Early Embryo Development Research Articles

Exploring the Impact of Controlled Ovarian Stimulation and Non-Invasive Oocyte Assessment in ART Treatments.

Invasive and noninvasive features are normally applied to select developmentally competent oocytes and embryos that can increase the take-home baby rates in assisted reproductive technology. The noninvasive approach mainly applied to determine oocyte and embryo competence has been, since the early days of IVF, the morphological evaluation of the mature cumulus-oocyte complex at the time of pickup, first polar body, zona pellucida thickness, perivitelline space and cytoplasm appearance. Morphological evaluation of oocyte quality is one of the options used to predict successful fertilization, early embryo development, uterine implantation and the capacity of an embryo to generate a healthy pregnancy to term. Thus, this paper aims to provide an analytical revision of the current literature relating to the correlation between ovarian stimulation procedures and oocyte/embryo quality. In detail, several aspects of oocyte quality such as morphological features, oocyte competence and its surrounding environment will be discussed. In addition, the main noninvasive features as well as novel approaches to biomechanical parameters of oocytes that might be correlated with the competence of embryos to produce a healthy pregnancy and live birth will be illustrated.

Molecular characterizations and functional roles of NANOG in early development of porcine embryos

Nanog homeobox (NANOG) is the gateway to the pluripotent ground state in mouse embryonic stem cells and early embryos. However, understanding of the molecular signatures and functional characteristics of porcine NANOG remains limited. In this study, we analyzed the gene structure and sequence characteristics of porcine NANOG and found that the porcine NANOG gene is localized on chromosome 5, while NANOG sequence on chromosome 1 is the processed pseudogene. We explored the expression pattern of NANOG in porcine early embryos by immunofluorescence staining and Realtime-PCR and RNA-seq, the results showed that transcription of porcine NANOG commences at the 4-cell stage, while expression of the NANOG protein is initially observed in the inner cell mass of blastocysts. Furthermore, we identified a NANOG splicing variant in porcine early embryos, which maintain the overall structure of the original NANOG mRNA, except for a deletion of 38 base pairs in the second exon. To further investigate the function of NANOG in early embryo development in pigs, we employed siRNA-mediated deletion of the two specific transcripts on porcine zygotes. The results showed that blastocyst rate was significantly reduced after NANOG deleting. A significant decrease in the expression of DNA methylation-related gene DNMT3B was also observed in D3 embryo from the NANOG deleting group. In conclusion, the porcine NANOG gene, accompanied by a single-exon processed pseudogene, exhibits two transcripts and plays a pivotal role in the development of early-stage embryos.

Modeling unveils sex differences of signaling networks in mouse embryonic stem cells.

For a short period during early development of mammalian embryos, both X chromosomes in females are active, before dosage compensation is ensured through X-chromosome inactivation. In female mouse embryonic stem cells (mESCs), which carry two active X chromosomes, increased X-dosage affects cell signaling and impairs differentiation. The underlying mechanisms, however, remain poorly understood. To dissect X-dosage effects on the signaling network in mESCs, we combine systematic perturbation experiments with mathematical modeling. We quantify the response to a variety of inhibitors and growth factors for cells with one (XO) or two X chromosomes (XX). We then build models of the signaling networks in XX and XO cells through a semi-quantitative modeling approach based on modular response analysis. We identify a novel negative feedback in the PI3K/AKT pathway through GSK3. Moreover, the presence of a single active X makes mESCs more sensitive to the differentiation-promoting Activin A signal and leads to a stronger RAF1-mediated negative feedback in the FGF-triggered MAPK pathway. The differential response to these differentiation-promoting pathways can explain the impaired differentiation propensity of female mESCs.

