Mouse Preimplantation Development Research Articles

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

NSUN5 is essential for proper cell proliferation and differentiation of mouse preimplantation embryos.

Proper early embryonic development in mammals relies on precise cellular signaling pathways. This study reveals that NSUN5 is crucial for the regulation of the Hippo pathway, ensuring normal proliferation and differentiation in mouse preimplantation embryos. NOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. Our data revealed an upregulation of Nsun5 at the two-cell stage of mouse preimplantation development, suggesting its significance in early embryonic development. Given m5C's important role in stabilizing rRNA and mRNA and the Hippo signaling pathway's critical function in lineage segregation during embryogenesis, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. To examine this hypothesis, we employed Nsun5-specific small interfering RNAs for targeted gene silencing in mouse preimplantation embryos. Nsun5 knockdown resulted in significant developmental impairments including reduced blastocyst formation, smaller size of blastocysts, and impaired hatching from the zona pellucida. Nsun5 knockdown also led to decreased cell numbers and increased apoptosis in embryos. We also observed diminished nuclear translocation of yes-associated protein 1 (YAP1) in Nsun5 knockdown embryos at the morula stage, indicating disrupted cell differentiation. This disruption was further evidenced by an altered ratio of CDX2-positive to OCT4-positive cells. Furthermore, Nsun5 depletion was found to upregulate the Hippo signaling-related key genes, Lats1 and Lats2 at the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway.

Influence of the storage conditions of embryo culture media on mouse development.

The culture of preimplantation embryos in vitro is an important method for human and mouse reproductive technology. This study aims to investigate the influence of different conditions of culture media on the preimplantation stage of mouse embryos cultured in vitro, and monitor the post-implantation development of new mice after embryo transfer to surrogate females. We demonstrated here that mouse embryos cultured in vitro in fresh M16, KSOM, Global, and HTF embryo culture media from one cell to the blastocyst stage and the subsequent embryo transfer to surrogate females are able to proceed through post-implantation development and, after birth, develop into healthy mice. However, culture of embryos in differently aged media shows various (often unpredictable) results. To find the optimal storage conditions of culture media, we suggest that the freezing and long-term storage of these media at - 80°C will not influence the quality of the media. To test this hypothesis, we grew embryos from one cell to blastocysts in vitro in the selected media after thawing and subsequently transferring them to surrogate females. Embryo culture in these four media after thawing does not affect preimplantation and postnatal mouse development. Thus, we have shown that storage of embryo culture media at low temperature (- 80°C) does not impact the quality of the media, and subsequently, it can be used for the culture of embryos for the full preimplantation period, the same as in fresh media.

ADNP modulates SINE B2-derived CTCF-binding sites during blastocyst formation in mice.

CTCF is crucial for chromatin structure and transcription regulation in early embryonic development. However, the kinetics of CTCF chromatin occupation in preimplantation embryos have remained unclear. In this study, we used CUT&RUN technology to investigate CTCF occupancy in mouse preimplantation development. Our findings revealed that CTCF begins binding to the genome prior to zygotic genome activation (ZGA), with a preference for CTCF-anchored chromatin loops. Although the majority of CTCF occupancy is consistently maintained, we identified a specific set of binding sites enriched in the mouse-specific short interspersed element (SINE) family B2 that are restricted to the cleavage stages. Notably, we discovered that the neuroprotective protein ADNP counteracts the stable association of CTCF at SINE B2-derived CTCF-binding sites. Knockout of Adnp in the zygote led to impaired CTCF binding signal recovery, failed deposition of H3K9me3, and transcriptional derepression of SINE B2 during the morula-to-blastocyst transition, which further led to unfaithful cell differentiation in embryos around implantation. Our analysis highlights an ADNP-dependent restriction of CTCF binding during cell differentiation in preimplantation embryos. Furthermore, our findings shed light on the functional importance of transposable elements (TEs) in promoting genetic innovation and actively shaping the early embryo developmental process specific to mammals.

TEAD4 regulates KRT8 and YAP in preimplantation embryos in mice but not in cattle.

