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Profiling combinations of histone modifications identifies gene regulatory elements in different states and discovers features controlling transcriptional and epigenetic programs. However, efforts to map chromatin states in complex, heterogeneous samples are hindered by the lack of methods that can profile multiple histone modifications together with transcriptomes in individual cells. Here, we describe single-cell multitargets and mRNA sequencing (scMTR-seq), a high-throughput method that enables simultaneous profiling of six histone modifications and transcriptome in single cells. We apply scMTR-seq to uncover dynamic and coordinated changes in chromatin states and transcriptomes during human endoderm differentiation. We also use scMTR-seq to produce lineage-resolved chromatin maps and gene regulatory networks in mouse blastocysts, revealing epigenetic asymmetries at gene regulatory regions between the three embryo lineages and identifying Trps1 as a potential repressor in epiblast cells of trophectoderm-associated enhancer networks and their target genes. Together, scMTR-seq enables investigation of combinatorial chromatin landscapes in a broad range of heterogeneous samples, providing insights into epigenetic regulatory systems.

P-Rex2 is a Rac guanine-nucleotide factor (Rac-GEF) that controls glucose homeostasis. This role is thought to be mediated through its adaptor function inhibiting Pten rather than through its Rac-GEF activity, but this remains to be demonstrated. To examine this question, we have investigated the roles of P-Rex2 in glucose homeostasis using Prex2 and catalytically-inactive Prex2 mice. We show that P-Rex2 is required for insulin sensitivity but limits glucose clearance, suppressing glucose uptake into liver and skeletal muscle independently of its catalytic activity. In hepatocytes, P-Rex2 suppresses Glut2 cell surface levels, mitochondrial membrane potential and mitochondrial ATP production. We identify the orphan GPCR Gpr21 as a P-Rex2 target and propose that P-Rex2 limits hepatic glucose clearance by controlling Gpr21 trafficking. In skeletal muscle cells, P-Rex2 suppresses glucose uptake through a separate adaptor function, independently of Gpr21. Additionally, P-Rex2 suppresses insulin secretion by pancreatic islets and plasma insulin levels. Finally, P-Rex2 plays distinct Rac-GEF activity dependent and independent roles in PIP production in liver and skeletal muscle, respectively. Together, our study identifies complex roles of P-Rex2 in glucose homeostasis, mediated through largely GEF-activity independent mechanisms which include the GPCR Gpr21 in hepatocytes and but are not obviously linked to the regulation of Pten.

T follicular helper (T) cells are a helper T-cell subset that is defined by their localisation to B-cell areas of secondary lymphoid tissues, enabling them to provide their B-cell helper function. Precursors of T cells migrate to the B-cell follicles by upregulating CXCR5 and downregulating CCR7, a process that can be blocked by S1PR1 overexpression. T cells and their precursors also express the early activation antigen CD69, which is a negative regulator of S1PR1. In this study, we tested the hypothesis that CD69 expression by T cells is important for their differentiation and localisation after immunization. Genetic deletion of CD69 on T cells and a proportion of their precursors did not alter their formation, nor their ability to support high-affinity B-cell responses. This demonstrates that although CD69 is expressed highly on T cells, it is not necessary for their formation or their B-cell helper functions in lymph nodes (LNs).

The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized the study of gene expression in individual cells, providing unprecedented insights into cellular heterogeneity and developmental processes. The application of scRNA-seq to oocyte biology has facilitated the identification of species-specific transcriptional signatures and developmental trajectories, enhancing our understanding of oogenesis. This chapter presents a detailed protocol for scRNA-seq analysis of growing bovine oocytes.

