apical proteins and the establishment of apical-basal polarity during development of the eight-cell embryo to the blastocyst. Overall, our study provides an invaluable resource about the remodelling of the endogenous lipidome in mammalian preimplantation embryo development and mechanistic insights into the regulation of embryogenesis and implantation by lipid unsaturation.

Transitions in development - an interview with Peng Du.

Peng Du is Associate Professor at Peking University College of Life Sciences, where he started his own lab in 2018. Peng's research focusses on post-transcriptional RNA regulatory pathways in early mammalian embryonic development and disease. We spoke to Peng over Zoom to find out more about his career path, his transition from plant to mammalian research and his experience becoming a group leader.

Roles and Regulation of H3K4 Methylation During Mammalian Early Embryogenesis and Embryonic Stem Cell Differentiation.

From generation of germ cells, fertilization, and throughout early mammalian embryonic development, the chromatin undergoes significant alterations to enable precise regulation of gene expression and genome use. Methylation of histone 3 lysine 4 (H3K4) correlates with active regions of the genome, and it has emerged as a dynamic mark throughout this timeline. The pattern and the level of H3K4 methylation are regulated by methyltransferases and demethylases. These enzymes, as well as their protein partners, play important roles in early embryonic development and show phenotypes in embryonic stem cell self-renewal and differentiation. The various roles of H3K4 methylation are interpreted by dedicated chromatin reader proteins, linking this modification to broader molecular and cellular phenotypes. In this review, we discuss the regulation of different levels of H3K4 methylation, their distinct accumulation pattern, and downstream molecular roles with an early embryogenesis perspective.

Single-cell RNA-seq and single-cell bisulfite-sequencing reveal insights into yak preimplantation embryogenesis

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders.

Chromosomal Aberrations As a Biological Phenomenon in Human Embryonic Development.

Frequent chromosomal abnormalities are a distinctive feature of early embryonic development in mammals, especially humans. Aneuploidy is considered as a contributing factor to failed embryo implantation and spontaneous abortions. In the case of chromosomal mosaicism, its effect on the potency of embryos to normally develop has not been sufficiently studied. Although, a significant percentage of chromosomal defects in early human embryos are currently believed to be associated with the features of clinical and laboratory protocols, in this review, we focus on the biological mechanisms associated with chromosomal abnormalities. In particular, we address the main events in oocyte meiosis that affects not only the genetic status of an unfertilized oocyte, but also further embryo viability, and analyze the features of first cleavage divisions and the causes of frequent chromosomal errors in early embryonic development. In addition, we discuss current data on self-correction of the chromosomal status in early embryos.

18S ribosomal RNA methyltransferase METTL5-mediated CDX2 translation regulates porcine early embryo development1

N6-methyladenosine (m6A) plays a key role in mammalian early embryonic development and cell lineage differentiation. However, the role and mechanisms of 18S ribosomal RNA (rRNA) m6A methyltransferase METTL5 in early embryonic development remain unclear. Here, we found that 18S rRNA m6A methyltransferase METTL5 plays an important role in porcine early embryonic development. METTL5 knockdown and overexpression significantly reduced the developmental efficiency of porcine early embryos and impaired cell lineage allocation. METTL5 knockdown apparently decreased the global translation efficiency in blastocyst, while METTL5 overexpression increased the global translation efficiency. Furthermore, METTL5 knockdown did not affect the abundance of CDX2 mRNA, but resulted in a significant reduction in CDX2 protein levels. Moreover, the low developmental efficiency and abnormal lineage distribution of METTL5 knockdown embryos could be rescued by CDX2 overexpression. Collectively, our results demonstrated that 18S rRNA methyltransferase METTL5 regulates porcine early embryonic development via modulating the translation of CDX2.

In Vitro Embryogenesis and Gastrulation Using Stem Cells in Mice and Humans.