Lineage specification plays a vital role in preimplantation development. TEAD4 is an essential transcription factor for trophectoderm lineage specification in mice but not in cattle. Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development.

Evidence for Functional Roles of MicroRNAs in Lineage Specification During Mouse and Human Preimplantation Development.

Proper formation of the blastocyst, including the specification of the first embryonic cellular lineages, is required to ensure healthy embryo development and can significantly impact the success of assisted reproductive technologies (ARTs). However, the regulatory role of microRNAs in early development, particularly in the context of preimplantation lineage specification, remains largely unknown. Taking a cross-species approach, this review aims to summarize the expression dynamics and functional significance of microRNAs in the differentiation and maintenance of lineage identity in both the mouse and the human. Findings are consolidated from studies conducted using in vitro embryonic stem cell models representing the epiblast, trophectoderm, and primitive endoderm lineages (modeled by naïve embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells, respectively) to provide insight on what may be occurring in the embryo. Additionally, studies directly conducted in both mouse and human embryos are discussed, emphasizing similarities to the stem cell models and the gaps in our understanding, which will hopefully lead to further investigation of these areas. By unraveling the intricate mechanisms by which microRNAs regulate the specification and maintenance of cellular lineages in the blastocyst, we can leverage this knowledge to further optimize stem cell-based models such as the blastoids, enhance embryo competence, and develop methods of non-invasive embryo selection, which can potentially increase the success rates of assisted reproductive technologies and improve the experiences of those receiving fertility treatments.

87 Determining the impact of CO2 concentration and pH on mouse pre-implantation embryo development

87 Determining the impact of CO2 concentration and pH on mouse pre-implantation embryo development

Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals.

Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos in vivo and in vitro. However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, which could be rescued by the addition of Lac-CoA and recapitulated by overexpression of H3K18R mutation. By profiling the landscape of H3K18lac during mouse preimplantation development, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and correlates with their expressions. In humans, H3K18lac is also enriched in ZGA markers and temporally concomitant with their expressions. Taken together, we profile the landscapes of H3K18lac in mouse and human preimplantation embryos, and demonstrate the important role for H3K18lac in major ZGA, showing that a conserved metabolic mechanism underlies preimplantation development of mammalian embryos.

MYC–MAX heterodimerization is essential for the induction of major zygotic genome activation and subsequent preimplantation development

In mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts in the embryo, begins in the S phase at the one-cell stage, with major ZGA occurring especially at the late two-cell stage. Myc is a transcription factor expressed in parallel with ZGA, but its direct association with major ZGA has not been clarified. In this study, we found that developmental arrest occurs at the two-cell stage when mouse embryos were treated with antisense oligonucleotides targeting Myc or MYC-specific inhibitors from the one-cell stage. To identify when MYC inhibition affects development, we applied time-limited inhibitor treatment and found that inhibition of MYC at the one-cell, four-cell, and morula stages had no effect on preimplantation development, whereas inhibitor treatment at the two-cell stage arrested development at the two-cell stage. Furthermore, transcriptome analysis revealed that when MYC function was inhibited, genes expressed in the major ZGA phase were suppressed. These results suggest that MYC is essential for the induction of major ZGA and subsequent preimplantation development. Revealing the function of MYC in preimplantation development is expected to contribute to advances in assisted reproductive technology.

P-802 TFAP2C is required for proper segregation of developmental markers regulating trophectoderm commitment during mouse preimplantation development

