Mouse Embryo Cells Research Articles

Influence of FGF4 and BMP4 on FGFR2 dynamics during the segregation of epiblast and primitive endoderm cells in the pre-implantation mouse embryo.

Specification of the epiblast (EPI) and primitive endoderm (PE) in the mouse embryo involves fibroblast growth factor (FGF) signaling through the RAS/MAP kinase pathway. FGFR1 and FGFR2 are thought to mediate this signaling in the inner cell mass (ICM) of the mouse blastocyst and BMP signaling can also influence PE specification. In this study, we further explored the dynamics of FGFR2 expression through an enhanced green fluorescent protein (eGFP) reporter mouse line (FGFR2-eGFP). We observed that FGFR2-eGFP is present in the late 8-cell stage; however, it is absent or reduced in the ICM of early blastocysts. We then statistically correlated eGFP expression with PE and EPI markers GATA6 and NANOG, respectively. We detected that eGFP is weakly correlated with GATA6 in early blastocysts, but this correlation quickly increases as the blastocyst develops. The correlation between eGFP and NANOG decreases throughout blastocyst development. Treatment with FGF from the morula stage onwards did not affect FGFR2-eGFP presence in the ICM of early blastocysts; however, late blastocysts presented FGFR2-eGFP in all cells of the ICM. BMP treatment positively influenced FGFR2-eGFP expression and reduced the number of NANOG-positive cells in late blastocysts. In conclusion, FGFR2 is not strongly associated with PE precursors in the early blastocyst, but it is highly correlated with PE cells as blastocyst development progresses, consistent with the proposed role for FGFR2 in maintenance rather than initiating the PE lineage.

Glucose promoting the early embryonic development by increasing the lipid synthesis at 2-cell stage.

The optimization of culture conditions is one of the main strategies to improve the embryo development competence in in vitro fertilization (IVF). Glucose is an important carbon source while also exists in the oviductal fluid in vivo, the effect of glucose in embryo development microenvironment is still unclear. Here we employed the LC-MS to detect and analyze the metabolites in the culture medium of different cleavage stages including 2-Cell, 4-Cell and 8-Cell mouse embryos, respectively. The effects of the external glucose were estimated by measuring the development rate at different glucose concentrations from 0 to 5mmol/L, and the gene expression changes were detected to explore the potential mechanism after the addition of glucose in the media. Our results indicated the 2-Cell and 8-Cell stages had defined characteristic metabolites, while 4-Cell stage was the transition state. Global and contiguous metabolic characteristics showed the glycometabolism play a critical role at each early cleavage stages during the embryo development. The 8-Cell rates demonstrated the addition of glucose in culture media significantly improve the embryo competence, the highest rate was 87.33% using 3mmol/L glucose in media, in contrast only 9.95% using the media without glucose. Meanwhile, the blocked embryos were mainly enriched at 2-Cell stage. Further transcriptome study found 3mmol/L glucose in media remarkably upregulated the gene expression of lipid biosynthesis at 2-Cell stage, the increased lipid was confirmed by nile red staining. These data indicated the glucose may promote the development competence through increasing the lipid biosynthesis to overcoming the 2-Cell block. Our findings were helpful for the further optimization of IVF culture media, as well as the estimation of embryo quality using metabolites in the culture media.

Single cell Hi-C identifies plastic chromosome conformations underlying the gastrulation enhancer landscape

Embryonic development involves massive proliferation and differentiation of cell lineages. This must be supported by chromosome replication and epigenetic reprogramming, but how proliferation and cell fate acquisition are balanced in this process is not well understood. Here we use single cell Hi-C to map chromosomal conformations in post-gastrulation mouse embryo cells and study their distributions and correlations with matching embryonic transcriptional atlases. We find that embryonic chromosomes show a remarkably strong cell cycle signature. Despite that, replication timing, chromosome compartment structure, topological associated domains (TADs) and promoter-enhancer contacts are shown to be variable between distinct epigenetic states. About 10% of the nuclei are identified as primitive erythrocytes, showing exceptionally compact and organized compartment structure. The remaining cells are broadly associated with ectoderm and mesoderm identities, showing only mild differentiation of TADs and compartment structures, but more specific localized contacts in hundreds of ectoderm and mesoderm promoter-enhancer pairs. The data suggest that while fully committed embryonic lineages can rapidly acquire specific chromosomal conformations, most embryonic cells are showing plastic signatures driven by complex and intermixed enhancer landscapes.

