Temporal dynamics of woolly mammoth genome erosion prior to extinction.

A number of species have recently recovered from near-extinction. Although these species have avoided the immediate extinction threat, their long-term viability remains precarious due to the potential genetic consequences of population declines, which are poorly understood on a timescale beyond a few generations. Woolly mammoths (Mammuthus primigenius) became isolated on Wrangel Island around 10,000 years ago and persisted for over 200 generations before becoming extinct around 4,000 years ago. To study the evolutionary processes leading up to the mammoths' extinction, we analyzed 21 Siberian woolly mammoth genomes. Our results show that the population recovered quickly from a severe bottleneck and remained demographically stable during the ensuing six millennia. We find that mildly deleterious mutations gradually accumulated, whereas highly deleterious mutations were purged, suggesting ongoing inbreeding depression that lasted for hundreds of generations. The time-lag between demographic and genetic recovery has wide-ranging implications for conservation management of recently bottlenecked populations.

Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo.

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity.

Cancer cells often overexpress CD47, which triggers the inhibitory receptor SIRPα expressed on macrophages, to elude phagocytosis and antitumor immunity. Pharmacological blockade of CD47 or SIRPα is showing promise as anticancer therapy, although CD47 blockade has been associated with hematological toxicities that may reflect its broad expression pattern on normal cells. Here we found that, in addition to triggering SIRPα, CD47 suppressed phagocytosis by a SIRPα-independent mechanism. This mechanism prevented phagocytosis initiated by the pro-phagocytic ligand, SLAMF7, on tumor cells, due to a cis interaction between CD47 and SLAMF7. The CD47-SLAMF7 interaction was disrupted by CD47 blockade and by a first-in-class agonist SLAMF7 antibody, but not by SIRPα blockade, thereby promoting antitumor immunity. Hence, CD47 suppresses phagocytosis not only by engaging SIRPα, but also by masking cell-intrinsic pro-phagocytic ligands on tumor cells and knowledge of this mechanism may influence the decision between CD47 blockade or SIRPα blockade for therapeutic purposes.

Engineered T cell therapy for central nervous system injury.

Traumatic injuries to the central nervous system (CNS) afflict millions of individuals worldwide1, yet an effective treatment remains elusive. Following such injuries, the site is populated by a multitude of peripheral immune cells, including T cells, but a comprehensive understanding of the roles and antigen specificity of these endogenous T cells at the injury site has been lacking. This gap has impeded the development of immune-mediated cellular therapies for CNS injuries. Here, using single-cell RNA sequencing, we demonstrated the clonal expansion of mouse and human spinal cord injury-associated T cells and identified that CD4+ T cell clones in mice exhibit antigen specificity towards self-peptides of myelin and neuronal proteins. Leveraging mRNA-based T cell receptor (TCR) reconstitution, a strategy aimed to minimize potential adverse effects from prolonged activation of self-reactive T cells, we generated engineered transiently autoimmune T cells. These cells demonstrated notable neuroprotective efficacy in CNS injury models, in part by modulating myeloid cells via IFNγ. Our findings elucidate mechanistic insight underlying the neuroprotective function of injury-responsive T cells and pave the way for the future development of T cell therapies for CNS injuries.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Hybrid speciation driven by multilocus introgression of ecological traits.

Hybridization allows adaptations to be shared among lineages and may trigger the evolution of new species1,2. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridization was crucial in generating reproductive isolation3. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to examine a case of hybrid speciation in Heliconius butterflies. We show that Heliconius elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, Heliconius pardalinus, which homogenizes 99% of their genomes. The remaining 1% introgressed from the other parent, Heliconius melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands contain multiple traits that are under disruptive selection, including colour pattern, wing shape, host plant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits, and that speciation with gene flow is possible with a multilocus genetic architecture.

Structural basis of NINJ1-mediated plasma membrane rupture in cell death.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.

Identification of astrocyte regulators by nucleic acid cytometry.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

Tropical forests are approaching critical temperature thresholds.

