Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Neural networks are typically defined by their synaptic connectivity, yet synaptic wiring diagrams often provide limited insight into network function. This is due partly to the importance of non-synaptic communication by neuromodulators, which can dynamically reconfigure circuit activity to alter its output. Here, we systematically map the patterns of neuromodulatory connectivity in a network that governs a developmentally critical behavioral sequence in Drosophila. This sequence, which mediates pupal ecdysis, is governed by the serial release of several key factors, which act both somatically as hormones and within the brain as neuromodulators. By identifying and characterizing the functions of the neuronal targets of these factors, we find that they define hierarchically organized layers of the network controlling the pupal ecdysis sequence: a modular input layer, an intermediate central pattern generating layer, and a motor output layer. Mapping neuromodulatory connections in this system thus defines the functional architecture of the network. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copulation is the goal of the courtship process, crucial to reproductive success and evolutionary fitness. Identifying the circuitry underlying copulation is a necessary step towards understanding universal principles of circuit operation, and how circuit elements are recruited into the production of ordered action sequences. Here, we identify key sex-specific neurons that mediate copulation in Drosophila, and define a sexually dimorphic motor circuit in the male abdominal ganglion that mediates the action sequence of initiating and terminating copulation. This sexually dimorphic circuit composed of three neuronal classes - motor neurons, interneurons and mechanosensory neurons - controls the mechanics of copulation. By correlating the connectivity, function and activity of these neurons we have determined the logic for how this circuitry is coordinated to generate this male-specific behavior, and sets the stage for a circuit-level dissection of active sensing and modulation of copulatory behavior. [ABSTRACT FROM AUTHOR]

Summary: 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. [Copyright &y& Elsevier]