Science Journals

We report a minimally disruptive labeling strategy for stress granule protein, G3BP Stress Granule Assembly Factor 1 (G3BP1), and ALS-linked protein, TAR DNA-binding protein 43 (TDP-43), using the fluorescent non-canonical amino acid Anap. By integrating the genetic code expansion (GCE) with rational site selection, we achieved precise incorporation of Anap that preserves protein structure and function. In live cells and neurons, Anap labeling faithfully recapitulated localization, stress-induced dynamics, and recovery behavior, outperforming conventional fluorescent tags, and enabling physiologically relevant visualization of protein pathobiology.

Cortical control of movement is a distributed computation spanning multiple densely interconnected regions. Although we have rich anatomical atlases and a coarse understanding of how function maps to areas and subregions, we lack a detailed account of how behaviorally relevant activity is organized across the cortical sheet. Here, we trained head-fixed mice to perform a 15-target reach-to-grasp task while we performed cellular-resolution, two-photon calcium imaging across five regions of sensorimotor cortex (>39,000 layer 2/3 neurons). We characterized each neuron’s trial-averaged peri-event activity with interpretable metrics and mapped these response properties across areas, revealing large-scale spatial structure. Neuronal response profiles often shifted abruptly at anatomical borders: motor areas showed sharper tuning and more linear relationships with target location, whereas somatosensory areas displayed more heterogeneous response patterns. Neural response properties also differed according to somatotopic representation. Nonlinear dimensionality reduction of the neural feature matrix revealed that areas varied in their average response profiles, but that areas did not have well-separated feature distributions; instead, each area contained subpopulations. Neurons in each subpopulation had characteristic response profiles and were distributed across multiple cortical areas. The spatial distributions of the subpopulations overlapped, with neurons from different subpopulations salt-and-pepper intermingled in the overlap zones. Together, these results describe novel activity structure across sensorimotor cortex and identify several distinct but spatially overlapping subpopulations with characteristic activity patterns during reach-to-grasp behavior.

Specialized anatomical structures in the mouth and feet of tsetse flies help them feed on blood from a variety of hosts.

The subthalamic nucleus (STN) is a part of the indirect and hyperdirect pathways in the basal ganglia (BG) and has been implicated in movement control, impulsivity, and decision-making. We recently demonstrated that, for perceptual decisions, the STN includes at least three subpopulations of neurons with different decision-related activity patterns (Branam et al., 2024). Here, we show that, for decisions that require both perceptual and reward-based processing, many STN neurons are sensitive to both sensory evidence and reward expectations. Within a drift-diffusion framework, three STN subpopulations show different relationships to model components reflecting the formation of the decision variable, dynamics of the decision bound, and non-decision-related processes. Many STN neurons also represent quantities related to decision evaluation, including choice accuracy and reward expectation. These results help to further delineate the multiple roles that STN plays in forming and evaluating complex decisions that combine multiple sources of information.

Eukaryotic mitochondria are characterized by several features that represent vestiges of their prokaryotic ancestry. One such feature is the N-terminal formylation of proteins encoded by mitochondrial DNA that undergo translation by mitochondrial ribosomes. N-formylated proteins are also released by bacteria and trigger activation of immune cells such as neutrophils. Growing evidence indicates that circulating levels of mitochondrial formyl proteins are elevated in the serum of patients with excessive inflammatory responses. However, the mechanisms by which they are released into circulation are not known. In this study, we have identified vascular endothelial cells as a source of Pink1-dependent release of mitochondrial formyl proteins in response to inflammatory mediators. Mechanistically, the mitophagy mediator Pink1 is stabilized by inflammatory activation of endothelial cells, promoting mitophagy and mitochondrial formyl peptide release both in mice and primary human endothelial cells. Using nanoparticle delivery of <i>Pink1</i>-targeting sgRNA in mice expressing endothelial-specific Cas9, we developed a mouse model in which <i>Pink1</i> is specifically depleted in the endothelium. Deletion of endothelial <i>Pink1</i> decreased circulating formyl peptide levels, lowered lung neutrophil infiltration and reduced mortality in mice. We thus propose that endothelial cells upregulate pro-inflammatory mitophagy in response to inflammation, leading to the release of mitochondrial formyl peptides and detrimental neutrophil recruitment into the lung.

