Science

Subplate neurons (SpNs) are among the earliest-generated cortical neurons and are essential for neocortical circuit assembly. Despite this central role, they have long been considered a mammalian innovation, yet their evolutionary origin remains unresolved. Here, using comparative single-cell and spatial transcriptomics across amniotes (mice, chicks, and turtles), we identify two distinct developmental and evolutionary origins of SpNs: atypical SpNs (aSpNs), an Nr4a2-negative population conserved across amniotes and originating from the medial pallium, and mammalian-type SpNs (mSpNs), an Nr4a2-positive population preferentially expanded in mammals and arising from early-born cortical neurons. Cross-species analyses show that early-born pallial neurons in non-mammalian amniotes differentiate into thalamic input neurons, whereas this ancestral program is repurposed in mammals, with early-born neurons transiently adopting a subplate identity. We further show that this fate switch is controlled by Zbtb18 repression linked to thalamic input. Collectively, these findings establish a dual-origin model for SpNs and provide a unifying framework for understanding neocortical evolution. One-Sentence SummaryDevelopmental rewiring of an ancestral input-neuron program gave rise to the mammalian subplate.

The genetic program of animal development is conserved, but its rate of execution varies across species. Heterochrony, shifts in the relative timing of developmental events, generates phenotypic variation, but its prevalence and origin are unclear. Here, we compare two vertebrates with a 3-fold difference in developmental rate, zebrafish (Danio rerio) and medaka (Oryzias latipes) and characterize heterochrony with embryo-scale single-cell genomics. We generated an atlas of >1.2M single-cell transcriptomes of medaka from blastula to hatch and developed a new approach to represent medaka development in "zebrafish time", uncovering many cryptic, cell-level timing shifts not predicted by medakas slower development. We confirm a divergent heterochrony in the medaka notochord using in vivo imaging, revealing that coupled acceleration and delay of sister cell types shapes the species-specific morphology of this tissue. Our results point to cell type-specific timing deviations as a reservoir of phenotypic variation and we propose that species-specific developmental rate can emerge from these cell-level differences.

Animals use nervous systems to sense and respond to their environment. Yet single-celled organisms can also detect cues to execute diverse behaviors, suggesting that core components for animal sensation predate multicellularity and nervous systems. Here, we report that choanoflagellates, the closest living animal relatives, use an ancient sensory receptor family to detect bacterial prey. These receptors are related to transient receptor potential ion channels but are distinguished by WD40 domains, defining TRPW. TRPW1 detects specific bacterial lipids to modulate flagellar beating, providing a mechanism for attraction towards prey. TRPW emerged in early eukaryotes and reveals ancestral architectural and ligand-binding features that predate animal somatosensory receptors. In multicellular choanoflagellates, TRPW1 elicits collective responses, linking bacterial ecology to the evolution of receptors, sensory organelles, and multicellular life.

Retro-1 and Retro-2 are structurally distinct small molecules that protect cells from diverse toxins and viruses by disrupting retrograde trafficking, yet their mechanism of action has remained elusive. We show that both compounds target Get3, the ATPase chaperone of the guided entry of tail-anchored proteins (GET) pathway, which mediates biogenesis of tail-anchored SNARE proteins required for retrograde transport to the ER membrane. Cryo-electron microscopy reveals that Retro compounds bind a cryptic pocket in Get3, allosterically stabilizing Get3 in a stalled complex with upstream pathway components. Our work uncovers the GET pathway as an unsuspected vulnerability in pathogen entry, provides clear routes toward compound optimization, and establishes stabilization of dynamic protein complexes as a therapeutic strategy.

AbstractThe systematic navigation of biocatalyst space is constrained by elusive structure-activity rules and a lack of evolutionary history. Here, we present IMUSE, a strategy integrating machine learning with ultra-high-throughput screening. By screening millions of droplet-encapsulated de novo enzymes, we generated massive synthetic sequence-structure datasets to train models that capture their complex fitness landscapes and biophysical principles. These models effectively guide functional exploration across both sequence and novel structure spaces. IMUSE identified synergistic triple mutations yielding [~]5-fold activity improvements and discovered active second-generation designs with novel catalytic pockets, boosting the experimental success rate >4.9-fold ([~]30%). This work demonstrates how synthetic fitness landscapes bridge the data gap in de novo enzyme space, transforming stochastic search into deterministic navigation to unlock highly proficient biocatalysts beyond natural boundaries.

