RESUMO
Immunity to infection has been extensively studied in humans and mice bearing naturally occurring or experimentally introduced germline mutations. Mouse studies are sometimes neglected by human immunologists, on the basis that mice are not humans and the infections studied are experimental and not natural. Conversely, human studies are sometimes neglected by mouse immunologists, on the basis of the uncontrolled conditions of study and small numbers of patients. However, both sides would agree that the infectious phenotypes of patients with inborn errors of immunity often differ from those of the corresponding mutant mice. Why is that? We argue that this important question is best addressed by revisiting and reinterpreting the findings of both mouse and human studies from a genetic perspective. Greater caution is required for reverse-genetics studies than for forward-genetics studies, but genetic analysis is sufficiently strong to define the studies likely to stand the test of time. Genetically robust mouse and human studies can provide invaluable complementary insights into the mechanisms of immunity to infection common and specific to these two species.
Assuntos
Doenças do Sistema Imunitário , Imunidade , Fenótipo , Animais , Humanos , Camundongos , Imunidade/genética , Doenças do Sistema Imunitário/genéticaRESUMO
Alphaviruses are emerging and reemerging viruses that cause disease syndromes ranging from incapacitating arthritis to potentially fatal encephalitis. While infection by arthritogenic and encephalitic alphaviruses results in distinct clinical manifestations, both virus groups induce robust innate and adaptive immune responses. However, differences in cellular tropism, type I interferon induction, immune cell recruitment, and B and T cell responses result in differential disease progression and outcome. In this review, we discuss aspects of immune responses that contribute to protective or pathogenic outcomes after alphavirus infection.
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Infecções por Alphavirus , Alphavirus , Interferon Tipo I , Infecções por Alphavirus/patologia , Animais , Humanos , Imunidade , TropismoRESUMO
Natural killer (NK) cells are innate lymphocytes that provide critical host defense against pathogens and cancer. Originally heralded for their early and rapid effector activity, NK cells have been recognized over the last decade for their ability to undergo adaptive immune processes, including antigen-driven clonal expansion and generation of long-lived memory. This review presents an overview of how NK cells lithely partake in both innate and adaptive responses and how this versatility is manifest in human NK cell-mediated immunity.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Animais , Humanos , Imunidade Celular , Células Matadoras NaturaisRESUMO
Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.
Assuntos
Senescência Celular , Humanos , Animais , Biomarcadores/metabolismo , Guias como Assunto , Neoplasias/patologiaRESUMO
Obesity causes significant morbidity and mortality globally. Research in the last three decades has delivered a step-change in our understanding of the fundamental mechanisms that regulate energy homeostasis, building on foundational discoveries in mouse models of metabolic disease. However, not all findings made in rodents have translated to humans, hampering drug discovery in this field. Here, we review how studies in mice and humans have informed our current framework for understanding energy homeostasis, discuss their challenges and limitations, and offer a perspective on how human studies may play an increasingly important role in the discovery of disease mechanisms and identification of therapeutic targets in the future.
Assuntos
Modelos Animais de Doenças , Doenças Metabólicas , Pesquisa Translacional Biomédica , Animais , Humanos , Camundongos , Doenças Metabólicas/metabolismo , Metabolismo Energético , Homeostase , Obesidade/metabolismoRESUMO
Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.
Assuntos
Prosencéfalo , Animais , Prosencéfalo/metabolismo , Prosencéfalo/embriologia , Camundongos , Ratos , Blastocisto/metabolismo , Feminino , Sistemas CRISPR-Cas/genética , Transcriptoma , Organogênese , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Masculino , Camundongos Endogâmicos C57BLRESUMO
The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.
RESUMO
Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.
Assuntos
COVID-19 , Influenza Humana , Animais , Humanos , Camundongos , COVID-19/virologia , COVID-19/genética , Influenza Humana/virologia , Replicação Viral , Macrófagos/metabolismo , Macrófagos/virologia , Feminino , Masculino , SARS-CoV-2 , Pulmão/virologia , Pulmão/patologia , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Ácido Oleico/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Camundongos Knockout , Carga Viral , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/genética , Infecções por Orthomyxoviridae/virologia , Infecções Respiratórias/virologia , CriançaRESUMO
Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.
