Controlling organoid symmetry breaking uncovers an excitable system underlying human axial elongation.

The human embryo breaks symmetry to form the anterior-posterior axis of the body. As the embryo elongates along this axis, progenitors in the tail bud give rise to tissues that generate spinal cord, skeleton, and musculature. This raises the question of how the embryo achieves axial elongation and patterning. While ethics necessitate in vitro studies, the variability of organoid systems has hindered mechanistic insights. Here, we developed a bioengineering and machine learning framework that optimizes organoid symmetry breaking by tuning their spatial coupling. This framework enabled reproducible generation of axially elongating organoids, each possessing a tail bud and neural tube. We discovered that an excitable system composed of WNT/FGF signaling drives elongation by inducing a neuromesodermal progenitor-like signaling center. We discovered that instabilities in the excitable system are suppressed by secreted WNT inhibitors. Absence of these inhibitors led to ectopic tail buds and branches. Our results identify mechanisms governing stable human axial elongation.

Multimodal charting of molecular and functional cell states via in situ electro-sequencing.

Paired mapping of single-cell gene expression and electrophysiology is essential to understand gene-to-function relationships in electrogenic tissues. Here, we developed in situ electro-sequencing (electro-seq) that combines flexible bioelectronics with in situ RNA sequencing to stably map millisecond-timescale electrical activity and profile single-cell gene expression from the same cells across intact biological networks, including cardiac and neural patches. When applied to human-induced pluripotent stem-cell-derived cardiomyocyte patches, in situ electro-seq enabled multimodal in situ analysis of cardiomyocyte electrophysiology and gene expression at the cellular level, jointly defining cell states and developmental trajectories. Using machine-learning-based cross-modal analysis, in situ electro-seq identified gene-to-electrophysiology relationships throughout cardiomyocyte development and accurately reconstructed the evolution of gene expression profiles based on long-term stable electrical measurements. In situ electro-seq could be applicable to create spatiotemporal multimodal maps in electrogenic tissues, potentiating the discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.

Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.

Cell-type-specific population dynamics of diverse reward computations.

Computational analysis of cellular activity has developed largely independently of modern transcriptomic cell typology, but integrating these approaches may be essential for full insight into cellular-level mechanisms underlying brain function and dysfunction. Applying this approach to the habenula (a structure with diverse, intermingled molecular, anatomical, and computational features), we identified encoding of reward-predictive cues and reward outcomes in distinct genetically defined neural populations, including TH+ cells and Tac1+ cells. Data from genetically targeted recordings were used to train an optimized nonlinear dynamical systems model and revealed activity dynamics consistent with a line attractor. High-density, cell-type-specific electrophysiological recordings and optogenetic perturbation provided supporting evidence for this model. Reverse-engineering predicted how Tac1+ cells might integrate reward history, which was complemented by in vivo experimentation. This integrated approach describes a process by which data-driven computational models of population activity can generate and frame actionable hypotheses for cell-type-specific investigation in biological systems.

Breaking NGF-TrkA immunosuppression in melanoma sensitizes immunotherapy for durable memory T cell protection.

Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system's immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF-TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.

Splicing Activation by Rbfox Requires Self-Aggregation through Its Tyrosine-Rich Domain.

Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.

Brain-wide Maps Reveal Stereotyped Cell-Type-Based Cortical Architecture and Subcortical Sexual Dimorphism.

The stereotyped features of neuronal circuits are those most likely to explain the remarkable capacity of the brain to process information and govern behaviors, yet it has not been possible to comprehensively quantify neuronal distributions across animals or genders due to the size and complexity of the mammalian brain. Here we apply our quantitative brain-wide (qBrain) mapping platform to document the stereotyped distributions of mainly inhibitory cell types. We discover an unexpected cortical organizing principle: sensory-motor areas are dominated by output-modulating parvalbumin-positive interneurons, whereas association, including frontal, areas are dominated by input-modulating somatostatin-positive interneurons. Furthermore, we identify local cell type distributions with more cells in the female brain in 10 out of 11 sexually dimorphic subcortical areas, in contrast to the overall larger brains in males. The qBrain resource can be further mined to link stereotyped aspects of neuronal distributions to known and unknown functions of diverse brain regions.

Spatial omics advances for in situ RNA biology.

