Hox gene-specific cellular targeting using split intein Trojan exons.

The Trojan exon method, which makes use of intronically inserted T2A-Gal4 cassettes, has been widely used in Drosophila to create thousands of gene-specific Gal4 driver lines. These dual-purpose lines provide genetic access to specific cell types based on their expression of a native gene while simultaneously mutating one allele of the gene to enable loss-of-function analysis in homozygous animals. While this dual use is often an advantage, the truncation mutations produced by Trojan exons are sometimes deleterious in heterozygotes, perhaps by creating translation products with dominant negative effects. Such mutagenic effects can cause developmental lethality as has been observed with genes encoding essential transcription factors. Given the importance of transcription factors in specifying cell type, alternative techniques for generating specific Gal4 lines that target them are required. Here, we introduce a modified Trojan exon method that retains the targeting fidelity and plug-and-play modularity of the original method but mitigates its mutagenic effects by exploiting the self-splicing capabilities of split inteins. "Split Intein Trojan exons" (siTrojans) ensure that the two truncation products generated from the interrupted allele of the native gene are trans-spliced to create a full-length native protein. We demonstrate the efficacy of siTrojans by generating a comprehensive toolkit of Gal4 and Split Gal4 lines for the segmentally expressed Hox transcription factors and illustrate their use in neural circuit mapping by targeting neurons according to their position along the anterior-posterior axis. Both the method and the Hox gene-specific toolkit introduced here should be broadly useful.

Distinctive features of the central synaptic organization of Drosophila larval proprioceptors.

Proprioceptive feedback is critically needed for locomotor control, but how this information is incorporated into central proprioceptive processing circuits remains poorly understood. Circuit organization emerges from the spatial distribution of synaptic connections between neurons. This distribution is difficult to discern in model systems where only a few cells can be probed simultaneously. Therefore, we turned to a relatively simple and accessible nervous system to ask: how are proprioceptors' input and output synapses organized in space, and what principles underlie this organization? Using the Drosophila larval connectome, we generated a map of the input and output synapses of 34 proprioceptors in several adjacent body segments (5-6 left-right pairs per segment). We characterized the spatial organization of these synapses, and compared this organization to that of other somatosensory neurons' synapses. We found three distinguishing features of larval proprioceptor synapses: (1) Generally, individual proprioceptor types display segmental somatotopy. (2) Proprioceptor output synapses both converge and diverge in space; they are organized into six spatial domains, each containing a unique set of one or more proprioceptors. Proprioceptors form output synapses along the proximal axonal entry pathway into the neuropil. (3) Proprioceptors receive few inhibitory input synapses. Further, we find that these three features do not apply to other larval somatosensory neurons. Thus, we have generated the most comprehensive map to date of how proprioceptor synapses are centrally organized. This map documents previously undescribed features of proprioceptors, raises questions about underlying developmental mechanisms, and has implications for downstream proprioceptive processing circuits.

The Role of Even-Skipped in Drosophila Larval Somatosensory Circuit Assembly.

Proper somatosensory circuit assembly is critical for processing somatosensory stimuli and for responding accordingly. In comparison to other sensory circuits (e.g., olfactory and visual), somatosensory circuits have unique anatomy and function. However, understanding of somatosensory circuit development lags far behind that of other sensory systems. For example, there are few identified transcription factors required for integration of interneurons into functional somatosensory circuits. Here, as a model, we examine one type of somatosensory interneuron, Even-skipped (Eve) expressing laterally placed interneurons (ELs) of the Drosophila larval nerve cord. Eve is a highly conserved, homeodomain transcription factor known to play a role in cell fate specification and neuronal axon guidance. Because marker genes are often functionally important in the cell types they define, we deleted eve expression specifically from EL interneurons. On the cell biological level, using single neuron labeling, we find eve plays several previously undescribed roles in refinement of neuron morphogenesis. Eve suppresses aberrant neurite branching, promotes axon elongation, and regulates dorsal-ventral dendrite position. On the circuit level, using optogenetics, calcium imaging, and behavioral analysis, we find eve expression is required in EL interneurons for the normal encoding of somatosensory stimuli and for normal mapping of outputs to behavior. We conclude that the eve gene product coordinately regulates multiple aspects of EL interneuron morphogenesis and is critically required to properly integrate EL interneurons into somatosensory circuits. Our data shed light on the genetic regulation of somatosensory circuit assembly.

