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.

Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons.

Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.

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.

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.

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.

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.

A Genetic Toolkit for Dissecting Dopamine Circuit Function in Drosophila.

The neuromodulator dopamine (DA) plays a key role in motor control, motivated behaviors, and higher-order cognitive processes. Dissecting how these DA neural networks tune the activity of local neural circuits to regulate behavior requires tools for manipulating small groups of DA neurons. To address this need, we assembled a genetic toolkit that allows for an exquisite level of control over the DA neural network in Drosophila. To further refine targeting of specific DA neurons, we also created reagents that allow for the conversion of any existing GAL4 line into Split GAL4 or GAL80 lines. We demonstrated how this toolkit can be used with recently developed computational methods to rapidly generate additional reagents for manipulating small subsets or individual DA neurons. Finally, we used the toolkit to reveal a dynamic interaction between a small subset of DA neurons and rearing conditions in a social space behavioral assay.

Functional dissection of a neuronal network required for cuticle tanning and wing expansion in Drosophila.

A subset of Drosophila neurons that expresses crustacean cardioactive peptide (CCAP) has been shown previously to make the hormone bursicon, which is required for cuticle tanning and wing expansion after eclosion. Here we present evidence that CCAP-expressing neurons (NCCAP) consist of two functionally distinct groups, one of which releases bursicon into the hemolymph and the other of which regulates its release. The first group, which we call NCCAP-c929, includes 14 bursicon-expressing neurons of the abdominal ganglion that lie within the expression pattern of the enhancer-trap line c929-Gal4. We show that suppression of activity within this group blocks bursicon release into the hemolymph together with tanning and wing expansion. The second group, which we call NCCAP-R, consists of NCCAP neurons outside the c929-Gal4 pattern. Because suppression of synaptic transmission and protein kinase A (PKA) activity throughout NCCAP, but not in NCCAP-c929, also blocks tanning and wing expansion, we conclude that neurotransmission and PKA are required in NCCAP-R to regulate bursicon secretion from NCCAP-c929. Enhancement of electrical activity in NCCAP-R by expression of the bacterial sodium channel NaChBac also blocks tanning and wing expansion and leads to depletion of bursicon from central processes. NaChBac expression in NCCAP-c929 is without effect, suggesting that the abdominal bursicon-secreting neurons are likely to be silent until stimulated to release the hormone. Our results suggest that NCCAP form an interacting neuronal network responsible for the regulation and release of bursicon and suggest a model in which PKA-mediated stimulation of inputs to normally quiescent bursicon-expressing neurons activates release of the hormone.

Facilitating Neuron-Specific Genetic Manipulations in Drosophila melanogaster Using a Split GAL4 Repressor.

Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter's ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely-used technique for refined cell type-specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS (Upstream Activating Sequence) system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the "Killer Zipper" (KZip+), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip+ acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip+ technology, we have developed a versatile set of LexAop-KZip+ fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. KZip+ significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used.

Probable involvement of p11 with interferon alpha induced depression.

Depression is one of the major side effects of interferon alpha (IFN-α) treatment, but the molecular mechanism underlying IFN-α-induced depression remains unclear. Several studies have shown that the serotonin receptors 5-HTR1b and 5-HTR4 play key roles in the anti-depression effects associated with p11 (S100A10). We investigated the effects of IFN-α on the regulation of p11, 5-HTR1b and 5-HTR4 in mice and human neuroblastoma cells (SH-sy5y). We found that intraperitoneal injection with IFN-α in Balb/c mice resulted in an increased immobility in FST and TST, and potently lowered the protein levels of p11, 5-HTR1b and 5-HTR4 in the hippocampus or cingulate gyrus. IFN-α significantly down-regulated the protein levels of p11, 5-HTR1b and 5-HTR4 in SH-sy5y cells, in a time- and dose-dependent manner. Our study revealed that over-expression of p11 could prevent the IFN-α-induced down-regulation of 5-HTR1b and 5-HTR4. The results indicated that IFN-α treatment resulted in p11 down-regulation, which subsequently decreased 5-HTR1b and 5-HTR4 in vitro or in vivo. Our findings suggested that p11 might be a potential regulator on 5-HTR1b and 5-HTR4 as well as a predictor of or a therapeutic target for IFN-α-induced depression.

Plug-and-play genetic access to drosophila cell types using exchangeable exon cassettes.

Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of "coding introns" (i.e., introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted "plug-and-play" cassettes (called "Trojan exons") that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette) transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system.

Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts.

The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.

The neural substrate of spectral preference in Drosophila.

Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here, we identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.