EMBO Journal最新文献

During development, stem and progenitor cells divide and transition through multipotent states to generate the diverse cell types by undergoing defined changes in biomolecular composition, which underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, the cardiac and pharyngeal muscle programs are jointly primed in multipotent cardiopharyngeal progenitors, and segregate in distinct daughter cells only after cell division. Here, using the tunicate Ciona, we showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single-cell RNA-sequencing (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S, and G2 phases. We refer to this process as "transcriptome maturation", and identified candidate mature genes, including the Rho GAP-coding gene Depdc1b, which peaks in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and by enabling asymmetric cell division that influences subsequent fate decisions, illustrating the concept of "behavioral competence". We show that both classic regulatory circuits and coupling with the G1-S transition drive transcriptome maturation, ensuring the timely deployment of lineage-specific programs.

DNA replication is essential to life and ensures the accurate transmission of genetic information, which is significantly disturbed during cancer development and chemotherapy. While DNA replication is tightly controlled in time and space, methods to visualise and quantify replication dynamics within 3D human cells are lacking. Here, we introduce 3D-Spatial Assay for Replication Kinetics (3D-SPARK), an approach enabling nanoscale analysis of DNA synthesis dynamics in situ. 3D-SPARK integrates optimised nucleotide analogue pulse labelling with super-resolution microscopy to detect, classify, and quantify replication nanostructures in single cells. By combining immunofluorescence techniques with click chemistry-based nascent DNA labelling and transfection of fluorescent nucleotide derivatives, we map multi-colour DNA synthesis events in relation to established replication proteins, local RNA-protein condensates or large subnuclear domains. We demonstrate quantitative changes in size, relative abundance and spatial arrangement of nanoscale DNA synthesis events upon chemotherapeutic treatment, CDC6 oncogene expression and loss of chromatin organiser RIF1. The flexibility, precision and modular design of 3D-SPARK helps bridging the gap between spatial cell biology, genomics, and 2D fibre-based replication studies in health and disease.

Entry into and exit from cellular quiescence require dynamic adjustments in nutrient acquisition, yet the mechanisms by which quiescent cells downregulate amino acid (AA) transport remain poorly understood. Here we show that cells entering quiescence selectively target plasma membrane-resident amino acid transporters for endocytosis and lysosomal degradation. This process matches amino acid uptake with reduced translational demand and promotes survival during extended periods of quiescence. Mechanistically, we identify the α-arrestin TXNIP as a key regulator of this metabolic adaptation, since it mediates the endocytosis of the SLC7A5-SLC3A2 (LAT1-4F2hc) AA transporter complex in response to reduced AKT signaling. To promote transporter ubiquitination, TXNIP interacts with NEDD4L and other HECT-type ubiquitin ligases. Loss of TXNIP disrupts this regulation, resulting in dysregulated amino acid uptake, sustained mTORC1 signaling, and ultimately cell death under prolonged quiescence. The characterization of a novel TXNIP loss-of-function variant in a patient with a severe metabolic disease further supports its role in nutrient homeostasis and human health. Together, these findings highlight TXNIP's central role in controlling nutrient acquisition and metabolic plasticity with implications for quiescence biology and diseases.

The host innate immune system provides the first line of protection against invading microbial pathogens, including fungi. Recognition of fungi by host pattern-recognition receptors (PRRs) is critical for their clearance. PRRs bind to pathogen-associated molecular patterns (PAMPs) that can be present on the fungal surface, secreted by them, or found in their genetic material, but also damage-associated molecular patterns (DAMPs) released by host cells as a result of fungal infection. These receptors can be located at the cell surface, the endosome, or in the cytosol of host cells. Depending on PRR location and the nature of the molecular patterns (PAMPs/DAMPs) they recognize, their activation induces specific signaling pathways culminating in tailored immune responses. There are two families of innate immune receptors that can principally sense fungi, namely membrane-bound Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). In addition, as phagocytosed fungal pathogens can escape the phagolysosome and reach the cytoplasm, cytosolic sensors such as Nod-like receptors (NLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are also important in fungal sensing and play essential roles in antifungal host protection. This review summarizes the cytosolic receptors and the signaling pathways involved in antifungal innate immunity.

