EMBO Reports最新文献

Hematopoietic stem and progenitor cells (HSPCs) emerge from arterial endothelial cells (ECs) through a process termed endothelial-to-hematopoietic-transition (EHT), a process induced by paracrine signals and driven by a transcriptional cascade. Despite inductive signals being broadly received by ECs in the dorsal aorta (DA), only a subset of ECs undergoes EHT, while others maintain their vascular identity. The molecular mechanisms that determine this selective fate decision remain poorly understood. Here, we discover Apelin signaling as a critical regulator of cell fates in the DA, acting as a molecular switch to balance vascular and hematopoietic identities. We show that Apelin receptor (Aplnr)-expressing ECs retain their arterial identity, while Aplnr non-expressing ECs are primed to become hemogenic endothelial cells (HECs) and transition into HSPCs. Loss of Apelin signaling leads to excessive EC-to-HEC conversion and increased HSPC numbers. Conversely, forced Aplnr expression abolishes HSPC formation by maintaining EC identity. These findings reveal that Apelin signaling regulates HSPC formation by preserving endothelial identity. In summary, our findings establish Apelin signaling as a critical regulator for balancing endothelial and hematopoietic fates.

The Arctic ground squirrel (AGS, Urocitellus parryii), an extreme hibernator, exhibits remarkable resilience to stressors like hypoxia and hypothermia, making it an ideal model for studying cellular metabolic adaptation. The underlying mechanisms of AGS resilience are largely unknown. Here, we use lipidomic and metabolomic profiling to discover specific downregulation of triglyceride lipids and upregulation of the lipid biosynthetic precursor malonic acid in AGS neural stem cells (NSC) versus murine NSCs. Inhibiting lipid biosynthesis recapitulates hypoxic resilience of squirrel NSCs. Extending this model, we find that acute exposure to hypoxia downregulates key lipid biosynthetic enzymes in C. elegans, while inhibiting lipid biosynthesis reduces mitochondrial fission and facilitates hypoxic survival. Moreover, inhibiting lipid biosynthesis protects against APOE4-induced pathologies and aging trajectories in C. elegans. These findings suggest triglyceride downregulation as a conserved metabolic resilience mechanism, offering insights into protective strategies for neural tissues under hypoxic or ischemic conditions, APOE4-induced pathologies and aging.

Cell surface glycoproteins Basigin or embigin form heterodimers with monocarboxylate transporters (MCTs), enhancing their membrane trafficking and modulating their transport functions. Cancer cells often reprogram their metabolism and depend on proton-coupled lactate transport mediated by MCTs to sustain their glycolytic state and to maintain intracellular pH. A deeper understanding of MCTs regulation may open avenues for the development of novel inhibitors, potentially applicable in clinical settings. Here, we determine the cryo-EM structures of the human MCT2-embigin complex in both apo and AR-C155858-bound states and observe that embigin engages in extensive interactions with MCT2, facilitating its localization to the plasma membrane and substrate transport. Given the high structural conservation among MCTs, we conduct virtual screening based on MCT1/2 structures and identify Tucatinib as an effective inhibitor of pyruvate transport mediated by both MCT1 and MCT2. We show that Tucatinib potently inhibits the proliferation and migration of cervical tumor cells in vitro and tumor growth in a mouse xenograft model, while exhibiting excellent biological safety. These findings offer molecular insights into the structural and functional mechanism of MCT2 and identify Tucatinib as novel dual inhibitor of both transporters.

Motile cilia are evolutionarily conserved protrusions critical for motility and homeostasis. Their rhythmic movements require the central pair microtubules (CP-MTs). While the initial CP-MT assembly in mammals is mediated by WDR47 and microtubule minus-end-binding CAMSAPs, the mechanism by which CP-MTs are stabilized remains unclear. Here, we demonstrate that WDR47 coordinates JHY and SPEF1 to maintain the stability of mammalian CP-MTs. By generating a proximity interactome of WDR47, we identify a group of CP-MT-associated proteins, including SPEF1 and JHY. WDR47 enriches JHY and SPEF1 to the central lumen and tip of nascent cilia, whereas SPEF1 recruits WDR47 and JHY to CP-MTs through direct interactions. Jhy deficiency in mice preferentially disrupts distal CP-MTs, resulting in rotatory ciliary beats. Phylogenetic analyses suggest conserved functions of WDR47 and SPEF1 in protozoa and metazoans, as well as a role for JHY in animals with radial or bilateral body symmetry. We propose that JHY emerges to further reinforce CP-MTs, enabling the transition from switchable to fixed ciliary waveforms in metazoan evolution.