The critical temperature beyond which photosynthetic machinery in tropical trees begins to fail averages approximately 46.7 °C (Tcrit)1. However, it remains unclear whether leaf temperatures experienced by tropical vegetation approach this threshold or soon will under climate change. Here we found that pantropical canopy temperatures independently triangulated from individual leaf thermocouples, pyrgeometers and remote sensing (ECOSTRESS) have midday peak temperatures of approximately 34 °C during dry periods, with a long high-temperature tail that can exceed 40 °C. Leaf thermocouple data from multiple sites across the tropics suggest that even within pixels of moderate temperatures, upper canopy leaves exceed Tcrit 0.01% of the time. Furthermore, upper canopy leaf warming experiments (+2, 3 and 4 °C in Brazil, Puerto Rico and Australia, respectively) increased leaf temperatures non-linearly, with peak leaf temperatures exceeding Tcrit 1.3% of the time (11% for more than 43.5 °C, and 0.3% for more than 49.9 °C). Using an empirical model incorporating these dynamics (validated with warming experiment data), we found that tropical forests can withstand up to a 3.9 ± 0.5 °C increase in air temperatures before a potential tipping point in metabolic function, but remaining uncertainty in the plasticity and range of Tcrit in tropical trees and the effect of leaf death on tree death could drastically change this prediction. The 4.0 °C estimate is within the 'worst-case scenario' (representative concentration pathway (RCP) 8.5) of climate change predictions2 for tropical forests and therefore it is still within our power to decide (for example, by not taking the RCP 6.0 or 8.5 route) the fate of these critical realms of carbon, water and biodiversity3,4.

Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells.

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.

Structure of the MRAS-SHOC2-PP1C phosphatase complex.

RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1-3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS-SHOC2-PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF-RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies-developmental syndromes caused by aberrant MAPK pathway activation6-14-and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)-RAS-driven tumours15-18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS-MAPK signalling, therefore providing the structural basis for therapeutic interventions.

Identification of environmental factors that promote intestinal inflammation.

Genome-wide association studies have identified risk loci linked to inflammatory bowel disease (IBD)1-a complex chronic inflammatory disorder of the gastrointestinal tract. The increasing prevalence of IBD in industrialized countries and the augmented disease risk observed in migrants who move into areas of higher disease prevalence suggest that environmental factors are also important determinants of IBD susceptibility and severity2. However, the identification of environmental factors relevant to IBD and the mechanisms by which they influence disease has been hampered by the lack of platforms for their systematic investigation. Here we describe an integrated systems approach, combining publicly available databases, zebrafish chemical screens, machine learning and mouse preclinical models to identify environmental factors that control intestinal inflammation. This approach established that the herbicide propyzamide increases inflammation in the small and large intestine. Moreover, we show that an AHR-NF-κB-C/EBPß signalling axis operates in T cells and dendritic cells to promote intestinal inflammation, and is targeted by propyzamide. In conclusion, we developed a pipeline for the identification of environmental factors and mechanisms of pathogenesis in IBD and, potentially, other inflammatory diseases.

Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.

Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.

Genomic evidence reveals three W-autosome fusions in Heliconius butterflies.

Sex chromosomes are evolutionarily labile in many animals and sometimes fuse with autosomes, creating so-called neo-sex chromosomes. Fusions between sex chromosomes and autosomes have been proposed to reduce sexual conflict and to promote adaptation and reproductive isolation among species. Recently, advances in genomics have fuelled the discovery of such fusions across the tree of life. Here, we discovered multiple fusions leading to neo-sex chromosomes in the sapho subclade of the classical adaptive radiation of Heliconius butterflies. Heliconius butterflies generally have 21 chromosomes with very high synteny. However, the five Heliconius species in the sapho subclade show large variation in chromosome number ranging from 21 to 60. We find that the W chromosome is fused with chromosome 4 in all of them. Two sister species pairs show subsequent fusions between the W and chromosomes 9 or 14, respectively. These fusions between autosomes and sex chromosomes make Heliconius butterflies an ideal system for studying the role of neo-sex chromosomes in adaptive radiations and the degeneration of sex chromosomes over time. Our findings emphasize the capability of short-read resequencing to detect genomic signatures of fusion events between sex chromosomes and autosomes even when sex chromosomes are not explicitly assembled.

Underlying and proximate drivers of biodiversity changes in Mesoamerican biosphere reserves.