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Embrião de Mamíferos , Células-Tronco Embrionárias , Animais , Técnicas de Cocultura , Macaca fascicularis , Células-Tronco Embrionárias/metabolismo , Diferenciação Celular , Endoderma/metabolismo , Linhagem da CélulaRESUMO
Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.
Assuntos
Metilação de DNA , Epigênese Genética , Camundongos , Humanos , Animais , Ilhas de CpG , Padrões de Herança , Mamíferos/genéticaRESUMO
Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.
Assuntos
Gastrulação , Mesoderma , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteínas Nucleares/metabolismo , Transdução de SinaisRESUMO
We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from â¼250 × 140 × 90 µm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.
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Neocórtex , Animais , Camundongos , Microscopia Eletrônica , Neocórtex/fisiologia , Organelas , Células Piramidais/fisiologia , Sinapses/fisiologiaRESUMO
Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.
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Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma , Camundongos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , TransgenesRESUMO
Mouse germline cysts, on average, develop into six oocytes supported by 24 nurse cells that transfer cytoplasm and organelles to generate a Balbiani body. We showed that between E14.5 and P5, cysts periodically activate some nurse cells to begin cytoplasmic transfer, which causes them to shrink and turnover within 2 days. Nurse cells die by a programmed cell death (PCD) pathway involving acidification, similar to Drosophila nurse cells, and only infrequently by apoptosis. Prior to initiating transfer, nurse cells co-cluster by scRNA-seq with their pro-oocyte sisters, but during their final 2 days, they cluster separately. The genes promoting oocyte development and nurse cell PCD are upregulated, whereas the genes that repress transfer, such as Tex14, and oocyte factors, such as Nobox and Lhx8, are under-expressed. The transferred nurse cell centrosomes build a cytocentrum that establishes a large microtubule aster in the primordial oocyte that organizes the Balbiani body, defining the earliest oocyte polarity.
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Linhagem da Célula , Cistos , Oócitos , Animais , Apoptose , Crescimento Celular , Cistos/genética , Cistos/metabolismo , Citoplasma/metabolismo , Drosophila melanogaster , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Ovário/citologia , Ovário/embriologia , Ovário/metabolismoRESUMO
Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.
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Neoplasias , Animais , Genes ras , Camundongos , Neoplasias/genética , Filogenia , Sequenciamento do ExomaRESUMO
Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.
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Organogênese , Transcriptoma , Animais , DNA/genética , Embrião de Mamíferos , Feminino , Perfilação da Expressão Gênica/métodos , Mamíferos/genética , Camundongos , Organogênese/genética , Gravidez , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genéticaRESUMO
Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.
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Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Animais , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas , Humanos , CamundongosRESUMO
Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.
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Desenvolvimento Embrionário/fisiologia , Gastrulação/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Feminino , Expressão Gênica , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas , Gravidez , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
Retrotransposons mediate gene regulation in important developmental and pathological processes. Here, we characterized the transient retrotransposon induction during preimplantation development of eight mammals. Induced retrotransposons exhibit similar preimplantation profiles across species, conferring gene regulatory activities, particularly through long terminal repeat (LTR) retrotransposon promoters. A mouse-specific MT2B2 retrotransposon promoter generates an N-terminally truncated Cdk2ap1ΔN that peaks in preimplantation embryos and promotes proliferation. In contrast, the canonical Cdk2ap1 peaks in mid-gestation and represses cell proliferation. This MT2B2 promoter, whose deletion abolishes Cdk2ap1ΔN production, reduces cell proliferation and impairs embryo implantation, is developmentally essential. Intriguingly, Cdk2ap1ΔN is evolutionarily conserved in sequence and function yet is driven by different promoters across mammals. The distinct preimplantation Cdk2ap1ΔN expression in each mammalian species correlates with the duration of its preimplantation development. Hence, species-specific transposon promoters can yield evolutionarily conserved, alternative protein isoforms, bestowing them with new functions and species-specific expression to govern essential biological divergence.
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Sequência Conservada , Desenvolvimento Embrionário/genética , Proteínas Quinases/metabolismo , Retroelementos/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Sequência de Bases , Blastocisto/metabolismo , Proliferação de Células , Evolução Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mamíferos/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismoRESUMO
SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.