P-127 Micro-Magnetic Resonance Spectroscopy of individual mammalian embryos: a safe and non-invasive diagnostic tool for embryo screening in assisted reproduction

Abstract Study question Is micro-magnetic resonance (micro-MRS) a safe tool to non-invasively unravel the metabolic fingerprint of single mammalian embryos? Summary answer We successfully tested the safety of micro-MRS in a mouse model. No long or short-term adverse effect was found in vitro and in vivo. What is known already Non-invasive selection of the best embryo to transfer is one of the most significant challenges for ART professionals. The chemical sensitivity, resolving power, and, more importantly, the non-invasive nature of MRS makes it an excellent candidate to investigate the building blocks of complex organisms. Although MRS is a well-established technique for the biochemical profiling of large organisms, handling small samples like embryos and 3D cell cultures and spheroids alongside sensitivity issues has prevented its adoption for clinical and research applications. Our group has overcome these limitations with microchip-based sensors to leverage non-invasive MRS technology down to the embryo scale. Study design, size, duration This safety study was divided into two main phases in order to test all the components needed to operate the micro-MRS analysis. In phase 1 we used > 800 2-cell embryos to test a) radiofrequency exposure, b) device activation, and c) static magnetic field exposure. In phase 2 we confirmed in-vivo that MF exposure was not affecting live animals (n267) over 3 generations of mice by assessing different IVF outcomes, natural mating, live parameters and histopathology. Participants/materials, setting, methods Cryopreserved 2-cell B6D2F1 mouse embryos were purchased cryopreserved from JanvierLabs. In phase1 embryos were thawed and used for testing micro-MRS main components' safety. Mouse embryo assay (MEA) was used to first assess micro-MRS adverse effects in-vitro. In phase 2 embryos were exposed to magnetic field in a 9.4T Magnet for 1h at 37 °C, then surgically transferred to surrogate mothers. Both mothers and progeny were tracked up to F3. Statistical significance was set at p < 0.05. Main results and the role of chance This is the first study ever conducted to assess the safety and efficacy of micro-MRS on pre-implantation mammalian embryos. Our results show, with statistical significance, that micro-MRS is safe for use in mammalians. In particular, we have de-risked the three main aspects/components that could affect the embryos: 1) Materials to which the embryos are exposed when loaded into the device, 2) Radiofrequency energy required to activate the device and perform a measurement, and 3) Magnetic field required to analyze the embryos biochemistry in a non-invasive way. Overall no adverse effect was observed when the primary IVF outcomes were assessed, such as implantation rate and live birth rate. In addition, no visible alteration of the overall appearance of ED14.5 foetuses, F1, F2 and F3 mice was observed when comparing the mice obtained from treated or control embryos. The surrogate mothers did not display any significant alteration or visible health issues after embryo transfer. F1 and F2 mice were able to naturally mate and conceive new progeny suggesting that the reproductive potential of the animals is not affected by the magnetic field exposure. Furthermore, histopathological analysis of 5 representative organs did not reveal adverse effects on both surrogate mothers or F1 pups. Limitations, reasons for caution A vital step towards establishing MRS as a clinical and research tool is the reliable detection of a wide range of signals from cellular components. Our method is ready for R&D studies, while its clinical application requires further safety studies and protocol optimization. Wider implications of the findings A non-invasive quick assay would provide the means to reveal the role of embryonic lipids throughout development. Micro-MRS can further develop into a safe embryo assay for selection before embryo transfer. This would apply to both human and animal ART, whose success rate is relatively low. Trial registration number NA

Antiproliferative Activity and Impact on Human Gut Microbiota of New O-Alkyl Derivatives of Naringenin and Their Oximes.

