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.

Enteric neurons increase maternal food intake during reproduction.

Reproduction induces increased food intake across females of many animal species1-4, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain.

split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80.

The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of "split-intein Gal4" in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type-specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm ("Two Against Background" or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.

What genetic model organisms offer the study of behavior and neural circuits.

The past decade has witnessed the development of powerful, genetically encoded tools for manipulating and monitoring neuronal function in freely moving animals. These tools are most readily deployed in genetic model organisms and efforts to map the circuits that govern behavior have increasingly focused on worms, flies, zebrafish, and mice. The traditional virtues of these animals for genetic studies in terms of small size, short generation times, and ease of animal husbandry in a laboratory setting have facilitated rapid progress, and the neural basis of an increasing number of behaviors is being established at cellular resolution in each of these animals. The depth and breadth of this analysis should soon offer a significantly more comprehensive understanding of how the circuitry underlying behavior is organized in particular animals and promises to help answer long-standing questions that have waited for such a brain-wide perspective on nervous system function. The comprehensive understanding achieved in genetic model animals is thus likely to make them into paradigmatic examples that will serve as touchstones for comparisons to understand how behavior is organized in other animals, including ourselves.

Model Organisms in G Protein-Coupled Receptor Research.

The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism.

Neural and hormonal control of postecdysial behaviors in insects.

The shedding of the old exoskeleton that occurs in insects at the end of a molt (a process called ecdysis) is typically followed by the expansion and tanning of a new one. At the adult molt, these postecdysial processes include expansion and hardening of the wings. Here we describe recent advances in understanding the neural and hormonal control of wing expansion and hardening, focusing on work using Drosophila melanogaster in which genetic manipulations have permitted detailed investigation of postecdysial processes and their modulation by sensory input. To place this work in context, we briefly review recent progress in understanding the neuroendocrine regulation of ecdysis, which appears to be largely conserved across insect species. Investigations into the neuroendocrine networks that regulate ecdysial and postecdysial behaviors provide insights into how stereotyped, yet environmentally responsive, sequences are generated and how they develop and evolve.

Command and compensation in a neuromodulatory decision network.

The neural circuits that mediate behavioral choices must not only weigh internal demands and environmental circumstances, but also select and implement specific actions, including associated visceral or neuroendocrine functions. Coordinating these multiple processes suggests considerable complexity. As a consequence, even circuits that support simple behavioral decisions remain poorly understood. Here we show that the environmentally sensitive wing expansion decision of adult fruit flies is coordinated by a single pair of neuromodulatory neurons with command-like function. Targeted suppression of these neurons using the Split Gal4 system abrogates the fly's ability to expand its wings in the face of environmental challenges, while stimulating them forces expansion by coordinately activating both motor and neuroendocrine outputs. The arbitration and implementation of the wing expansion decision by this neuronal pair may illustrate a general strategy by which neuromodulatory neurons orchestrate behavior. Interestingly, the decision network exhibits a plasticity that is unmasked under conducive environmental conditions in flies lacking the function of the command-like neuromodulatory neurons. Such flies can often expand their wings using a motor program distinct from that of wild-type animals and controls. This compensatory program may be the vestige of an ancestral, environmentally insensitive program used for wing expansion that existed before the evolution of the environmentally adaptive program currently used by Drosophila and other cyclorrhaphan flies.

Eclosion gates progression of the adult ecdysis sequence of Drosophila.

Animal behavior is often organized into stereotyped sequences that promote the goals of reproduction, development and survival. However, for most behaviors, the neural mechanisms that govern the order of execution of the motor programs within a sequence are poorly understood. An important model in understanding the hormonal determinants of behavioral sequencing is the ecdysis sequence, which is performed by insects at each developmental transition, or molt. The adult ecdysis sequence in Drosophila includes the emergence of the insect from the pupal case followed by expansion and hardening of the wings. Wing expansion is governed by the hormone bursicon, and stimulation of the bursicon-expressing neurons in newly eclosed flies induces rapid wing expansion. Here we show that that such stimulation delivered prior to eclosion has no immediate effect, but does cause rapid wing expansion after eclosion if the stimulus is delivered within 40 min of that event. We observe a similar delayed effect upon stimulation of a single pair of bursicon-expressing neurons previously identified as command neurons for wing expansion. We conclude that command neuron stimulation enables the motor output pathway for wing expansion, but that this pathway is blocked prior to eclosion. By manipulating the time of eclosion, we demonstrate that some physiological process tightly coupled to adult ecdysis releases the block on wing expansion. Eclosion thus serves as a behavioral checkpoint and complements hormonal mechanisms to ensure that wing expansion strictly follows eclosion in the ecdysis sequence.

split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80.

