Sctensor detects many-to-many cell-cell interactions from single cell RNA-sequencing data.

BACKGROUND: Complex biological systems are described as a multitude of cell-cell interactions (CCIs). Recent single-cell RNA-sequencing studies focus on CCIs based on ligand-receptor (L-R) gene co-expression but the analytical methods are not appropriate to detect many-to-many CCIs. RESULTS: In this work, we propose scTensor, a novel method for extracting representative triadic relationships (or hypergraphs), which include ligand-expression, receptor-expression, and related L-R pairs. CONCLUSIONS: Through extensive studies with simulated and empirical datasets, we have shown that scTensor can detect some hypergraphs that cannot be detected using conventional CCI detection methods, especially when they include many-to-many relationships. scTensor is implemented as a freely available R/Bioconductor package.

Distinctiveness and continuity in transcriptome and connectivity in the anterior-posterior axis of the paraventricular nucleus of the thalamus.

The paraventricular nucleus of the thalamus (PVT) projects axons to multiple areas, mediates a wide range of behaviors, and exhibits regional heterogeneity in both functions and axonal projections. Still, questions regarding the cell types present in the PVT and the extent of their differences remain inadequately addressed. We applied single-cell RNA sequencing to depict the transcriptomic characteristics of mouse PVT neurons. We found that one of the most significant variances in the PVT transcriptome corresponded to the anterior-posterior axis. While the single-cell transcriptome classified PVT neurons into five types, our transcriptomic and histological analyses showed continuity among the cell types. We discovered that anterior and posterior subpopulations had nearly non-overlapping projection patterns, while another population showed intermediate patterns. In addition, these subpopulations responded differently to appetite-related neuropeptides, with their activation showing opposing effects on food consumption. Our studies unveiled the contrasts and the continuity of PVT neurons that underpin their function.

WUSCHEL-RELATED HOMEOBOX 13 suppresses de novo shoot regeneration via cell fate control of pluripotent callus.

Plants can regenerate their bodies via de novo establishment of shoot apical meristems (SAMs) from pluripotent callus. Only a small fraction of callus cells is eventually specified into SAMs but the molecular mechanisms underlying fate specification remain obscure. The expression of WUSCHEL (WUS) is an early hallmark of SAM fate acquisition. Here, we show that a WUS paralog, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), negatively regulates SAM formation from callus in Arabidopsis thaliana. WOX13 promotes non-meristematic cell fate via transcriptional repression of WUS and other SAM regulators and activation of cell wall modifiers. Our Quartz-Seq2-based single cell transcriptome revealed that WOX13 plays key roles in determining cellular identity of callus cell population. We propose that reciprocal inhibition between WUS and WOX13 mediates critical cell fate determination in pluripotent cell population, which has a major impact on regeneration efficiency.

Single-cell transcriptomics uncovers EGFR signaling-mediated gastric progenitor cell differentiation in stomach homeostasis.

Defects in gastric progenitor cell differentiation are associated with various gastric disorders, including atrophic gastritis, intestinal metaplasia, and gastric cancer. However, the mechanisms underlying the multilineage differentiation of gastric progenitor cells during healthy homeostasis remain poorly understood. Here, using a single-cell RNA sequencing method, Quartz-Seq2, we analyzed the gene expression dynamics of progenitor cell differentiation toward pit cell, neck cell, and parietal cell lineages in healthy adult mouse corpus tissues. Enrichment analysis of pseudotime-dependent genes and a gastric organoid assay revealed that EGFR-ERK signaling promotes pit cell differentiation, whereas NF-κB signaling maintains gastric progenitor cells in an undifferentiated state. In addition, pharmacological inhibition of EGFR in vivo resulted in a decreased number of pit cells. Although activation of EGFR signaling in gastric progenitor cells has been suggested as one of the major inducers of gastric cancers, our findings unexpectedly identified that EGFR signaling exerts a differentiation-promoting function, not a mitogenic function, in normal gastric homeostasis.

WormTensor: a clustering method for time-series whole-brain activity data from C. elegans.

