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Here we delineate the ontogeny of the mammalian endoderm by generating 112,217 single-cell transcriptomes, which represent all endoderm populations within the mouse embryo until midgestation. We use graph-based approaches to model differentiating cells, which provides a spatio-temporal characterization of developmental trajectories and defines the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncover a relationship between descendants of these two lineages, in which epiblast cells differentiate into endoderm at two distinct time points—before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates and as they spatially converged within the nascent gut endoderm, which revealed these cells to be globally similar but retain aspects of their lineage history. We observed the regionalized identity of cells along the anterior–posterior axis of the emergent gut tube, which reflects their embryonic or extra-embryonic origin, and the coordinated patterning of these cells into organ-specific territories. The developing mouse gut endoderm, mapped at single-cell resolution, reveals trajectories of cell differentiation before and during gastrulation and the emergence of regionalized cell identities along the anterior–posterior axis of the gut tube.

The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. By using RNA-seq and immunofluorescence, we note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. For the purpose of viral entry studies, we used fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, and we observe robust infection of trophectoderm cells. This permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or ‘hubness’, positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors. Here, the authors describe 3D hubs as regulatory subunits of gene expression in the three essential lineages of embryogenesis. They develop a computational model that can predict novel enhancers and they validate such enhancers in the context of specific lineages.

Understanding how pluripotency is established and maintained is a cornerstone question in stem cell and developmental biology. Pluripotency in vivo is ascribed to the epiblast (EPI) lineage of the early mammalian embryo. The EPI and its sister lineage, the primitive (extraembryonic) endoderm (PrE), are specified from a common progenitor population. Despite being well studied, the molecular mechanisms underlying this cell fate decision remain poorly understood. The FGF/ERK signaling axis has been implicated in driving EPI-PrE lineage specification. We therefore investigated the individual and combinatorial roles of Fgfr1 and Fgfr2 receptors in mediating lineage specification. We combined single cell approaches for quantitative immunofluorescence, gene expression profiling and characterization of a novel reporter of FGF signaling to evaluate receptor function. We found distinct requirements for each receptor in relaying the FGF signal and defined a predominant role for FGFR1, rather than FGFR2, in regulating fate choice, contrary to established models. Cellular signaling converges on the transcriptional state of cells to determine cell fate. We therefore leveraged single-cell RNA sequencing (scRNA-seq) to characterize the transcriptional dynamics underlying EPI and PrE specification in vivo. Our analysis revealed two sub-states within the uncommitted progenitor population, and identified the transcriptional dynamics that define a temporal window for FGF signaling to mediate fate specification. To overcome the limited biological material available from embryos, we then established a scalable in vitro model of EPI-PrE specification. We employed a cellular reprogramming approach between embryonic stem (ES) and eXtraembryonic ENdoderm (XEN) stem cells – in vitro counterparts of the EPI and PrE, respectively, to further asses their lineage relationship. We showed that expression of Oct4, Klf4 and Sox2 successfully reprograms XEN cells to ES. However, the low efficiency and slow kinetics are in stark contrast to Gata4-mediated ES-to-XEN conversion, highlighting the differential plasticity of these lineages. We coupled this bi-directional conversion strategy with scRNA-seq to identify potential drivers and roadblocks of this lineage switch, and potentially of lineage specification in vivo. The present work has advanced our understanding of EPI-PrE specification and provides a platform for further mechanistic analyses of genetic and epigenetic factors that establish and maintain lineage identity.

There is an ongoing unmet need of early identification and discussion regarding the sexual and urinary dysfunction in the peri-operative period to improve the quality of life (QoL), particularly in young rectal cancer survivors. Retrospective analysis of prospectively maintained database was done. Male patients less than 60 years who underwent nerve preserving, sphincter sparing rectal cancer surgery between January 2013 and December 2019, were screened. International Index of Erectile Function (IIEF-5) questionnaire was given to assess erectile dysfunction (ED). Patients were asked questions regarding their sexual and urinary function from the EORTC-QL CRC 38 questionnaire, and responses were recorded. Patients were also asked to report any retrograde ejaculation in post-operative period. Sixty-two patients were included in the study. Fifty-four patients (87.1%) received a diversion stoma. Sixteen patients (29.6%) felt stoma was interfering with their sexual function. Six patients (9.7%) reported retrograde ejaculation. Only 5 patients (8.06%) had moderate to severe ED, and the rest had none to mild ED. On univariate and multivariate analysis, only age predicted the development of clinically significant ED. Ten patients (16.1%) had significantly reduced sexual urges, and 23 patients (37.1%) had significant decrease in sexual satisfaction after surgery. Five patients (8.06%) reported having minor urinary complaints. No patient reported having major complaint pertaining to urinary health. While long-term urinary complaints are infrequent, almost half the patient suffered from erectile dysfunction in some form. There is a weak but significant association of age and ED. Follow-up clinic visits provide an ideal opportunity to counsel patients and provide any medical intervention, when necessary.

Mammalian preimplantation development is associated with marked metabolic robustness, and embryos can develop under a wide variety of nutrient conditions, including even the complete absence of soluble amino acids. Here we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of preimplantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis, alongside a robust lysosomal digestive system. Following transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant on exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the preimplantation epiblast. More broadly, we find that all lineages of preimplantation blastocysts exhibit constitutive macropinocytic protein uptake and digestion. Taken together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of preimplantation development and provide insight into the catabolic strategies that enable embryos to sustain viability before implantation. Todorova et al. characterize the strategies through which embryos secure amino acid supply during the early phases of development. Their findings show that, in the preimplantation phase, embryos uptake whole proteins through macropinocytosis and, over time, they shift towards soluble amino acid uptake. This strategy may contribute to protecting embryos from nutrient fluctuations.

Cell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signaling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors and are critical mediators of FGF signaling in cell lineage specification and maturation in mouse embryos. We show that loss of compromises primitive endoderm formation at pre-implantation stages. Furthermore, deficiency delays naïve pluripotency exit and epiblast maturation, leading to elevated NANOG and reduced OTX2 expression within the blastocyst epiblast. As a consequence of delayed pluripotency progression, deficient embryos exhibit anterior visceral endoderm migration defects post-implantation, a process essential for coordinated embryonic patterning and gastrulation initiation. Our results demonstrate the successive roles of these FGF signaling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or \"hubness\" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors.

Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and e traembryonic doderm (XEN) stem cells - counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4, KLF4 and SOX2-induced XEN-to-iPS reprogramming progressed with diminished efficiency and kinetics. The dominant PrE transcriptional program, safeguarded by , and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE states without transitioning through the ICM.