The split-Gal4 system allows for intersectional genetic labeling of highly specific cell-types and tissues in Drosophila . However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a new split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is fully repressible by Gal80. We demonstrate the potent inducibility of "split-intein Gal4" in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell-type specific genetic drivers based on in silico predictions generated by single cell RNAseq (scRNAseq) datasets, and we describe a new algorithm ("Two Against Background" or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible. Significance statement: The split-Gal4 system allows Drosophila researchers to drive transgene expression with extraordinary cell type specificity. However, the existing split-Gal4 system cannot be controlled temporally, and therefore cannot be applied to many important areas of research. Here, we present a new split-Gal4 system based on a self-excising split-intein, which is fully controllable by Gal80, as well as a related drug-inducible split GeneSwitch system. This approach can both leverage and inform single-cell RNAseq datasets, and we introduce an algorithm to identify pairs of genes that precisely and narrowly mark a desired cell cluster. Our split-intein Gal4 system will be of value to the Drosophila research community, and allow for the creation of highly specific genetic drivers that are also inducible/repressible.

Sleep in Drosophila is regulated by adult mushroom bodies.

Sleep is one of the few major whole-organ phenomena for which no function and no underlying mechanism have been conclusively demonstrated. Sleep could result from global changes in the brain during wakefulness or it could be regulated by specific loci that recruit the rest of the brain into the electrical and metabolic states characteristic of sleep. Here we address this issue by exploiting the genetic tractability of the fruitfly, Drosophila melanogaster, which exhibits the hallmarks of vertebrate sleep. We show that large changes in sleep are achieved by spatial and temporal enhancement of cyclic-AMP-dependent protein kinase (PKA) activity specifically in the adult mushroom bodies of Drosophila. Other manipulations of the mushroom bodies, such as electrical silencing, increasing excitation or ablation, also alter sleep. These results link sleep regulation to an anatomical locus known to be involved in learning and memory.

Neuromodulation and the toolkit for behavioural evolution: can ecdysis shed light on an old problem?

The geneticist Thomas Dobzhansky famously declared: 'Nothing in biology makes sense except in the light of evolution'. A key evolutionary adaptation of Metazoa is directed movement, which has been elaborated into a spectacularly varied number of behaviours in animal clades. The mechanisms by which animal behaviours have evolved, however, remain unresolved. This is due, in part, to the indirect control of behaviour by the genome, which provides the components for both building and operating the brain circuits that generate behaviour. These brain circuits are adapted to respond flexibly to environmental contingencies and physiological needs and can change as a function of experience. The resulting plasticity of behavioural expression makes it difficult to characterize homologous elements of behaviour and to track their evolution. Here, we evaluate progress in identifying the genetic substrates of behavioural evolution and suggest that examining adaptive changes in neuromodulatory signalling may be a particularly productive focus for future studies. We propose that the behavioural sequences used by ecdysozoans to moult are an attractive model for studying the role of neuromodulation in behavioural evolution.

Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila.

Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution, we here describe a multiphasic behavioral sequence in Drosophila. Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution.

How do we find out how the brain works? One way is to use imaging techniques to visualise an animal's brain in action as it performs simple behaviours: as the animal moves, parts of its brain light up under the microscope. For laboratory animals like fruit flies, which have relatively small brains, this lets us observe their brain activity right down to the level of individual brain cells. The brain directs movements via collective activity of the body's muscles. Our ability to track the activity of individual muscles is, however, more limited than our ability to observe single brain cells: even modern imaging technology still cannot monitor the activity of all the muscle cells in an animal's body as it moves about. Yet this is precisely the information that scientists need to fully understand how the brain generates behaviour. Fruit flies perform specific behaviours at certain stages of their life cycle. When the fly pupa begins to metamorphose into an adult insect, it performs a fixed sequence of movements involving a set number of muscles, which is called the pupal ecdysis sequence. This initial movement sequence and the rest of metamorphosis both occur within the confines of the pupal case, which is a small, hardened shell surrounding the whole animal. Elliott et al. set out to determine if the fruit fly pupa's ecdysis sequence could be used as a kind of model, to describe a simple behaviour at the level of individual muscles. Imaging experiments used fly pupae that were genetically engineered to produce an activity-dependent fluorescent protein in their muscle cells. Pupal cases were treated with a chemical to make them transparent, allowing easy observation of their visually 'labelled' muscles. This yielded a near-complete record of muscle activity during metamorphosis. Initially, individual muscles became active in small groups. The groups then synchronised with each other over the different regions of the pupa's body to form distinct movements, much as syllables join to form words. This synchronisation was key to progression through metamorphosis and was co-ordinated at each step by specialised nerve cells that produce or respond to specific hormones. These results reveal how the brain might direct muscle activity to produce movement patterns. In the future, Elliott et al. hope to compare data on muscle activity with comprehensive records of brain cell activity, to shed new light on how the brain, muscles, and other factors work together to control behaviour.

Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression.

Selective genetic manipulation of neuronal function in vivo requires techniques for targeting gene expression to specific cells. Existing systems accomplish this using the promoters of endogenous genes to drive expression of transgenes directly in cells of interest or, in "binary" systems, to drive expression of a transcription factor or recombinase that subsequently activates the expression of other transgenes. All such techniques are constrained by the limited specificity of the available promoters. We introduce here a combinatorial system in which the DNA-binding (DBD) and transcription-activation (AD) domains of a transcription factor are independently targeted using two different promoters. The domains heterodimerize to become transcriptionally competent and thus drive transgene expression only at the intersection of the expression patterns of the two promoters. We use this system to dissect a neuronal network in Drosophila by selectively targeting expression of the cell death gene reaper to subsets of neurons within the network.

Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel.

After emergence, adult flies and other insects select a suitable perch and expand their wings. Wing expansion is governed by the hormone bursicon and can be delayed under adverse environmental conditions. How environmental factors delay bursicon release and alter perch selection and expansion behaviors has not been investigated in detail. Here we provide evidence that in Drosophila the motor programs underlying perch selection and wing expansion have different environmental dependencies. Using physical manipulations, we demonstrate that the decision to perch is based primarily on environmental valuations and is incrementally delayed under conditions of increasing perturbation and confinement. In contrast, the all-or-none motor patterns underlying wing expansion are relatively invariant in length regardless of environmental conditions. Using a novel technique for targeted activation of neurons, we show that the highly stereotyped wing expansion motor patterns can be initiated by stimulation of N(CCAP), a small network of central neurons that regulates the release of bursicon. Activation of this network using the cold-sensitive rat TRPM8 channel is sufficient to trigger all essential behavioral and somatic processes required for wing expansion. The delay of wing expansion under adverse circumstances thus couples an environmentally sensitive decision network to a command-like network that initiates a fixed action pattern. Because N(CCAP) mediates environmentally insensitive ecdysis-related behaviors in Drosophila development before adult emergence, the study of wing expansion promises insights not only into how networks mediate behavioral choices, but also into how decision networks develop.

The Drosophila Split Gal4 System for Neural Circuit Mapping.

The diversity and dense interconnectivity of cells in the nervous system present a huge challenge to understanding how brains work. Recent progress toward such understanding, however, has been fuelled by the development of techniques for selectively monitoring and manipulating the function of distinct cell types-and even individual neurons-in the brains of living animals. These sophisticated techniques are fundamentally genetic and have found their greatest application in genetic model organisms, such as the fruit fly Drosophila melanogaster. Drosophila combines genetic tractability with a compact, but cell-type rich, nervous system and has been the incubator for a variety of methods of neuronal targeting. One such method, called Split Gal4, is playing an increasingly important role in mapping neural circuits in the fly. In conjunction with functional perturbations and behavioral screens, Split Gal4 has been used to characterize circuits governing such activities as grooming, aggression, and mating. It has also been leveraged to comprehensively map and functionally characterize cells composing important brain regions, such as the central complex, lateral horn, and the mushroom body-the latter being the insect seat of learning and memory. With connectomics data emerging for both the larval and adult brains of Drosophila, Split Gal4 is also poised to play an important role in characterizing neurons of interest based on their connectivity. We summarize the history and current state of the Split Gal4 method and indicate promising areas for further development or future application.

Cre-assisted fine-mapping of neural circuits using orthogonal split inteins.

Existing genetic methods of neuronal targeting do not routinely achieve the resolution required for mapping brain circuits. New approaches are thus necessary. Here, we introduce a method for refined neuronal targeting that can be applied iteratively. Restriction achieved at the first step can be further refined in a second step, if necessary. The method relies on first isolating neurons within a targeted group (i.e. Gal4 pattern) according to their developmental lineages, and then intersectionally limiting the number of lineages by selecting only those in which two distinct neuroblast enhancers are active. The neuroblast enhancers drive expression of split Cre recombinase fragments. These are fused to non-interacting pairs of split inteins, which ensure reconstitution of active Cre when all fragments are expressed in the same neuroblast. Active Cre renders all neuroblast-derived cells in a lineage permissive for Gal4 activity. We demonstrate how this system can facilitate neural circuit-mapping in Drosophila.