Dogs and their closest extant relative, the grey wolf, diverged around 30k years ago, but have since experienced complex histories of gene flow involving other canids, adaptive pressures due to close association with humans, and changing climates. We infer joint genealogies of dogs, grey wolves, and a coyote using available whole genomes to reconstruct the evolutionary forces shaping the dog genome. These genealogies reveal multiple strong mutation-rate pulses unique to dogs, including signals detectable across ancient dogs from the past 10,000 years. We further detect pervasive genealogical signatures of purifying selection and find that GC-biased gene conversion is a major driver of diversity patterns around gene promoters in dogs. We introduce a new genealogy-based selection scan, TwigScan, that computes time-stratified differentiation, increasing power over traditional FST-based approaches. Applying this framework, alongside a second single-population test for detecting more recent selection within dogs, we identify multiple known and novel loci with signatures of positive selection. Among these, the region surrounding the amylase 2B locus shows evidence of introgression from a deeply divergent, unsampled canid lineage with divergence comparable to that of dholes. AMY2B duplications appear to occur exclusively on this introgressed haplotype which increased in frequency approximately 7,000-8,000 years ago, coinciding with increased reliance on starch-rich diets in human populations. Together, these results show how mutation-rate variation, gene conversion, selection, and inter-species gene flow have jointly shaped the dog genome, highlighting the power of genealogical approaches for resolving complex evolutionary histories. Significance StatementDogs were likely the first animal domesticated by humans, yet the evolutionary processes shaping the modern dog genome remain unresolved. Recent advances now enable reconstruction of genome-wide genealogies from genetic variation, enabling evolutionary histories to be traced along ancestral lineages through time. Applied to dogs, grey wolves, and coyotes, we investigate how mutation, selection, and interspecies gene flow shaped canine evolution. We identify mutation-rate pulses unique to dogs--only the second clear example of such a phenomenon in mammals--and quantify pervasive effects of purifying selection and GC-biased gene conversion. We identify regions of excess dog-wolf divergence and show that the selected AMY2B starch-digestion locus in dogs likely originated through introgression from a deeply divergent ghost canid lineage.

Generative AI-based protein design can create diverse structures for desired functions but offers human designers limited control over three-dimensional architectures. We developed ProteinSketch, a bimanual virtual reality platform in which immersive backbone sketches and volumetric envelopes are directly created and translated into constraints for diffusion-based protein generation. RFdiffusion-based real-time refinement enabled interactive exploration and construction of user-specified protein topologies. Volumetric conditioning of sketched envelopes enabled high-fidelity control of anisometric geometries, confirmed by cryo-electron microscopy. These user-defined volumetric constraints enabled functional-binder design and extension of pre-existing minibinders to surfaces otherwise difficult to access within complex molecular environments using conventional design approaches. This collaborative human-AI framework integrates human spatial reasoning with generative AI for spatially directed, shape-controlled design of protein structure and function. One-sentence summaryImmersive ProteinSketch embeds human spatial reasoning in AI-guided design of protein topology, shape, and function.

The Neolithic period in western Eurasia involved major dispersal events, including the translocation of livestock into Europe. These domestic animal translocations likely featured founder effects, fundamentally reshaping their genetic landscape. One well-known example is the Mediterranean dispersal, hypothesised to involve rapid maritime translocation with small founding herds, yet the genetic consequences for the livestock themselves remain poorly understood. To investigate this, we generated 12 palaeogenomes of sheep and goat from La Draga and Cova de lAvellaner, and coanalyzed these with other Neolithic Mediterranean assemblages, including 4 additional novel genomes. We observe a significantly lower genetic diversity and higher runs of homozygosity (ROH) in Iberian sheep and goat populations compared to other ancient European and southwest Asian populations. We quantify the magnitude of the bottleneck in goats at [~]205 Ne, highlighting the severity of the founder event. We further identify species-specific demographic trajectories: goats experienced a severe and prolonged bottleneck, likely driven by regional isolation within the western Mediterranean, intensive exploitation strategies, and small initial founding herds, while sheep were likely more frequently exchanged across the region. Lastly, identity-by-descent (IBD) indicates connectivity along the Mediterranean coast compatible with a cabotage model. This suggests that seaways facilitated rapid initial dispersal but not sustained livestock transfer from southwest Asia, shaping the genetic foundation of Iberian livestock herds. SignificanceThe Neolithic transition in western Eurasia fundamentally restructured human societies, yet the genetic consequences for translocated livestock remain poorly characterized. Analyzing 16 novel palaeogenomes from Mediterranean Neolithic sheep and goats, we demonstrate that maritime dispersal imposed severe founder effects on early Iberian livestock, reducing effective population sizes to as low as [~]205 in goats. Species-specific demographic trajectories reveal that goats experienced prolonged genetic isolation within the western Mediterranean, while sheep maintained broader regional connectivity. Identity-by-descent analyses support a cabotage model of coastal dispersal, indicating that Mediterranean seaways facilitated rapid initial colonization but not sustained genetic exchange with southwest Asian source populations. These findings reveal that the earliest livestock introductions into Europe were demographically constrained events whose genetic signatures persist in the foundations of Iberian pastoral heritage.