Adaptive evolution enables species to survive and thrive under changing environmental conditions. In the face of accelerating global climate change, thermal stress represents a major challenge to the persistence of terrestrial arthropods. Understanding the genetic mechanisms underlying thermal adaptation is therefore critical for predicting species’ evolutionary potential and future success. Here, we combine experimental evolution, phenotypic assays, and multi-omics analyses to investigate the adaptive responses of the diamondback moth (<i>Plutella xylostella</i>), a globally destructive pest of cruciferous crops, to contrasting thermal environments. Populations evolved under hot (32 °C/27 °C) and cold (15 °C/10 °C) regimes exhibited distinct life history and fitness traits relative to those maintained under favorable conditions (26 °C). The hot strain showed accelerated development, higher fecundity, and increased survival under extreme heat, while the cold strain exhibited lower supercooling and freezing points, indicating enhanced cold hardiness. Integrated transcriptomic and metabolomic analyses revealed extensive transcriptional reprogramming and convergent metabolic adjustments, notably a reduction in lipid metabolism to conserve energy under thermal stress. Crucially, non-synonymous mutations in <i>PxSODC</i> enhance superoxide scavenging efficiency, enabling effective oxidative stress management at lower gene expression levels. Furthermore, we identified epigenetic regulation via DNA methylation as a key mediator of this thermal tolerance. Together, these coordinated mutational, epigenetic, and metabolic insights highlight this arthropod’s capacity for global dispersal and likely persistence under climate change, establishing a framework for understanding equivalent effects in other species.

The first 10 million years (Myr) following the Cretaceous-Paleogene (K-Pg) mass extinction marked a period of global greenhouse conditions and dramatic rise of placental mammals. Because ~80% of known terrestrial sections capturing post-K-Pg mammal recovery come from North America, a substantial knowledge gap exists in the tempo and mode of recovery in Asia, where only 3% of global sites are located and most contain species found nowhere else. We show that isolated Paleocene eutherian assemblages from China (1) exhibited high mean tooth size and disparity early in the Paleocene, (2) shifted in their dental shape in parallel with regional and global environmental changes later in the Paleocene, and (3) achieved maximum dental shape-performance covariation near the end of the first 10 Myr post-K-Pg. This ‘brawn before bite’ transformation, coupled with prolonged dental shape versus performance variability, favors a scenario whereby many living orders of eutherian mammals were borne out of phenotypically and functionally plastic ancestral assemblages, including those in tropical South China, during the Paleocene.

Keratinocytes, the dominant cell type in the melanoma microenvironment during tumor initiation, exhibit diverse effects on melanoma progression. Using a zebrafish model of melanoma and human cell co-cultures, we observed that keratinocytes undergo an epithelial-mesenchymal transition (EMT)-like transformation in the presence of melanoma, reminiscent of their behavior during wound healing. Surprisingly, overexpression of the EMT-transcription factor Twist in keratinocytes led to improved overall survival in zebrafish melanoma models, despite no change in tumor initiation rates. This survival benefit was attributed to reduced melanoma invasion, as confirmed by human cell co-culture assays. Single-cell RNA-sequencing revealed a unique melanoma cell cluster in the Twist-overexpressing condition, exhibiting a more differentiated, less invasive phenotype. Further analysis nominated homotypic jam3b–jam3b and pgrn–sort1a interactions between Twist-overexpressing keratinocytes and melanoma cells as potential mediators of the invasive restraint. Our findings suggest that EMT in the tumor microenvironment may paradoxically limit melanoma invasion through altered cell–cell interactions.