During early mammalian embryonic development, fertilized one-cell embryos develop into pre-implantation blastocysts and subsequently establish three germ layers through gastrulation during post-implantation development. In recent years, stem cells have emerged as a powerful tool to study embryogenesis and gastrulation without the need for eggs, allowing for the generation of embryo-like structures known as synthetic embryos or embryoids. These in vitro models closely resemble early embryos in terms of morphology and gene expression and provide a faithful recapitulation of early pre- and post-implantation embryonic development. Synthetic embryos can be generated through a combinatorial culture of three blastocyst-derived stem cell types, such as embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm cells, or totipotent-like stem cells alone. This review provides an overview of the progress and various approaches in studying in vitro embryogenesis and gastrulation in mice and humans using stem cells. Furthermore, recent findings and breakthroughs in synthetic embryos and gastruloids are outlined. Despite ethical considerations, synthetic embryo models hold promise for understanding mammalian (including humans) embryonic development and have potential implications for regenerative medicine and developmental research.

Research progress on the study of transcriptome-wide poly(A) tails in mammalian oocytes and early embryos.

During mammalian oocyte-to-embryo transition, before zygotic genome activation, the transcription in oocytes and embryos is silenced, so the post-transcriptional regulation of mRNA plays an essential role in this process. Poly(A) tail is an important post-transcriptional modification that affects mRNA metabolism and translation efficiency. With the development of sequencing technology and analysis tools, especially the methods based on third-generation sequencing technology, the length and composition of poly(A) tails can be accurately measured, greatly expanding our understanding of poly(A) tails in mammalian early embryonic development. This review focuses on the achievements of poly(A) tail sequencing methods and the research progress of poly(A) tail in regulating oocyte-to-embryo transition, discussing the future applications for the investigation of mammalian early embryonic development and infertility related diseases.

The RNA m6A landscape of mouse oocytes and preimplantation embryos.

Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Here we apply low-input methyl RNA immunoprecipitation and sequencing to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development.

Overview of junctional complexes during mammalian early embryonic development.

Cell-cell junctions form strong intercellular connections and mediate communication between blastomeres during preimplantation embryonic development and thus are crucial for cell integrity, polarity, cell fate specification and morphogenesis. Together with cell adhesion molecules and cytoskeletal elements, intercellular junctions orchestrate mechanotransduction, morphokinetics and signaling networks during the development of early embryos. This review focuses on the structure, organization, function and expressional pattern of the cell-cell junction complexes during early embryonic development. Understanding the importance of dynamic junction formation and maturation processes will shed light on the molecular mechanism behind developmental abnormalities of early embryos during the preimplantation period.

Establishment of human induced trophoblast stem cells via reprogramming of fibroblasts.

During early mammalian embryonic development, trophoblast cells play an essential role in establishing cell-cell interactions at the maternal-fetal interface to ensure a successful pregnancy. In a recent study, we showed that human fibroblasts can be reprogrammed into induced trophoblast stem (iTS) cells by transcription factor-mediated nuclear reprogramming using the Yamanaka factors OCT4, KLF4, SOX2 and c-MYC (OKSM) and a selection of TS cell culture conditions. The derivation of TS cells from human blastocysts or first-trimester placenta can be limited by difficulties in obtaining adequate material as well as ethical implications. By contrast, the described approach allows the generation of iTS cells from the adult cells of individuals with diverse genetic backgrounds, which are readily accessible to many laboratories around the world. Here we describe a step-by-step protocol for the generation and establishment of human iTS cells directly from dermal fibroblasts using a non-integrative reprogramming method. The protocol consists of four main sections: (1) recovery of cryopreserved human dermal fibroblasts, (2) somatic cell reprogramming, (3) passaging of reprogramming intermediates and (4) derivation of iTS cell cultures followed by routine maintenance of iTS cells. These iTS cell lines can be established in 2-3 weeks and cultured long term over 50 passages. We also discuss several characterization methods that can be performed to validate the iTS cells derived using this approach. Our protocol allows researchers to generate patient-specific iTS cells to interrogate the trophoblast and placenta biology as well as their interactions with embryonic cells in health and diseases.

Functional evaluation of LTR-derived lncRNAs in porcine oocytes and zygotes with RNA-seq and small RNA-seq.

Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of early embryonic development in mammals. However, they are seldom investigated in pigs. Here, to annotate full-length RNA transcripts, we performed annotation using a newly developed computational pipeline—an RNA-seq and small RNA-seq combined strategy—using our previously obtained RNA-seq and small RNA-seq data from porcine oocytes and zygotes. As evidenced by the length comparison, the frequency of the core promoter, and the polyadenylation signal motifs, the transcripts appear to be full-length. Furthermore, our strategy allowed the identification of a large number of endogenous retrovirus-associated lncRNAs (ERV-lncRNAs) and found that some of them were highly expressed in porcine zygotes, as compared to oocytes. Through the knockdown strategy, two ERV-lncRNAs (TCONS_00035465 and TCONS_00031520) were identified as playing potential roles in the early embryo development of pigs, laying a foundation for future research.

ELF4 is critical to zygotic gene activation and epigenetic reprogramming during early embryonic development in pigs.

Zygotic gene activation (ZGA) and epigenetic reprogramming are critical in early embryonic development in mammals, and transcription factors are involved in regulating these events. However, the effects of ELF4 on porcine embryonic development remain unclear. In this study, the expression of ELF4 was detected in early porcine embryos and different tissues. By knocking down ELF4, the changes of H3K9me3 modification, DNA methylation and ZGA-related genes were analyzed. Our results showed that ELF4 was expressed at all stages of early porcine embryos fertilized in vitro (IVF), with the highest expression level at the 8-cell stage. The embryonic developmental competency and blastocyst quality decreased after ELF4 knockdown (20.70% control vs. 17.49% si-scramble vs. 2.40% si-ELF4; p < 0.001). Knockdown of ELF4 induced DNA damage at the 4-cell stage. Interfering with ELF4 resulted in abnormal increases in H3K9me3 and DNA methylation levels at the 4-cell stage and inhibited the expression of genes related to ZGA. These results suggest that ELF4 affects ZGA and embryonic development competency in porcine embryos by maintaining genome integrity and regulating dynamic changes of H3K9me3 and DNA methylation, and correctly activating ZGA-related genes to promote epigenetic reprogramming. These results provide a theoretical basis for further studies on the regulatory mechanisms of ELF4 in porcine embryos.

H3K27ac mediated SS18/BAFs relocation regulates JUN induced pluripotent-somatic transition

BackgroundThe exit from pluripotency or pluripotent-somatic transition (PST) landmarks an event of early mammalian embryonic development, representing a model for cell fate transition.ResultsIn this study, using a robust JUN-induced PST within 8 h as a model, we investigate the chromatin accessibility dynamics (CAD) as well as the behaviors of corresponding chromatin remodeling complex SS18/BAFs, to probe the key events at the early stage of PST. Here, we report that, JUN triggers the open of 34661 chromatin sites within 4 h, accomplished with the activation of somatic genes, such as Anxa1, Fosl1. ChIP-seq data reveal a rapid relocation of SS18/BAFs from pluripotent loci to AP-1 associated ones. Consistently, the knockdown of Brg1, core component of BAF complexes, leads to failure in chromatin opening but not closing, resulting in delay for JUN induced PST. Notably, the direct interaction between SS18/BAFs and JUN-centric protein complexes is undetectable by IP-MS. Instead, we show that H3K27ac deposited by cJUN dependent process regulates SS18/BAFs complex to AP1-containing loci and facilitate chromatin opening and gene activation.ConclusionsThese results reveal a rapid transfer of chromatin remodeling complexes BAF from pluripotent to somatic loci during PST, revealing a simple mechanistic aspect of cell fate control.

In vitro investigation of mammalian early embryonic development.

Mammalian embryonic development starts from a fertilized egg, which cleaves to form morula and blastocyst. At the same time, the early embryo is transported from the fallopian tube to the uterus for implantation. After implantation, the embryo undergoes gastrulation and forms a gastrula, further developing a new individual. The development of embryo in the uterus causes the difficulties in sampling and observation, hindering the understanding of mammalian embryonic development. Therefore, it is necessary to develop the technology to overcome the barrier of in vivo embryonic development. In December 2021, "Embryo 'husbandry' opens windows into early development" was selected as one of Science's 2021 breakthroughs. This review focuses on the achievements of in vitro mammalian embryos and discusses their limitations and the future applications for the investigation of mammalian embryonic development and human related diseases.