Naringenin is a 5,7,4'-trihydroxyflavanone naturally occurring mainly in citrus fruits, characterized by a wide spectrum of biological activity. Chemical modifications based on alkylation and oximation in most cases increase its bioactivity. The aim of our research was to evaluate the antiproliferative activity and influence on selected representatives of the human gut microbiota of new synthesized O-alkyl derivatives (A1-A10) and their oximes (B1-B10), which contain hexyl, heptyl, octyl, nonyl and undecyl chains attached to the C-7 or to both the C-7 and C-4' positions in naringenin. To the best of our knowledge, compounds A3, A4, A6, A8-A10 and B3-B10 have not been described in the scientific literature previously. The anticancer activity was tested on human colon cancer cell line HT-29 and mouse embryo fibroblasts 3T3-L1 using the sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. We also determined the impacts of all compounds on the growth of Gram-positive and Gram-negative bacterial strains, such as Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. The antimicrobial activity was expressed in terms of minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) values. For 7,4'-di-O-hexylnaringenin (A2), 7-O-undecylnaringenin (A9) and their oximes (B2, B9), which were safe for microbiota (MIC > 512 µg/mL) and almost all characterized by high cytotoxicity against the HT-29 cell line (A2: IC50 > 100 µg/mL; A9: IC50 = 17.85 ± 0.65 µg/mL; B2: IC50 = 49.76 ± 1.63 µg/mL; B9: IC50 = 11.42 ± 1.17 µg/mL), apoptosis assays were performed to elucidate their mechanisms of action. Based on our results, new compound B9 induced an apoptotic process via caspase 3/7 activation, which proved its potential as an anticancer agent.

Antibacterial and antibiofilm activity of permanently ionized quaternary ammonium fluoroquinolones

A series of quaternary ammonium fluoroquinolones was obtained by exhaustive methylation of the amine groups present at the 7-position of fluoroquinolones, including ciprofloxacin, enoxacin, gatifloxacin, lomefloxacin, and norfloxacin. The synthesized molecules were tested for their antibacterial and antibiofilm activities against Gram-positive and Gram-negative human pathogens, i.e. Staphylococcus aureus and Pseudomonas aeruginosa. The study showed that the synthesized compounds are potent antibacterial agents (MIC values at the lowest 6.25 μM) with low cytotoxicity in vitro as assessed on the BALB 3T3 mouse embryo cell line. Further experiments proved that the tested derivatives are able to bind to the DNA gyrase and topoisomerase IV active sites in a fluoroquinolone-characteristic manner. The most active quaternary ammonium fluoroquinolones, in contrast to ciprofloxacin, reduce the total biomass of P. aeruginosa ATCC 15442 biofilm in post-exposure experiments. The latter effect may be due to the dual mechanism of action of the quaternary fluoroquinolones, which also involves disruption of bacterial cell membranes. IAM-HPLC chromatographic experiments with immobilized artificial membranes (phospholipids) showed that the most active compounds were those with moderate lipophilicity and containing a cyclopropyl group at the N1 nitrogen atom in the fluoroquinolone core.

Estrogen signaling encourages blastocyst development and implantation potential.

Estrogen is well-known for preparing uterine receptivity. However, its roles in regulating embryo development and implantation are unclear. Our objective was to characterize estrogen receptor 1 (ESR1) in human and mouse embryos and determine the effect of estradiol (E2) supplementation on pre- and peri-implantation blastocyst development. Mouse embryos, 8-cell through hatched blastocyst stages, and human embryonic days 5-7 blastocysts were stained for ESR1 and imaged using confocal microscopy. We then treated 8-cell mouse embryos with 8nM E2 during in vitro culture (IVC) and examined embryo morphokinetics, blastocyst development, and cell allocation into the inner cell mass (ICM) and trophectoderm (TE). Finally, we disrupted ESR1, using ICI 182,780, and evaluated peri-implantation development. ESR1 exhibits nuclear localization in early blastocysts followed by aggregation, predominantly in the TE of hatching and hatched blastocysts, in human and mouse embryos. During IVC, most E2 was absorbed by the mineral oil, and no effect on embryo development was found. When IVC was performed without an oil overlay, embryos treated with E2 exhibited increased blastocyst development and ICM:TE ratio. Additionally, embryos treated with ICI 182,780 had significantly decreased trophoblast outgrowth during extended embryo culture. Similar ESR1 localization in mouse and human blastocysts suggests a conserved role in blastocyst development. These mechanisms may be underappreciated due to the use of mineral oil during conventional IVC. This work provides important context for how estrogenic toxicants may impact reproductive health and offers an avenue to further optimize human-assisted reproductive technology (ART) to treat infertility.

