Peri-implantation Development Research Articles

ZIC2 and ZIC3 promote SWI/SNF recruitment to safeguard progression towards human primed pluripotency

The primed epiblast acts as a transitional stage between the relatively homogeneous naïve epiblast and the gastrulating embryo. Its formation entails coordinated changes in regulatory circuits driven by transcription factors and epigenetic modifications. Using a multi-omic approach in human embryonic stem cell models across the spectrum of peri-implantation development, we demonstrate that the transcription factors ZIC2 and ZIC3 have overlapping but essential roles in opening primed-specific enhancers. Together, they are essential to facilitate progression to and maintain primed pluripotency. ZIC2/3 accomplish this by recruiting SWI/SNF to chromatin and loss of ZIC2/3 or degradation of SWI/SNF both prevent enhancer activation. Loss of ZIC2/3 also results in transcriptome changes consistent with perturbed Polycomb activity and a shift towards the expression of genes linked to differentiation towards the mesendoderm. Additionally, we find an intriguing dependency on the transcriptional machinery for sustained recruitment of ZIC2/3 over a subset of primed-hESC specific enhancers. Taken together, ZIC2 and ZIC3 regulate highly dynamic lineage-specific enhancers and collectively act as key regulators of human primed pluripotency.

Assembly of a stem cell-derived human postimplantation embryo model.

The embryonic and extraembryonic tissue interactions underlying human embryogenesis at implantation stages are not currently understood. We have generated a pluripotent stem cell-derived model that mimics aspects of peri-implantation development, allowing tractable experimentation otherwise impossible in the human embryo. Activation of the extraembryonic lineage-specific transcription factors GATA6 and SOX17 (hypoblast factors) or GATA3 and TFAP2C (encoding AP2γ; trophoblast factors) in human embryonic stem (ES) cells drive conversion to extraembryonic-like cells. When combined with wild-type ES cells, self-organized embryo-like structures form in the absence of exogenous factors, termed human inducible embryoids (hiEmbryoids). The epiblast-like domain of hiEmbryoids polarizes and differentiates in response to extraembryonic-secreted extracellular matrix and morphogen cues. Extraembryonic mesenchyme, amnion and primordial germ cells are specified in hiEmbryoids in a stepwise fashion. After establishing stable inducible ES lines and converting ES cells to RSeT culture media, the protocol takes 7-10 d to generate hiEmbryoids. Generation of hiEmbryoids can be performed by researchers with basic expertise in stem cell culture.

O-030 The impact of aneuploidy on human peri-implantation development: findings from the extended in vitro culture of 191 human embryos