Desert Hedgehog (Dhh) mutations cause Leydig cell dysfunction, yet the mechanisms governing Leydig lineage commitment through Dhh-mediated receptor selectivity, transcriptional effector specificity, and steroidogenic coupling remain elusive. In this study, using CRISPR/Cas9-mediated gene knockout and stem Leydig cells (SLCs) transplantation, we identified a critical Dhh/Patched 2 (Ptch2)/Glioma-associated oncogene homolog 1 (Gli1)/steroidogenic factor 1 (Sf1) signaling axis essential for SLC differentiation in Nile tilapia (<i>Oreochromis niloticus</i>). Dhh deficiency resulted in defective adult Leydig cells and androgen insufficiency. Rescue experiments involving 11-ketotestosterone administration and a Dhh agonist treatment, combined with SLCs transplantation, demonstrated that Dhh regulates SLC differentiation, not survival. In vitro knockout of <i>ptch1</i> and <i>ptch2</i> in SLCs revealed that Ptch2 likely acts as the functional receptor for Dhh. This was further supported by in vivo genetic rescue experiments, where <i>ptch2</i> mutation did not impair testicular development, yet completely rescued the testicular defects in <i>dhh</i> mutants—consistent with Ptch2 acting as an inhibitory receptor whose loss alleviates Dhh pathway suppression. Luciferase assays in Gli-knockout SLCs demonstrated that Gli1 acts as the primary transcriptional effector and transactivates <i>sf1</i> expression. Additionally, functional transplantation assays confirmed that Sf1 is indispensable for SLC differentiation, as Sf1-overexpressing SLCs rescued differentiation, whereas <i>sf1</i>-mutant SLCs failed. Overall, our work delineates the Dhh-Ptch2-Gli1-Sf1 axis and provides fundamental insights into the endocrine regulation of Leydig cell lineage development.

African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic pigs and wild boars, which poses severe threats to the global pork industry. Despite the promise of live attenuated vaccines (LAVs), their narrow margin between efficacy and residual virulence presents major safety challenges. This study bridges a critical knowledge gap in ASF vaccinology by identifying innate and adaptive correlates of protection. This was achieved by using an established model with two groups of pigs differing in baseline immunological status (farm and specific pathogen-free [SPF]). The animals were immunized with an attenuated ASFV strain and subsequently challenged with a related, highly virulent genotype II strain. By applying a systems immunology approach, we correlated kinetic data, including serum cytokines, blood transcription modules (BTMs), T-cell responses, and antibody levels, with clinical outcomes to track protective and detrimental immune responses to the virus over time. Key innate correlates of protection included early and sustained IFN-α response, activation of antigen presentation BTMs, and controlled IL-8 levels during immunization. Lower baseline immune activation observed in SPF pigs in steady state was linked to increased protection. Adaptive correlates encompassed cell cycle, plasma cell, and T-cell BTM responses lasting until day 15 post-immunization. Consequently, an effective response from ASFV-specific T_h cells prior to challenge indicated protection. After the challenge, an early IFN-α response, along with low levels of pro-inflammatory cytokines and a strong induction of memory T_h and T_c cells, correlated with improved clinical outcomes. The model highlights the critical role of host-specific factors in vaccine efficacy and provides a valuable framework for optimizing ASFV vaccine design while distinguishing between protective and detrimental immune responses.

Exogenous attention is a rapid, involuntary mechanism that automatically reallocates processing resources toward salient stimuli. It enhances visual sensitivity in the vicinity of the salient stimulus, both in extrafoveal regions and within the high-acuity foveola. While the spatial frequencies (SFs) modulated by exogenous attention in extrafoveal vision are well characterized, it remains unknown how this mechanism operates within the foveola, which can resolve SFs up to 30 cycles per degree (CPD). Here, we examined which SFs were enhanced by fine-grained deployments of exogenous attention within this highest-acuity region of the visual field. Using high-precision eye-tracking to precisely localize gaze during attentional allocation, we found that exogenous attention at the foveal scale selectively enhances contrast sensitivity for low- to mid-range SFs (4–8 CPD), with no significant benefits for higher SFs (12–20 CPD). In contrast, attention-related benefits on asymptotic performance at the highest contrast were observed across a wide range of SFs. These results indicate that, despite the high-resolution capacity of the foveola, exogenous attention remains an inflexible mechanism that, even at this scale, selectively enhances contrast gain for lower SFs—mirroring its behavior in extrafoveal vision.