Acute exposure of triclocarban affects early embryo development in mouse through disrupting maternal-to-zygotic transition and epigenetic modifications

Triclocarban (TCC) is a broad-spectrum antibacterial agent used globally, and high concentrations of this harmful chemical exist in the environment. The human body is directly exposed to TCC through skin contact. Moreover, TCC is also absorbed through diet and inhaled through breathing, which results in its accumulation in the body. The safety profile of TCC and its potential impact on human health are still not completely clear; therefore, it becomes imperative to evaluate the reproductive toxicity of TCC. Here, we explored the effect of TCC on the early embryonic development of mice and its associated mechanisms. We found that acute exposure of TCC affected the early embryonic development of mice in a dose-dependent manner. Approximately 7600 differentially expressed genes (DEGs) were obtained by sequencing the transcriptome of 2-cell mouse embryos; of these, 3157 genes were upregulated and 4443 genes were downregulated in the TCC-treated embryos. GO and KEGG analysis revealed that the enriched genes were mainly involved in redox processes, RNA synthesis, DNA damage, apoptosis, mitochondria, endoplasmic reticulum, Golgi apparatus, cytoskeleton, peroxisome, RNA polymerase, and other components or processes. Moreover, the Venn analysis showed that the zygotic genome activation (ZGA) was affected and the degradation of maternal effector genes was inhibited. TCC induced changes in the epigenetic modification of 2-cell embryos. The level of DNA methylation increased significantly. Further, the levels of H3K27ac, H3K9ac, and H3K27me3 histone modifications decreased significantly, whereas those of H3K4me3 and H3K9me3 modifications increased significantly. Additionally, TCC induced oxidative stress and DNA damage in the 2-cell embryos. In conclusion, acute exposure of TCC affected early embryo development, destroyed early embryo gene expression, interfered with ZGA and maternal gene degradation, induced changes in epigenetic modification of early embryos, and led to oxidative stress and DNA damage in mouse early embryos.

Glycolytic flux-signaling controls mouse embryo mesoderm development.

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

Metallic Nanoparticle-Doped Oxide Semiconductor Film for Bone Tumor Suppression and Bone Regeneration.

Bone implants with the photothermal effect are promising for the treatment of bone tumor defects. Noble metal-based photothermal nanoagents are widely studied for their stable photothermal effect, but they are expensive and difficult to directly grow on implant surfaces. In contrast, non-noble metal photothermal nanoagents are economical but unstable. Herein, to develop a stable and economical photothermal film on bone implants, a Ni nanoparticle-doped oxide semiconductor film was grown in situ on Nitinol via the reduction of Ni-Ti-layered double hydroxides. Ni nanoparticles remained stable in the NiTiO3 structure even when immersed in fluid for 1 month, and thus, the film presented a reliable photothermal effect under near-infrared light irradiation. The film also showed excellent in vitro and in vivo antitumor performance. Moreover, the nanostructure on the film allowed bone differentiation of mouse embryo cells (C3H10T1/2), and the released Ni ions supported the angiogenesis behavior of human vein endothelial cells. Bone implantation experiments further showed the enhancement of osteointegration of the modified Nitinol implant in vivo. This novel multifunctional Nitinol bone implant design offers a promising strategy for the therapy of bone tumor-related defects.

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station.

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at -80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

Shoutai Wan Improves Embryo Survival by Regulating Aerobic Glycolysis of Trophoblast Cells in a Mouse Model of Recurrent Spontaneous Abortion.