Abstract Study question How does aneuploidy affect lineage specification and the developmental potential of human embryos cultured up to 12 days post fertilization (D12)? Summary answer While distinct types of aberrations carry their own risk of embryonic arrest, prevailing aneuploidies appear to compromise lineage specification beyond the blastocyst stage of development. What is known already Chromosomal instability is a hallmark of human preimplantation development, with up to 50% of embryos diagnosed as aneuploid following preimplantation genetic testing (PGT). Aneuploidies are a major cause of implantation failure, pregnancy loss and human congenital defects. Yet despite their clinical relevance, the impact of distinct aberrations on early human development remains poorly understood. Innovative platforms for culturing human embryos up to D12 provide an important opportunity for investigating chromosomal instability. Understanding the effects of specific aneuploidies on human peri-implantation development will be key for advancing human embryo research and improving the interpretation of findings from in vitro embryo models. Study design, size, duration A total of 191 PGT blastocysts obtained from 103 patients were included in the study. Blastocysts were selected based on their original diagnosis, including embryos with single whole-chromosome aneuploidies (single trisomies, n = 37 and single monosomies, n = 71), single segmental aberrations (n = 22), embryos with complex chromosomal constitutions (two or more abnormalities, n = 44), as well as control euploid embryos (n = 17). Participants/materials, setting, methods Vitrified blastocysts were warmed and cultured up to D12 using an optimized protocol to generate embryo outgrowths. We correlated the original PGT diagnoses to developmental outcomes, including outgrowth viability, morphology and outgrowth size. We evaluated blastocyst quality using the Gardner grading system. Following culture, we immunostained 38 viable outgrowths for lineage-specific markers, including OCT4 (epiblast), GATA6 (hypoblast) and CK-7 (trophoblast). Fisher’s exact test was used for statistical analysis (p < 0.05 was considered significant). Main results and the role of chance Following extended culture, 43.5% of embryos remained viable and attached, while 56.5% degenerated and detached. No significant correlation was found between embryo grade and attachment rate. Euploid embryos exhibited the highest attachment rate, with 88.2% (15/17) forming viable outgrowths. Euploid embryos also generated significantly larger outgrowths than trisomic and monosomic embryos (p = 0.01). Embryos diagnosed with trisomies and segmental aberrations exhibited attachment rates comparable to euploid embryos, at 70.3% (p = 0.18) and 68.2% (p = 0.25), respectively. However, embryos with monosomies and complex chromosomal constitutions arrested more readily (28.2% and 15.9% viable, <0.00001, respectively). All euploid embryos (6/6) retained a discernible OCT4+ epiblast-like structure. This was similar to embryos with segmental aberrations and trisomies, with 75% (3/4) and 53.8% (7/13) of outgrowths containing OCT4+ cells, respectively. Nonetheless, distinct trisomies exerted varying effects on peri-implantation development in vitro. For instance, viable trisomies (13, 18, 21) developed similarly to euploid embryos, while outgrowths carrying a trisomy 22 had fewer OCT4+ cells (p = 0.02) and a higher number of GATA6+ cells (p < 0.01), suggesting a potential lineage proliferation defect. Monosomies had the most profound effects on development. Compared to euploid embryos, monosomic embryos formed significantly fewer OCT4+ outgrowths (28.6%, p = 0.02) and contained fewer epiblast (p < 0.0001) and hypoblast-like (p = 0.003) cells. Limitations, reasons for caution The limited number of embryos in certain groups warrants careful interpretation. The central drawback of the models is that embryo outgrowths are predominately flattened, which confounds identification of three-dimensional structures formed during normal embryogenesis. Future studies should encompass additional lineage specification markers and potential culture refinements to enhance our interpretation. Wider implications of the findings Our findings underscore the critical importance of reporting the chromosomal composition of human embryos utilized for research. This practice will enable a more nuanced interpretation of results from in vitro embryo models and ultimately advance our capabilities in human embryo research. Trial registration number not applicable

Single-cell transcriptional landscapes of bovine peri-implantation development

Supporting healthy pregnancy outcomes requires a comprehensive understanding of the molecular and cellular programs of peri-implantation development, when most pregnancy failure occurs. Here, we present single-cell transcriptomes of bovine peri-implantation embryo development at day 12, 14, 16, and 18 post-fertilization. We defined the cellular composition and gene expression of embryonic disc, hypoblast, and trophoblast lineages in bovine peri-implantation embryos, and identified markers and pathway signaling that represent distinct stages of bovine peri-implantation lineages; the expression of selected markers was validated in peri-implantation embryos. Using detailed time-course transcriptomic analyses, we revealed a previously unrecognized primitive trophoblast cell lineage. We also characterized conserved and divergence peri-implantation lineage programs between bovine and other mammalian species. Finally, we established cell-cell communication signaling underlies embryonic and extraembryonic cell interaction to ensure proper early development. These data provide foundational information to discover essential biological signaling underpinning bovine peri-implantation development.

Cell specification and functional interactions in the pig blastocyst inferred from single-cell transcriptomics and uterine fluids proteomics

The embryonic development of the pig comprises a long in utero pre- and peri-implantation development, which dramatically differs from mice and humans. During this peri-implantation period, a complex series of paracrine signals establishes an intimate dialogue between the embryo and the uterus. To better understand the biology of the pig blastocyst during this period, we generated a large dataset of single-cell RNAseq from early and hatched blastocysts, spheroid and ovoid conceptus and proteomic datasets from corresponding uterine fluids. Our results confirm the molecular specificity and functionality of the three main cell populations. We also discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent progenitor cells, and the other, expressing pro-apoptotic markers, which could correspond to the Rauber's layer. Our work provides new insights into the biology of these populations, their reciprocal functional interactions, and the molecular dialogue with the maternal uterine environment.