Abstract Study question What is the effect of CRISPR/Cas9-mediated knock-out of trophectoderm markers TFAP2A/C on polarization and lineage commitment in mouse? Summary answer CRISPR/Cas9-mediated KO revealed discrepancies in TFAP2A/C function, wherein TFAP2C is dispensable for blastocyst formation but controls the segregation of transcription factors in the mouse embryo. What is known already TFAP2C is an important transcription factor that is expressed solely in trophectoderm (TE) in mouse, and both TE and epiblast in human. Recently, genetic ablation of both TFAP2C and TEAD4 (player of the HIPPO-pathway) completely abolished polarization in mouse, which indicates that TFAP2C might already act before the first lineage segregation takes place. In addition, TFAP2C RNA depletion experiments in mouse embryos revealed a compensatory upregulation of TFAP2A, which indicates functional redundancy. Nonetheless, the question remains how exactly TFAP2C regulates polarization on a molecular level in mouse and whether there is also a functional implication of its isoform TFAP2A. Study design, size, duration Guide RNAs were designed targeting exon 5 of Tfap2c and exon 2 of the Tfap2a gene. CRISPR/Cas9 ribonucleoprotein complexes were delivered into mouse zygotes via electroporation. Additionally, appropriate non-targeted and scramble (inactive crRNA) control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing (NGS) were applied to check for gene editing efficiency and the impact of KO on embryonic development and lineage segregation. Participants/materials, setting, methods Targeted mouse embryos and controls were cultured for a maximum of 4.5 days in vitro. They were stained for different developmental markers, including CDX2 (TE), SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE) at different stages of development, such as polarization (E2.5), first (E3.5) and second (E4.5) lineage segregation. Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency. Main results and the role of chance CRISPR/Cas9-mediated electroporation of mouse zygotes generated efficiently complete knock-out embryos. Of the 39 mouse zygotes targeted for TFAP2C, 38 (97%) of them were edited. From the 38 embryos edited for TFAP2C, 24 (62%) embryos displayed 100% frameshift mutations, and were considered KO. TFAP2C KO mouse embryos cultured until E4.5 were still able to form blastocysts (13/15, 87%), but were associated with inferior blastocyst quality compared to the control groups. Furthermore, TFAP2c-null blastocysts could hatch but herniated at multiple places, in contrast to wild-type blastocysts, which typically herniate at one place. In full frameshift TFAP2C KO mouse embryos, we observed delayed CDX2 expression at E2.5 (n = 7). At E3.5 (n = 5), however, CDX2 expression could still be pertained in KO embryos, whereas nuclear localization of SOX2 was disturbed. At E4.5 (n = 6), some primitive endoderm cells were observed in the presumed ICM (SOX17-positive), whereas no NANOG-positive (EPI) cells could be detected. However, some cells of presumed TE of E4.5 TFAP2C-KO embryos displayed erroneous SOX17 or NANOG expression, indicating that they adopted another cell fate irrespective of cell localization. Secondly, we generated TFAP2A KO mouse embryos (3/5, 60%) which exhibited complete morula arrest (100%; n = 3) at E4.5. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mouse embryonic stem cells. The observations of the study will be consolidated by increasing the sample size. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the TFAP2-transcription factor family could improve our fundamental understanding on the spatial segregation of key transcription factors, which is crucial for successful implantation. Trial registration number Not applicable

O-058 Metabolism, histone modification and early embryo development

Abstract Cellular metabolism provides fundamental components for chromatin dynamics and epigenetics. However, metabolic features, and their impact on epigenetic remodeling during mammalian pre-implantation development, remain poorly understood. In this study, we established the metabolic landscape of mouse pre-implantation embryos from zygote to blastocyst, and quantified their absolute carbohydrate metabolites. We found that amino acid/carbohydrate/lipid metabolism increased during pre-implantation development, whereas nucleotide metabolism increased at the 4- and 8-cell stages. We further demonstrated that oxidized nicotinamide adenine dinucleotide (NAD+) is indispensable for mouse pre-implantation development. Mechanistically, NAD+ avoids excessive minor zygotic gene activation (ZGA) by cooperating with deacylase SIRT1 to remove zygotic H3K27ac. In human, NAD+ supplement can promote the removal of zygotic H3K27ac and benefit preimplantation development. Our findings demonstrate that precise and timely regulation of minor ZGA is controlled by metabolic dynamics, and enhance our understanding of the metabolism of mammalian early embryos.