BACKGROUND: In the field of neuroscience, neural modules and circuits that control biological functions have been found throughout entire neural networks. Correlations in neural activity can be used to identify such neural modules. Recent technological advances enable us to measure whole-brain neural activity with single-cell resolution in several species including [Formula: see text]. Because current neural activity data in C. elegans contain many missing data points, it is necessary to merge results from as many animals as possible to obtain more reliable functional modules. RESULTS: In this work, we developed a new time-series clustering method, WormTensor, to identify functional modules using whole-brain activity data from C. elegans. WormTensor uses a distance measure, modified shape-based distance to account for the lags and the mutual inhibition of cell-cell interactions and applies the tensor decomposition algorithm multi-view clustering based on matrix integration using the higher orthogonal iteration of tensors (HOOI) algorithm (MC-MI-HOOI), which can estimate both the weight to account for the reliability of data from each animal and the clusters that are common across animals. CONCLUSION: We applied the method to 24 individual C. elegans and successfully found some known functional modules. Compared with a widely used consensus clustering method to aggregate multiple clustering results, WormTensor showed higher silhouette coefficients. Our simulation also showed that WormTensor is robust to contamination from noisy data. WormTensor is freely available as an R/CRAN package https://cran.r-project.org/web/packages/WormTensor .

Maternal epigenetic factors in embryonic and postnatal development.

Maternal factors present in oocytes and surrounding granulosa cells influence early development of embryos. In this study, we searched for epigenetic regulators that are expressed in oocytes and/or granulosa cells. Some of the 120 epigenetic regulators examined were expressed specifically in oocytes and/or granulosa cells. When their expression was examined in young versus aged oocytes or granulosa cells, many were significantly up- or downregulated in aged cells. The maternal role of six genes in development was investigated by generating oocyte-specific knock-out (MKO) mice. Two genes (Mllt10, Kdm2b) did not show maternal effects on later development, whereas maternal effects were evident for Kdm6a, Kdm4a, Prdm3, and Prdm16 for MKO female mice. Offspring from Kdm6a MKO mice underwent perinatal lethality at a higher rate. Pups derived from Prdm3;Prdm16 double MKO showed a higher incidence of postnatal death. Finally, embryos derived from Kdm4a MKO mice showed early developmental defects as early as the peri-implantation stage. These results suggest that many of maternal epigenetic regulators undergo differential expression upon aging. Some, such as Kdm4a, Kdm6a, Prdm3, and Prdm16, have maternal role in later embryonic or postnatal development.

Enhanced transcriptional heterogeneity mediated by NF-κB super-enhancers.

The transcription factor NF-κB, which plays an important role in cell fate determination, is involved in the activation of super-enhancers (SEs). However, the biological functions of the NF-κB SEs in gene control are not fully elucidated. We investigated the characteristics of NF-κB-mediated SE activity using fluorescence imaging of RelA, single-cell transcriptome and chromatin accessibility analyses in anti-IgM-stimulated B cells. The formation of cell stimulation-induced nuclear RelA foci was abolished in the presence of hexanediol, suggesting an underlying process of liquid-liquid phase separation. The gained SEs induced a switch-like expression and enhanced cell-to-cell variability in transcriptional response. These properties were correlated with the number of gained cis-regulatory interactions, while switch-like gene induction was associated with the number of NF-κB binding sites in SE. Our study suggests that NF-κB SEs have an important role in the transcriptional regulation of B cells possibly through liquid condensate formation consisting of macromolecular interactions.

A common epigenetic mechanism across different cellular origins underlies systemic immune dysregulation in an idiopathic autism mouse model.

Immune dysregulation plays a key role in the pathogenesis of autism. Changes occurring at the systemic level, from brain inflammation to disturbed innate/adaptive immune in the periphery, are frequently observed in patients with autism; however, the intrinsic mechanisms behind them remain elusive. We hypothesize a common etiology may lie in progenitors of different types underlying widespread immune dysregulation. By single-cell RNA sequencing (sc-RNA seq), we trace the developmental origins of immune dysregulation in a mouse model of idiopathic autism. It is found that both in aorta-gonad-mesonephros (AGM) and yolk sac (YS) progenitors, the dysregulation of HDAC1-mediated epigenetic machinery alters definitive hematopoiesis during embryogenesis and downregulates the expression of the AP-1 complex for microglia development. Subsequently, these changes result in the dysregulation of the immune system, leading to gut dysbiosis and hyperactive microglia in the brain. We further confirm that dysregulated immune profiles are associated with specific microbiota composition, which may serve as a biomarker to identify autism of immune-dysregulated subtypes. Our findings elucidate a shared mechanism for the origin of immune dysregulation from the brain to the gut in autism and provide new insight to dissecting the heterogeneity of autism, as well as the therapeutic potential of targeting immune-dysregulated autism subtypes.

Erratum: Derepression of inflammation-related genes link to microglia activation and neural maturation defect in a mouse model of Kleefstra syndrome.

[This corrects the article DOI: 10.1016/j.isci.2021.102741.].

Local states of chromatin compaction at transcription start sites control transcription levels.

The 'open' and 'compact' regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels.

Derepression of inflammation-related genes link to microglia activation and neural maturation defect in a mouse model of Kleefstra syndrome.