Mouse embryonic stem cells (mESCs), in addition to differentiating into the three germ layers, can reverse typical developmental trajectories, as exemplified by their ability to de-differentiate into 2-cell-like cells (2CLCs) that resemble the mammalian embryo during zygotic genome activation (ZGA). This unique property offers the opportunity to elucidate the molecular principles that govern the pre-implantation stages of mammalian development. Here, we dissect the functions of the chromatin repressor EHMT2, a candidate antagonist of the mESC-to-2CLC transition, by leveraging a multipurpose allele for acute protein depletion and efficient immunoprecipitation. Our experiments revealed distinct principles of EHMT2-mediated gene repression in mESCs based on specific chromatin binding patterns and protein co-factors. Most notably, EHMT2 directly represses large clusters of co-regulated gene loci that comprise a significant fraction of the 2CLC-specific transcriptome by initiating H3K9me2 spreading from distal LINE-1 elements. EHMT2 counteracts the recruitment of the activator DPPA2/4 to promoter-proximal endogenous retroviral elements (ERVs) at 2CLC genes. EHMT2 depletion enhances the expression of ZGA-associated transcripts in 2CLCs and synergizes with spliceosome inhibition and retinoic acid signaling to facilitate the mESC-to-2CLC transition. In contrast to ZGA-associated genes, the repression of germ layer-associated transcripts by EHMT2 occurs outside of gene clusters, in collaboration with ZFP462, and involves binding to non-repetitive candidate enhancers. Our observations provide novel mechanistic insight into how pluripotent cells achieve attenuation of their bidirectional differentiation potential and reveal unique transcriptional features of murine totipotent cells.

The mechanisms regulating gamete fusion and preventing polyspermy in mammalian fertilization remain incompletely understood. This study combines real-time imaging, confocal microscopy and statistical analysis to investigate fertilization and polyspermy prevention dynamics in mice. By tracking the behavior of over one hundred spermatozoa entering the perivitelline space of oocytes, we dissect the respective contributions of oocyte structures (zona pellucida (ZP), perivitelline space (PVS), oolemma) and sperm components (head, flagellum) to fertilization and polyspermy prevention. We find that fertilization requires specific sperm head movements on the oolemma, driven by flagellar beating and facilitated by trapping the flagellum in the ZP, revealing a novel role for this structure. Our kinetic analysis characterizes a slow "penetration block" that gradually limits sperm entry into the PVS and a faster "fusion block" that prevents further fusion events. As the penetration block becomes significant after the fusion block is established, only the latter effectively prevents polyspermy in mice. We propose that it acts through neutralization of excess sperm in the PVS by oocyte-derived proteins CD9 and JUNO coating non-fertilizing spermatozoa.

Cryptorchidism is a common congenital abnormality that increases infertility and testicular cancer risk in adulthood. However, a few mammals exhibit naturally undescended testes while maintaining normal reproduction. The mechanisms underlying this natural cryptorchidism remain unclear. Here, we found evolutionary relaxation in INSL3 and RXFP2 of cryptorchid mammals, with the highest dN/dS ratio observed in cetaceans. Cellular experiments demonstrated that cetacean INSL3 downregulated the cAMP-PKA-CREB pathway, thereby reducing gubernacular cell proliferation and contraction. Cetacean INSL3 knock-in mice exhibited groin-located testes, nearly perfectly mimicking cryptorchid phenotypes in cetaceans and other mammals with incompletely descended testes. Collagen and muscle fibers in the gubernaculum of transgenic mice were reduced, with differentially expressed genes enriched in muscle development and contraction pathways. Additionally, the knock-in mice displayed male sterility, impaired testicular development, and upregulated inflammatory pathways in the testes. Our findings reveal how evolutionary changes in the INSL3/RXFP2 pathway contribute to natural cryptorchidism in mammals and provide insights for investigating reproductive health and cancer resistance in cryptorchid species.

Sphingolipids govern diverse cellular processes; their dysregulation underlies numerous diseases. Despite extensive characterizations, understanding the orchestration of the sphingolipid network within living organisms remains challenging. We established a versatile genetic platform of CRISPR-engineered reporters of 52 sphingolipid regulators, recapitulating endogenous gene activity and protein distribution. This platform further allows conditional protein degradation for functional characterization. In addition, we developed the biosensor OlyA^w to detect ceramide phosphoethanolamine and visualize membrane raft dynamics in vivo. Using this platform, we established comprehensive profiles of the sphingolipid metabolic network in the brain at the transcriptional and translational levels. The highly heterogeneous patterns indicate extensive coordination between distinct cell types and regions, suggesting the brain functions as a coherent unit to execute specific steps of sphingolipid metabolism. As a proof-of-concept application, we showed cell type-specific requirements of sphingomyelinases, including CG6962/dSMPD4 and CG3376/aSMase, degrading distinct subcellular pools of ceramide phosphoethanolamine to maintain brain function. These findings establish a foundation for future studies on brain sphingolipid metabolism and showcase the utilization of this genetic platform in elucidating in vivo mechanisms of sphingolipid metabolism.

Brain organoids are a promising model for studying human neurodevelopment and disease. Despite the potential, their 3D structure exhibits high variability during differentiation across batches and cell lines, presenting a significant challenge for biomedical applications. During development, organoids are exposed to fluid flow shear stress (fFSS) generated by the flow of culture media over the developing tissue. This stress is thought to disrupt cellular integrity and morphogenesis, leading to variation in organoids architecture, ultimately affecting reproducibility. Understanding the interplay between tissue morphology, cell identity and organoid development is therefore essential for advancing the use of brain organoids. Here, we demonstrate that reducing fFSS, by employing a vertically rotating chamber during neuronal induction, a critical phase for organoid morphogenesis, along with an extended cell aggregation phase to minimize fusions, significantly improves the reproducibility of brain organoids. Remarkably, reducing fFSS minimizes morphological structure variation and preserves transcriptional signature fidelity across differentiation batches and cell lines. This approach could enhance the reliability of brain organoid models, with important implications for neurodevelopmental research and preclinical studies.