Asymmetric expression of maternal mRNA governs first cell-fate decision.

The goal of preimplantation development is to establish the fates of the embryonic and extra-embryonic cells. However, when and how cell fates are determined during early mammalian embryonic development remains unclear. We report that the high mobility group (HMG) protein family member HMGA1 was distributed differentially in mouse two-cell blastomeres. Knockdown of Hmga1 expression in one of the two cells reduced the number of cells contributing to the inner cell mass (ICM), suggesting that differential distribution of HMGA1 in the blastomeres in two-cell mouse embryos affected the selection of embryonic cell lineages. Mechanistically, HMGA1 promotes the expression of the ICM-specific gene Sox2. The results of this study show that mouse embryos demonstrate heterogeneity as early as the two-cell stage, and that these differences are related to cell-fate differentiation in early mouse embryos.

P–803 Novel culture conditions for the improvement of the in vitro expansion of human male fetal germ cells

Abstract Study question Do different ECMs/substrates and growth media culture conditions improve in vitro male human primordial germ cell (hPGC) expansion? Summary answer We achieved in vitro expansion improvement of male hPGCs with specific growth factors such as LIF, EGF, FGF2 and GDNF on gelatin- and vitronectin-coating cultures. What is known already PGCs are the precursors of male and female gametes, which are specified during early mammalian post-implantation embryonic development. PGCs undergo sequential cell divisions to differentiate into pro-spermatogonia (pSPG). In vitro propagation of pSPG could be important to understand the transition to spermatogonial stem cells (SSCs), important for fertility preservation in patients with infertility. Here, we aimed at performing a comparative analysis on in vitro feeder-free culture systems, based on different extracellular matrix (ECM) and growth media culture conditions, to support the expansion of the male germline stem cell populations using second trimester human male gonads as primary material. Study design, size, duration We collected human 2nd trimester male fetal gonads from elective abortions. Male gonads were dissected in saline solution (0.9% NaCl) and were either fixed overnight in 4% paraformaldehyde (PFA) for immunohistochemistry or disaggregated by enzymatic digestion for in vitro culture. Participants/materials, setting, methods After differential plating, fetal cells were cultured for 6 days. Disaggregated gonads were cultured with two different growing media (Medium 1 supplemented with LIF, EGF, FGF–2 and GDNF and Medium 2 supplemented with RA, BMP 4 and Activin A) on gelatin, laminin, vitronectin or matrigel coated plates. Cultured cells were immunostained, quantified for the expression of DDX4 and POU5F1 after 3 days (D3) and 6 days (D6) of culture. Main results and the role of chance We pursued to evaluate whether germ cells dissociated from a pool of male fetal gonads could propagate in vitro when cultured for D6 in different conditions. We observed that expansion of POU5F1-positive early PGCs and DDX4-positive late PGCs was only observed when cells were plated on gelatin or vitronectin and cultured with Medium 1, containing the growth factors LIF, EGF, FGF2 and GDNF. However, a reduced percentage of PGCs was observed in all four different coatings when grown with Medium 2, containing RA, BMP4 and Activin A. We analyzed the relative expression of the PGC markers POU5F1, DDX4 and MAGEA4 in histological sections of gonads from embryos at 18.5 weeks of gestation. Two populations of hPGCs were observed: ∼10–30% of the gonadal cells expressed solely DDX4 (late PGCs), whereas less than 10% of gonadal cells expressed POU5F1 (early PGCs). SOX9 and STARD1 expression was evaluated, confirming the presence of Sertoli cells and Leydig cells, respectively. Limitations, reasons for caution Due to the limited and difficulty to obtain human fetal tissue, a limited number of samples were used. Wider implications of the findings: We expanded human male fetal germ cells in vitro for D6 on gelatin and vitronectin coated plates with Medium 1, containing growth factors LIF, EGF, FGF2 and GDNF. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to pSPGs and eventually SSCs. Trial registration number Not applicable