Arsenite exposure disturbs maternal-to-zygote transition by attenuating H3K27ac during mouse preimplantation development

Arsenite is commonly used as an insecticide, antiseptic and herbicide. It can enter the food chain via through soil contamination, and harm human health, including the reproductive systems. Early embryos, as the initial stage of mammalian life, are very sensitive to the environmental toxins and pollutants. However, whether and how arsenite disturbs the early embryo development remains unclear. Our study used mouse early embryos as a model and revealed that arsenite exposure did not cause reactive oxygen species production, DNA damage or apoptosis. However, arsenite exposure arrested embryonic development at the 2-cell stage by altering gene expression patterns. The transcriptional profile in the disrupted embryos showed abnormal maternal-to-zygote transition (MZT). More importantly, arsenite exposure attenuated H3K27ac modification enrichment at the promoter region of Brg1, a key gene for MZT, which inhibited its transcription, and further affected MZT and early embryonic development. In conclusion our study highlights arsenite exposure affects MZT by reducing the enrichment of H3K27ac on the embryonic genome, and ultimately induces early embryonic development arrest at the 2-cell stage.

Reading and writing of mRNA m6A modification orchestrate maternal-to-zygotic transition in mice

N6-methyladenosine (m6A) modification has been shown to regulate RNA metabolism. Here, we investigate m6A dynamics during maternal-to-zygotic transition (MZT) in mice through multi-omic analysis. Our results show that m6A can be maternally inherited or de novo gained after fertilization. Interestingly, m6A modification on maternal mRNAs not only correlates with mRNA degradation, but also maintains the stability of a small group of mRNAs thereby promoting their translation after fertilization. We identify Ythdc1 and Ythdf2 as key m6A readers for mouse preimplantation development. Our study reveals a key role of m6A mediated RNA metabolism during MZT in mammals.

O3-4 - Effects of conditioned media from polarized macrophages on mouse pre-implantation embryonic development and outgrowth of blastocysts

O3-4 - Effects of conditioned media from polarized macrophages on mouse pre-implantation embryonic development and outgrowth of blastocysts

Cross-species comparison of mouse and human preimplantation development with an emphasis on lineage specification.

Human embryogenesis still remains largely unexplored. This review helps identify some of our current gaps in knowledge pertaining to preimplantation development, which may have implications for understanding fundamental aspects of human development, assisted reproductive technologies, and stem cell biology. Preimplantation development is arguably one of the most critical stages of embryogenesis. Beginning with the formation of the totipotent zygote post-fertilization, a series of cell divisions, and a complex coordination of physical cues, molecular signals and changes in gene expression lead to the formation of the blastocyst, a structure capable of implanting into the uterine wall. The blastocyst is composed of more specified cellular lineages, which will give rise to every tissue of the developing organism as well as the extra-embryonic lineages which support fetal growth. While the mouse has been used as a model to understand the events of preimplantation development for decades, in recent years, an expanding body of work has been conducted using the human embryo. These studies have identified some crucial species differences, particularly in the transcriptional and spatio-temporal expression of lineage markers and responses to cell signaling perturbations. This review compares recent findings on preimplantation development in mouse and human, with a focus on the specification of the first cellular lineages. Highlighting differences and noting mechanisms that require further examination in the human embryo is of critical importance for both the accurate translation of results from the mouse model and our overall understanding of mammalian development. We further highlight the latest advancement in reproductive research, the development of the 3D stem cell-based models known as 'blastoids'. The knowledge discussed in this review has major clinical implications for assisted reproductive technologies such as in vitro fertilization and for applications in stem cell biology.

Involvement of linker histone variant H1a in the regulation of early preimplantation development in mice.

Linker histone variants regulate higher-order chromatin structure and various cellular processes. It has been suggested that linker histone variant H1a loosens chromatin structure and activates transcription. However, its role in early mouse development remains to be elucidated. We investigated the functions of H1a during preimplantation development using H1a gene-deleted mice. Although H1a homozygous knockout (KO) mice were born without any abnormalities, the number of offspring were reduced when the mothers but not fathers were homozygous KO animals. Maternal H1a KO compromised development during the morula and blastocyst stages, but not differentiation of the inner cell mass or trophectoderm. Thus, maternal linker histone H1a is important in early development.