During gastrulation, mouse epiblast cells form the three germ layers that establish the body plan and initiate organogenesis. While single-cell atlases have advanced our understanding of lineage diversification, spatial aspects of differentiation remain poorly defined. Here, we applied spatial transcriptomics to mouse embryos at embryonic (E) E7.25 and E7.5 days and integrated these data with existing E8.5 spatial and E6.5-E9.5 single-cell RNA-seq atlases. This resulted in a spatiotemporal atlas of over 150,000 cells with 82 refined cell-type annotations. The resource enables exploration of gene expression dynamics across anterior-posterior and dorsal-ventral axes, uncovering spatial logic guiding mesodermal fate decisions in the primitive streak. We also developed a computational pipeline to project additional single-cell datasets into this framework for comparative analysis. Freely accessible through an interactive web portal, this atlas offers a valuable tool for the developmental and stem cell biology communities to investigate mouse embryogenesis in a spatial and temporal context.

Although the regulation of branching morphogenesis by spatially distributed cues is well established, the underlying intracellular signaling mechanisms are not well understood. The development of the lacrimal gland is driven by fibroblast growth factor (FGF) signaling, which activates phospholipase C gamma (PLCγ). Here, we showed that mutating the PLCγ1 binding site on Fgfr2 leads to ectopic branching and hyperplasia in the lacrimal gland, which was phenocopied by either deleting PLCγ1 or disabling any of its SH2 domains. PLCγ1 inactivation did not change the level of Fgfr2 or affect mitogen-activated protein kinase (MAPK) signaling but instead led to sustained AKT phosphorylation due to increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) production. Consistent with this, the PLCγ1 mutant phenotype can be reproduced by the elevation of phosphatidylinositol 3-kinase (PI3K) signaling in Pten knockout and attenuated by blocking AKT signaling. Our findings demonstrate that FGF-activated PLCγ modulates PI3K signaling by shifting phosphoinositide metabolism, revealing the crucial role of PLCγ in branching morphogenesis and organ size control.

Breeding management in laboratory rodents is challenging, particularly around parturition and the neonatal period, where cage disturbance is often avoided in an attempt to limit neonatal mortality. Nevertheless, cage-side observations and single daily checks frequently underestimate pup numbers born and miss parturition complications. Home Cage Monitoring (HCM) systems are gaining popularity in animal facilities, detecting critical events such as food availability and activity levels. Parturition is a complex event involving specific patterns of behaviour, activity and vocalisations. In this study, audio and video data were collected from parturition events of single-housed C57BL/6J females and breeding pairs housed in a prototype rack with integrated microphones. Vocalisations were detected during parturition in both housing conditions, with minimal vocalisations observed prior to parturition, except for ultrasonic sounds in pair-housed mice (). After parturition, all vocalisations gradually decreased. Despite limitations such as the need for post-event analysis and the focus on a single mouse strain, this study suggests that detecting vocalisations can be a promising basis for developing automated parturition detection. This highlights the potential of HCM systems for improving breeding management and welfare in laboratory rodent colonies.

Embryonic development requires the accurate spatiotemporal execution of cell lineage-specific gene expression programs, which are controlled by transcriptional enhancers. Developmental enhancers adopt a primed chromatin state prior to their activation. How this primed enhancer state is established and maintained and how it affects the regulation of developmental gene networks remains poorly understood.

Metastatic uveal melanoma is an aggressive disease with limited effective therapeutic options. To comprehensively map monogenic and digenic dependencies, we performed CRISPR-Cas9 screening in ten extensively profiled human uveal melanoma cell line models. Analysis involved genome-wide single-gene and combinatorial paired-gene CRISPR libraries. Among our 76 uveal melanoma-specific essential genes and 105 synthetic lethal gene pairs, we identified and validated the CDP-diacylglycerol synthase 2 gene (CDS2) as a genetic dependency in the context of low CDP-diacylglycerol synthase 1 gene (CDS1) expression. We further demonstrate that CDS1/CDS2 forms a synthetic lethal interaction in vivo and reveal that CDS2 knockout results in the disruption of phosphoinositide synthesis and increased cellular apoptosis and that re-expression of CDS1 rescues this cell fitness defect. We extend our analysis using pan-cancer data, confirming increased CDS2 essentiality in diverse tumor types with low CDS1 expression. Thus, the CDS1/CDS2 axis is a therapeutic target across a range of cancers.