Having a blast(oid): Modeling human embryo peri-implantation development with blastoids.

Studying human embryo development, most particularly around the time of implantation, is a challenge, yet it is necessary to improve assisted reproduction techniques. In this issue, Yu etal.1 and Karvas etal.2 improve integrated stem cell models, called blastoids, to model the peri-implantation human embryo.

Inhibition of Wnt activity improves peri-implantation development of somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells into totipotency. Although pre-implantation development of SCNT embryos has greatly improved, most SCNT blastocysts are still arrested at the peri-implantation stage, and the underlying mechanism remains elusive. Here, we develop a 3D in vitro culture system for SCNT peri-implantation embryos and discover that persistent Wnt signals block the naïve-to-primed pluripotency transition of epiblasts with aberrant H3K27me3 occupancy, which in turn leads to defects in epiblast transformation events and subsequent implantation failure. Strikingly, manipulating Wnt signals can attenuate the pluripotency transition and H3K27me3 deposition defects in epiblasts and achieve up to a 9-fold increase in cloning efficiency. Finally, single-cell RNA-seq analysis reveals that Wnt inhibition markedly enhances the lineage developmental trajectories of SCNT blastocysts during peri-implantation development. Overall, these findings reveal diminished potentials of SCNT blastocysts for lineage specification and validate a critical peri-implantation barrier for SCNT embryos.

Embryo-uterine interaction coordinates mouse embryogenesis during implantation.

Embryo implantation into the uterus marks a key transition in mammalian development. In mice, implantation is mediated by the trophoblast and is accompanied by a morphological transition from the blastocyst to the egg cylinder. However, the roles of trophoblast-uterine interactions in embryo morphogenesis during implantation are poorly understood due to inaccessibility in utero and the remaining challenges to recapitulate it ex vivo from the blastocyst. Here, we engineer a uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo and reveal essential roles of the physical embryo-uterine interaction. We demonstrate that adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. Modeling the implanting embryo as a wetting droplet links embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Light-sheet live imaging and the experimental control of the engineered uterine geometry and trophoblast velocity uncovers the coordination between trophoblast motility and embryo growth, where the trophoblast delineates space for embryo morphogenesis.

Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids

Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage development as well as underlying mechanisms remains obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling pathways synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast development and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights into human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis.

O-157 Modelling human early development

Abstract Understanding human peri-implantation development is necessary to improve in vitro fertilisation. Technical progress, such as scRNAseq and integrated stem cell models have dramatically improved our ability to understand human peri-implantation development. However, in-depth analysis and systematic validation in human embryos remains necessary to build a reliable reference. Here, we started by integrating pre- and post-implantation scRNAseq embryo datasets to generate a peri-implantation reference, highlighting progression of fates during early human development. Second, we identified the dynamics of genes modules, groups of genes with similar expression pattern. In particular, this highlighted the hierarchy of transcription factor potentially driving cell fate progression. Importantly, we performed prolonged embryo culture to validate by immunofluorescence progression of EPI and TE fates. Moreover, we compared the expression profile of the identified markers in stem cell models. Finally, we used the combination of gene modules to benchmark blastoid models. Altogether, our study paves the way for modelling human peri-implantation development with stem cell models. Trial registration number NA Study funding: Funding source:

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.

Transient suppression of SUMOylation in embryonic stem cells generates embryo-like structures

Recent advances in synthetic embryology have opened new avenues for understanding the complex events controlling mammalian peri-implantation development. Here, we show that mouse embryonic stem cells (ESCs) solely exposed to chemical inhibition of SUMOylation generate embryo-like structures comprising anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs give rise to spheroids that self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in a droplet microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell transcriptomics reveals various cellular lineages, including properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures. Transient SUMOylation suppression gradually increases DNA methylation genome wide and repressive mark deposition at Nanog. Interestingly, cell-to-cell variations in SUMOylation levels occur during early embryogenesis. Our approach provides a proof of principle for potentially powerful strategies to explore early embryogenesis by targeting chromatin roadblocks of cell fate change.

Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts classified according to developmental potential, maternal age and morphology.

Selection of high-quality embryos is important to achieve successful pregnancy in assisted reproductive technology (ART). Recently, it has been debated whether RNA-sequencing (RNA-Seq) should be applied to ART to predict embryo quality. However, information on genes that can serve as markers for pregnant expectancy is limited. Furthermore, there is no information on which transcriptome of trophectoderm (TE) or inner cell mass (ICM) is more highly correlated with pregnant expectancy. Here, we performed RNA-Seq analysis of TE and ICM of human blastocysts, the pregnancy expectation of which was retrospectively determined using the clinical outcomes of 1,890 cases of frozen-thawed blastocyst transfer. We identified genes that were correlated with the expected pregnancy rate in ICM and TE, respectively, with a larger number of genes identified in TE than in ICM. Downregulated genes in the TE of blastocysts that were estimated to have lower expectation of pregnancy included tight junction-related genes such as CXADR and ATP1B1, which have been implicated in peri-implantation development. Moreover, we identified dozens of differentially expressed genes by regrouping the blastocysts based on the maternal age and the Gardner score. Additionally, we showed that aneuploidy estimation using RNA-Seq datasets does not correlate with pregnancy expectation. Thus, our study provides an expanded list of candidate genes for the prediction of pregnancy in human blastocyst embryos.

Progression of the pluripotent epiblast depends upon the NMD factor UPF2.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that degrades RNAs harboring in-frame stop codons in specific contexts. Loss of NMD factors leads to embryonic lethality in organisms spanning the phylogenetic scale, but the mechanism remains unknown. Here, we report that the core NMD factor, UPF2, is required for expansion of epiblast cells within the inner cell mass of mice in vivo. We identify NMD target mRNAs in mouse blastocysts - both canonical and alternatively processed mRNAs - including those encoding cell cycle arrest and apoptosis factors, raising the possibility that NMD is essential for embryonic cell proliferation and survival. In support, the inner cell mass of Upf2-null blastocysts rapidly regresses with outgrowth and is incompetent for embryonic stem cell derivation in vitro. In addition, we uncovered concordant temporal- and lineage-specific regulation of NMD factors and mRNA targets, indicative of a shift in NMD magnitude during peri-implantation development. Together, our results reveal developmental and molecular functions of the NMD pathway in the early embryo.

EXTENDED IN VITRO CULTURE OF HUMAN EMBRYOS DEMONSTRATES THE POOR PREDICTIVE VALUE OF DIAGNOSING CHROMOSOMAL MOSAICISM FROM A TROPHECTODERM BIOPSY

Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into clinical practice to improve outcomes. However, diagnostic accuracy remains controversial, particularly regarding chromosomal mosaicism and de novo segmental variants. Ultimately, the interpretation of these diagnoses is exceedingly complex due to the absence of a precise clinical phenotype. Here, we use an extended embryo in vitro culture model to establish the positive predictive value (PPV) of PGT-A. Our platform allows the culture of human embryos up to 12 days post fertilization (D), providing a unique opportunity for assessing chromosomal (in)stability during human peri-implantation development.

Dynamic enlargement and mobilization of lipid droplets in pluripotent cells coordinate morphogenesis during mouse peri-implantation development

Mammalian pre-implantation embryos accumulate substantial lipids, which are stored in lipid droplets (LDs). Despite the fundamental roles of lipids in many cellular functions, the significance of building-up LDs for the developing embryo remains unclear. Here we report that the accumulation and mobilization of LDs upon implantation are causal in the morphogenesis of the pluripotent epiblast and generation of the pro-amniotic cavity in mouse embryos, a critical step for all subsequent development. We show that the CIDEA protein, found abundantly in adipocytes, enhances lipid storage in blastocysts and pluripotent stem cells by promoting LD enlargement through fusion. The LD-stored lipids are mobilized into lysosomes at the onset of lumenogenesis, but without CIDEA are prematurely degraded by cytosolic lipases. Loss of lipid storage or inactivation of lipophagy leads to the aberrant formation of multiple cavities within disorganised epithelial structures. Thus, our study reveals an unexpected role for LDs in orchestrating tissue remodelling and uncovers underappreciated facets of lipid metabolism in peri-implantation development.