Background During embryo implantation, the blastocyst exhibits a high capacity for aerobic glycolysis, which results in a unique microenvironment of high lactate/low pH at the maternal-fetal interface. Shoutai Wan (STW) is an effective Chinese herbal formula widely used in the clinical treatment of recurrent spontaneous abortion (RSA). However, the specific molecular mechanism by which STW prevents abortion is yet to be elucidated. Methods Female CBA/J mice were allocated into six groups randomly and then mated with BALB/c mice as the control group, DBA/2 mice as the RSA model, CBA/J×DBA/2 mice treated with dydrogesterone as the DQYT group, or CBA/J×DBA/2 mice treated with low, medium, and high-dose STW as the STW-L, STW-M, and STW-H groups, respectively. Drug administration started 14 days before mating and ended on the 14th day of pregnancy. The embryo loss rate of each group was calculated on day 14 of gestation, and the pregnancy outcomes of the mice in each group were observed. The mouse serum was collected to determine the levels of progesterone (P) and chorionic gonadotropin (CG). The activities of HK2, PKM2, and LDHA, the key glycolytic enzymes in each group, were detected. The expressions of lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, and GPR81 as well as the morphology of trophoblast cells were examined. Results The embryo loss rate and adverse pregnancy outcomes were significantly increased (P < 0.05) in the RSA model group. After dydrogesterone or different doses of STW treatment, the embryo loss rate and adverse pregnancy outcomes were rescued to varying degrees (P < 0.05). Interestingly, there was no significant difference among the groups in terms of serum P and CG (P < 0.05). Moreover, the activities of key glycolytic enzymes, lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, GPR81 protein or mRNA expression, and morphological abnormalities of trophoblast cells improved significantly in the RSA mice after dydrogesterone or different doses of STW treatment (P < 0.05). Conclusion STW can promote aerobic glycolysis in trophoblast cells of RSA mouse embryos, thereby improving the microenvironment of the maternal-fetal interface and enhancing embryo implantation.

Effects of nylon mesh vitrification on the cytoskeleton, mitochondria, and aquaporins of mouse 2-cell embryos.

Effects of nylon mesh vitrification on the cytoskeleton, mitochondria, and aquaporins of mouse 2-cell embryos.

Stepwise pluripotency transitions in mouse stem cells.

Pluripotent cells in mouse embryos, which first emerge in the inner cell mass of the blastocyst, undergo gradual transition marked by changes in gene expression, developmental potential, polarity, and morphology as they develop from the pre-implantation until post-implantation gastrula stage. Recent studies of cultured mouse pluripotent stem cells (PSCs) have clarified the presence of intermediate pluripotent stages between the naïve pluripotent state represented by embryonic stem cells (ESCs-equivalent to the pre-implantation epiblast) and the primed pluripotent state represented by epiblast stem cells (EpiSCs-equivalent to the late post-implantation gastrula epiblast). In this review, we discuss these recent findings in light of our knowledge on peri-implantation mouse development and consider the implications of these new PSCs to understand their temporal sequence and the feasibility of using them as model system for pluripotency.

Cell fate determining molecular switches and signaling pathways in Pax7-expressing somitic mesoderm

During development, different cell types originate from a common progenitor at well-defined time points. Previous lineage-tracing of Pax7+ progenitors from the somitic mesoderm has established its developmental trajectory towards the dermis, brown adipocytes, and skeletal muscle in the dorsal trunk; yet the molecular switches and mechanisms guiding the differentiation into different lineages remain unknown. We performed lineage-tracing of Pax7-expressing cells in mouse embryos at E9.5 and profiled the transcriptomes of Pax7-progenies on E12.5, E14.5, and E16.5 at single-cell level. Analysis of single-cell transcriptomic data at multiple time points showed temporal-specific differentiation events toward muscle, dermis, and brown adipocyte, identified marker genes for putative progenitors and revealed transcription factors that could drive lineage-specific differentiation. We then utilized a combination of surface markers identified in the single-cell data, Pdgfra, Thy1, and Cd36, to enrich brown adipocytes, dermal fibroblasts, and progenitors specific for these two cell types at E14.5 and E16.5. These enriched cell populations were then used for further culture and functional assays in vitro, in which Wnt5a and Rgcc are shown to be important factors that could alter lineage decisions during embryogenesis. Notably, we found a bipotent progenitor population at E14.5, having lineage potentials towards both dermal fibroblasts and brown adipocytes. They were termed eFAPs (embryonic fibro/adipogenic progenitors) as they functionally resemble adult fibro/adipogenic progenitors. Overall, this study provides further understanding of the Pax7 lineage during embryonic development using a combination of lineage tracing with temporally sampled single-cell transcriptomics.

