Mouse Cell Types Research Articles

Source cell-type epigenetic memory persists in induced pluripotent cells but is lost in subsequently derived germline cells.

Introduction: Retention of source cell-type epigenetic memory may mitigate the potential for induced pluripotent stem cells (iPSCs) to fully achieve transitions in cell fate in vitro. While this may not preclude the use of iPSC-derived somatic cell types for therapeutic applications, it becomes a major concern impacting the potential use of iPSC-derived germline cell types for reproductive applications. The transition from a source somatic cell type to iPSCs and then on to germ-cell like cells (GCLCs) recapitulates two major epigenetic reprogramming events that normally occur during development in vivo-embryonic reprogramming in the epiblast and germline reprogramming in primordial germ cells (PGCs). We examined the extent of epigenetic and transcriptomic memory persisting first during the transition from differentiated source cell types to iPSCs, and then during the transition from iPSCs to PGC-like cells (PGCLCs). Methods: We derived iPSCs from four differentiated mouse cell types including two somatic and two germ cell types and tested the extent to which each resulting iPSC line resembled a) a validated ES cell reference line, and b) their respective source cell types, on the basis of genome-wide gene expression and DNA methylation patterns. We then induced each iPSC line to form PGCLCs, and assessed epigenomic and transcriptomic memory in each compared to endogenous PGCs/M-prospermatogonia. Results: In each iPSC line, we found residual gene expression and epigenetic programming patterns characteristic of the corresponding source differentiated cell type from which each was derived. However, upon deriving PGCLCs, we found very little evidence of lingering epigenetic or transcriptomic memory of the original source cell type. Discussion: This result indicates that derivation of iPSCs and then GCLCs from differentiated source cell types in vitro recapitulates the two-phase epigenetic reprogramming that normally occurs in vivo, and that, to a significant extent, germline cell types derived in vitro from pluripotent cells accurately recapitulate epigenetic programming and gene expression patterns corresponding to equivalent endogenous germ cell types, suggesting that they have the potential to form the basis of in vitro gametogenesis as a useful therapeutic strategy for treatment of infertility.

Hypomorphic human SEL1L and HRD1 variants uncouple multilayered ER-associated degradation machinery.

The suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) complex of the endoplasmic reticulum-associated degradation (ERAD) machinery is a key cellular proteostasis pathway. Although previous studies have shown ERAD as promoting the development and maintenance of many cell types in mice, its importance to human physiology remained undetermined. In two articles in this issue of the JCI, Qi and colleagues describe four biallelic hypomorphic SEL1L and HRD1 variants that were associated with neurodevelopment disorders, locomotor dysfunction, impaired immunity, and premature death in patients. These pathogenic SEL1L-HRD1 variants shine a light on the critical importance of ERAD in humans and pave the way for future studies dissecting ERAD mechanisms in specific cell types.

Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry.

Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.

Col1α2-Cre-mediated recombination occurs in various cell types due to Cre expression in epiblasts

The Cre-LoxP system has been commonly used for cell-specific genetic manipulation. However, many Cre strains exhibit excision activity in unexpected cell types or tissues. Therefore, it is important to identify the cell types in which recombination takes place. Fibroblasts are a cell type that is inadequately defined due to a lack of specific markers to detect the entire cell lineage. Here, we investigated the Cre recombination induced by Col1α2-iCre, one of the most common fibroblast-mesenchymal Cre driver lines, by using a double-fluorescent Cre reporter line in which GFP is expressed when recombination occurs. Our results indicated that Col1α2-iCre activity was more extensive across cell types than previously reported: Col1α2-iCre-mediated recombination was found in not only cells of mesenchymal origin but also those of other lineages, including haematopoietic cells, myocardial cells, lung and intestinal epithelial cells, and neural cells. In addition, study of embryos revealed that recombination by Col1α2-iCre was observed in the early developmental stage before gastrulation in epiblasts, which would account for the recombination across various cell types in adult mice. These results offer more insights into the activity of Col1α2-iCre and suggest that experimental results obtained using Col1α2-iCre should be carefully interpreted.