Short Communication: Maternal obesity alters ovine endometrial gene expression during peri-implantation development.

Exposure to maternal obesity in utero is associated with marked developmental effects in offspring that may not be evident until adulthood. Mechanisms regulating the programming effects of maternal obesity on fetal development have been reported, but little is known about how maternal obesity affects the earliest periods of embryonic development. This work explored how obesity influences endometrial gene expression during the peri-implantation period using a sheep model. Ewes were assigned randomly to diets that produced an obese state or maintained a lean state. After 4 mo, obese and lean ewes were bred and then euthanized at day 14 post-breeding. The uterus was excised, conceptuses were flushed, and endometrial tissue was collected. Isolated RNA from endometrial tissues (n = 6 ewes/treatment) were sequenced using an Illumina-based platform. Reads were mapped to the Ovis aries genome (Oar_4.0). Differential gene expression was determined, and results were filtered (false discovery rate ≤ 0.05 and ≥2-fold change, ≥0.2 reads/kilobase/million reads). Differentially expressed genes (DEGs) were identified (n = 699), with 171 downregulated and 498 upregulated in obese vs. lean endometrium, respectively. The most pronounced gene ontology categories identified were cellular process, metabolic process, and biological regulation. Enrichments were detected within the DEGs for genes involved with immune system processes, negative regulation of apoptosis, cell growth, and cell adhesion. A literature search revealed that 125 DEGs were associated with either the trophoblast lineage or the placenta. Genes within this grouping were involved with wingless/integrated signaling, angiogenesis, and integrin signaling. In summary, these data indicate that the peri-implantation endometrium is responsive to maternal obesity. Transcript profile analyses suggest that the endometrial immune response, adhesion, and angiogenesis may be especially susceptible to obesity. Thus, alterations in uterine transcript profiles during early embryogenesis may be a mechanism responsible for developmental programming following maternal obesity exposure in utero.

P-138 Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts with expected pregnancy rates

Abstract Study question Is it possible to identify the molecular factors that contribute to the implantation potential of blastocysts? Summary answer Genes correlated with expected pregnancy rate in trophectoderm (TE) and inner cell mass (ICM) respectively were identified, and aneuploidy alone couldn’t predict the pregnancy expectation. What is known already The selection of suitable embryos for transfer is critical for achieving successful pregnancy outcomes in assisted reproductive technology (ART). Although pre-implantation genetic testing for aneuploidy (PGT-A) as well as morphological and chronological evaluation of embryos, have been conducted in clinical practice, they do not fully guarantee successful pregnancy. Recently, transcriptional events in early human embryonic development have been analyzed using RNA-sequencing (RNA-seq) and researchers are attempting to apply this information to ART. Study design, size, duration To determine the correlation between blastocyst evaluation and pregnancy rate, we retrospectively analyzed 1,890 cases underwent frozen-thawed blastocyst transfer from March 2018 to December 2020. A total of 13 blastocysts that were cryopreserved for clinical use between February 2011 and September 2018, then scheduled for disposal and with consented for research, were subjected to RNA-seq without distinguishing between conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI). Participants/materials, setting, methods Blastocysts were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic with informed consent under ethical approval. TE and ICM cells were collected from blastocysts by using a micromanipulator and then subjected to RNA-seq. Gene expression analysis and digital karyotyping using RNA-seq were performed simultaneously for TE and ICM cells, respectively. One-way analysis of variance, chi-square test and Tukey's multiple comparison test were used for this study. Main results and the role of chance Blastocysts were classified into three groups to correlate with pregnancy rates based on the diameter of the blastocyst and the time to reach this size: those taking less than 130 h to reach a diameter of > 170 μm (Group 1, n = 676), those taking more than 140 h to reach a diameter of < 180 μm (Group 2, n = 158), and the rest (Group 3, n = 1,056). The pregnancy rates of Groups 1, 2 and 3 were 59.0%, 16.5%, and 34.2%, respectively (p < 0.01). Assessing the differences in overall transcripts correlated between Group 1 (n = 5), Group 2 (n = 4), and Group 3 (n = 4), 26 and 67 differentially expressed genes (DEGs) were identified in ICM and TE cells, respectively. Importantly, downregulated genes in TE of blastocysts with lower expectation of pregnancy included tight junction-related genes, such as CXADR, CLDN10, and ATP1B1, which were implicated in peri-implantation development. Digital karyotyping revealed karyotypic abnormalities and mosaicism in all groups with no common abnormalities observed, suggesting that aneuploidy alone cannot predict the pregnancy expectation. Limitations, reasons for caution Although 93 genes potentially related to implantation have been identified, it is still unclear how these genes are involved in implantation. In vitro implantation models using human embryos and artificial embryos currently under development are expected to contribute to the elucidation of the functions of these genes. Wider implications of the findings Our results provide reliable candidates for genes that could allow for non-invasive selection of high-quality blastocysts for ART and add to the knowledge base of transcriptional events in human peri-implantation development. Trial registration number not applicable