Development of motor circuits: From neuronal stem cells and neuronal diversity to motor circuit assembly.

In this review, we discuss motor circuit assembly starting from neuronal stem cells. Until recently, studies of neuronal stem cells focused on how a relatively small pool of stem cells could give rise to a large diversity of different neuronal identities. Historically, neuronal identity has been assayed in embryos by gene expression, gross anatomical features, neurotransmitter expression, and physiological properties. However, these definitions of identity are largely unlinked to mature functional neuronal features relevant to motor circuits. Such mature neuronal features include presynaptic and postsynaptic partnerships, dendrite morphologies, as well as neuronal firing patterns and roles in behavior. This review focuses on recent work that links the specification of neuronal molecular identity in neuronal stem cells to mature, circuit-relevant identity specification. Specifically, these studies begin to address the question: to what extent are the decisions that occur during motor circuit assembly controlled by the same genetic information that generates diverse embryonic neuronal diversity? Much of the research addressing this question has been conducted using the Drosophila larval motor system. Here, we focus largely on Drosophila motor circuits and we point out parallels to other systems. And we highlight outstanding questions in the field. The main concepts addressed in this review are: (1) the description of temporal cohorts-novel units of developmental organization that link neuronal stem cell lineages to motor circuit configuration and (2) the discovery that temporal transcription factors expressed in neuronal stem cells control aspects of circuit assembly by controlling the size of temporal cohorts and influencing synaptic partner choice.

RNA-binding protein syncrip regulates starvation-induced hyperactivity in adult Drosophila.

How to respond to starvation determines fitness. One prominent behavioral response is increased locomotor activities upon starvation, also known as Starvation-Induced Hyperactivity (SIH). SIH is paradoxical as it promotes food seeking but also increases energy expenditure. Despite its importance in fitness, the genetic contributions to SIH as a behavioral trait remains unexplored. Here, we examined SIH in the Drosophila melanogaster Genetic Reference Panel (DGRP) and performed genome-wide association studies. We identified 23 significant loci, corresponding to 14 genes, significantly associated with SIH in adult Drosophila. Gene enrichment analyses indicated that genes encoding ion channels and mRNA binding proteins (RBPs) were most enriched in SIH. We are especially interested in RBPs because they provide a potential mechanism to quickly change protein expression in response to environmental challenges. Using RNA interference, we validated the role of syp in regulating SIH. syp encodes Syncrip (Syp), an RBP. While ubiquitous knockdown of syp led to semi-lethality in adult flies, adult flies with neuron-specific syp knockdown were viable and exhibited decreased SIH. Using the Temporal and Regional Gene Expression Targeting (TARGET) system, we further confirmed the role of Syp in adult neurons in regulating SIH. To determine how syp is regulated by starvation, we performed RNA-seq using the heads of flies maintained under either food or starvation conditions. RNA-seq analyses revealed that syp was alternatively spliced under starvation while its expression level was unchanged. We further generated an alternatively-spliced-exon-specific knockout (KO) line and found that KO flies showed reduced SIH. Together, this study demonstrates a significant genetic contribution to SIH as a behavioral trait, identifies syp as a SIH gene, and highlights the significance of RBPs and post-transcriptional processes in the brain in regulating behavioral responses to starvation.

Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships.

How circuit wiring is specified is a key question in developmental neurobiology. Previously, using the Drosophila motor system as a model, we found the classic temporal transcription factor Hunchback acts in NB7-1 neuronal stem cells to control the number of NB7-1 neuronal progeny form functional synapses on dorsal muscles (Meng et al., 2019). However, it is unknown to what extent control of motor neuron-to-muscle synaptic partnerships is a general feature of temporal transcription factors. Here, we perform additional temporal transcription factor manipulations-prolonging expression of Hunchback in NB3-1, as well as precociously expressing Pdm and Castor in NB7-1. We use confocal microscopy, calcium imaging, and electrophysiology to show that in every manipulation there are permanent alterations in neuromuscular synaptic partnerships. Our data show temporal transcription factors, as a group of molecules, are potent determinants of synaptic partner choice and therefore ultimately control circuit membership.

How prolonged expression of Hunchback, a temporal transcription factor, re-wires locomotor circuits.