Generation of a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette in the AAVS1 locus by CRISPR/Cas9-mediated homologous recombination

Diphtheria toxin A (DTA) is an exotoxin secreted by Corynebacterium diphtheriae. After entering the cell through receptor-mediated manner, DTA can trigger the programmed cell death mechanism and lead to cell death. In 2001, Michiko Saito established a Diphtheria toxin receptor–mediated cell knockout system, which can conditional deplete specific cell type in transgenic mice. This system is not only very useful in the pathogenesis study of human diseases, but also has a wide application prospect in the study of organ development and regeneration. In 2008, David Voehringer described a newly generated mouse strain that encodes DTA under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus. Thereby, it can be used in combination with tissue-specific and/or inducible Cre-expressing mouse strains to achieve toxin-mediated cell ablation in vivo. The application of DTA–mediated cell knockout system in mice has been widely reported, but it has rarely been used in human cells. Accordingly, we generated a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette (loxp-stop-loxp-DTA, LSL-DTA) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human organoid, which may provide a model for achieving conditional cell ablation in human tissues and organs.

N6-Methyladenosine mRNA Modification: From Modification Site Selectivity to Neurological Functions.

The development of various chemical methods has enabled scientists to decipher the distribution features and biological functions of RNA modifications in the past decade. In addition to modifying noncoding RNAs such as tRNAs and rRNAs, N6-methyladenosine (m6A) has been proven to be the most abundant internal chemical modification on mRNAs in eukaryotic cells and is also the most widely studied mRNA modification to date. Extensive studies have repeatedly demonstrated the important functions of m6A in various biological conditions, ranging from embryonic organ development to adult organ function and pathogenesis. Unlike DNA methylation which is relatively stable, the reversible m6A modification on mRNA is highly dynamic and easily influenced by various internal or external factors, such as cell type, developmental stage, nutrient supply, circadian rhythm, and environmental stresses.In this Account, we review our previous findings on the site selectivity mechanisms regulating m6A formation, as well as the physiological roles of m6A modification in cerebellum development and long-term memory consolidation. In our initial efforts to profile m6A in various types of mouse and human cells, we surprisingly found that the sequence motifs surrounding m6A sites were often complementary with the seed sequences of miRNAs. By manipulating the abundance of the miRNA biogenesis enzyme Dicer or individual miRNAs or mutating miRNA sequences, we were able to reveal a new role of nucleus localized miRNAs, which is to guide the m6A methyltransferase METTL3 to bind to mRNAs and to promote m6A formation. As a result, we partially answered the question of why only a small proportion of m6A motifs within an mRNA could have m6A modification at a certain time point. We further explored the functions of m6A modification in regulating brain development and brain functions. We found that cerebellum had the most severe defects when Mettl3 was knocked out in developing mouse embryonic brain and revealed that the underlying mechanisms could be attributed to aberrant mRNA splicing and enhanced cell apoptosis under m6A deficit conditions. On the other hand, knocking out Mettl3 in postnatal hippocampus did not cause morphological defects in the mouse brain but impaired the efficacy of long-term memory consolidation. Under learning stimuli, formation of m6A modifications could be detected on transcripts encoding proteins related to dendrite growth, synapse formation, and other memory related functions. Loss of m6A modifications on these transcripts would result in translation deficiency and reduced protein production, particularly in the translation of early response genes, and therefore would compromise the efficacy of long-term memory consolidation. Interestingly, excessive training sessions or increased training intensity could overcome such m6A deficiency related memory defects, which is likely due to the longer turnover cycle and the cumulative abundance of proteins throughout the training process. In addition to revealing the roles of m6A modification in regulating long-term memory formation, our work also demonstrated an effective method for studying memory formation efficacy. As the lack of an appropriate model for studying memory formation efficacy has been a long-lasting problem in the field of neural science, our hippocampus-specific postnatal m6A knockout model could also be utilized to study other questions related to memory formation efficacy.

IL-12 sensing in neurons induces neuroprotective CNS tissue adaptation and attenuates neuroinflammation in mice.

Interleukin-12 (IL-12) is a potent driver of type 1 immunity. Paradoxically, in autoimmune conditions, including of the CNS, IL-12 reduces inflammation. The underlying mechanism behind these opposing properties and the involved cellular players remain elusive. Here we map IL-12 receptor (IL-12R) expression to NK and T cells as well as neurons and oligodendrocytes. Conditionally ablating the IL-12R across these cell types in adult mice and assessing their susceptibility to experimental autoimmune encephalomyelitis revealed that the neuroprotective role of IL-12 is mediated by neuroectoderm-derived cells, specifically neurons, and not immune cells. In human brain tissue from donors with multiple sclerosis, we observe an IL-12R distribution comparable to mice, suggesting similar mechanisms in mice and humans. Combining flow cytometry, bulk and single-nucleus RNA sequencing, we reveal an IL-12-induced neuroprotective tissue adaption preventing early neurodegeneration and sustaining trophic factor release during neuroinflammation, thereby maintaining CNS integrity in mice.