Adverse effects of plastic leachate and its component 2,4-DTBP on the early development of zebrafish embryos

Plastic waste has become a global environmental problem threatening the health of aquatic organisms especially via leachate. In this study, the test of zebrafish embryo showed adverse effects of leachate from some agricultural mulching films after UV light aging for 60 h. A typical phenolic antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) was detected in the leachate and tested further for the zebrafish embryo biotoxicity. The microplastic leachate (6, 8 g/L, mass concentration measured by weight of plastic) increased the death and malformation rates, and reduced the hatching rate, heart rate, and body length of zebrafish larvae in the 96-hour early development period. Similar adverse effects were also caused by the 2,4-DTBP (0.01, 0.1, 1.0 mg/L, corresponding to 0.049, 0.49, and 4.85 μM) to some degree but could not completely explain the significant influences caused by the plastic leachate. Transcriptome analysis of zebrafish embryos exposed to the 2,4-DTBP for 96 h showed that the protein, fat, and carbohydrate digestion and absorption pathways, pancreatic secretion, PPAR signaling pathway, tryptophan metabolism, and adipocytokine signaling pathway were considerably down-regulated, but the cholesterol metabolism pathway was up-regulated in larval zebrafish. The altered transcriptional expression of mRNA at early development stage (96 h post fertilization) of zebrafish suggested that the 2,4-DTBP caused reduction of digestive capacity and pancreatic secretory function, and adversely affected processes associated with energy metabolism and glycolipid metabolism of larval zebrafish. This study helps us further understanding the effects of plastic leachate on the early development of fishes.

Sperm-borne lncRNA loc100847420 improves development of early bovine embryos

Long non-coding RNAs (lncRNAs) act as competing endogenous RNAs (ceRNAs) that play a significant role in bovine embryo development; but the influence of sperm-borne lncRNA on the preimplantation development of bovine embryos has not been reported in detail. In this study, we aimed to clarify how sperm-borne lncRNAs can act to regulate early development of bovine embryos. Utilizing high-throughput sequencing technology and quantitative real-time PCR (qPCR), we found that the lncRNA, loc100847420, was highly enriched in bovine sperm and was carried into the oocyte during fertilization. Introduction of wild-type loc100847420 had no effect on cleavage rate of parthenogenetic embryos compared with injection of mutant loc100847420 (70.58 ± 2.85% vs 70.46 ± 1.98%, p > 0.05), but significantly improved the blastocyst rate (33.67 ± 2.40% vs 28.35 ± 3.06%, p < 0.05), total numbers of cells (p < 0.05), numbers of inner cell mass (ICM) cells (p < 0.05) and numbers of trophoblast (TE) cells (p < 0.05). In summary, the sperm-borne lncRNA, loc100847420, can improve the developmental potential of early bovine embryos.

Specc1l deficiency leads to abnormal oocyte meiosis and reduced blastocyst development in mouse.

This study aimed to investigate the potential role of the mouse Specc1l (Sperm antigen with calponin homology and coiled-coil domains 1 like), a microtubule and actin cytoskeleton-associated protein during oocyte meiotic maturation and its potential effects on preimplantation development. This study shows that the transcriptional levels of Specc1l did not significantly change from the germinal vesicle (GV) stage to the metaphase II (MII) stage, but maternal transcripts rapidly and gradually degraded after fertilization. Specc1l was detected in both the cytoplasm and GV, but not in the nucleolus-like body in the GV intact oocyte. At the MII stages, Specc1l was widely distributed in the cytoplasm but did not co-localize with chromatin. Knockdown of Specc1l expression in oocytes resulted in abnormal spindle morphology and misaligned chromosomes, as well as a decrease in the rate of polar body extrusion and a reduced developmental competence of oocytes, leading to decreased blastocyst formation rate. In conclusion, this study provides evidence that Specc1l plays a critical role in mouse oocyte meiotic maturation and early embryo development, specifically in proper bipolar spindle assembly and extrusion of the first polar body.

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle-dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased invitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production invitro.

KLF4 facilitates chromatin accessibility remodeling in porcine early embryos.