Regulated biogenesis of photosynthetic proteins by the chloroplast ribosomes is central to plant development and environmental adaptation. Here, we describe changes to the ribosome that occurred during evolution of chloroplasts from cyanobacteria in the photosynthetic lineage based on structural, proteomic and bioinformatic analyses. We identify structural features common to oxygenic photosynthetic organisms and distinct from other bacteria, as well as the gain and loss of ribosomal proteins, rRNA modifications and hibernation factors upon endosymbiosis. We uncover structural variation among land plant lineages at sites that remodel the peptide exit tunnel and features associated with translation initiation. The data challenge the prevailing view of ribosomal features that are specific to the chloroplast by demonstrating that many originated earlier, in cyanobacteria, or later, during land plant evolution.

Temperature shapes reproductive strategies across animals, yet how individuals switch between sexual and asexual reproduction remains unknown. We establish the planarian Phagocata morgani as a model for temperature-dependent reproductive plasticity and adapt multiplexed single-cell transcriptomics to profile >1 million nuclei from >300 animals across body sizes and temperatures. Leveraging individual variation in cell composition, we reconstruct an organism-wide trajectory that bifurcates toward alternative reproductive fates. Temperature extremes constrain worms to one fate, whereas intermediate conditions permit probabilistic commitment to either. At the bifurcation, temperature gates a stem cell pool: warmth suppresses differentiation and promotes progenitor accumulation, whereas cold transcriptionally activates this pool for de novo sexual organogenesis. These findings reveal how environmental inputs act on stem cells to couple body size, temperature, and reproductive fate.

All proteins can begin to fold on the ribosome, and many proteins critically rely on co-translational folding to attain their native conformation. The molecular details underlying this crucial process, however, remain largely unknown and are not accounted for by structure predictions such as AlphaFold. To probe high-resolution folding during active translation, we develop a novel application of hydrogen-deuterium pulse labeling. We show that two proteins sequentially adopt stable structure during elongation, while a third protein only has time to loosely fold during active elongation. This loose folding kinetically traps the N-terminus and alters the post-translational folding pathway, allowing it to circumvent an aggregation-prone intermediate. These results highlight the crucial non-equilibrium coupling between translation and folding and reveal diverse strategies to promote robust co-translational folding.

Because they can bind many strains of influenza, antibodies targeting the hemagglutinin (HA) stem have been attractive targets for vaccine development. Many monoclonal antibodies (mAbs) directed at the HA stem have been isolated from humans, and these mAbs have mediated broad protection in animal models. We describe here HA stem-directed mAbs isolated from rhesus macaques immunized with an "ordinary" H1 HA trimer. All immunized rhesus macaques developed high serum titers with broad reactivity to diverse H1N1 and H5N1 viruses, and 7 isolated mAbs strongly blocked canonical stem antibody CR6261 binding to H1. MAb DH726.1 robustly protected mice from lethal challenge with H1N1 and H5N1 viruses, and cryo-EM showed the binding footprint overlapped that of some human mAbs. These findings suggest that vaccination with the standard, trimeric HA immunogens may be sufficient to elicit stem antibodies at titers adequate to protect against zoonotic H5N1 influenza. In BriefEfforts to achieve broad influenza protection have largely emphasized increasingly sophisticated immunogen designs to redirect antibody responses toward conserved epitopes. Here, we show that a single, conventional HA immunogen readily elicits antibodies targeting the conserved HA stem, suggesting that routine influenza vaccination may provide broader protection than previously appreciated. HighlightsO_LIMonovalent HA immunization induces strong, stem-directed immune responses in rhesus macaques C_LIO_LIEpitope on the HA stem confirmed by Cryo-EM C_LIO_LIFc-mediated activation of immune effector cell by rhesus-derived stem mAb C_LIO_LIHA stem-directed antibody protects mice against H1N1 and H5N1 viruses C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/738984v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@508b9org.highwire.dtl.DTLVardef@7fabbeorg.highwire.dtl.DTLVardef@1e47b84org.highwire.dtl.DTLVardef@1693bfb_HPS_FORMAT_FIGEXP M_FIG C_FIG