Flux balance network expansion predicts stage-specific human peri_implantation embryo metabolism.

Metabolism is an essential cellular process for the growth and maintenance of organisms. A better understanding of metabolism during embryogenesis may shed light on the developmental origins of human disease. Metabolic networks, however, are vastly complex with many redundant pathways and interconnected circuits. Thus, computational approaches serve as a practical solution for unraveling the genetic basis of embryo metabolism to help guide future experimental investigations. RNA-sequencing and other profiling technologies make it possible to elucidate metabolic genotype-phenotype relationships and yet our understanding of metabolism is limited. Very few studies have examined the temporal or spatial metabolomics of the human embryo, and prohibitively small sample sizes traditionally observed in human embryo research have presented logistical challenges for metabolic studies, hindering progress towards the reconstruction of the human embryonic metabolome. We employed a network expansion algorithm to evolve the metabolic network of the peri-implantation embryo metabolism and we utilized flux balance analysis (FBA) to examine the viability of the evolved networks. We found that modulating oxygen uptake promotes lactate diffusion across the outer mitochondrial layer, providing in-silico support for a proposed lactate-malate-aspartate shuttle. We developed a stage-specific model to serve as a proof-of-concept for the reconstruction of future metabolic models of development. Our work shows that it is feasible to model human metabolism with respect to time-dependent changes characteristic of peri-implantation development.

ATG7-mediated autophagy facilitates embryonic stem cell exit from naive pluripotency and marks commitment to differentiation

ABSTRACT Macroautophagy/autophagy is a conserved cellular mechanism to degrade unneeded cytoplasmic proteins and organelles to recycle their components, and it is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Whereas autophagy is essential for early development of embryos, no information exists regarding its functions during the transition from naive-to-primed pluripotency. Here, by using an in vitro transition model of ESCs to epiblast-like cells (EpiLCs), we find that dynamic changes in ATG7-dependent autophagy are critical for the naive-to-primed transition, and are also necessary for germline specification. RNA-seq and ATAC-seq profiling reveal that NANOG acts as a barrier to prevent pluripotency transition, and autophagy-dependent NANOG degradation is important for dismantling the naive pluripotency expression program through decommissioning of naive-associated active enhancers. Mechanistically, we found that autophagy receptor protein SQSTM1/p62 translocated into the nucleus during the pluripotency transition period and is preferentially associated with K63 ubiquitinated NANOG for selective protein degradation. In vivo, loss of autophagy by ATG7 depletion disrupts peri-implantation development and causes increased chromatin association of NANOG, which affects neuronal differentiation by competitively binding to OTX2-specific neuroectodermal development-associated regions. Taken together, our findings reveal that autophagy-dependent degradation of NANOG plays a critical role in regulating exit from the naive state and marks distinct cell fate allocation during lineage specification. Abbreviations: 3-MA: 3-methyladenine; EpiLC: epiblast-like cell; ESC: embryonic stem cell; PGC: primordial germ cell