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A 3D digital atlas of C. elegans and its application to single-cell analyses.
We built a digital nuclear atlas of the newly hatched, first larval stage (L1) of the wild-type hermaphrodite of Caenorhabditis elegans at single-cell resolution from confocal image stacks of 15 individual worms. The atlas quantifies the stereotypy of nuclear locations and provides other statistics on the spatial patterns of the 357 nuclei that could be faithfully segmented and annotated out of the 558 present at this developmental stage. We then developed an automated approach to assign cell names to each nucleus in a three-dimensional image of an L1 worm. We achieved 86% accuracy in identifying the 357 nuclei automatically. This computational method will allow high-throughput single-cell analyses of the post-embryonic worm, such as gene expression analysis, or ablation or stimulation of cells under computer control in a high-throughput functional screen.
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The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.
Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 μm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.
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Distinct systemic impacts of Aβ42 and Tau revealed by whole-organism snRNA-seq.
Both neuronal and peripheral tissues become disrupted in Alzheimer's disease (AD). However, a comprehensive understanding of how AD impacts different tissues across the whole organism is lacking. Using Drosophila, we generated an AD Fly Cell Atlas (AD-FCA) based on whole-organism single-nucleus transcriptomes of 219 cell types from flies expressing AD-associated proteins, either human amyloid-β 42 peptide (Aβ42) or Tau, in neurons. We found that Aβ42 primarily affects the nervous system, including sensory neurons, while Tau induces accelerated aging in peripheral tissues. We identified a neuronal cluster enriched in Aβ42 flies, which has high lactate dehydrogenase (LDH) expression. This LDH-high cluster is conserved in 5XFAD mouse and human AD datasets. We found a conserved defect in fat metabolism from both fly and mouse tauopathy models. The AD-FCA offers new insights into how Aβ42 or Tau systemically and differentially affects a whole organism and provides a valuable resource for understanding brain-body communication in neurodegeneration.
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Single-cell transcriptional profiling identifies the swimming crab Portunus trituberculatus in response to bacterial infection.
Crustaceans rely entirely on innate immunity, yet the cellular composition, functional specialization, and pathogen-induced remodeling of their immune system remain poorly resolved. Here, we generated a high-resolution single-cell transcriptomic atlas of hemocytes from the swimming crab Portunus trituberculatus following Vibrio parahaemolyticus infection using 10× Genomics scRNA-seq. Seven putatively distinct hemocyte clusters were identified, including granulocytes, semigranular hemocytes, prohemocytes, unresolved hemocytes, hyalinocyte-like hemocytes, biosynthetically active secretory hemocytes, and regulatory hemocytes. Although the overall cellular composition remained relatively stable after infection, hemocytes exhibited pronounced cluster-specific transcriptional reprogramming involving Toll/NF-κB signaling, antimicrobial peptide synthesis and metabolic rewiring. By integrating single-cell and bulk transcriptomes, we identified multiple anti-lipopolysaccharide factors (ALFs) as key secretory effectors and experimentally validated their antibacterial activities. FITC-based bacterial engulfment assays and RNA-seq of sorted phagocytes demonstrated that phagocytic capability was shared across multiple hemocyte clusters. Notably, the immunoglobulin superfamily receptor DSCAM displayed extensive alternative splicing and strong infection-induced activation in unresolved hemocytes. Immune-training experiments showed that prior bacterial exposure was associated with altered DSCAM expression and reduced early cumulative mortality upon secondary challenge, suggesting a memory-like immune phenotype. These findings provide a foundational framework for understanding crustacean immunity and advancing disease-resistant breeding in aquaculture.
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Single-cell transcriptomic profiling of the mouse cochlea: An atlas for targeted therapies.
Functional molecular characterization of the cochlea has mainly been driven by the deciphering of the genetic architecture of sensorineural deafness. As a result, the search for curative treatments, which are sorely lacking in the hearing field, has become a potentially achievable objective, particularly via cochlear gene and cell therapies. To this end, a complete inventory of cochlear cell types, with an in-depth characterization of their gene expression profiles right up to their final differentiation, is indispensable. We therefore generated a single-cell transcriptomic atlas of the mouse cochlea based on an analysis of more than 120,000 cells on postnatal day 8 (P8), during the prehearing period, P12, corresponding to hearing onset, and P20, when cochlear maturation is almost complete. By combining whole-cell and nuclear transcript analyses with extensive in situ RNA hybridization assays, we characterized the transcriptomic signatures covering nearly all cochlear cell types and developed cell type-specific markers. Three cell types were discovered; two of them contribute to the modiolus which houses the primary auditory neurons and blood vessels, and the third one consists in cells lining the scala vestibuli. The results also shed light on the molecular basis of the tonotopic gradient of the biophysical characteristics of the basilar membrane that critically underlies cochlear passive sound frequency analysis. Finally, overlooked expression of deafness genes in several cochlear cell types was also unveiled. This atlas paves the way for the deciphering of the gene regulatory networks controlling cochlear cell differentiation and maturation, essential for the development of effective targeted treatments.
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Single-cell RNA sequencing reveals the Müller subtypes and inner blood-retinal barrier regulatory network in early diabetic retinopathy.