Chromatin accessibility remodeling driven by pioneer factors is critical for the development of early embryos. Current studies have illustrated several pioneer factors as being important for agricultural animals, but what are the pioneer factors and how the pioneer factors remodel the chromatin accessibility in porcine early embryos is not clear. By employing low-input DNase-seq (liDNase-seq), we profiled the landscapes of chromatin accessibility in porcine early embryos and uncovered a unique chromatin accessibility reprogramming pattern during porcine preimplantation development. Our data revealed that KLF4 played critical roles in remodeling chromatin accessibility in porcine early embryos. Knocking down of KLF4 led to the reduction of chromatin accessibility in early embryos, whereas KLF4 overexpression promoted the chromatin openness in porcine blastocysts. Furthermore, KLF4 deficiency resulted in mitochondrial dysfunction and developmental failure of porcine embryos. In addition, we found that overexpression of KLF4 in blastocysts promoted lipid droplet accumulation, whereas knockdown of KLF4 disrupted this process. Taken together, our study revealed the chromatin accessibility dynamics and identified KLF4 as a key regulator in chromatin accessibility and cellular metabolism during porcine preimplantation embryo development.

Depletion of TIAR impairs embryogenesis via inhibiting zygote genome transcribe.

T cell intracellular antigen 1 related protein (TIAR), an RNA-binding protein (RBP), regulates pre-messenger RNA (pre-mRNA) alternative splicing, has been suggested to affect the maturation of primordial germ cells and early mouse embryo development. However, the underlying mechanism remains elusive. In this study, we revealed that TIAR was primarily located in the nucleus at the 2-cell stage embryo, accompanied by highly active transcription. Using immunofluorescence staining and western blotting, we first described the localization and expression level of TIAR during the whole period of oocyte matured and embryogenesis. Knocked down of TIAR could significantly inhibit transcribed and blocked the early mouse embryo development. Combined with RNAP II inhibitor and pre-RNA splicing inhibitor treatment, we further supposed that TIAR might affect transcription at 2-cell via regulating pre-mRNA splicing, and then regulate early mouse embryo development. Collectively, our results provided a novel and potential understanding of TIAR in embryogenesis, suggesting TIAR is required for transcription and embryonic development.

Loss of ACTL7A causes small head sperm by defective acrosome-acroplaxome-manchette complex

BackgroundActin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. In this study, we aim to explore the additional functions of ACTL7A beyond the process of acrosome biogenesis and investigate the possible underlying mechanisms.MethodsNuclear morphology analysis was used to observe the sperm head shape of ACTL7A-mutated patients. Actl7a knock-out (KO) mouse model was generated. Immunofluorescence and transmission electron microscopy (TEM) were performed to analyze the structure of spermatids during spermiogenesis. Tandem mass tags labeling quantitative proteomics strategy was employed to explore the underlying molecular mechanisms. The expression levels of key proteins in the pathway were analyzed by western blotting. Intracytoplasmic sperm injection (ICSI)-artificial oocyte activation (AOA) technology was utilized to overcome fertilization failure in male mice with a complete knockout of Actl7a.ResultsThe new phenotype of small head sperm associated with loss of ACTL7A in patients was discovered, and further confirmed in Actl7a-KO mice. Immunofluorescence and TEM analyses revealed that the deletion of ACTL7A damaged the formation of acrosome-acroplaxome-manchette complex, leading to abnormalities in the shaping of sperm heads. Moreover, a proteomic analysis of testes from WT and Actl7a-KO mice revealed that differentially expressed genes were notably enriched in PI3K/AKT/mTOR signaling pathway which is strongly associated with autophagy. Inhibition of autophagy via PI3K/AKT/mTOR signaling pathway activation leading to PDLIM1 accumulation might elucidate the hindered development of manchette in Actl7a-KO mice. Remarkably, AOA successfully overcame fertilization failure and allowed for the successful production of healthy offspring from the Actl7a complete knockout male mice.ConclusionsLoss of ACTL7A causes small head sperm as a result of defective acrosome-acroplaxome-manchette complex via autophagy inhibition. ICSI-AOA is an effective technique to rescue male infertility resulting from ACTL7A deletion. These findings provide essential evidence for the diagnosis and treatment of patients suffering from infertility.