How circuits assemble starting from stem cells is a fundamental question in developmental neurobiology. We test the hypothesis that, in neuronal stem cells, temporal transcription factors predictably control neuronal terminal features and circuit assembly. Using the Drosophila motor system, we manipulate expression of the classic temporal transcription factor Hunchback (Hb) specifically in the NB7-1 stem cell, which produces U motor neurons (MNs), and then we monitor dendrite morphology and neuromuscular synaptic partnerships. We find that prolonged expression of Hb leads to transient specification of U MN identity, and that embryonic molecular markers do not accurately predict U MN terminal features. Nonetheless, our data show Hb acts as a potent regulator of neuromuscular wiring decisions. These data introduce important refinements to current models, show that molecular information acts early in neurogenesis as a switch to control motor circuit wiring, and provide novel insight into the relationship between stem cell and circuit.

Direction Selectivity in Drosophila Proprioceptors Requires the Mechanosensory Channel Tmc.

Drosophila Transmembrane channel-like (Tmc) is a protein that functions in larval proprioception. The closely related TMC1 protein is required for mammalian hearing and is a pore-forming subunit of the hair cell mechanotransduction channel. In hair cells, TMC1 is gated by small deflections of microvilli that produce tension on extracellular tip-links that connect adjacent villi. How Tmc might be gated in larval proprioceptors, which are neurons having a morphology that is completely distinct from hair cells, is unknown. Here, we have used high-speed confocal microscopy both to measure displacements of proprioceptive sensory dendrites during larval movement and to optically measure neural activity of the moving proprioceptors. Unexpectedly, the pattern of dendrite deformation for distinct neurons was unique and differed depending on the direction of locomotion: ddaE neuron dendrites were strongly curved by forward locomotion, while the dendrites of ddaD were more strongly deformed by backward locomotion. Furthermore, GCaMP6f calcium signals recorded in the proprioceptive neurons during locomotion indicated tuning to the direction of movement. ddaE showed strong activation during forward locomotion, while ddaD showed responses that were strongest during backward locomotion. Peripheral proprioceptive neurons in animals mutant for Tmc showed a near-complete loss of movement related calcium signals. As the strength of the responses of wild-type animals was correlated with dendrite curvature, we propose that Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain. Our findings begin to explain how distinct cellular systems rely on a common molecular pathway for mechanosensory responses.

Temporal Cohorts of Lineage-Related Neurons Perform Analogous Functions in Distinct Sensorimotor Circuits.

Neuronal stem cell lineages are the fundamental developmental units of the brain, and neuronal circuits are the fundamental functional units of the brain. Determining lineage-circuitry relationships is essential for deciphering the developmental logic of circuit assembly. While the spatial distribution of lineage-related neurons has been investigated in a few brain regions [1-9], an important, but unaddressed question is whether temporal information that diversifies neuronal progeny within a single lineage also impacts circuit assembly. Circuits in the sensorimotor system (e.g., spinal cord) are thought to be assembled sequentially [10-14], making this an ideal brain region for investigating the circuit-level impact of temporal patterning within a lineage. Here, we use intersectional genetics, optogenetics, high-throughput behavioral analysis, single-neuron labeling, connectomics, and calcium imaging to determine how a set of bona fide lineage-related interneurons contribute to sensorimotor circuitry in the Drosophila larva. We show that Even-skipped lateral interneurons (ELs) are sensory processing interneurons. Late-born ELs contribute to a proprioceptive body posture circuit, whereas early-born ELs contribute to a mechanosensitive escape circuit. These data support a model in which a single neuronal stem cell can produce a large number of interneurons with similar functional capacity that are distributed into different circuits based on birth timing. In summary, these data establish a link between temporal specification of neuronal identity and circuit assembly at the single-cell level.

The Hunchback temporal transcription factor establishes, but is not required to maintain, early-born neuronal identity.