Single-cell analysis of the miRNA activities in tuberculous meningitis (TBM) model mice injected with the BCG vaccine

BackgroundOur previous study revealed the transcriptome atlas of specific cell types in tuberculous meningitis (TBM) model mice injected with the BCG vaccine via scRNA sequencing. However, the activities of miRNAs in TBM at single-cell resolution remain to be explored. MethodCell type-specific miRNA activities were investigated by using motif enrichment analyses (miReact) on the transcriptome data of 15 cell types. The target mRNAs of miRNAs were predicted and subjected to enrichment analysis. Furthermore, miRNAs and their target mRNAs with opposite expression trends were chosen to construct functional networks. Besides, qRT-PCR and RNA scope were performed to verify the expression level of representative miRNA. ResultsThe tSNE dimensionality reduction presented 15 cell types in TBM model mice, in which microglia and endothelial cells accounted for the majority. Target mRNAs of each cell type were predicted for verification or network construction. The immune and inflammation-related miRNA-mRNA networks of macrophages and microglia, oxidative phosphorylation-related miRNA-mRNA networks of neurons, ion and protein transport-related networks of epididymal cells, and angiogenesis-related miRNA-mRNA networks of VSMCs were constructed. The miRNA activity analysis revealed that miR-21a-3p activity was increased in microglia, macrophages, neurons and epididymal cells. The result of qRT-PCR and RNA scope indicate that miR-21a-3p was significantly higher-expressed in TBM brain tissue compared with normal brain tissue. ConclusionIn our study, an in-depth exploration of the mRNA expression and miRNA activity of macrophages, microglia, epididymal cells, neurons and vascular smooth muscle cells during TBM progression was conducted using scRNA-Seq, which provided novel insights into the immune cell engagement in TBM patients.

Acute murine cytomegalovirus infection boosts cell-type specific response and lipid metabolism changes in the liver of infant mice.

Human cytomegalovirus (HCMV) infection in infants can lead to severe diseases, including neonatal hepatitis. The single-cell dimensional changes in immune cells after the initial CMV infection remain elusive, as do the effects of CMV infection on hepatic lipid metabolism. We employed single-cell RNA-sequencing to investigate the changes in liver cell types and immune responses in infant mice following murine CMV (MCMV) infection. Additionally, we examined alterations in protein expression profiles related to lipid metabolism in hepatocytes and the role of the key transcription factor PPAR-γ in hepatocytes during CMV infection. Our study revealed that MCMV infects most liver cell types in infant mice, leading to an increase in the proportion of proliferating CD8 effector T cells and a subset of Nos2+ monocytes, potentially playing an essential role in early anti-viral responses. Furthermore, MCMV infection resulted in altered protein expression of lipid metabolism in hepatocytes. Knocking down the transcription factor PPAR-γ in hepatocytes effectively inhibited CMV infection. Our findings underscore the immune system's response to early-stage MCMV infection and the subsequent impact on hepatic lipid metabolism in infant mice. This research provides new insights into the mechanisms of CMV infection and could pave the way for novel therapeutic strategies.

SCENIC+: single-cell multiomic inference of enhancers and gene regulatory networks

Joint profiling of chromatin accessibility and gene expression in individual cells provides an opportunity to decipher enhancer-driven gene regulatory networks (GRNs). Here we present a method for the inference of enhancer-driven GRNs, called SCENIC+. SCENIC+ predicts genomic enhancers along with candidate upstream transcription factors (TFs) and links these enhancers to candidate target genes. To improve both recall and precision of TF identification, we curated and clustered a motif collection with more than 30,000 motifs. We benchmarked SCENIC+ on diverse datasets from different species, including human peripheral blood mononuclear cells, ENCODE cell lines, melanoma cell states and Drosophila retinal development. Next, we exploit SCENIC+ predictions to study conserved TFs, enhancers and GRNs between human and mouse cell types in the cerebral cortex. Finally, we use SCENIC+ to study the dynamics of gene regulation along differentiation trajectories and the effect of TF perturbations on cell state. SCENIC+ is available at scenicplus.readthedocs.io.