Spatial regulation of RNA plays a critical role in gene expression regulation and cellular function. Understanding spatially resolved RNA dynamics and translation is vital for bringing new insights into biological processes such as embryonic development, neurobiology, and disease pathology. This review explores past studies in subcellular, cellular, and tissue-level spatial RNA biology driven by diverse methodologies, ranging from cell fractionation, in situ and proximity labeling, imaging, spatially indexed next-generation sequencing (NGS) approaches, and spatially informed computational modeling. Particularly, recent advances have been made for near-genome-scale profiling of RNA and multimodal biomolecules at high spatial resolution. These methods enabled new discoveries into RNA's spatiotemporal kinetics, RNA processing, translation status, and RNA-protein interactions in cells and tissues. The evolving landscape of experimental and computational strategies reveals the complexity and heterogeneity of spatial RNA biology with subcellular resolution, heralding new avenues for RNA biology research.

ALKBH1-Mediated tRNA Demethylation Regulates Translation.

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

ATR inhibition induces synthetic lethality in mismatch repair-deficient cells and augments immunotherapy.

The mismatch repair (MMR) deficiency of cancer cells drives mutagenesis and offers a useful biomarker for immunotherapy. However, many MMR-deficient (MMR-d) tumors do not respond to immunotherapy, highlighting the need for alternative approaches to target MMR-d cancer cells. Here, we show that inhibition of the ATR kinase preferentially kills MMR-d cancer cells. Mechanistically, ATR inhibitor (ATRi) imposes synthetic lethality on MMR-d cells by inducing DNA damage in a replication- and MUS81 nuclease-dependent manner. The DNA damage induced by ATRi is colocalized with both MSH2 and PCNA, suggesting that it arises from DNA structures recognized by MMR proteins during replication. In syngeneic mouse models, ATRi effectively reduces the growth of MMR-d tumors. Interestingly, the antitumor effects of ATRi are partially due to CD8+ T cells. In MMR-d cells, ATRi stimulates the accumulation of nascent DNA fragments in the cytoplasm, activating the cGAS-mediated interferon response. The combination of ATRi and anti-PD-1 antibody reduces the growth of MMR-d tumors more efficiently than ATRi or anti-PD-1 alone, showing the ability of ATRi to augment the immunotherapy of MMR-d tumors. Thus, ATRi selectively targets MMR-d tumor cells by inducing synthetic lethality and enhancing antitumor immunity, providing a promising strategy to complement and augment MMR deficiency-guided immunotherapy.

Theory of the Multiregional Neocortex: Large-Scale Neural Dynamics and Distributed Cognition.

The neocortex is a complex neurobiological system with many interacting regions. How these regions work together to subserve flexible behavior and cognition has become increasingly amenable to rigorous research. Here, I review recent experimental and theoretical work on the modus operandi of a multiregional cortex. These studies revealed several general principles for the neocortical interareal connectivity, low-dimensional macroscopic gradients of biological properties across cortical areas, and a hierarchy of timescales for information processing. Theoretical work suggests testable predictions regarding differential excitation and inhibition along feedforward and feedback pathways in the cortical hierarchy. Furthermore, modeling of distributed working memory and simple decision-making has given rise to a novel mathematical concept, dubbed bifurcation in space, that potentially explains how different cortical areas, with a canonical circuit organization but gradients of biological heterogeneities, are able to subserve their respective (e.g., sensory coding versus executive control) functions in a modularly organized brain.

N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in mammalian mRNA. This modification is reversible and non-stoichiometric and adds another layer to the dynamic control of mRNA metabolism. The stability of m(6)A-modified mRNA is regulated by an m(6)A reader protein, human YTHDF2, which recognizes m(6)A and reduces the stability of target transcripts. Looking at additional functional roles for the modification, we find that another m(6)A reader protein, human YTHDF1, actively promotes protein synthesis by interacting with translation machinery. In a unified mechanism of m(6)A-based regulation in the cytoplasm, YTHDF2-mediated degradation controls the lifetime of target transcripts, whereas YTHDF1-mediated translation promotion increases translation efficiency, ensuring effective protein production from dynamic transcripts that are marked by m(6)A. Therefore, the m(6)A modification in mRNA endows gene expression with fast responses and controllable protein production through these mechanisms.