The ability to dynamically adjust a behavioral response to a stimulus depending on context is of critical importance for animals. To investigate the neural basis supporting context-dependent sensory processing, we developed a behavioral task in which mice changed their response to a single whisker deflection according to a continuously present contextual cue. Through unbiased optogenetic inactivation mapping, we found that neuronal activity in sensory and motor cortices contributed to task execution and, interestingly, we uncovered an unexpected role of the retrosplenial cortex (RSC) for contextual integration. Widefield calcium imaging revealed that the RSC was the first dorsal cortical area to show context discrimination in response to whisker stimulation, followed by the whisker motor cortex. Finally, we combined optogenetic inactivation with calcium imaging to define causal context-dependent changes in sensorimotor processing. Our cortex-wide mapping experiments thus begin to define key cortical nodes for context-dependent sensorimotor transformation and highlight an important contribution of RSC.

As the resident tissue macrophage of the liver, Kupffer cells (KCs) play an important role in homeostasis and tissue support. However, current in vitro liver models often ignore the contribution of these KCs towards the proper response and function of the tissue. This is especially relevant when we consider the implications of immune-mediated drug injuries. To address this issue, we developed an isogenic co-culture system utilising iPSC-derived macrophages (iMacs) and hepatocytes (iHeps). Directly co-culturing iHeps with iMacs improved the differentiation and maturation of the iHeps, with significant downregulation of fetal hepatocyte markers as well as upregulation of cytochrome genes. Furthermore, the co-culture also imparted stronger KC identity to the iMacs in a contact-dependent manner, with iMacs cultured in iHep conditioned media alone showing weaker expression of key KC markers. Finally, challenging the iHep-iMac co-culture system with seven paradigm hepatotoxic compounds showed dose-dependent cytokine response in the five compounds associated with immune-mediated liver injuries while no significant changes were observed in the two compounds with no reported immune-dependent complications. This effect was also not recapitulated when the co-culture was instead performed with human peripheral blood monocyte-derived macrophages, suggesting that iMacs are essential for liver toxicity response. Taken together, our study shows not only the importance of macrophages in tissue systems, but also that the source of macrophages is critical to the development of accurate in vitro human models.

Metabolic dysfunction-associated steatotic liver disease (MASLD) progression involves the replacement of protective embryo-derived Kupffer cells (KCs) by inflammatory monocyte-derived macrophages (MoMFs), yet the regulatory mechanisms remain unclear. Here, we identify chitinase 3-like 1 (Chi3l1/YKL-40) as a critical metabolic regulator of hepatic macrophage fate. We observed high expression of Chi3l1 in both KCs and MoMFs during MASLD development. Genetic deletion of Chi3l1 specifically in KCs significantly exacerbated MASLD severity and metabolic dysfunction, whereas MoMF-specific Chi3l1 deletion showed minimal metabolic effects. Mechanistic studies revealed that this cell type-specific regulation arises from differential metabolic requirements: KCs display elevated glucose metabolism compared to MoMFs. Chi3l1 directly interacts with glucose to inhibit its cellular uptake, thereby selectively protecting glucose-dependent KCs from metabolic stress-induced cell death while having negligible effects on less glucose-dependent MoMFs. These findings uncover a novel Chi3l1-mediated metabolic checkpoint that preferentially maintains KCs populations through glucose metabolism modulation, providing important new insights into the pathogenesis of MASLD and potential therapeutic strategies targeting macrophage-specific metabolic pathways.