Extracellular messaging elicited by exercise

The exercise-induced secretomes of multiple cell types in mice are characterized.

Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs Rps27 and Rps27l in vertebrates.

The formation of paralogs through gene duplication is a core evolutionary process. For paralogs that encode components of protein complexes such as the ribosome, a central question is whether they encode functionally distinct proteins or whether they exist to maintain appropriate total expression of equivalent proteins. Here, we systematically tested evolutionary models of paralog function using the ribosomal protein paralogs Rps27 (eS27) and Rps27l (eS27L) as a case study. Evolutionary analysis suggests that Rps27 and Rps27l likely arose during whole-genome duplication(s) in a common vertebrate ancestor. We show that Rps27 and Rps27l have inversely correlated mRNA abundance across mouse cell types, with the highest Rps27 in lymphocytes and the highest Rps27l in mammary alveolar cells and hepatocytes. By endogenously tagging the Rps27 and Rps27l proteins, we demonstrate that Rps27- and Rps27l-ribosomes associate preferentially with different transcripts. Furthermore, murine Rps27 and Rps27l loss-of-function alleles are homozygous lethal at different developmental stages. However, strikingly, expressing Rps27 protein from the endogenous Rps27l locus or vice versa completely rescues loss-of-function lethality and yields mice with no detectable deficits. Together, these findings suggest that Rps27 and Rps27l are evolutionarily retained because their subfunctionalized expression patterns render both genes necessary to achieve the requisite total expression of two equivalent proteins across cell types. Our work represents the most in-depth characterization of a mammalian ribosomal protein paralog to date and highlights the importance of considering both protein function and expression when investigating paralogs.

Specific Methyl-CpG Configurations Define Cell Identity through Gene Expression Regulation.

Cell identity is determined by the chromatin structure and profiles of gene expression, which are dependent on chromatin accessibility and DNA methylation of the regions critical for gene expression, such as enhancers and promoters. These epigenetic modifications are required for mammalian development and are essential for the establishment and maintenance of the cellular identity. DNA methylation was once thought to be a permanent repressive epigenetic mark, but systematic analyses in various genomic contexts have revealed a more dynamic regulation than previously thought. In fact, both active DNA methylation and demethylation occur during cell fate commitment and terminal differentiation. To link methylation signatures of specific genes to their expression profiles, we determined the methyl-CpG configurations of the promoters of five genes switched on and off during murine postnatal brain differentiation by bisulfite-targeted sequencing. Here, we report the structure of significant, dynamic, and stable methyl-CpG profiles associated with silencing or activation of the expression of genes during neural stem cell and brain postnatal differentiation. Strikingly, these methylation cores mark different mouse brain areas and cell types derived from the same areas during differentiation.

Dynamic Patterns of Mammalian Mitochondrial DNA Replication Uncovered Using SSiNGLe-5'ES.

The proper replication of mitochondrial DNA is key to the maintenance of this crucial organelle. Multiple studies aimed at understanding the mechanisms of replication of the mitochondrial genome have been conducted in the past several decades; however, while highly informative, they were conducted using relatively low-sensitivity techniques. Here, we established a high-throughput approach based on next-generation sequencing to identify replication start sites with nucleotide-level resolution and applied it to the genome of mitochondria from different human and mouse cell types. We found complex and highly reproducible patterns of mitochondrial initiation sites, both previously annotated and newly discovered in this work, that showed differences among different cell types and species. These results suggest that the patterns of the replication initiation sites are dynamic and might reflect, in some yet unknown ways, the complexities of mitochondrial and cellular physiology. Overall, this work suggests that much remains unknown about the details of mitochondrial DNA replication in different biological states, and the method established here opens up a new avenue in the study of the replication of mitochondrial and potentially other genomes.