Major construction entails major demolition.

Embryonic cells of the early mouse embryo become hypersensitive to apoptotic stimuli before gastrulation. In this issue of Developmental Cell, Pernaute etal. show that this switch in sensitivity is a result of a change in mitochondrial dynamics and mitophagy levels controlled by DRP1, a regulator of mitochondrial fission.

Derivation of totipotent-like stem cells with blastocyst-like structure forming potential

It is challenging to derive totipotent stem cells in vitro that functionally and molecularly resemble cells from totipotent embryos. Here, we report that a chemical cocktail enables the derivation of totipotent-like stem cells, designated as totipotent potential stem (TPS) cells, from 2-cell mouse embryos and extended pluripotent stem cells, and that these TPS cells can be stably maintained long term in vitro. TPS cells shared features with 2-cell mouse embryos in terms of totipotency markers, transcriptome, chromatin accessibility and DNA methylation patterns. In vivo chimera formation assays show that these cells have embryonic and extraembryonic developmental potentials at the single-cell level. Moreover, TPS cells can be induced into blastocyst-like structures resembling preimplantation mouse blastocysts. Mechanistically, inhibition of HDAC1/2 and DOT1L activity and activation of RARγ signaling are important for inducing and maintaining totipotent features of TPS cells. Our study opens up a new path toward fully capturing totipotent stem cells in vitro.

Expanding the Editing Window of Cytidine Base Editors With the Rad51 DNA-Binding Domain in Rice.

Recently developed base editors provide a powerful tool for plant research and crop improvement. Although a number of different deaminases and Cas proteins have been used to improve base editors the editing efficiency, and editing window are still not optimal. Fusion of a non-sequence-specific single-stranded DNA-binding domain (DBD) from the human Rad51 protein between Cas9 nickase and the deaminase has been reported to dramatically increase the editing efficiency and expand the editing window of base editors in the mammalian cell lines and mouse embryos. We report the use of this strategy in rice, by fusing a rice codon-optimized human Rad51 DBD to the cytidine base editors AncBE4max, AncBE4max-NG, and evoFERNY. Our results show that the addition of Rad51 DBD did not increase editing efficiency in the major editing window but the editing range was expanded in all the three systems. Replacing the human Rad51 DBD with the rice Rad51 DBD homolog also expanded the editing window effectively.

Cell size and polarization determine cytokinesis furrow ingression dynamics in mouse embryos

Cytokinesis is the final step of cell division during which a contractile ring forms a furrow that partitions the cytoplasm in two. How furrow ingression is spatiotemporally regulated and how it is adapted to complex cellular environments and developmental transitions remain poorly understood. Here, we examine furrow ingression dynamics in the context of the early mouse embryo and find that cell size is a powerful determinant of furrow ingression speed during reductive cell divisions. In addition, the emergence of cell polarity and the assembly of the apical domain in outer cells locally inhibits the recruitment of cytokinesis components and thereby negatively regulates furrow ingression specifically on one side of the furrow. We show that this biasing of cytokinesis is not dependent upon cell–cell adhesion or shape but rather is cell intrinsic and is caused by a paucity of cytokinetic machinery in the apical domain. The results thus reveal that in the mouse embryo cell polarity directly regulates the recruitment of cytokinetic machinery in a cell-autonomous manner and that subcellular organization can instigate differential force generation and constriction speed in different zones of the cytokinetic furrow.

Transcription of rRNA in early mouse embryos promotes chromatin reorganization and expression of major satellite repeats

During the first cell cycles of early development, the chromatin of the embryo is highly reprogrammed while the embryonic genome starts its own transcription. The spatial organization of the genome is an important process that contributes to regulating gene transcription in time and space. It has, however, been poorly studied in the context of early embryos. To study the cause-and-effect link between transcription and spatial organization in embryos, we focused on ribosomal genes, which are silent initially but start to be transcribed in 2-cell mouse embryos. We demonstrated that ribosomal sequences and early unprocessed rRNAs are spatially organized in a very particular manner between 2-cell and 16-cell stage. By using drugs that interfere with ribosomal DNA transcription, we showed that this organization - which is totally different in somatic cells - depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors transcription of major satellite sequences once the 4-cell stage has been reached.