Expression patterns and biological function of BCL2L10 during mouse preimplantation development

BCL2-like 10 (BCL2L10) is abundantly expressed in mammalian oocytes and plays a crucial role in the completion of oocyte meiosis. However, the expression patterns of BCL2L10 and its biological functions during preimplantation development have not been well characterized. Here, we investigated the spatiotemporal expressions of Bcl2l10 during mouse preimplantation development using RT-qPCR and immunofluorescence and its biological function using siRNA and morpholino injection into pronuclear embryos. Results from RT-qPCR showed that Bcl2l10 was highly expressed in the metaphase Ⅱ-stage oocytes and pronuclear-stage embryos, but expression markedly decreased from the two-cell stage onwards and was no longer detected at the four-cell stage and beyond. Immunofluorescence staining showed that BCL2L10 was detectable throughout preimplantation development and localized in the cytoplasm and nuclei. Knocking down Bcl2l10 resulted in a reduced blastocyst formation rate (P < 0.01) and decreased expression of OCT4, NANOG, and SOX17 (P < 0.05). We concluded that the role of BCL2L10 is strongly associated with developmental competence of preimplantation mouse embryos.

Bisphenol Exposure Disrupts Cytoskeletal Organization and Development of Pre-Implantation Embryos.

The endocrine disrupting activity of bisphenol compounds is well documented, but less is known regarding their impact on cell division and early embryo formation. Here, we tested the effects of acute in vitro exposure to bisphenol A (BPA) and its common substitute, bisphenol F (BPF), during critical stages of mouse pre-implantation embryo development, including the first mitotic division, cell polarization, as well as morula and blastocyst formation. Timing of initial cleavage was determined by live-cell imaging, while subsequent divisions, cytoskeletal organization and lineage marker labeling were assessed by high-resolution fluorescence microscopy. Our analysis reveals that brief culture with BPA or BPF impeded cell division and disrupted embryo development at all stages tested. Surprisingly, BPF was more detrimental to the early embryo than BPA. Notably, poor embryo development was associated with cytoskeletal disruptions of the actomyosin network, apical domain formation during cell polarization, actin ring zippering for embryo sealing and altered cell lineage marker profiles. These results underscore that bisphenols can disrupt cytoskeletal integrity and remodeling that is vital for early embryo development and raise concerns regarding the use of BPF as a ‘safe’ BPA substitute.

Expression and localization of NRF2/Keap1 signalling pathway genes in mouse preimplantation embryos exposed to free fatty acids

Obese women experience greater incidence of infertility, with reproductive tracts exposing preimplantation embryos to elevated free fatty acids (FFA) such as palmitic acid (PA) and oleic acid (OA). PA treatment impairs mouse preimplantation development in vitro, while OA co-treatment rescues blastocyst development of PA treated embryos. In the present study, we investigated the effects of PA and OA treatment on NRF2/Keap1 localization, and relative antioxidant enzyme (Glutathione peroxidase; Gpx1, Catalase; Cat, Superoxide dismutase; Sod1 and γ-Glutamylcysteine ligase catalytic unit; Gclc) mRNA levels, during in vitro mouse preimplantation embryo development. Female mice were superovulated, mated, and embryos cultured in the presence of bovine Serum albumin (BSA) control or PA, or OA, alone (each at 100 μM) or PA + OA combined (each at 100 μM) treatment. NRF2 displayed nuclear localization at all developmental stages, whereas Keap1 primarily displayed cytoplasmic localization throughout control mouse preimplantation development in vitro. Relative transcript levels of Nrf2, Keap1, and downstream antioxidants significantly increased throughout control mouse preimplantation development in vitro. PA treatment significantly decreased blastocyst development and the levels of nuclear NRF2, while OA and PA + OA treatments did not. PA and OA treatments did not impact relative mRNA levels of Nrf2, Keap1, Gpx1, Cat, Sod1 or Gclc. Our outcomes demonstrate that cultured mouse embryos display nuclear NRF2, but that PA treatment reduces nuclear NRF2 and thus likely impacts NRF2/KEAP1 stress response mechanisms. Further studies should investigate whether free fatty acid effects on NRF2/KEAP1 contribute to the reduced fertility displayed by obese patients.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.