The covariance environment defines cellular niches for spatial inference.

A key challenge of analyzing data from high-resolution spatial profiling technologies is to suitably represent the features of cellular neighborhoods or niches. Here we introduce the covariance environment (COVET), a representation that leverages the gene-gene covariate structure across cells in the niche to capture the multivariate nature of cellular interactions within it. We define a principled optimal transport-based distance metric between COVET niches that scales to millions of cells. Using COVET to encode spatial context, we developed environmental variational inference (ENVI), a conditional variational autoencoder that jointly embeds spatial and single-cell RNA sequencing data into a latent space. ENVI includes two decoders: one to impute gene expression across the spatial modality and a second to project spatial information onto single-cell data. ENVI can confer spatial context to genomics data from single dissociated cells and outperforms alternatives for imputing gene expression on diverse spatial datasets.

ZFP281 controls transcriptional and epigenetic changes promoting mouse pluripotent state transitions via DNMT3 and TET1.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

Amino acid intake strategies define pluripotent cell states.

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.

3D Enhancer-promoter networks provide predictive features for gene expression and coregulation in early embryonic lineages.

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.

Tgfbr1 regulates lateral plate mesoderm and endoderm reorganization during the trunk to tail transition.

During the trunk to tail transition the mammalian embryo builds the outlets for the intestinal and urogenital tracts, lays down the primordia for the hindlimb and external genitalia, and switches from the epiblast/primitive streak to the tailbud as the driver of axial extension. Genetic and molecular data indicate that Tgfbr1 is a key regulator of the trunk to tail transition. Tgfbr1 has been shown to control the switch of the neuro mesodermal-competent cells from the epiblast to the chordo-neural hinge to generate the tail bud. We now show that Tgfbr1 signaling also controls the remodeling of the lateral plate mesoderm (LPM) and of the embryonic endoderm associated with the trunk to tail transition. In the absence of Tgfbr1 the two LPM layers do not converge at the end of the trunk, extending instead as separate layers enclosing the celomic cavity until the caudal embryonic extremity, and failing to activate markers of primordia for the hindlimb and external genitalia. However, this extended LPM, does not exhibit the molecular signatures characteristic of this tissue in the trunk. The vascular remodeling involving the dorsal aorta and the umbilical artery leading to the connection between embryonic and extraembryonic circulation was also affected in the Tgfbr1 mutant embryos. Similar alterations in the LPM and vascular system were also observed in Isl1 null mutants, indicating that this factor acts in the regulatory cascade downstream of Tgfbr1 in LPM-derived tissues. In addition, in the absence of Tgfbr1 the embryonic endoderm fails to expand to form the endodermal cloaca and to extend posteriorly to generate the tail gut. We present evidence suggesting that the remodeling activity of Tgfbr1 in the LPM and endoderm results from the control of the posterior primitive streak fate after its regression during the trunk to tail transition. Our data, together with previously reported observations, place Tgfbr1 at the top of the regulatory processes controlling the trunk to tail transition.

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

Systematic mapping and modeling of 3D enhancer-promoter interactions in early mouse embryonic lineages reveal regulatory principles that determine the levels and cell-type specificity of gene expression.

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 in silico 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.

RNA polymerase II dynamics shape enhancer-promoter interactions.

How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we investigated enhancer-promoter communication by integrating data from nucleosome-resolution genomic contact maps, nascent transcription and perturbations affecting either RNA polymerase II (Pol II) dynamics or the activity of thousands of candidate enhancers. Integration of new Micro-C experiments with published CRISPRi data demonstrated that enhancers spend more time in close proximity to their target promoters in functional enhancer-promoter pairs compared to nonfunctional pairs, which can be attributed in part to factors unrelated to genomic position. Manipulation of the transcription cycle demonstrated a key role for Pol II in enhancer-promoter interactions. Notably, promoter-proximal paused Pol II itself partially stabilized interactions. We propose an updated model in which elements of transcriptional dynamics shape the duration or frequency of interactions to facilitate enhancer-promoter communication.

A ratchet-like apical constriction drives cell ingression during the mouse gastrulation EMT.

Epithelial-to-mesenchymal transition (EMT) is a fundamental process whereby epithelial cells acquire mesenchymal phenotypes and the ability to migrate. EMT is the hallmark of gastrulation, an evolutionarily conserved developmental process. In mammals, epiblast cells ingress at the primitive streak to form mesoderm. Cells ingress and exit the epiblast epithelial layer and the associated EMT is dynamically regulated and involves a stereotypical sequence of cell behaviors. 3D time-lapse imaging of gastrulating mouse embryos combined with cell and tissue scale data analyses revealed the asynchronous ingression of epiblast cells at the primitive streak. Ingressing cells constrict their apical surfaces in a pulsed ratchet-like fashion through asynchronous shrinkage of apical junctions. A quantitative analysis of the distribution of apical proteins revealed the anisotropic and reciprocal enrichment of members of the actomyosin network and Crumbs2 complexes, potential regulators of asynchronous shrinkage of cell junctions. Loss of function analyses demonstrated a requirement for Crumbs2 in myosin II localization and activity at apical junctions, and as a candidate regulator of actomyosin anisotropy.