As the basic pathological changes of diabetic retinopathy (DR), the destruction of the blood-retina barrier (BRB) and vascular leakage have attracted extensive attention. Without timely intervention, BRB damage will eventually lead to serious visual impairment. However, due to the delicate structure and complex function of the BRB, the mechanism underlying damage to the BRB in DR has not been fully clarified. Here, we used single-cell RNA sequencing (RNA-seq) technology to analyze 35,910 cells from the retina of healthy and streptozotocin (STZ)-induced diabetic rats, focusing on the degeneration of the main cells constituting the rat BRB in DR and the new definition of two subpopulations of Müller cells at the cell level, Ctxn3 +Müller and Ctxn3 -Müller cells. We analyzed the characteristics and significant differences between the two groups of Müller cells and emphasized the importance of the Ctxn3 +Müller subgroup in diseases. In endothelial cells, we found possible mechanisms of self-protection and adhesion and recruitment to pericytes. In addition, we constructed a communication network between endothelial cells, pericytes, and Müller subsets and clarified the complex regulatory relationship between cells. In summary, we constructed an atlas of the iBRB in the early stage of DR and elucidate the degeneration of its constituent cells and Müller cells and the regulatory relationship between them, providing a series of potential targets for the early treatment of DR.
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Human subcutaneous and visceral adipocyte atlases uncover classical and nonclassical adipocytes and depot-specific patterns.
Human adipose depots are functionally distinct. Yet, recent single-nucleus RNA sequencing (snRNA-seq) analyses largely uncovered overlapping or similar cell-type landscapes. We hypothesized that adipocyte subtypes, differentiation trajectories and/or intercellular communication patterns could illuminate this depot similarity-difference gap. For this, we performed snRNA-seq of human subcutaneous or visceral adipose tissues (five or ten samples, respectively). Of 27,665 adipocyte nuclei in both depots, most were 'classical', namely enriched in lipid metabolism pathways. However, we also observed 'nonclassical' adipocyte subtypes, enriched in immune-related, extracellular matrix deposition (fibrosis), vascularization or angiogenesis or ribosomal and mitochondrial processes. Pseudo-temporal analysis showed a developmental trajectory from adipose progenitor cells to classical adipocytes via nonclassical adipocytes, suggesting that the classical state stems from loss, rather than gain, of specialized functions. Last, intercellular communication routes were consistent with the different inflammatory tone of the two depots. Jointly, these findings provide a high-resolution view into the contribution of cellular composition, differentiation and intercellular communication patterns to human fat depot differences.
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Single-cell landscape reveals active cell subtypes and their interaction in the tumor microenvironment of gastric cancer.
Background: Gastric cancer remains the third most common cause of cancer-related death worldwide. The development of novel therapeutic strategies for gastric cancer requires a deep understanding of the tumor cells and microenvironment of gastric cancer. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine untreated non-metastatic gastric cancer patients. The transcriptomic atlas and ligand-receptor-based intercellular communication networks of the single cells were characterized. Results: Here, we profiled the transcriptomes of 47,304 cells from nine patients with gastric cancer. Tregs cells were significantly enriched in the gastric tumor tissues with increased expression of immune suppression related genes, which suggest a more immunosuppressive microenvironment. We also observed the absence of separate exhausted CD8+ T cell cluster, and the low expression level of exhaustion markers PDCD1, CTLA4, HAVCR2, LAG-3, and TIGIT in those specific cells. These may serve as molecular-level evidence for the limited benefit of immunotherapy among gastric cancer patients. In addition, we found ACKR1 specifically expressed in tumor endothelial cells, associated with poor prognosis in the cohort data and potentially provided a novel target of gastric cancer treatment. Furthermore, the tight interaction between endothelial cells and fibroblast implied the important roles of fibroblast in tumor angiogenesis and the maintenance of tumor vasculature. Conclusions: In conclusion, this single-cell atlas provide understanding the cellular heterogeneity from molecular level in gastric cancer and will serve as a valuable resource for developing innovative early and companion diagnostics, as well as discovering novel targeted therapies for gastric cancer.
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Pan-cancer analysis and single-cell analysis identifies the CENPN as a biomarker for survival prognosis and immunotherapy.
BACKGROUND: Centromere protein N (CENPN), located on chromosome 16q23.2, encodes vital nucleosome-associated complexes that are essential for dynamic assembly processes. CENPN plays a pivotal role in regulating cell proliferation and cell cycle progression by influencing mitotic events. Despite its potential importance, the precise functional role and regulatory mechanisms of CENPN in diverse malignancies remain largely unexplored. This study aimed to elucidate the role of CENPN in human cancers and evaluate its prognostic significance. METHODS: Investigate the role of CENPN in various malignancies, we leveraged data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. We employed a comprehensive suite of web platforms and software tools for data analysis, including R, Cytoscape, an integrated repository portal for tumor-immune system interactions (TISIDB), CBio Cancer Genomics Portal (cBioPortal), Search Tool for the Retrieval of Interaction Gene/Proteins (STRING), Gene Set Cancer Analysis (GSCALite), and a cancer single-cell state atlas (CancerSEA). RESULTS: The findings demonstrated that CENPN expression was elevated in the majority of cancer types and differentially expressed across molecular and immune subtypes. Functional enrichment analysis in multiple tumors also identified possible pathways of CENPN involvement in tumorigenesis. Its expression positively correlated with Th2 and Tcm cells in most cancers. It is also correlated with genetic markers of immunomodulators in various cancers. CONCLUSIONS: Overall, CENPN expression is closely related to cancers and has the potential to act as a cancer biomarker.
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A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.
Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.