Investigating cell-specific effects of FMRP deficiency on spiny projection neurons in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS), resulting from a mutation in the Fmr1 gene, is the most common monogenic cause of autism and inherited intellectual disability. Fmr1 encodes the Fragile X Messenger Ribonucleoprotein (FMRP), and its absence leads to cognitive, emotional, and social deficits compatible with the nucleus accumbens (NAc) dysfunction. This structure is pivotal in social behavior control, consisting mainly of spiny projection neurons (SPNs), distinguished by dopamine D1 or D2 receptor expression, connectivity, and associated behavioral functions. This study aims to examine how FMRP absence differentially affects SPN cellular properties, which is crucial for categorizing FXS cellular endophenotypes. We utilized a novel Fmr1-/y::Drd1a-tdTomato mouse model, which allows in-situ identification of SPN subtypes in FXS mice. Using RNA-sequencing, RNAScope and ex-vivo patch-clamp in adult male mice NAc, we comprehensively compared the intrinsic passive and active properties of SPN subtypes. Fmr1 transcripts and their gene product, FMRP, were found in both SPNs subtypes, indicating potential cell-specific functions for Fmr1. The study found that the distinguishing membrane properties and action potential kinetics typically separating D1- from D2-SPNs in wild-type mice were either reversed or abolished in Fmr1-/y::Drd1a-tdTomato mice. Interestingly, multivariate analysis highlighted the compound effects of Fmr1 ablation by disclosing how the phenotypic traits distinguishing each cell type in wild-type mice were altered in FXS. Our results suggest that the absence of FMRP disrupts the standard dichotomy characterizing NAc D1- and D2-SPNs, resulting in a homogenous phenotype. This shift in cellular properties could potentially underpin select aspects of the pathology observed in FXS. Therefore, understanding the nuanced effects of FMRP absence on SPN subtypes can offer valuable insights into the pathophysiology of FXS, opening avenues for potential therapeutic strategies.

Common factors among three types of cells aged in mice.

The greatest risk factor for the formation of numerous significant chronic disorders is aging. Understanding the core molecular underpinnings of aging can help to slow down the inevitable process. Systematic study of gene expression or DNA methylation data is possible at the transcriptomics and epigenetics levels. DNA methylation and gene expression are both affected by aging. Gene expression is an important element in the aging of Homo sapiens. In this work, we evaluated the expression of differentially expressed genes (DEGs), proteins, and transcription factors (TFs) in three different types of cells in mice: antibody-secreting cells, cardiac mesenchymal stromal cells, and skeletal muscle cells. The goal of this article is to uncover a common cause during aging among these cells in order to increase understanding about establishing complete techniques for preventing aging and improving people's quality of life. We conducted a comprehensive network-based investigation to establish which genes and proteins are shared by the three different types of aged cells. Our findings clearly indicated that aging induces gene dysregulation in immune, pharmacological, and apoptotic pathways. Furthermore, our research developed a list of hub genes with differential expression in aging responses that should be investigated further to discover viable anti-aging treatments.

Intestinal cell type-specific communication networks underlie homeostasis and response to Western diet.

The small intestine plays a key role in immunity and mediates inflammatory responses to high fat diets. We have used single-cell RNA-sequencing (scRNA-seq) and statistical modeling to examine gaps in our understanding of the dynamic properties of intestinal cells and underlying cellular mechanisms. Our scRNA-seq and flow cytometry studies of different layers of intestinal cells revealed new cell subsets and modeled developmental trajectories of intestinal intraepithelial lymphocytes, lamina propria lymphocytes, conventional dendritic cells, and enterocytes. As compared to chow-fed mice, a high-fat high-sucrose (HFHS) "Western" diet resulted in the accumulation of specific immune cell populations and marked changes to enterocytes nutrient absorption function. Utilizing ligand-receptor analysis, we profiled high-resolution intestine interaction networks across all immune cell and epithelial structural cell types in mice fed chow or HFHS diets. These results revealed novel interactions and communication hubs among intestinal cells, and their potential roles in local as well as systemic inflammation.

Characterization of chromatin accessibility patterns in different mouse cell types using machine learning methods at single-cell resolution.

Chromatin accessibility is a generic property of the eukaryotic genome, which refers to the degree of physical compaction of chromatin. Recent studies have shown that chromatin accessibility is cell type dependent, indicating chromatin heterogeneity across cell lines and tissues. The identification of markers used to distinguish cell types at the chromosome level is important to understand cell function and classify cell types. In the present study, we investigated transcriptionally active chromosome segments identified by sci-ATAC-seq at single-cell resolution, including 69,015 cells belonging to 77 different cell types. Each cell was represented by existence status on 20,783 genes that were obtained from 436,206 active chromosome segments. The gene features were deeply analyzed by Boruta, resulting in 3897 genes, which were ranked in a list by Monte Carlo feature selection. Such list was further analyzed by incremental feature selection (IFS) method, yielding essential genes, classification rules and an efficient random forest (RF) classifier. To improve the performance of the optimal RF classifier, its features were further processed by autoencoder, light gradient boosting machine and IFS method. The final RF classifier with MCC of 0.838 was constructed. Some marker genes such as H2-Dmb2, which are specifically expressed in antigen-presenting cells (e.g., dendritic cells or macrophages), and Tenm2, which are specifically expressed in T cells, were identified in this study. Our analysis revealed numerous potential epigenetic modification patterns that are unique to particular cell types, thereby advancing knowledge of the critical functions of chromatin accessibility in cell processes.