How mature cells actively defend their identity against alternative fates is poorly understood. Here we show that two types of fully differentiated Drosophila somatosensory neurons, derived from distinct lineages, are continuously prevented from transdifferentiating into one another by a post-mitotic bistable switch. Two homeodomain transcription factors, Hmx and Cut, are co-expressed in both neuronal types but adopt opposite stoichiometric dominances through mutual repression, each defining one identity. Disrupting this balance in mature neurons triggers deterministic, lineage-crossing reprogramming of transcriptome, dendritic and axonal architecture, and sensory behavior without reversion to a progenitor state. This regulatory logic is evolutionarily conserved: Drosophila sensory identities share transcriptomic signatures with distinct human A{delta} mechanoreceptors, and human CUX and HMX proteins functionally replace their fly counterparts. Mature neuronal identity is thus not a fixed endpoint but a poised equilibrium actively maintained by ongoing competition between opposing selectors. TEASERMature somatosensory identity is a poised equilibrium actively maintained by continuous competition between opposing terminal selectors.

Protein oligomers are ubiquitous in biological systems and essential for function. However, the de novo design of oligomers that controllably assemble in response to exogenous stimuli remains challenging. Here, we present an AI-based generative approach that leverages an interface-seeded strategy for designing responsive homo-oligomers from isolated interaction modules. Experimentally validated designs are highly accurate and explore new-to-nature topologies. We show that designs effectively respond to their chemical triggers with conditional oligomerization or to phosphorylation-driven conformational changes with reversible oligomerization. We further functionalized our responsive assemblies to build ligand-dependent membrane binding systems and phosphorylation-controlled gene regulatory switches. Our framework enables the generalizable design of responsive protein complexes, opening novel possibilities for the engineering of biosynthetic systems with sophisticated regulatory mechanisms.

Enhancers are key regulators of mammalian gene expression, yet how they interact with promoters in space (contact vs. action-at-a-distance) and in time (transient vs. stable) remains poorly understood. Recent studies suggest that enhancers can activate promoters across distances exceeding 200 nanometers, challenging classical contact models, but limited spatiotemporal resolution has obscured the mechanistic details of enhancer-promoter (E-P) interactions and their link to transcription. Here, we engineered a synthetic biology platform optimized for the simultaneous visualization of E-P 3D distance and nascent transcription using super-resolution live-cell imaging. By applying five complementary approaches integrating imaging, 3D genomics, and gene expression data across cell lines, we estimate that transcriptional activation is mediated by [~]25-42 nanometer contacts on the seconds timescale. Our results support a transient contact mechanism for E-P-mediated gene activation.

The plant kingdom is rich with medicinal natural products that are complex and difficult to access. Discovering how plants build these molecules can be challenging, especially for biosynthetic pathways that have unusual chemical transformations or require intricate coordination among cellular compartments. Here, we leveraged 300 million years of metabolic conservation to uncover how medicinal clubmosses organize extracellular and intracellular alkaloid biosynthesis to produce the Alzheimers disease therapeutic huperzine A (HupA). We reveal not only scaffold-forming enzymes that form key precursors to hundreds of clubmoss alkaloids, but also an essential transporter that connects metabolism across the plasma membrane to enable complete HupA biosynthesis. Our results demonstrate how ancient evolutionary conservation can be used to identify cryptic biosynthetic components and unexpected cellular organization in plant specialized metabolism.

Complex, multicellular extremophiles face intense challenges in coordinating their cells, tissues, and organs to function in harsh environments. Tidestromia oblongifolia is a desert plant that thrives in Death Valley, where air temperatures exceed 50 {degrees}C. Using natural collections of T. oblongifolia seed, we identified individuals that survive in daily air temperature regimens at the eukaryotic upper thermal limit of 60 {degrees}C. Genome-wide association testing revealed genetic variation for survival in extreme heat, and transcriptomics identified pathways regulating physiological cooling. High-throughput infrared imaging showed that survival was enabled by extreme leaf cooling, a physiological mechanism not previously reported in a thermophilic organism. These discoveries provide insights into mechanisms of extreme heat adaptation in a complex organism that could be leveraged to engineer heat-resilient crops.