BACKGROUND: Drosophila and mammalian neural progenitors typically generate a diverse family of neurons in a stereotyped order. Neuronal diversity can be generated by the sequential expression of temporal transcription factors. In Drosophila, neural progenitors (neuroblasts) sequentially express the temporal transcription factors Hunchback (Hb), Kruppel, Pdm, and Castor. Hb is necessary and sufficient to specify early-born neuronal identity in multiple lineages, and is maintained in the post-mitotic neurons produced during each neuroblast expression window. Surprisingly, nothing is currently known about whether Hb acts in neuroblasts or post-mitotic neurons (or both) to specify first-born neuronal identity. METHODS: Here we selectively remove Hb from post-mitotic neurons, and assay the well-characterized NB7-1 and NB1-1 lineages for defects in neuronal identity and function. RESULTS: We find that loss of Hb from embryonic and larval post-mitotic neurons does not affect neuronal identity. Furthermore, removing Hb from post-mitotic neurons throughout the entire CNS has no effect on larval locomotor velocity, a sensitive assay for motor neuron and pre-motor neuron function. CONCLUSIONS: We conclude that Hb functions in progenitors (neuroblasts/GMCs) to establish heritable neuronal identity that is maintained by a Hb-independent mechanism. We suggest that Hb acts in neuroblasts to establish an epigenetic state that is permanently maintained in early-born neurons.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex, including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs.

Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines-chosen for sparse neuronal expression-to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program.

Even-Skipped(+) Interneurons Are Core Components of a Sensorimotor Circuit that Maintains Left-Right Symmetric Muscle Contraction Amplitude.

Bilaterally symmetric motor patterns--those in which left-right pairs of muscles contract synchronously and with equal amplitude (such as breathing, smiling, whisking, and locomotion)--are widespread throughout the animal kingdom. Yet, surprisingly little is known about the underlying neural circuits. We performed a thermogenetic screen to identify neurons required for bilaterally symmetric locomotion in Drosophila larvae and identified the evolutionarily conserved Even-skipped(+) interneurons (Eve/Evx). Activation or ablation of Eve(+) interneurons disrupted bilaterally symmetric muscle contraction amplitude, without affecting the timing of motor output. Eve(+) interneurons are not rhythmically active and thus function independently of the locomotor CPG. GCaMP6 calcium imaging of Eve(+) interneurons in freely moving larvae showed left-right asymmetric activation that correlated with larval behavior. TEM reconstruction of Eve(+) interneuron inputs and outputs showed that the Eve(+) interneurons are at the core of a sensorimotor circuit capable of detecting and modifying body wall muscle contraction.

Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience.

A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.

A resource for manipulating gene expression and analyzing cis-regulatory modules in the Drosophila CNS.

Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data sets are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizing interneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.

Characterization of Drosophila larval crawling at the level of organism, segment, and somatic body wall musculature.

Understanding rhythmic behavior at the developmental and genetic levels has important implications for neurobiology, medicine, evolution, and robotics. We studied rhythmic behavior--larval crawling--in the genetically and developmentally tractable organism, Drosophila melanogaster. We used narrow-diameter channels to constrain behavior to simple, rhythmic crawling. We quantified crawling at the organism, segment, and muscle levels. We showed that Drosophila larval crawling is made up of a series of periodic strides. Each stride consists of two phases. First, while most abdominal segments remain planted on the substrate, the head, tail, and gut translocate; this "visceral pistoning" moves the center of mass. The movement of the center of mass is likely powered by muscle contractions in the head and tail. Second, the head and tail anchor while a body wall wave moves each abdominal segment in the direction of the crawl. These two phases can be observed occurring independently in embryonic stages before becoming coordinated at hatching. During forward crawls, abdominal body wall movements are powered by simultaneous contraction of dorsal and ventral muscle groups, which occur concurrently with contraction of lateral muscles of the adjacent posterior segment. During reverse crawls, abdominal body wall movements are powered by phase-shifted contractions of dorsal and ventral muscles; and ventral muscle contractions occur concurrently with contraction of lateral muscles in the adjacent anterior segment. This work lays a foundation for use of Drosophila larva as a model system for studying the genetics and development of rhythmic behavior.

An image-free opto-mechanical system for creating virtual environments and imaging neuronal activity in freely moving Caenorhabditis elegans.

Non-invasive recording in untethered animals is arguably the ultimate step in the analysis of neuronal function, but such recordings remain elusive. To address this problem, we devised a system that tracks neuron-sized fluorescent targets in real time. The system can be used to create virtual environments by optogenetic activation of sensory neurons, or to image activity in identified neurons at high magnification. By recording activity in neurons of freely moving C. elegans, we tested the long-standing hypothesis that forward and reverse locomotion are generated by distinct neuronal circuits. Surprisingly, we found motor neurons that are active during both types of locomotion, suggesting a new model of locomotion control in C. elegans. These results emphasize the importance of recording neuronal activity in freely moving animals and significantly expand the potential of imaging techniques by providing a mean to stabilize fluorescent targets.