Imported malaria is a critical obstacle to achieving elimination in low transmission settings, but importation classification tools combining human mobility and parasite genomics are lacking. A Bayesian model combining epidemiological, human mobility, and parasite genetic data was developed to estimate malaria importation and geographic origins of <i>Plasmodium falciparum</i> cases. Using microhaplotype-based genetic relatedness from 1605 samples across nine Mozambican provinces in 2022, the study focused on two low-transmission districts in the south: Magude and Matutuine. Parasites from southern Mozambique showed lower genetic relatedness to those from northern/central regions (0.021) than the national average (0.034, p&lt;0.001), indicating limited connectivity. Overall, 42% (88/207) of infections in these districts were classified as imported, mainly originating from Inhambane province (63% [55/88]). Imported cases showed higher parasite complexity than local ones (odds ratios [OR] = 1.3). Importation rates differed markedly between districts – Matutuine (48.60%, 87/179) was far more affected than Magude (10.71%, 3/28) – highlighting the need for localised rather than uniform elimination strategies. In Matutuine, importation appears to be actively sustaining transmission, suggesting that reducing malaria burden in source regions (particularly Inhambane) and targeting travellers from central and northern Mozambique would have the greatest elimination impact.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is critical for viral fusion and entry into host cells and remains a primary target for vaccine and antiviral drug development. Advances in soluble gp140 trimer design have provided insight into the ectodomain structure and dynamics. While structural information is available for the membrane-proximal external region (MPER) and transmembrane domain (TMD), these regions remain comparatively understudied. Furthermore, high-resolution structural information for the cytoplasmic tail (CT), particularly within the context of the intact trimer, is limited and largely uncertain. Additionally, previous studies have typically treated the ectodomain and TMD as separate entities. To investigate the trimeric gp120–gp41 as a complete entity and its structural flexibility, we built a full-length model of the gp120–gp41 trimer that is fully glycosylated with N-linked glycans and embedded in a lipid bilayer, and performed all-atom molecular dynamics simulations. Our results show that the ectodomain maintains a rigid internal structure stable in the prefusion state, whereas the intrinsic flexibility of the MPER enables the ectodomain to adopt a range of tilted orientations, potentially enhancing spatial alignment for receptor engagement. The centrally positioned R696 residue in the TMD interacts with lipid headgroups, ions, and CT residues, resulting in conformational variability in the TMD and perturbations in the surrounding membrane that may facilitate the fusion process. Finally, we demonstrate how simulation trajectories can be leveraged to evaluate the accessibility of antibody epitopes across different regions of the protein.

The rapid cross-species transmission of highly pathogenic avian influenza presents a significant zoonotic threat. Elucidating the avian interferon (IFN) system, the primary antiviral defense in chickens, is critical for controlling the virus at its source and preventing its spillover into humans and other species. We engineered type I (IFN-α/β) and type III (IFN-λ) IFN receptor knockout chickens to dissect the role of IFNs in viral infections. Results revealed that type I IFN predominantly modulates innate immune cell populations, T cell subsets, and their contribution to antibody production following immunization under physiological conditions. In ovo and in vivo challenge experiments utilizing diverse influenza A virus strains demonstrated strain-specific roles of both IFN-α/β and IFN-λ in orchestrating viral pathogenesis, immunological responses, and tissue-tropism effects. Notably, type I IFN was particularly crucial in the initial defense mechanisms against H3N1 avian influenza A virus infection. These novel models offer unprecedented insights into avian IFN biology within the context of avian influenza, which is essential for developing more effective strategies to prevent and control this public health challenge.