In humans ­ as well as flies and most other animals ­ the brain controls how we move and behave, and regulates heartbeat, breathing and other core processes. To perform these different roles, cells known as neurons form large networks that quickly carry messages around the brain and to other parts of the body. In order to fully understand how the brain works, it is important to first understand how individual neurons connect to each other and operate within these networks. Fruit flies and other animals with small brains are often used as models to study how the brain works. There are several methods currently available that allow researchers to manipulate small groups of fruit fly neurons for study, and in some cases it is even possible to target individual neurons. However, it remains an aspirational goal to be able to target every neuron in the fly brain individually. The Gal4-UAS system is a way of manipulating gene activity widely used to study neurons in fruit flies. The system consists of two parts: a protein that can bind DNA and control the activity of genes (Gal4); and a genetic sequence (the UAS) that tells Gal4 where to bind and therefore which genes to activate. Fruit flies can be genetically engineered so that only specific cells make Gal4. This makes it possible, for example, to limit the activity of a gene under the control of the UAS to a specific set of neurons and therefore to identify or target these neurons. Luan et al. developed a new technique named SpaRCLIn that allows the targeting of a subset of neurons within a group already identified with the Gal4-UAS system. During embryonic development, all neurons originate from a small pool of cells called neuroblasts, and it is possible to target the descendants of particular neuroblasts. SpaRCLIn exploits this strategy to limit the activity of Gal4 to smaller and smaller numbers of neuroblast descendants. In this way, Luan et al. found that SpaRCLIn was routinely capable of limiting patterns of Gal4 activity to one, or a few, neurons at a time. Further experiments used SpaRCLIn to identify two pairs of neurons that trigger a well-known feeding behavior in fruit flies. Luan et al. also developed a SpaRCLIn toolkit that will form the basis of a community resource other researchers can use to study neurons in fruit flies. These findings could also benefit researchers developing similar tools in mice and other animals.

Non-canonical Eclosion Hormone-Expressing Cells Regulate Drosophila Ecdysis.

Eclosion hormone (EH) was originally identified as a brain-derived hormone capable of inducing the behavioral sequences required for molting across insect species. However, its role in this process (called ecdysis) has since been confounded by discrepancies in the effects of genetic and cellular manipulations of EH function in Drosophila. Although knock-out of the Eh gene results in severe ecdysis-associated deficits accompanied by nearly complete larval lethality, ablation of the only neurons known to express EH (i.e. Vm neurons) is only partially lethal and surviving adults emerge, albeit abnormally. Using new tools for sensitively detecting Eh gene expression, we show that EH is more widely expressed than previously thought, both within the nervous system and in somatic tissues, including trachea. Ablating all Eh-expressing cells has effects that closely match those of Eh gene knock-out; developmentally suppressing them severely disrupts eclosion. Our results thus clarify and extend the scope of EH action.

Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death.

Hormones are often responsible for synchronizing somatic physiological changes with changes in behavior. Ecdysis (i.e., the shedding of the exoskeleton) in insects has served as a useful model for elucidating the molecular and cellular mechanisms of this synchronization, and has provided numerous insights into the hormonal coordination of body and behavior. An example in which the mechanisms have remained enigmatic is the neurohormone bursicon, which, after the final molt, coordinates the plasticization and tanning of the initially folded wings with behaviors that drive wing expansion. The somatic effects of the hormone are governed by bursicon that is released into the blood from neurons in the abdominal ganglion (the B(AG)), which die after wing expansion. How bursicon induces the behavioral programs required for wing expansion, however, has remained unknown. Here we show by targeted suppression of excitability that a pair of bursicon-immunoreactive neurons distinct from the B(AG) and located within the subesophageal ganglion in Drosophila (the B(SEG)) is involved in controlling wing expansion behaviors. Unlike the B(AG), the B(SEG) arborize widely in the nervous system, including within the abdominal neuromeres, suggesting that, in addition to governing behavior, they also may modulate the B(AG.) Indeed, we show that animals lacking bursicon receptor function have deficits both in the humoral release of bursicon and in posteclosion apoptosis of the B(AG). Our results reveal novel neuromodulatory functions for bursicon and support the hypothesis that the B(SEG) are essential for orchestrating both the behavioral and somatic processes underlying wing expansion.

Combinatorial methods for refined neuronal gene targeting.

Methods for the selective and reproducible expression of genetically encoded tools in targeted subsets of cells are required to facilitate studies of neuronal development, connectivity, and function in living animals. In the absence of techniques for synthesizing promoters that target defined cell groups, current methods exploit the regulatory elements of endogenous genes to achieve specificity of transgene expression. However, single promoters often have expression patterns too broad to pinpoint the functional roles of specific neurons. In this review, we describe emerging combinatorial techniques that make transgene expression contingent not upon a single promoter, but upon two or more promoters. Although only a few such techniques are currently available, recent developments promise rapid growth in this area in the coming years.