Protected areas are of paramount relevance to conserving wildlife and ecosystem contributions to people. Yet, their conservation success is increasingly threatened by human activities including habitat loss, climate change, pollution, and species overexploitation. Thus, understanding the underlying and proximate drivers of anthropogenic threats is urgently needed to improve protected areas' effectiveness, especially in the biodiversity-rich tropics. We addressed this issue by analyzing expert-provided data on long-term biodiversity change (last three decades) over 14 biosphere reserves from the Mesoamerican Biodiversity Hotspot. Using multivariate analyses and structural equation modeling, we tested the influence of major socioeconomic drivers (demographic, economic, and political factors), spatial indicators of human activities (agriculture expansion and road extension), and forest landscape modifications (forest loss and isolation) as drivers of biodiversity change. We uncovered a significant proliferation of disturbance-tolerant guilds and the loss or decline of disturbance-sensitive guilds within reserves causing a "winner and loser" species replacement over time. Guild change was directly related to forest spatial changes promoted by the expansion of agriculture and roads within reserves. High human population density and low nonfarming occupation were identified as the main underlying drivers of biodiversity change. Our findings suggest that to mitigate anthropogenic threats to biodiversity within biosphere reserves, fostering human population well-being via sustainable, nonfarming livelihood opportunities around reserves is imperative.

Comparative analysis of integrative classification methods for multi-omics data.

Recent advances in sequencing, mass spectrometry, and cytometry technologies have enabled researchers to collect multiple 'omics data types from a single sample. These large datasets have led to a growing consensus that a holistic approach is needed to identify new candidate biomarkers and unveil mechanisms underlying disease etiology, a key to precision medicine. While many reviews and benchmarks have been conducted on unsupervised approaches, their supervised counterparts have received less attention in the literature and no gold standard has emerged yet. In this work, we present a thorough comparison of a selection of six methods, representative of the main families of intermediate integrative approaches (matrix factorization, multiple kernel methods, ensemble learning, and graph-based methods). As non-integrative control, random forest was performed on concatenated and separated data types. Methods were evaluated for classification performance on both simulated and real-world datasets, the latter being carefully selected to cover different medical applications (infectious diseases, oncology, and vaccines) and data modalities. A total of 15 simulation scenarios were designed from the real-world datasets to explore a large and realistic parameter space (e.g. sample size, dimensionality, class imbalance, effect size). On real data, the method comparison showed that integrative approaches performed better or equally well than their non-integrative counterpart. By contrast, DIABLO and the four random forest alternatives outperform the others across the majority of simulation scenarios. The strengths and limitations of these methods are discussed in detail as well as guidelines for future applications.

The TLK-ASF1 histone chaperone pathway plays a critical role in IL-1ß-mediated AML progression.

ABSTRACT: Identifying and targeting microenvironment-driven pathways that are active across acute myeloid leukemia (AML) genetic subtypes should allow the development of more broadly effective therapies. The proinflammatory cytokine interleukin-1ß (IL-1ß) is abundant in the AML microenvironment and promotes leukemic growth. Through RNA-sequencing analysis, we identify that IL-1ß-upregulated ASF1B (antisilencing function-1B), a histone chaperone, in AML progenitors compared with healthy progenitors. ASF1B, along with its paralogous protein ASF1A, recruits H3-H4 histones onto the replication fork during S-phase, a process regulated by Tousled-like kinase 1 and 2 (TLKs). Although ASF1s and TLKs are known to be overexpressed in multiple solid tumors and associated with poor prognosis, their functional roles in hematopoiesis and inflammation-driven leukemia remain unexplored. In this study, we identify that ASF1s and TLKs are overexpressed in multiple genetic subtypes of AML. We demonstrate that depletion of ASF1s significantly reduces leukemic cell growth in both in vitro and in vivo models using human cells. Using a murine model, we show that overexpression of ASF1B accelerates leukemia progression. Moreover, Asf1b or Tlk2 deletion delayed leukemia progression, whereas these proteins are dispensable for normal hematopoiesis. Through proteomics and phosphoproteomics analyses, we uncover that the TLK-ASF1 pathway promotes leukemogenesis by affecting the cell cycle and DNA damage pathways. Collectively, our findings identify the TLK1-ASF1 pathway as a novel mediator of inflammatory signaling and a promising therapeutic target for AML treatment across diverse genetic subtypes. Selective inhibition of this pathway offers potential opportunities to intervene effectively, address intratumoral heterogeneity, and ultimately improve clinical outcomes in AML.

Target-selective vertebrate motor axon regeneration depends on interaction with glial cells at a peripheral nerve plexus.

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from 3 nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real-time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.