EMBO Reports最新文献

Primordial germ cells (PGCs) are the precursors of gametes, and the ability to derive PGC-like cells (PGCLCs) from pluripotent stem cells has transformed germline research. A key limitation remains producing PGCLCs in sufficient numbers for large-scale applications. Here, we show that overexpression of Nanog plus three PGC master regulators - Prdm1, Prdm14, and Tfap2c - in mouse epiblast-like cells and formative embryonic stem cells yields abundant and highly enriched PGCLCs without costly recombinant cytokines. Nanog enhances the PGC regulatory network, suppresses somatic differentiation, and stabilizes PGCLC fate. Transcriptomically, these PGCLCs are developmentally more advanced than cytokine-induced counterparts and can be sustained long-term or differentiated into spermatogonia-like cells. Using this platform, we conduct a CRISPRi screen of 701 epigenetic genes to identify those needed for PGCLC formation. Downregulation of Ncor2, a histone deacetylase (HDAC) recruiter, has the greatest impact. Additionally, the HDAC inhibitors valproic acid and sodium butyrate suppress PGCLC formation and sperm counts of in utero-exposed animals. This work establishes a scalable system for functional screening of genes that influence germline development.

Gastrulation is a fundamental developmental process during which germ layers are formed and the body axes are defined by the precise orchestration of cell movements and fate specification. Here, we identify the SOXE transcription factor Sox8 as a pivotal regulator of Xenopus laevis gastrulation. We show that Sox8 is expressed in the ventrolateral mesoderm, and that its depletion-via CRISPR-DiCas7-11-leads to blastopore closure defects and impaired AP axis elongation. Transcriptomic analysis reveals that Sox8 modulates Wnt signalling, in part by directly activating transcription of kremen2, a Wnt inhibitor. Indeed, chromatin immunoprecipitation confirms direct binding of Sox8 to the kremen2 promoter. Consequently, Sox8 or Kremen2 knockdown results in an abnormal ventral expansion of wnt11b mRNA that was consistent with increased nuclear β-catenin and reduced BMP signalling. These treatments also led to disruptions in axial and paraxial mesodermal patterning. Together, our data provide new insights into the molecular control of vertebrate gastrulation and invite researchers to assess whether this Sox8/Kremen2 regulatory axis is involved in other biological processes.

Notch signalling is a major signalling pathway coordinating cellular processes between neighbouring animal cells. In Drosophila, two E3 ubiquitin ligases, Neuralized (Neur) and Mindbomb1 (Mib1), regulate Notch ligand activation and are essential for development. However, the mammalian orthologs of Neur, Neuralized-like (NEURL) 1 and 1B, appear to be dispensable for development, as double knock-out mice show no overt developmental defects. Thus, it is unclear if and how NEURL proteins regulate the mammalian Notch ligands. To address this question, we examined NEURL proteins' ability to activate Notch ligands in a humanized Drosophila model and mammalian cell culture. We found that, unlike MIB1, NEURL proteins activate Notch only with a subset of mammalian ligands, which contain a Neuralized binding motif. This motif has the consensus sequence NxxN and is present only in Notch ligands DLL1 and JAG1, but not in DLL4 and JAG2. Thus, our results reveal a differential regulatory mechanism of Notch activation in mammals, which can potentially explain the limited role of NEURL proteins in mammalian development and homeostasis.

GPR164 is a free fatty acid receptor, activated by both short-chain fatty acids and medium-chain fatty acids, and expressed throughout the gastrointestinal tract. Although GPR164 is reported to be involved in the release of gut hormones, the physiological functions of this receptor in the maintenance of intestinal homeostasis remain unclear. In this study, we explore the role of GPR164 in regulating intestinal barrier function using mice lacking Gpr164 gene (Gpr164^-/-). A loss-of-function mutation in Gpr164 promotes cell proliferation and disrupts the intestinal barrier function in both Caco-2 cells and mice. Genome-wide RNA-seq analysis reveals that Gpr164 deletion causes aberrant Wnt/β-catenin signaling, and the intraperitoneal injection of the Wnt/β-catenin inhibitor PNU-74654 ameliorates intestinal hyperproliferation, differentiation and barrier permeability phenotypes of Gpr164^-/- mice. Gpr164^-/- mice also exhibit gut microbial dysbiosis and inflammation. Thus, our findings uncover the pivotal role of GPR164 in the maintenance of intestinal homeostasis through regulating barrier function.

Acid sensing is essential for various biological processes in animals, yet it exhibits species-specific characteristics. In this study, we identified a proton-dissociation-permeated sodium channel (PDPNaC1) in the antennal sensory neurons of the centipede Scolopendra subspinipes mutilans. PDPNaC1, which is permeable to monovalent cations, assembles as a homotrimer. Unlike most proton-gated channels, where proton binding induces currents, PDPNaC1's transient ion-permeable state is triggered by proton dissociation. By resolving the high-resolution cryo-electron microscopy (cryo-EM) structure of PDPNaC1, combined with mutagenesis and electrophysiological analyses, we identified Gly378, rather than the Gly-Ala-Ser tract, as a key determinant of ion selectivity. Furthermore, Ser376, located in the ion-permeable pathway, likely serves as a proton-binding site, leading to an H⁺-blocking effect that results in proton-dissociated currents. Thus, the identification of PDPNaC1 suggests the remarkable diversity of proton responses and molecular mechanisms in DEG/ENaC family.

The mechanical and metabolic states of progenitor and stem cells are emerging as key regulators of cell fate decisions. Lineage specification of pancreatic endocrine cells is promoted by reduced mechanical tension in vitro, but the underlying mechanism is poorly understood. Here, we show that heterogeneously deposited low-adhesion extracellular matrix (ECM) components, such as the laminin isoform LN411, trigger a local "soft" environment by broadly reducing the expression of integrins. Mimicking this low-tension state by in vitro knockdown and in vivo gene targeting of the LN-binding integrins Itga3 and Itga6 reveal their importance in inducing endocrinogenesis. Unexpectedly, the cell responds to this change in tensile forces by engaging a major metabolic enzyme, PDK4, to execute the resulting cell fate decision. PDK4 achieves this through two distinct mechanisms: a non-canonical action controlling YAP activity and a canonical metabolic function maintaining PDX1 expression. In sum, we believe our findings have broad relevance for how local changes in mechanical tension governs cell behaviour in many developmental and disease contexts.

In addition to their role in canonical autophagy, autophagy proteins (ATG) contribute to various cellular processes, including phagocytosis, membrane remodeling, and vesicle secretion. Several viruses also exploit components of the autophagy pathway for their own replication. Here, we explore the role of ATG proteins in HIV-1 assembly. Postulating that host proteins crucial for virion assembly are present at the assembly site and can be incorporated within virions, we analyze the proteome of HIV-1 preparations using mass spectrometry. We identify an enrichment of macroautophagy-related terms, notably 3 of the 6 ATG8 (LC3/GABARAP) proteins. Functional studies reveal that GABARAP proteins are critical for the production of infectious virions. Knockout of GABARAP proteins reduces the packaging of viral genomic RNA (gRNA) into particles, impairing virion infectivity. GABARAPL1 associates with gRNA and interacts with Gag in an RNA-dependent manner. Additionally, GABARAP knockout increases cellular Gag:gRNA complexes and decreases gRNA association with membranes, suggesting that GABARAP proteins regulate gRNA fate during HIV-1 assembly by facilitating its packaging. This study uncovers a novel role for GABARAP proteins in HIV-1 genome packaging.