Innate immune receptors often induce activation of conventional dendritic cells (cDCs) and enhance antigen (cross-)presentation, favouring immune responses. DNGR-1 (CLEC9A), a receptor expressed by type 1 cDCs (cDC1s) and implicated in immune responses to viruses and cancer, recognises F-actin exposed on dead cell remnants and promotes cross-presentation of associated antigens. Here, we show that recruitment of phosphatase SHIP1, a process governed by a single amino acid residue adjacent to the signalling motif of the receptor, partly explains how DNGR-1 fails to trigger cDC1 activation in vitro. Substituting this residue converts DNGR-1 into an activating receptor but decreases induction of cross-presentation of dead cell-associated antigens. Introducing the reverse mutation into the related receptor Dectin-1 impairs its activation capacity while enhancing its ability to promote cross-presentation. These findings reveal a functional trade-off in receptor signalling and suggest that DNGR-1 has evolved to prioritise antigen cross-presentation over cellular activation, possibly to minimise inflammatory responses to dead cells.

TET1, TET2 and TET3 are DNA demethylases with important roles in development and differentiation. To assess the contributions of TET proteins to cell function during early development, single and compound knockouts of Tet genes in mouse pluripotent embryonic stem cells (ESCs) were generated. Here, we show that TET proteins are not required to transit between naïve, formative and primed pluripotency states. Moreover, ESCs with double knockouts of Tet1 and Tet2 or triple knockouts of Tet1, Tet2 and Tet3 are phenotypically indistinguishable. TET1,2,3-deficient ESCs exhibit differentiation defects and fail to activate somatic gene expression, retaining expression of pluripotency transcription factors. Therefore, TET1 and TET2, but not TET3 act redundantly to facilitate somatic differentiation. Importantly however, TET-deficient ESCs can differentiate into primordial germ cell-like cells (PGCLCs), and do so at high efficiency in the presence or absence of PGC-promoting cytokines. Moreover, acquisition of a PGCLC transcriptional programme occurs more rapidly in TET-deficient cells. These results establish that TET proteins act at the juncture between somatic and germline fates: without TET proteins, epiblast cell differentiation defaults to the germline.

The maternal-to-zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screen, in which we targeted mRNAs encoding kinases and phosphatases or proteins regulating them in zebrafish. This screen identified branched-chain ketoacid dehydrogenase kinase, Bckdk, as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA, and expression of a phospho-mimetic mutant of Phf10 rescued the developmental defects observed after bckdk mRNA depletion, as well as restored H3K27ac levels. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.

Animals host symbiotic microbial communities that shape gut health. However, how the host immune system and microbiota interact to regulate epithelial homeostasis, particularly during early development, remains largely unclear. Human interleukin-26 (IL-26) is associated with gut inflammation and has intrinsic bactericidal activity in vitro, yet its in vivo functions are largely unknown, primarily due to its absence in rodents. To examine the role of IL-26 in early life, we used zebrafish and found that gut epithelial cells in il26-/- larvae exhibited increased proliferation, faster turnover, elevated DNA damage, and altered cell population abundance. This epithelial dysregulation occurred independently of the IL-26 canonical receptor and resulted from dysbiosis in il26-/- larvae. Moreover, IL-26 bactericidal activity was conserved in zebrafish, suggesting a potential role of this property in regulating microbiota composition. We further identified innate lymphoid cells (ILCs) as the primary source of IL-26 at this developmental stage. These findings establish IL-26 as a central player in a regulatory circuit linking the microbiota, ILCs, and intestinal epithelial cells to maintain gut homeostasis during early life.

Prostaglandin E2 (PGE2) signaling through EP2 and EP4 receptors is crucial in regulating inflammation, pain, and cancer progression. While selective and dual antagonists for these receptors hold therapeutic potential, their binding mechanisms and selectivity have remained unclear. In this study, we present cryo-electron microscopy (cryo-EM) structures of human EP2 and EP4 receptors in complex with selective antagonists PF-04418948 and grapiprant, as well as with the dual antagonist TG6-129. These structures reveal distinct binding pockets and interaction networks that dictate antagonist selectivity and efficacy. Notably, TG6-129 displays a novel binding mode, engaging deeply with EP2 while interacting more superficially with EP4 in a two-warhead manner. Furthermore, comparisons of active and inactive receptor structures elucidate the mechanisms underlying EP2 activation and antagonism. Overall, these findings provide a structural framework for understanding prostanoid receptor pharmacology and offer valuable insights for the rational design of improved selective and dual antagonists targeting EP2 and EP4 receptors.