Experimental models and epidemiological data suggest that environmental factors, for example, adverse nutrition prior to conception, can lead to phenotypes in offspring of exposed parents in the absence of continued exposure. As a result these phenotypes have been described as epigentically inherited. The mechanistic basis for such phenomena has not been established in most cases. In this review, we consider possible contributing mechanisms for environmentaly induced epigenetic inheritance, with a focus on maternally transmitted effects and by comparing to paradigms of epigenetic inheritance with a clear mechanistic understanding. Genomic imprinting has provided an important conceptual framework for how the epigenetic states of parental germlines can determine allelic expression in offspring, yet, generally speaking, imprinted genes appear resilient to epigenetic disruption from altered parental environments. Metastable epialleles are environmentally sensitive and variably expressed loci that can impact organism phenotype, but the nature of any epigenetic marker at these loci transferred to offspring is unclear. Studies of examples across these forms of epigenetic inheritance show predominant effects are mediated by oocyte factors involved inreprogramming of the genome post-fertilization, rather than direct effects on gametic DNA methylation, with the exception of genomic imprinting. The potential contribution of additional oocyte chromatin features to the specific liability of phenotypic effector genes and their potential to persist through this reprogramming, however, remains to be investigated.

Solid-organ transplant (SOT) recipients are at enhanced risk of infection and to poorly respond to vaccination due to comorbidities and immunosuppression. We performed a systems vaccinology study in 59 kidney and 31 lung transplant recipients who received 3 doses of COVID-19 mRNA BNT162b2 vaccine. We were able to characterize a baseline configuration associated with an effective humoral response to 3 doses, characterized by an innate and activated B cell profile, whereas a T cell signature was associated with a poorer response. We observed a distinct configuration associated with a detectable humoral response to 2 doses, partly mediated by double negative B cell subsets. These results suggest that, despite their immunosuppression, some SOT recipients can induce an effective humoral response to 3 doses of vaccine supported by a baseline configuration close to the healthy phenotype. Baseline immune phenotyping may help identify SOT recipients at the greatest risk of a poor vaccine response.

In vitro maturation is an essential tool in reproductive technologies, though its impact on oocyte quality remains a concern. This study shows that in vitro maturation alters gene expression and DNA methylation in bovine oocytes compared to in vivo matured oocytes, potentially compromising oocyte quality and developmental competence.

During mouse gastrulation, extraembryonic mesoderm (ExEM) contributes to the extraembryonic yolk sac (YS) and allantois, both of which are essential for successful gestation. Although the genetic networks coordinating intra-embryonic mesodermal subtype specification are well studied, ExEM diversification remains poorly understood. Here, we identify that embryoid body (EB) in vitro differentiation generates distinct lineages of mesodermal cells, matching YS and allantois development. Combining in vitro and in vivo mouse models, we discover that Eomesodermin (Eomes) controls the formation of YS-fated ExEM but is dispensable for allantois formation. Furthermore, simultaneous disruption of Eomes and T impedes the specification of any YS or allantois mesoderm, indicating compensatory roles for T during allantois formation upon Eomes depletion. Our study highlights previously unrecognized functional and mechanistic diversity in ExEM diversification and endothelial development and introduces a tractable EB model to dissect the signaling pathways and transcriptional networks driving the formation of key extraembryonic tissues.

