Wing dimorphisms have long served as models for examining the ecological and evolutionary tradeoffs associated with alternative phenotypes. Here, we investigated the genetic cause of the pea aphid () male wing dimorphism, wherein males exhibit one of two morphologies that differ in correlated traits that include the presence or absence of wings. We mapped this trait difference to a single genomic region and, using third generation, long-read sequencing, we identified a 120 kb insertion in the wingless allele. This insertion includes a duplicated gene, which is a strong candidate gene in the minimal mapped interval to cause the dimorphism. We found that both alleles were present prior to pea aphid biotype lineage diversification, we estimated that the insertion occurred millions of years ago, and we propose that both alleles have been maintained in the species, likely due to balancing selection.

All animals must transform ambiguous sensory data into successful behavior. This requires sensory representations that accurately reflect the statistics of natural stimuli and behavior. Multiple studies show that visual motion processing is tuned for accuracy under naturalistic conditions, but the sensorimotor circuits extracting these cues and implementing motion-guided behavior remain unclear. Here we show that the larval zebrafish retina extracts a diversity of naturalistic motion cues, and the retinorecipient pretectum organizes these cues around the elements of behavior. We find that higher-order motion stimuli, gliders, induce optomotor behavior matching expectations from natural scene analyses. We then image activity of retinal ganglion cell terminals and pretectal neurons. The retina exhibits direction-selective responses across glider stimuli, and anatomically clustered pretectal neurons respond with magnitudes matching behavior. Peripheral computations thus reflect natural input statistics, whereas central brain activity precisely codes information needed for behavior. This general principle could organize sensorimotor transformations across animal species.

The human pathogen targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable and mature focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Super-resolution microscopy revealed in infected cells the vertical displacement of paxillin and FAK from the signaling layer of focal adhesions; while vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions, required a post-invasion interaction with the host protein vinculin through a specific domain at the C-terminus of TarP. This interaction is repurposed from an actin-recruiting and -remodeling complex to one that mediates nano-architectural and dynamic changes of focal adhesions. The consequence of -stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, inserts TarP within focal adhesions to alter their organization and stability.

We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.

Animals can store information about experiences by activating specific neuronal populations, and subsequent reactivation of these neural ensembles can lead to recall of salient experiences. In the hippocampus, granule cells of the dentate gyrus participate in such memory engrams; however, whether there is an underlying logic to granule cell participation has not been examined. Here, we find that a range of novel experiences preferentially activates granule cells of the suprapyramidal blade relative to the infrapyramidal blade. Motivated by this, we identify a suprapyramidal-blade-enriched population of granule cells with distinct spatial, morphological, physiological, and developmental properties. Via transcriptomics, we map these traits onto a sparse and discrete granule cell subtype that is recruited at a 10-fold greater frequency than expected by subtype prevalence, constituting the majority of all recruited granule cells. Thus, in behaviors known to involve hippocampal-dependent memory formation, a rare and spatially localized subtype dominates overall granule cell recruitment.

Drosophila melanogaster is an established model for neuroscience research with relevance in biology and medicine. Until recently, research on the Drosophila brain was hindered by the lack of a complete and uniform nomenclature. Recognizing this, Ito et al. (2014) produced an authoritative nomenclature for the adult insect brain, using Drosophila as the reference. Here, we extend this nomenclature to the adult thoracic and abdominal neuromeres, the ventral nerve cord (VNC), to provide an anatomical description of this major component of the Drosophila nervous system. The VNC is the locus for the reception and integration of sensory information and involved in generating most of the locomotor actions that underlie fly behaviors. The aim is to create a nomenclature, definitions, and spatial boundaries for the Drosophila VNC that are consistent with other insects. The work establishes an anatomical framework that provides a powerful tool for analyzing the functional organization of the VNC.

Until recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

Recent advances in automatic image segmentation and synapse prediction in electron microscopy (EM) datasets of the brain enable more efficient reconstruction of neural connectivity. In these datasets, a single neuron can span thousands of images containing complex tree-like arbors with thousands of synapses. While image segmentation algorithms excel within narrow fields of views, the algorithms sometimes struggle to correctly segment large neurons, which require large context given their size and complexity. Conversely, humans are comparatively good at reasoning with large objects. In this paper, we introduce several semi-automated strategies that combine 3D visualization and machine guidance to accelerate connectome reconstruction. In particular, we introduce a strategy to quickly correct a segmentation through merging and cleaving, or splitting a segment along supervoxel boundaries, with both operations driven by affinity scores in the underlying segmentation. We deploy these algorithms as streamlined workflows in a tool called Neu3 and demonstrate superior performance compared to prior work, thus enabling efficient reconstruction of much larger datasets. The insights into proofreading from our work clarify the trade-offs to consider when tuning the parameters of image segmentation algorithms.

The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid with some molecules returning to the plasma membrane with a <5 minutes. Existing methods to study these trafficking pathways utilize chemical, radioactive, or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay, based on a newly designed cell-impermeable, fluorogenic ligand for HaloTag: 'Janelia Fluor 635i' (JFi; i=impermeant) which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed 'actin chromobodies' (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.