TimeTalk uses single-cell RNA-seq datasets to decipher cell-cell communication during early embryo development

Early embryonic development is a dynamic process that relies on proper cell-cell communication to form a correctly patterned embryo. Early embryo development-related ligand-receptor pairs (eLRs) have been shown to guide cell fate decisions and morphogenesis. However, the scope of eLRs and their influence on early embryo development remain elusive. Here, we developed a computational framework named TimeTalk from integrated public time-course mouse scRNA-seq datasets to decipher the secret of eLRs. Extensive validations and analyses were performed to ensure the involvement of identified eLRs in early embryo development. Process analysis identified that eLRs could be divided into six temporal windows corresponding to sequential events in the early embryo development process. With the interpolation strategy, TimeTalk is powerful in revealing paracrine settings and studying cell-cell communication during early embryo development. Furthermore, by using TimeTalk in the blastocyst and blastoid models, we found that the blastoid models share the core communication pathways with the epiblast and primitive endoderm lineages in the blastocysts. This result suggests that TimeTalk has transferability to other bio-dynamic processes. We also curated eLRs recognized by TimeTalk, which may provide valuable clues for understanding early embryo development and relevant disorders.

Substrate nesting guides cyst morphogenesis of human pluripotent stem cells without 3D extracellular matrix overlay

Understanding the principles underlying the self-organization of stem cells into tissues is fundamental for deciphering human embryo development. Here, we report that, without three-dimensional (3D) extracellular matrix (ECM) overlay, human pluripotent stem cells (hPSCs) cultured on two-dimensional soft elastic substrates can self-organize into 3D cysts resembling the human epiblast sac in a stiffness-dependent manner. Our theoretical modeling predicts that this cyst organization is facilitated and guided by the spontaneous nesting of the soft substrate, which results from the adhesion-dependent mechanical interaction between cells and substrate. Such substrate nesting is sufficient for the 3D assembly and polarization of hPSCs required for cyst organization, even without 3D ECM overlay. Furthermore, we identify that the reversible substrate nesting and cyst morphogenesis also require appropriate activation of ROCK-Myosin II pathway. This indicates a unique set of tissue morphomechanical signaling mechanisms that clearly differ from the canonical cystogenic mechanism previously reported in 3D ECM. Our findings highlight an unanticipated synergy between mechanical microenvironment and mechanotransduction in controlling tissue morphogenesis and suggest a mechanics-based strategy for generation of hPSCs-derived models for early human embryogenesis. Statement of significanceSoft substrates can induce the self-organization of human pluripotent stem cells (hPSCs) into cysts without three-dimensional (3D) extracellular matrix (ECM) overlay. However, the underlying mechanisms by which soft substrate guides cystogenesis are largely unknown. This study shows that substrate nesting, resulting from cell–substrate interaction, plays an important role in cyst organization, including 3D assembly and apical–basal polarization. Additionally, actomyosin contractility mediated by the ROCK-Myosin II pathway also contributes to the substrate deformation and cyst morphology. These findings demonstrate the interplay between the mechanical microenvironment and cells in tissue morphogenesis, suggesting a mechanics-based strategy in building hPSC-derived models for early human embryo development.

The landscape of implantation and placentation: deciphering the function of dynamic RNA methylation at the maternal-fetal interface.

The maternal-fetal interface is defined as the interface between maternal tissue and sections of the fetus in close contact. RNA methylation modifications are the most frequent kind of RNA alterations. It is effective throughout both normal and pathological implantation and placentation during pregnancy. By influencing early embryo development, embryo implantation, endometrium receptivity, immune microenvironment, as well as some implantation and placentation-related disorders like miscarriage and preeclampsia, it is essential for the establishment of the maternal-fetal interface. Our review focuses on the role of dynamic RNA methylation at the maternal-fetal interface, which has received little attention thus far. It has given the mechanistic underpinnings for both normal and abnormal implantation and placentation and could eventually provide an entirely novel approach to treating related complications.