Human stem cell-based embryo models (SCBEMs) are a research technology with the potential to facilitate our understanding of human embryogenesis, improve assisted reproductive technology outcomes, elucidate the causes of early pregnancy failure, and provide a clearer understanding of the developmental origins of disease. Given that human SCBEMs are designed to model specific phenotypic features and developmental processes of human embryos, they raise distinct concerns from other stem cell models, such as organoids. The International Society for Stem Cell Research (ISSCR) Guidelines for Stem Cell Research and Clinical Translation, published in 2021, made recommendations for research oversight of SCBEMs and established different categories of review based on involvement of embryonic and extraembryonic lineages. However, recent progress has enabled unexpected ways to create increasingly complex models, as well as more efficient means of doing so without including all major extraembryonic lineages. A working group was tasked by the ISSCR executive to undertake a thorough reexamination of the guidelines in the light of these advances. The three main recommendations of the working group are that all research involving organized 3-dimensional human SCBEMs (1) should be subject to appropriate review, (2) must have a clear scientific rationale, and (3) must be subject to limited timelines. The proposed modifications to the ISSCR guidelines are intended to bring more clarity to the field, help guide the deliberations of researchers, oversight committees and other relevant stakeholders, and ensure continued public confidence.

Enhancer elements interact with target genes at a distance to modulate their expression, but the molecular details of enhancer-promoter interaction are incompletely understood. G-quadruplex DNA secondary structures (G4s) have recently been shown to co-occur with 3D chromatin interactions; however, the functional importance of G4s within enhancers remains unclear.

NLRP2 is a subcortical maternal complex (SCMC) protein of mammalian oocytes and preimplantation embryos. SCMC proteins are encoded by maternal effect genes and play a pivotal role in the maternal-to-zygotic transition (MZT), early embryogenesis, and epigenetic (re)programming. Maternal inactivation of genes encoding SCMC proteins has been linked to infertility and subfertility in mice and humans, but the underlying molecular mechanisms for the diverse functions of SCMC proteins, and specifically the role of NLRP2, are incompletely understood.

Germinal center (GC) B cells are pivotal in establishing a robust humoral immune response and long-term serological immunity while maintaining antibody self-tolerance. GC B cells rely on autophagy for antigen presentation and homeostatic maintenance. However, these functions, primarily associated with the light zone, cannot explain the spatiotemporal autophagy upregulation in the dark zone of GCs. Here, combining imaging, molecular, and genomic approaches, we defined a functional mechanism controlling chromatin accessibility in GC B cells during their dark zone transition. This mechanism links autophagy and nuclear lamin B1 dynamics with their downstream effects, including somatic hypermutation and antibody affinity maturation. Moreover, the autophagy-lamin B1 axis is highly active in the aberrant ectopic GCs in the salivary glands of Sjögren's disease, defining its role in autoimmunity.

In this article for the Highlights of 2024 series, we review the latest advances in the biology of the germinal center response. These discoveries provide key insights into germinal center function and dysregulation, uncovering new opportunities for the development of more effective vaccines.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis, are often associated with mutations in stress granule proteins. Aberrant stress granule condensate formation is associated with disease, making it a potential target for pharmacological intervention. Here, we identified lipoamide, a small molecule that specifically prevents cytoplasmic condensation of stress granule proteins. Thermal proteome profiling showed that lipoamide stabilizes intrinsically disordered domain-containing proteins, including SRSF1 and SFPQ, which are stress granule proteins necessary for lipoamide activity. SFPQ has redox-state-specific condensate dissolving behavior, which is modulated by the redox-active lipoamide dithiolane ring. In animals, lipoamide ameliorates aging-associated aggregation of a stress granule reporter protein, improves neuronal morphology and recovers motor defects caused by amyotrophic lateral sclerosis-associated FUS and TDP-43 mutants. Thus, lipoamide is a well-tolerated small-molecule modulator of stress granule condensation, and dissection of its molecular mechanism identified a cellular pathway for redox regulation of stress granule formation.