Krug J, Perner B, Albertz C, Mörl H, Hopfenmüller VL, Englert C. 2023. Generation of a transparent killifish line through multiplex CRISPR/Cas9mediated gene inactivation. eLife 12:e81549. doi: 10.7554/eLife.81549. Published 23 February 2023 Prompted by a readers’ request we became aware that due to an oversight we provided two incorrect sequences for oligonucleotides. We sincerely apologize for this oversight and for any confusion this mistake may have caused. We have corrected the relevant sentence in the Materials and Methods; underline is used to highlight differences. Corrected text: To induce a DNA double-strand break in close proximity to the intended site of insertion, the following oligonucleotides for sgRNA synthesis were used: sg_cdkn1a_1: 5’-TAGG-GGGAGTGATATTTCCTTTGA-3’ sg_cdkn1a_2: 5’-AAAC-TCAAAGGAAATATCACTCCC-3’. Synthesis of this sgRNA was done as described above. Original text: To induce a DNA double-strand break in close proximity to the intended site of insertion, the following oligonucleotides for sgRNA synthesis were used: sg_cdkn1a_1: 5’-TAGG-AATATCACTCCCCGGATTTC-3’ sg_cdkn1a_2: 5’-AAAC-GAAATCCGGGGAGTGATATT-3’. Synthesis of this sgRNA was done as described above. The article has been corrected accordingly. Author details © 2026, Krug et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. - - 0 - citations Views, downloads and citations are aggregated across all versions of this paper published by eLife.

In open societies, disinformation is often considered a threat to the very fabric of democracy. However, we know little about how disinformation exerts its impact, especially its influence on individual learning processes. Guided by the notion that disinformation exerts its pernicious effects by capitalizing on learning biases, we ask which aspects of learning from potential disinformation align with ideal ‘Bayesian’ principles, and which exhibit biases deviating from these standards. To this end, we harnessed a reinforcement learning framework, offering computationally tractable models capable of estimating latent aspects of a learning process as well as identifying biases in learning. In two experiments, participants completed a two-armed bandit task, where they repeatedly chose between two lotteries and received outcome-feedback from sources of varying credibility, who occasionally disseminated disinformation by lying about true choice outcome (e.g., reporting non-reward when a reward was truly earned or vice versa). Computational modelling indicated that learning increased in tandem with source credibility, consistent with ideal-Bayesian principles. However, we also observed striking biases reflecting divergence from idealized Bayesian learning patterns. Notably, in one experiment individuals learned from sources that should have been ignored, as these were known to be fully unreliable. Additionally, the presence of disinformation elicited exaggerated learning from trustworthy information (akin to jumping to conclusions) and exacerbated a normalized measure of ‘positivity bias’ whereby individuals self-servingly boost their learning from positive, relative to negative, choice feedback. Thus, in the face of disinformation we identify specific cognitive mechanisms underlying learning biases, with potential implications for societal strategies aimed at mitigating its harmful impacts.

Understanding the organization and regulation of neurotransmission at the level of individual neurons and synapses requires tools that can track and manipulate transmitter-specific vesicles in vivo. Here, we present SynaptoTagMe, a suite of genetic tools in <i>Caenorhabditis elegans</i> to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the <i>C. elegans</i> nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use in vivo. The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication in vivo.

In the low Reynolds number regime, active biological systems utilize nonreciprocal cyclic activities to achieve motility, as seen in the spinning of bacterial flagella and the beating of cilia. Coupling among these active mechanical components leads to synchronization and emergence of metachronal waves. Here, we report that biofilms of <i>Pseudomonas nitroreducens</i> form active carpet-like surfaces textured with diverse topological defects, generating Mexican-wave-like collective behavior in which bacteria periodically lift up. On these active surfaces, non-reciprocally coupled extension and retraction activities of bacterial pili drive these collective oscillations. Surprisingly, this collective behavior exhibits left-right asymmetry across the biofilm driving unidirectionally propagating waves. We discover that this directionality is primarily governed by an aging-related frequency gradient across the biofilm. Leveraging these insights, we further demonstrate the ability to control the collective dynamics of these waves, including symmetry breaking, transitions from spiral waves into target and propagating plane waves by manipulating the elastic properties of biofilms. Overall, our findings illuminate the fundamental role of nonreciprocally interacting active components in regulating synchronization, collective dynamics, and symmetry-breaking phenomena in biological systems.