Embryo Rescue in Plant Breeding.

Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.

A Single-Cell Atlas of an Early Mongolian Sheep Embryo.

Cell types have been established during organogenesis based on early mouse embryos. However, our understanding of cell types and molecular mechanisms in the early embryo development of Mongolian sheep has been hampered. This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep. Thirteen major cell types were identified at E16 in Ujumqin sheep, and eight major cell types were identified at E16 in Hulunbuir short-tailed sheep. Function enrichment analysis showed that several pathways were significantly enriched in the TGF-beta signaling pathway, the Hippo signaling pathway, the platelet activation pathway, the riboflavin metabolism pathway, the Wnt signaling pathway, regulation of the actin cytoskeleton, and the insulin signaling pathway in the notochord cluster. Glutathione metabolism, glyoxylate, and dicarboxylate metabolism, the citrate cycle, thyroid hormone synthesis, pyruvate metabolism, cysteine and methionine metabolism, thermogenesis, and the VEGF signaling pathway were significantly enriched in the spinal cord cluster. Steroid biosynthesis, riboflavin metabolism, the cell cycle, the Hippo signaling pathway, the Hedgehog signaling pathway, the FoxO signaling pathway, the JAK-STAT signaling pathway, and the Wnt signaling pathway were significantly enriched in the paraxial mesoderm cluster. The notochord cluster, spinal cord cluster, and paraxial mesoderm cluster were found to be highly associated with tail development. Pseudo-time analysis demonstrated that the mesenchyme can translate to the notochord in Ujumqin sheep. Molecular assays revealed that the Hippo signaling pathway was enriched in Ujumqin sheep. This comprehensive single-cell map revealed previously unrecognized signaling pathways that will further our understanding of the mechanism of short-tailed sheep formation.

Monomethyl Phthalate Causes Early Embryo Development Delay, Apoptosis, and Energy Metabolism Disruptions Through Inducing Redox Imbalance.

Phthalates are a class of environmental endocrine disrupting chemicals which can cause reproductive system damages. However, data about reproductive toxicity spectrum of phthalate metabolites among Chinese women undergoing in vitro fertilization (IVF) treatments are scarce yet. Previous studies regarding underlying embryo toxicities focused on oxidative stress and apoptosis, while energy metabolism abnormality might be another key cause for embryo developmental disruptions. Here, we found that among the measured eight phthalate metabolites, monomethyl phthalate (MMP) had the second highest urinary concentration in women receiving IVF. Compare to the lowest exposure level group, MMP in tertile 3 was associated with fewer counts of oocyte retrieved and good-quality embryos, and MMP in tertile 2 was correlated with reduced good-quality embryo rate. The direct embryo toxicities of MMP were studied using mouse 2-cell embryos. Consistent to results found in human populations, exposure to MMP induced mouse early embryo developmental delay. Furthermore, MMP exposure led to excessive reactive oxygen species production in early embryos, and antioxidant can partially rescue the early embryo development slow down. Embryo apoptosis could also be caused by oxidative stress. To be noted, elevated apoptosis level was not found in live "slow" embryos but dead embryos, which suggested that apoptosis was not related to early embryo developmental delay. Additionally, MMP exposure depleted adenosine triphosphate (ATP) synthesis of early embryos, which could be reversed by antioxidant. In conclusion, MMP, as the newly found embryonic toxicant in Chinese women, resulted in early embryo development delay, apoptosis, and energy metabolism disruptions via inducing redox imbalance.

Insulin-like Growth Factor 1, Growth Hormone, and Anti-Müllerian Hormone Receptors Are Differentially Expressed during GnRH Neuron Development.