RNA binding proteins (RBP) of the ZFP36 family limit the differentiation and effector functions of CD4 and CD8 T cells, but little is known of their expression or function in regulatory T (Treg) cells. By using Treg cell-restricted deletion of Zfp36 family members we identify the role of Zfp36l1 and Zfp36l2 in Treg cells to maintain immune homeostasis. Mice with Treg cells deficient in these RBP display an inflammatory phenotype with an expansion in the numbers of type-2 conventional dendritic cells, T effector cells, T follicular helper and germinal center B cells and elevated serum cytokines and immunoglobulins. In the absence of Zfp36l1 and Zfp36l2, the pool of cycling CTLA-4 in naïve Treg cells is reduced, Treg cells are less sensitive to IL-2 and IL-7 but are more sensitive to IFNγ. In mice lacking both RBP in Treg cells, the deletion of a single allele of Ifng is sufficient to ameliorate the pathology. Our results indicate that ZFP36L1 and ZFP36L2 regulate the availability of IFNγ and are required for the maintenance of Treg cell stability. Thus, ZFP36L1 and ZFP36L2 regulate multiple pathways that enable Treg cells to enforce immune homeostasis.

Public dialogue is crucial for understanding societal views on human embryo research, and the complexity and sensitivity of this topic require special considerations of how such dialogues are facilitated. Here, we identify enablers of effective dialogue, which can improve the design and delivery of engagement exercises related to embryo research.

Porcine respiratory coronavirus (PRCV) is a naturally occurring pneumotropic coronavirus in the pig, providing a valuable large animal model to study acute respiratory disease. PRCV pathogenesis and the resulting immune response were investigated in pigs, the natural large animal host. We compared 2 strains, ISU-1 and 135, which induced differing levels of pathology in the respiratory tract to elucidate the mechanisms leading to mild or severe disease. The 135 strain induced greater pathology which was associated with higher viral load and stronger spike-specific antibody and T-cell responses. In contrast, the ISU-1 strain triggered mild pathology with a more balanced immune response and greater abundance of T regulatory cells. A higher frequency of putative T follicular helper cells was observed in animals infected with strain 135 at 11 days postinfection. Single-cell RNA-sequencing of bronchoalveolar lavage revealed differential gene expression in B and T cells between animals infected with 135 and ISU-1 at 1 day postinfection. These genes were associated with cell adhesion, migration, and immune regulation. Along with increased IL-6 and IL-12 production, these data indicate that heightened inflammatory responses to the 135 strain may contribute to pronounced pneumonia. Among bronchoalveolar lavage (BAL) immune cell populations, B cells and plasma cells exhibited the most gene expression divergence between pigs infected with different PRCV strains, highlighting their role in maintaining immune homeostasis in the respiratory tract. These findings indicate the potential of the PRCV model for studying coronavirus-induced respiratory disease and identifying mechanisms that determine infection outcomes.

The oncomutation lysine 27-to-methionine in histone H3 (H3K27M) is frequently identified in tumors of patients with diffuse midline glioma-H3K27 altered (DMG-H3K27a). H3K27M inhibits the deposition of the histone mark H3K27me3, which affects the maintenance of transcriptional programs and cell identity. Cells expressing H3K27M are also characterized by defects in genome integrity, but the mechanisms linking expression of the oncohistone to DNA damage remain mostly unknown. In this study, we demonstrate that expression of H3.1K27M in the model plant Arabidopsis thaliana interferes with post-replicative chromatin maturation mediated by the H3.1K27 methyltransferases ATXR5 and ATXR6. As a result, H3.1 variants on nascent chromatin remain unmethylated at K27 (H3.1K27me0), leading to ectopic activity of TONSOKU (TSK/TONSL), which induces DNA damage and genomic alterations. Elimination of TSK activity suppresses the genome stability defects associated with H3.1K27M expression, while inactivation of specific DNA repair pathways prevents survival of H3.1K27M-expressing plants. Overall, our results suggest that H3.1K27M disrupts the chromatin-based mechanisms regulating TSK activity, which causes genomic instability and may contribute to the etiology of DMG-H3K27a.