Mars's induced magnetosphere can degenerate.

The interaction between planets and stellar winds can lead to atmospheric loss and is, thus, important for the evolution of planetary atmospheres1. The planets in our Solar System typically interact with the solar wind, whose velocity is at a large angle to the embedded stellar magnetic field. For planets without an intrinsic magnetic field, this interaction creates an induced magnetosphere and a bow shock in front of the planet2. However, when the angle between the solar wind velocity and the solar wind magnetic field (cone angle) is small, the interaction is very different3. Here we show that when the cone angle is small at Mars, the induced magnetosphere degenerates. There is no shock on the dayside, only weak flank shocks. A cross-flow plume appears and the ambipolar field drives planetary ions upstream. Hybrid simulations with a 4° cone angle show agreement with observations by the Mars Atmosphere and Volatile Evolution mission4 and Mars Express5. Degenerate, induced magnetospheres are complex and not yet explored objects. It remains to be studied what the secondary effects are on processes like atmospheric loss through ion escape.

A hot-emitter transistor based on stimulated emission of heated carriers.

Hot-carrier transistors are a class of devices that leverage the excess kinetic energy of carriers. Unlike regular transistors, which rely on steady-state carrier transport, hot-carrier transistors modulate carriers to high-energy states, resulting in enhanced device speed and functionality. These characteristics are essential for applications that demand rapid switching and high-frequency operations, such as advanced telecommunications and cutting-edge computing technologies1-5. However, the traditional mechanisms of hot-carrier generation are either carrier injection6-11 or acceleration12,13, which limit device performance in terms of power consumption and negative differential resistance14-17. Mixed-dimensional devices, which combine bulk and low-dimensional materials, can offer different mechanisms for hot-carrier generation by leveraging the diverse potential barriers formed by energy-band combinations18-21. Here we report a hot-emitter transistor based on double mixed-dimensional graphene/germanium Schottky junctions that uses stimulated emission of heated carriers to achieve a subthreshold swing lower than 1 millivolt per decade beyond the Boltzmann limit and a negative differential resistance with a peak-to-valley current ratio greater than 100 at room temperature. Multi-valued logic with a high inverter gain and reconfigurable logic states are further demonstrated. This work reports a multifunctional hot-emitter transistor with significant potential for low-power and negative-differential-resistance applications, marking a promising advancement for the post-Moore era.

Earth's early continental crust formed from wet and oxidizing arc magmas.

Formation of continental crust has shaped the surface and interior of our planet and generated the land and mineral resources on which we rely. However, how the early continental crust of Earth formed is still debated1-7. Modern continental crust is largely formed from wet and oxidizing arc magmas at subduction zones, in which oceanic lithosphere and water recycle into the mantle8-10. The magmatic H2O content and redox state of ancient rocks that constitute the early continental crust, however, are difficult to quantify owing to ubiquitous metamorphism. Here we combine two zircon oxybarometers11,12 to simultaneously determine magmatic oxygen fugacity (fO2) and H2O content of Archaean (4.0-2.5 billion years ago) granitoids that dominate the early continental crust. We show that most Archaean granitoid magmas were ≥1 log unit more oxidizing than Archaean ambient mantle-derived magmas13,14 and had high magmatic H2O contents (6-10 wt%) and high H2O/Ce ratios (>1,000), similar to modern arc magmas. We find that magmatic fO2, H2O contents and H2O/Ce ratios of Archaean granitoids positively correlate with depth of magma formation, requiring transport of large amounts of H2O to the lower crust and mantle. These observations can be readily explained by subduction but are difficult to reconcile with non-subduction models of crustal formation3-7. We note an increase in magmatic fO2 and H2O content between 4.0 and 3.6 billion years ago, probably indicating the onset of subduction during this period.

Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Spatial predictors of immunotherapy response in triple-negative breast cancer.