In this study, we investigated how iron limitation alters central metabolism in <i>Mycobacterium tuberculosis</i> using metabolomics and stable isotope tracing. Our findings reveal a well-orchestrated metabolic programme to enable Krebs cycle activity despite the inefficient action of its iron-dependent enzymes. Under such conditions, carbon flux through the oxidative branch of the Krebs cycle is stalled, resulting in the accumulation of metabolites that are partially secreted. As a result, carbon flux from glycolysis is partially diverted to the reductive branch of the Krebs cycle to support the production of oxaloacetate and malate through the activity of phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Both branches terminate with the synthesis of malate, which is secreted. This unprecedented split of the Krebs cycle and malate secretion in a bacterial pathogen facilitates the continuous flow of carbon through the core of carbon metabolism, overcoming the metabolic stalling triggered by iron starvation.

The outflow tract (OFT) of the heart carries blood away from the heart into the great arteries. During embryogenesis, the OFT divides to form the aorta and pulmonary trunk, creating the double circulation present in mammals. Defects in this area account for one-third of all congenital heart defect cases. Here, we present comprehensive transcriptomic data on the developing OFT at two distinct time points (embryonic and fetal) and its adult derivatives, the aortic valves, and use spatial transcriptomics to define the distribution of cell populations. We uncover that distinctive embryonic signatures persist in adult cells and can be used as labels to retrospectively attribute relationships between cells separated by a large timescale. Single-cell regulatory network inference identifies GATA6, a transcription factor linked to common arterial trunk and bicuspid aortic valve, as a key regulator of valve precursor cells. Its downstream network reveals candidate drivers of human cardiac defects and illuminates the molecular mechanisms of both normal and pathological valve development. Our findings define the cellular and molecular signatures of the human OFT and its distinct cell lineages, which is critical for understanding congenital heart defects and developing cardiac tissue for regenerative medicine.

Picornaviruses employ internal ribosome entry sites (IRESs) in their genomic RNA to hijack the host’s translational machinery. The picornavirus, encephalomyocarditis virus, employs a type 2 IRES present in its 5’ untranslated region (5’UTR) and requires 43S ribosomal preinitiation complex (PIC), the central domain of eukaryotic initiation factor (eIF) 4G, eIF4A, and an essential ITAF (IRES trans-acting factor)-polypyrimidine tract binding protein 1 (PTB1) to form 48S PIC. In this study, we have used cryo-electron microscopy (cryo-EM) to determine the structure of encephalomyocarditis virus (EMCV) IRES-bound mammalian 48S PIC in a scanning-arrested closed state at the start codon. The EMCV IRES domains contact initiator tRNA (tRNA_i) and 40S head at the inter-subunit interface, which reveals an altogether unique mechanism used by viruses to capture host translational machinery for its protein synthesis. The tRNA_i is held away from the 40S body in contrast to canonical cap-dependent translation while the domain I apical region of EMCV IRES mimics 28S rRNA of 60S to interact with 40S ribosomal head proteins uS13 and uS19. The structural analysis accounts for numerous previously reported biochemical studies on type 2 IRES and shows how type 2 IRES interacts with 43S PIC to form 48S PIC. This study provides mechanistic insights for understanding EMCV IRES-mediated translation initiation, which could be extrapolated to other IRESs sharing similar motifs and factor requirements, including type 1 viral IRESs.

Neuronal intrinsic excitability is a mechanism implicated in learning and memory that is distinct from synaptic plasticity. Prior work in songbirds established that intrinsic properties (IPs) of premotor basal-ganglia-projecting neurons (HVC_X) relate to learned song. Here, we find that temporal song structure is related to specific HVC_X IPs: HVC_X from birds who sang longer songs, including longer invariant vocalizations (harmonic stacks), had IPs that reflected increased post-inhibitory rebound. This suggests a rebound excitation mechanism underlying the ability of HVC_X neurons to integrate over long periods of time throughout the song and represent sequence information. To explore this, we constructed a network model of realistic neurons showing how in vivo HVC bursting properties link rebound excitation to network structure and behavior. These results demonstrate an explicit link between neuronal IPs and learned behavior. We propose that sequential behaviors exhibiting temporal regularity require IPs to be included in realistic network-level descriptions.