Haploinsufficiency of EHMT1, which encodes histone H3 lysine 9 (H3K9) methyltransferase G9a-like protein (GLP), causes Kleefstra syndrome (KS), a complex disorder of developmental delay and intellectual disability. Here, we examined whether postnatal supply of GLP can reverse the neurological phenotypes seen in Ehmt1 Δ/+ mice as a KS model. Ubiquitous GLP supply from the juvenile stage ameliorated behavioral abnormalities in Ehmt1 Δ/+ mice. Postnatal neuron-specific GLP supply was not sufficient for the improvement of abnormal behaviors but still reversed the reduction of H3K9me2 and spine number in Ehmt1 Δ/+ mice. Interestingly, some inflammatory genes, including IL-1ß (Il1b), were upregulated and activated microglial cells increased in the Ehmt1 Δ/+ brain, and such phenotypes were also reversed by neuron-specific postnatal GLP supply. Il1b inactivation canceled the microglial and spine number phenotypes in the Ehmt1 Δ/+ mice. Thus, H3K9me2 and some neurological phenotypes are reversible, but behavioral abnormalities are more difficult to improve depending on the timing of GLP supply.

Single-oocyte transcriptome analysis reveals aging-associated effects influenced by life stage and calorie restriction.

Chromosome segregation errors in oocytes lead to the production of aneuploid eggs, which are the leading cause of pregnancy loss and of several congenital diseases such as Down syndrome. The frequency of chromosome segregation errors in oocytes increases with maternal age, especially at a late stage of reproductive life. How aging at various life stages affects oocytes differently remains poorly understood. In this study, we describe aging-associated changes in the transcriptome profile of mouse oocytes throughout reproductive life. Our single-oocyte comprehensive RNA sequencing using RamDA-seq revealed that oocytes undergo transcriptome changes at a late reproductive stage, whereas their surrounding cumulus cells exhibit transcriptome changes at an earlier stage. Calorie restriction, a paradigm that reportedly prevents aging-associated egg aneuploidy, promotes a transcriptome shift in oocytes with the up-regulation of genes involved in chromosome segregation. This shift is accompanied by the improved maintenance of chromosomal cohesin, the loss of which is a hallmark of oocyte aging and causes chromosome segregation errors. These findings have implications for understanding how oocytes undergo aging-associated functional decline throughout their reproductive life in a context-dependent manner.

Tracing the origin of hair follicle stem cells.

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.

Developmental excitation-inhibition imbalance underlying psychoses revealed by single-cell analyses of discordant twins-derived cerebral organoids.

Despite extensive genetic and neuroimaging studies, detailed cellular mechanisms underlying schizophrenia and bipolar disorder remain poorly understood. Recent progress in single-cell RNA sequencing (scRNA-seq) technologies enables identification of cell-type-specific pathophysiology. However, its application to psychiatric disorders is challenging because of methodological difficulties in analyzing human brains and the confounds due to a lifetime of illness. Brain organoids derived from induced pluripotent stem cells (iPSCs) of the patients are a powerful avenue to investigate the pathophysiological processes. Here, we generated iPSC-derived cerebral organoids from monozygotic twins discordant for psychosis. scRNA-seq analysis of the organoids revealed enhanced GABAergic specification and reduced cell proliferation following diminished Wnt signaling in the patient, which was confirmed in iPSC-derived forebrain neuronal cells. Two additional monozygotic twin pairs discordant for schizophrenia also confirmed the excess GABAergic specification of the patients' neural progenitor cells. With a well-controlled genetic background, our data suggest that unbalanced specification of excitatory and inhibitory neurons during cortical development underlies psychoses.

The Number of Transcription Factors at an Enhancer Determines Switch-like Gene Expression.

NF-κB is a transcription factor that activates super enhancers (SEs) and typical enhancers (TEs) and triggers threshold and graded gene expression, respectively. However, the mechanisms by which NF-κB selectively participates in these enhancers remain unclear. Here we show using mouse primary B lymphocytes that SE activity simultaneously associates with chromatin opening and enriched NF-κB binding, resulting in a higher fold change and threshold expression upon B cell receptor (BCR) activation. The higher fold change results from longer DNA, whereas the threshold response is explained by synergy in DNA-NF-κB binding and is supported by the coexistence of PU.1 and NF-κB in a SE before cell stimulation. This model indicates that the pre-existing NF-κB functions as a seed and triggers its processive binding upon BCR activation. Our mathematical modeling of the single-cell transcriptome reveals an additional role for SEs in divergent clonal responses in B cells.

Benchmarking single-cell RNA-sequencing protocols for cell atlas projects.

Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas.

Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.

Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.