Immune checkpoint blockade (ICB) benefits some patients with triple-negative breast cancer, but what distinguishes responders from non-responders is unclear1. Because ICB targets cell-cell interactions2, we investigated the impact of multicellular spatial organization on response, and explored how ICB remodels the tumour microenvironment. We show that cell phenotype, activation state and spatial location are intimately linked, influence ICB effect and differ in sensitive versus resistant tumours early on-treatment. We used imaging mass cytometry3 to profile the in situ expression of 43 proteins in tumours from patients in a randomized trial of neoadjuvant ICB, sampled at three timepoints (baseline, n = 243; early on-treatment, n = 207; post-treatment, n = 210). Multivariate modelling showed that the fractions of proliferating CD8+TCF1+T cells and MHCII+ cancer cells were dominant predictors of response, followed by cancer-immune interactions with B cells and granzyme B+ T cells. On-treatment, responsive tumours contained abundant granzyme B+ T cells, whereas resistant tumours were characterized by CD15+ cancer cells. Response was best predicted by combining tissue features before and on-treatment, pointing to a role for early biopsies in guiding adaptive therapy. Our findings show that multicellular spatial organization is a major determinant of ICB effect and suggest that its systematic enumeration in situ could help realize precision immuno-oncology.

Ectosomal PKM2 Promotes HCC by Inducing Macrophage Differentiation and Remodeling the Tumor Microenvironment.

Tumor-derived extracellular vesicles are important mediators of cell-to-cell communication during tumorigenesis. Here, we demonstrated that hepatocellular carcinoma (HCC)-derived ectosomes remodel the tumor microenvironment to facilitate HCC progression in an ectosomal PKM2-dependent manner. HCC-derived ectosomal PKM2 induced not only metabolic reprogramming in monocytes but also STAT3 phosphorylation in the nucleus to upregulate differentiation-associated transcription factors, leading to monocyte-to-macrophage differentiation and tumor microenvironment remodeling. In HCC cells, sumoylation of PKM2 induced its plasma membrane targeting and subsequent ectosomal excretion via interactions with ARRDC1. The PKM2-ARRDC1 association in HCC was reinforced by macrophage-secreted cytokines/chemokines in a CCL1-CCR8 axis-dependent manner, further facilitating PKM2 excretion from HCC cells to form a feedforward regulatory loop for tumorigenesis. In the clinic, ectosomal PKM2 was clearly detected in the plasma of HCC patients. This study highlights a mechanism by which ectosomal PKM2 remodels the tumor microenvironment and reveals ectosomal PKM2 as a potential diagnostic marker for HCC.

Large-scale across species transcriptomic analysis identifies genetic selection signatures associated with longevity in mammals.

Lifespan varies significantly among mammals, with more than 100-fold difference between the shortest and longest living species. This natural difference may uncover the evolutionary forces and molecular features that define longevity. To understand the relationship between gene expression variation and longevity, we conducted a comparative transcriptomics analysis of liver, kidney, and brain tissues of 103 mammalian species. We found that few genes exhibit common expression patterns with longevity in the three organs analyzed. However, pathways related to translation fidelity, such as nonsense-mediated decay and eukaryotic translation elongation, correlated with longevity across mammals. Analyses of selection pressure found that selection intensity related to the direction of longevity-correlated genes is inconsistent across organs. Furthermore, expression of methionine restriction-related genes correlated with longevity and was under strong selection in long-lived mammals, suggesting that a common strategy is utilized by natural selection and artificial intervention to control lifespan. Our results indicate that lifespan regulation via gene expression is driven through polygenic and indirect natural selection.

DIS3 ribonuclease is essential for spermatogenesis and male fertility in mice.

Spermatogonial stem cell (SSC) self-renewal and differentiation provide foundational support for long-term, steady-state spermatogenesis in mammals. Here, we have investigated the essential role of RNA exosome associated DIS3 ribonuclease in maintaining spermatogonial homeostasis and facilitating germ cell differentiation. We have established male germ-cell Dis3 conditional knockout (cKO) mice in which the first and subsequent waves of spermatogenesis are disrupted. This leads to a Sertoli cell-only phenotype and sterility in adult male mice. Bulk RNA-seq documents that Dis3 deficiency partially abolishes RNA degradation and causes significant increases in the abundance of transcripts. This also includes pervasively transcribed PROMoter uPstream Transcripts (PROMPTs), which accumulate robustly in Dis3 cKO testes. In addition, scRNA-seq analysis indicates that Dis3 deficiency in spermatogonia significantly disrupts RNA metabolism and gene expression, and impairs early germline cell development. Overall, we document that exosome-associated DIS3 ribonuclease plays crucial roles in maintaining early male germ cell lineage in mice.