Single-Cell RNA Transcriptome Profiling of Liver Cells of Short-Term Alcoholic Liver Injury in Mice

Alcoholic liver disease (ALD) is currently considered a global healthcare problem with limited pharmacological treatment options. There are abundant cell types in the liver, such as hepatocytes, endothelial cells, Kupffer cells and so on, but little is known about which kind of liver cells play the most important role in the process of ALD. To obtain a cellular resolution of alcoholic liver injury pathogenesis, 51,619 liver single-cell transcriptomes (scRNA-seq) with different alcohol consumption durations were investigated, 12 liver cell types were identified, and the cellular and molecular mechanisms of the alcoholic liver injury were revealed. We found that more aberrantly differential expressed genes (DEGs) were present in hepatocytes, endothelial cells, and Kupffer cells than in other cell types in alcoholic treatment mice. Alcohol promoted the pathological processes of liver injury; the specific mechanisms involved: lipid metabolism, oxidative stress, hypoxia, complementation and anticoagulation, and hepatocyte energy metabolism on hepatocytes; NO production, immune regulation, epithelial and cell migration on endothelial cells; antigen presentation and energy metabolism on Kupffer cells, based on the GO analysis. In addition, our results showed that some transcription factors (TFs) are activated in alcohol-treated mice. In conclusion, our study improves the understanding of liver cell heterogeneity in alcohol-fed mice at the single-cell level. It has potential value for understanding key molecular mechanisms and improving current prevention and treatment strategies for short-term alcoholic liver injury.

Single-nucleus Atlas of Sevoflurane-induced Hippocampal Cell Type- and Sex-specific Effects during Development in Mice.

Multiple neonatal exposures to sevoflurane induce neurocognitive dysfunctions in rodents. The lack of cell type-specific information after sevoflurane exposure limits the mechanistic understanding of these effects. In this study, the authors tested the hypothesis that sevoflurane exposures alter the atlas of hippocampal cell clusters and have neuronal and nonneuronal cell type-specific effects in mice of both sexes. Neonatal mice were exposed to 3% sevoflurane for 2 h at postnatal days 6, 8, and 10 and analyzed for the exposure effects at postnatal day 37. Single-nucleus RNA sequencing was performed in the hippocampus followed by in situ hybridization to validate the results of RNA sequencing. The Morris Water Maze test was performed to test neurocognitive function. The authors found sex-specific distribution of hippocampal cell types in control mice alongside cell type- and sex-specific effects of sevoflurane exposure on distinct hippocampal cell populations. There were important changes in male but not in female mice after sevoflurane exposure regarding the proportions of cornu ammonis 1 neurons (control vs. sevoflurane, males: 79.9% vs. 32.3%; females: 27.3% vs. 24.3%), dentate gyrus (males: 4.2% vs. 23.4%; females: 36.2% vs. 35.8%), and oligodendrocytes (males: 0.6% vs. 6.9%; females: 5.9% vs. 7.8%). In male but not in female mice, sevoflurane altered the number of significantly enriched ligand-receptor pairs in the cornu ammonis 1, cornu ammonis 3, and dente gyrus trisynaptic circuit (control vs. sevoflurane, cornu ammonis 1-cornu ammonis 3: 18 vs. 42 in males and 15 vs. 21 in females; cornu ammonis 1-dentate gyrus: 21 vs. 35 in males and 12 vs. 20 in females; cornu ammonis 3-dentate gyrus: 25 vs. 45 in males and 17 vs. 20 in females), interfered with dentate gyrus granule cell neurogenesis, hampered microglia differentiation, and decreased cornu ammonis 1 pyramidal cell diversity. Oligodendrocyte differentiation was specifically altered in females with increased expressions of Mbp and Mag. In situ hybridization validated the increased expression of common differentially expressed genes. This single-nucleus RNA sequencing study reveals the hippocampal atlas of mice, providing a comprehensive resource for the neuronal and nonneuronal cell type- and sex-specific effects of sevoflurane during development.