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Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies 1 – 4 . Because individual cells appear to be highly variable at all these levels 5 , it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form ‘fixed points’ in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3–4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems. Multiplexed imaging of 3,660 chromosomal loci in individual mouse embryonic stem cells by DNA seqFISH+ with immunofluorescence of 17 chromatin marks and subnuclear structures reveals invariant organization of loci within individual cells, and heterogeneous and long-lived distinct combinatorial chromatin states in cellular subpopulations.

During post-implantation development of the mouse embryo, descendants of the inner cell mass in the early epiblast transit from the naive to primed pluripotent state 1 . Concurrently, germ layers are formed and cell lineages are specified, leading to the establishment of the blueprint for embryogenesis. Fate-mapping and lineage-analysis studies have revealed that cells in different regions of the germ layers acquire location-specific cell fates during gastrulation 2 – 5 . The regionalization of cell fates preceding the formation of the basic body plan—the mechanisms of which are instrumental for understanding embryonic programming and stem-cell-based translational study—is conserved in vertebrate embryos 6 – 8 . However, a genome-wide molecular annotation of lineage segregation and tissue architecture of the post-implantation embryo has yet to be undertaken. Here we report a spatially resolved transcriptome of cell populations at defined positions in the germ layers during development from pre- to late-gastrulation stages. This spatiotemporal transcriptome provides high-resolution digitized in situ gene-expression profiles, reveals the molecular genealogy of tissue lineages and defines the continuum of pluripotency states in time and space. The transcriptome further identifies the networks of molecular determinants that drive lineage specification and tissue patterning, supports a role of Hippo–Yap signalling in germ-layer development and reveals the contribution of visceral endoderm to the endoderm in the early mouse embryo. Spatially resolved transcriptomes of cell populations at defined positions in the early mouse embryo reveal molecular bases of lineage specification and tissue patterning.

The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems. Throughput of in vivo genetic screens is a barrier to efficient application. Here the authors use a high-throughput CRISPR-based in vivo forward genetic screen in mice to identify transcriptional regulators of cardiomyocyte maturation, including the epigenetic modifiers RNF20 and RNF40.

In this Journal Club, Shengbao Suo highlights two publications that underscore the importance of genetic regulation of endogenous retrovirus expression in tumours.

Spatially resolved transcriptomic technologies have emerged as pivotal tools for elucidating molecular regulation and cellular interplay within the intricate tissue microenvironment, but hampered by insufficient gene recovery or challenges in achieving intact single-cell resolution. Here, we develop Cellular Mapping of Attributes with Position (CMAP), a method that efficiently maps large-scale individual cells to their precise spatial locations by integrating single-cell and spatial data through a divide­-and-­conquer strategy. Analysis of both simulated and real datasets shows that CMAP performs effectively and is adaptable across diverse data types and sequencing platforms. Particularly, CMAP handles scenarios well where discrepancies exist between single-cell and spatial transcriptomics data. Our findings underscore CMAP’s capacity to endow single-cells with exact spatial coordinates, facilitating the dissection of nuanced spatial-organ-specific endothelial cell heterogeneity, as well as the intricate cancer immune microenvironments that elude conventional single-cell or spatial data analysis. CMAP is a method that maps large-scale individual cells to their precise spatial locations by integrating single-cell and spatial transcriptomics data through a divide-and-conquer strategy, supporting diverse data types and mapping scenarios.

Adult skeletal muscle regeneration is mainly driven by muscle stem cells (MuSCs), which are highly heterogeneous. Although recent studies have started to characterize the heterogeneity of MuSCs, whether a subset of cells with distinct exists within MuSCs remains unanswered. Here, we find that a population of MuSCs, marked by Gli1 expression, is required for muscle regeneration. The Gli1 + MuSC population displays advantages in proliferation and differentiation both in vitro and in vivo. Depletion of this population leads to delayed muscle regeneration, while transplanted Gli1 + MuSCs support muscle regeneration more effectively than Gli1− MuSCs. Further analysis reveals that even in the uninjured muscle, Gli1 + MuSCs have elevated mTOR signaling activity, increased cell size and mitochondrial numbers compared to Gli1 − MuSCs, indicating Gli1 + MuSCs are displaying the features of primed MuSCs. Moreover, Gli1 + MuSCs greatly contribute to the formation of G Alert cells after muscle injury. Collectively, our findings demonstrate that Gli1 + MuSCs represents a distinct MuSC population which is more active in the homeostatic muscle and enters the cell cycle shortly after injury. This population functions as the tissue-resident sentinel that rapidly responds to injury and initiates muscle regeneration. Adult skeletal muscle regeneration is mainly driven by muscle stem cells (MuSCs), which are highly heterogeneous. Here, the authors found that a population of MuSCs, marked by Gli1 expression, is key contributor to muscle regeneration.

Background Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has been associated with neurological and neuropsychiatric illness in many individuals. We sought to further our understanding of the relationship between brain tropism, neuro-inflammation, and host immune response in acute COVID-19 cases. Methods Three brain regions (dorsolateral prefrontal cortex, medulla oblongata, and choroid plexus) from 5 patients with severe COVID-19 and 4 controls were examined. The presence of the virus was assessed by western blot against viral spike protein, as well as viral transcriptome analysis covering > 99% of SARS-CoV-2 genome and all potential serotypes. Droplet-based single-nucleus RNA sequencing (snRNA-seq) was performed in the same samples to examine the impact of COVID-19 on transcription in individual cells of the brain. Results Quantification of viral spike S1 protein and viral transcripts did not detect SARS-CoV-2 in the postmortem brain tissue. However, analysis of 68,557 single-nucleus transcriptomes from three distinct regions of the brain identified an increased proportion of stromal cells, monocytes, and macrophages in the choroid plexus of COVID-19 patients. Furthermore, differential gene expression, pseudo-temporal trajectory, and gene regulatory network analyses revealed transcriptional changes in the cortical microglia associated with a range of biological processes, including cellular activation, mobility, and phagocytosis. Conclusions Despite the absence of detectable SARS-CoV-2 in the brain at the time of death, the findings suggest significant and persistent neuroinflammation in patients with acute COVID-19.

Mammalian embryos exhibit sophisticated cellular patterning that is intricately orchestrated at both molecular and cellular level. It has recently become apparent that cells within the animal body display significant heterogeneity, both in terms of their cellular properties and spatial distributions. However, current spatial transcriptomic profiling either lacks three-dimensional representation or is limited in its ability to capture the complexity of embryonic tissues and organs. Here, we present a spatial transcriptomic atlas of all major organs at embryonic day 13.5 in the mouse embryo, and provide a three-dimensional rendering of molecular regulation for embryonic patterning with stacked sections. By integrating the spatial atlas with corresponding single-cell transcriptomic data, we offer a detailed molecular annotation of the dynamic nature of organ development, spatial cellular interactions, embryonic axes, and divergence of cell fates that underlie mammalian development, which would pave the way for precise organ engineering and stem cell-based regenerative medicine. Qu et al. present a detailed three-dimensional spatial transcriptome atlas of all major organs in the mouse embryo at E13.5, providing a better understanding of organ development and cellular interactions during mammalian development.

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell–dependent and –independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiota-independent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response.