Gonadotropin-releasing hormone (GnRH) neurons are key neuroendocrine cells in the brain as they control reproduction by regulating hypothalamic-pituitary-gonadal axis function. In this context, anti-Müllerian hormone (AMH), growth hormone (GH), and insulin-like growth factor 1 (IGF1) were shown to improve GnRH neuron migration and function in vitro. Whether AMH, GH, and IGF1 signaling pathways participate in the development and function of GnRH neurons in vivo is, however, currently still unknown. To assess the role of AMH, GH, and IGF1 systems in the development of GnRH neuron, we evaluated the expression of AMH receptors (AMHR2), GH (GHR), and IGF1 (IGF1R) on sections of ex vivo mice at different development stages. The expression of AMHR2, GHR, and IGF1R was assessed by immunofluorescence using established protocols and commercial antibodies. The head sections of mice were analyzed at E12.5, E14.5, and E18.5. In particular, at E12.5, we focused on the neurogenic epithelium of the vomeronasal organ (VNO), where GnRH neurons, migratory mass cells, and the pioneering vomeronasal axon give rise. At E14.5, we focused on the VNO and nasal forebrain junction (NFJ), the two regions where GnRH neurons originate and migrate to the hypothalamus, respectively. At E18.5, the median eminence, which is the hypothalamic area where GnRH is released, was analyzed. At E12.5, double staining for the neuronal marker ß-tubulin III and AMHR2, GHR, or IGF1R revealed a signal in the neurogenic niches of the olfactory and VNO during early embryo development. Furthermore, IGF1R and GHR were expressed by VNO-emerging GnRH neurons. At E14.5, a similar expression pattern was found for the neuronal marker ß-tubulin III, while the expression of IGF1R and GHR began to decline, as also observed at E18.5. Of note, hypothalamic GnRH neurons labeled for PLXND1 tested positive for AMHR2 expression. Ex vivo experiments on mouse sections revealed differential protein expression patterns for AMHR2, GHR, and IGF1R at any time point in development between neurogenic areas and hypothalamic compartments. These findings suggest a differential functional role of related systems in the development of GnRH neurons.

Young LINE-1 transposon 5' UTRs marked by elongation factor ELL3 function as enhancers to regulate naïve pluripotency in embryonic stem cells.

LINE-1s are the major clade of retrotransposons with autonomous retrotransposition activity. Despite the potential genotoxicity, LINE-1s are highly activated in early embryos. Here we show that a subset of young LINE-1s, L1Md_Ts, are marked by the RNA polymerase II elongation factor ELL3, and function as enhancers in mouse embryonic stem cells. ELL3 depletion dislodges the DNA hydroxymethylase TET1 and the co-repressor SIN3A from L1Md_Ts, but increases the enrichment of the Bromodomain protein BRD4, leading to loss of 5hmC, gain of H3K27ac, and upregulation of the L1Md_T nearby genes. Specifically, ELL3 occupies and represses the L1Md_T-based enhancer located within Akt3, which encodes a key regulator of AKT pathway. ELL3 is required for proper ERK activation and efficient shutdown of naïve pluripotency through inhibiting Akt3 during naïve-primed transition. Our study reveals that the enhancer function of a subset of young LINE-1s controlled by ELL3 in transcription regulation and mouse early embryo development.

Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

Biliary atresia is associated with polygenic susceptibility in ciliogenesis and planar polarity effector genes

Biliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA. We performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models. A GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p= 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p= 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p= 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hepatobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA-associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA. BA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes. Liver transplantation is needed to cure most children born with biliary atresia, a poorly understood rare disease. Transplant immunosuppression increases the likelihood of life-threatening infections and cancers. To improve care by preventing this disease and its progression to transplantation, we examined its genetic basis. We find that this disease is associated with both common and rare mutations in highly specialized genes which maintain normal communication and movement of cells, and their organization into bile ducts and blood vessels during early development of the human embryo. Because defects in these genes also cause other birth defects, our findings could lead to preventive strategies to lower the incidence of biliary atresia and potentially other birth defects.