The covariance environment defines cellular niches for spatial inference.

The tsunami of new multiplexed spatial profiling technologies has opened a range of computational challenges focused on leveraging these powerful data for biological discovery. A key challenge underlying computation is a suitable representation for features of cellular niches. Here, we develop the covariance environment (COVET), a representation that can capture the rich, continuous multivariate nature of cellular niches by capturing the gene-gene covariate structure across cells in the niche, which can reflect the cell-cell communication between them. We define a principled optimal transport-based distance metric between COVET niches and develop a computationally efficient approximation to this metric that can scale to millions of cells. Using COVET to encode spatial context, we develop environmental variational inference (ENVI), a conditional variational autoencoder that jointly embeds spatial and single-cell RNA-seq data into a latent space. Two distinct decoders either impute gene expression across spatial modality, or project spatial information onto dissociated single-cell data. We show that ENVI is not only superior in the imputation of gene expression but is also able to infer spatial context to disassociated single-cell genomics data.

Distinct tumor architectures for metastatic colonization of the brain.

Brain metastasis is a dismal cancer complication, hinging on the initial survival and outgrowth of disseminated cancer cells. To understand these crucial early stages of colonization, we investigated two prevalent sources of cerebral relapse, triple-negative (TNBC) and HER2+ breast cancer (HER2BC). We show that these tumor types colonize the brain aggressively, yet with distinct tumor architectures, stromal interfaces, and autocrine growth programs. TNBC forms perivascular sheaths with diffusive contact with astrocytes and microglia. In contrast, HER2BC forms compact spheroids prompted by autonomous extracellular matrix components and segregating stromal cells to their periphery. Single-cell transcriptomic dissection reveals canonical Alzheimer's disease-associated microglia (DAM) responses. Differential engagement of tumor-DAM signaling through the receptor AXL suggests specific pro-metastatic functions of the tumor architecture in both TNBC perivascular and HER2BC spheroidal colonies. The distinct spatial features of these two highly efficient modes of brain colonization have relevance for leveraging the stroma to treat brain metastasis.

Single-cell analysis of bidirectional reprogramming between early embryonic states reveals mechanisms of differential lineage plasticities.

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 eXtraembryonic ENdoderm (XEN) stem cells - in vitro 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 Gata4, and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping in vitro trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM.

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. We found that unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites. This proto-paused-like state transitioned to a longer, focused pause in derived metazoans which coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF reverts the mammalian focal pause to a proto-pause-like state and compromises transcriptional activation for a set of heat shock genes. Collectively, this work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

ZFP281 coordinates DNMT3 and TET1 for transcriptional and epigenetic control in pluripotent state transitions.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naïive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

Journey of the mouse primitive endoderm: from specification to maturation.

The blastocyst is a conserved stage and distinct milestone in the development of the mammalian embryo. Blastocyst stage embryos comprise three cell lineages which arise through two sequential binary cell fate specification steps. In the first, extra-embryonic trophectoderm (TE) cells segregate from inner cell mass (ICM) cells. Subsequently, ICM cells acquire a pluripotent epiblast (Epi) or extra-embryonic primitive endoderm (PrE, also referred to as hypoblast) identity. In the mouse, nascent Epi and PrE cells emerge in a salt-and-pepper distribution in the early blastocyst and are subsequently sorted into adjacent tissue layers by the late blastocyst stage. Epi cells cluster at the interior of the ICM, while PrE cells are positioned on its surface interfacing the blastocyst cavity, where they display apicobasal polarity. As the embryo implants into the maternal uterus, cells at the periphery of the PrE epithelium, at the intersection with the TE, break away and migrate along the TE as they mature into parietal endoderm (ParE). PrE cells remaining in association with the Epi mature into visceral endoderm. In this review, we discuss our current understanding of the PrE from its specification to its maturation. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Generation of knock-in degron tags for endogenous proteins in mice using the dTAG system.

Controlling the abundance of a protein of interest in vivo is crucial to study its function. Here, we provide a step-by-step protocol for generating genetically engineered mouse (GEM) models harboring a degradation tag (dTAG) fused to endogenous proteins to enable their degradation. We discuss considerations for the overall design and details for vectors generation. Then, we include steps for generation and validations of edited mouse embryonic stem cells followed by mouse colony establishment via chimeric mouse generation. For complete details on the use and execution of this protocol, please refer to Abuhashem et al. (2022c).

SARS-CoV-2 can infect human embryos.

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.