Leucine is a central nutrient signal and a ketogenic amino acid that fuels metabolism, yet how it is imported into mitochondria remains incompletely defined. Sideroflexins are conserved inner mitochondrial membrane proteins implicated in amino acid transport, but their mechanism and substrate specificity remain unclear. Using cryogenic electron microscopy, we determined the structure of SFXN1 in its matrix-open conformation. AlphaFold and Boltz co-folding of SFXN1 with a library of human metabolites identified leucine as a candidate substrate, findings supported by thermal stability measurements and mitochondrial transport assays. Comparison with a cytoplasmic-open-model reveals an alternating-access "toggle-switch" mechanism of transport. Together, these findings uncover the molecular basis of leucine transport by SFXN1 and provide a framework for understanding its role in metabolism and disease.

Alphaviruses are arthropod-borne viruses that recognize cellular receptors in both vertebrate hosts and mosquito vectors to complete their transmission cycle, yet how they maintain recognition of receptors across evolutionarily divergent host species remains unresolved. Among alphaviruses, chikungunya virus (CHIKV), which is primarily vectored in urban settings by Aedes species mosquitoes, is the most widespread, and causes explosive outbreaks that can involve hundreds of thousands to millions of cases annually. The cell adhesion protein Lachesin is a mosquito-specific cellular receptor for CHIKV and multiple other arthritogenic alphaviruses. The envelope E2-E1 glycoproteins of these alphaviruses broadly recognize Lachesin orthologs from diverse mosquito species, but not other insects or arachnids. Lachesin genetic manipulation to prevent mosquito virus infection without interfering with endogenous receptor function could have a major impact for CHIKV control. Here, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of alphaviruses bound to Aedes albopictus Lachesin. Comparative analysis of Lachesin-bound CHIKV, Semliki Forest virus (SFV), and Middelburg virus (MIDV) revealed that these three genetically divergent viruses use a similar surface to recognize Lachesin domain 1, but with reorganized E2-E1 glycoprotein contact residues. We show that a soluble Ae. albopictus Lachesin receptor decoy protein blocks the E2-E1-mediated entry of CHIKV and other arthritogenic alphaviruses into mammalian cells with greater breadth than a vertebrate receptor MXRA8 decoy and protects against lethal SFV challenge and CHIKV pathogenesis in murine models. Additionally, we identified a naturally occurring single residue Lachesin polymorphism that is found in some Anopheles (malaria vector) mosquitoes, and fully ablates CHIKV E2-E1 recognition, informing strategies for mosquito-targeted genetic interventions that could prevent mosquito vector infection and virus transmission. These findings define distinct determinants of receptor binding in mosquitoes and humans for arthritogenic alphaviruses, with implications for countermeasure development and outbreak preparedness.

Sequence-programmable directed evolution systems have great potential to accelerate bioengineering. Diversity-generating retroelements (DGRs) are natural hypermutation systems widely distributed in prokaryotes and bacteriophages with the capacity to introduce diverse mutations at template-specified sites of target genes. Here, we show that DGRs can be installed in E. coli and reprogrammed for the continuous, iterative mutagenesis of user-defined target genes. We show that the DGR template RNA can be reprogrammed for gene- and residue- specific mutagenesis, leaving untargeted, adjacent residues unchanged. Furthermore, we establish continuous DGR-enabled mutagenesis with F-plasmid-mediated horizontal gene transfer of target genes (HGT-DGR) into a new host for the progressive accumulation of target- specific mutations. Iterative HGT-DGR mutagenesis over seven cycles yielded an average mutation load of approximately 6% across adenine positions in the target segment, generating a diverse library of variants comprising 40% mutant sequences, with a median pairwise Hamming distance of 4 among mutant variants. HGT-DGR enables iterative diversification of either the same or different user-specified segments of the target gene, as demonstrated with the directed evolution of the M. mazei pyrrolysyl-tRNA synthetase for non-canonical amino acid incorporation. HGT-DGR provides a simple, low-cost, sequence-programmable system that enables iterative, position-specific and tunable in vivo mutagenesis of any target sequence for applications in biotechnology and medicine.