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.
Display omitted
•Plug-and-play reagents for gene- and cell-type-specific transgene expression in vivo•Binary and intersectional targeting is enabled for multiple transcriptional factors•Trojan exons couple to “off-the-shelf” Drosophila stocks bearing intronic MiMIC transposons•T-GEM Trojan exon can be used with the Crispr/Cas system to target introns in arbitrary genes of interest
Genetic manipulation and monitoring of cellular function in living animals requires tools for targeting transgene expression to specific cell types. Diao et al. present a “Trojan exon” approach that removes a common bottleneck in transgene targeting by facilitating the rapid production of transgenic organisms that can express transcriptional effectors in targeted cell types. The method uses off-the-shelf components in Drosophila and is modular, simple, fast, and precise.
In Drosophila, the Gal4-UAS system permits a transgene to be expressed in the same pattern as a gene of interest by placing the Gal4 transcription factor under control of the gene's DNA regulatory ...elements. If these regulatory elements are not known, however, expression of Gal4 in the desired pattern may be difficult or impossible. To solve this problem, we have developed a method for co-expressing Gal4 with the endogenous gene by exploiting the "ribosomal skipping" mechanism of the viral T2A peptide. This method requires explicit knowledge only of the endogenous gene's open reading frame and not its regulatory elements.
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.
Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream.
Here we report a novel ...function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts—beyond its known roles in development—as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2, the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP.
We therefore identify a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells.
Display omitted
•Enteroendocrine cells control the local stem cell niche via Bursicon•Enteroendocrine cells express the Bursicon ligand, and the muscle its receptor, DLGR2•Bursicon/DLGR2 signaling increases cAMP in visceral muscle to control EGF-like Vein•Bursicon signaling in the adult midgut instructs intestinal stem cell quiescence
Scopelliti et al. show that in the adult Drosophila midgut, enteroendocrine cells act locally—using Bursicon/DLGR2 signaling—to restrain stem cell proliferation via paracrine signaling to the mesenchymal stem cell niche.
Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila's preference for UV over visible light requires UV-sensing R7 ...photoreceptors and specific wide-field amacrine neurons called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8 and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8's dendritic field but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s→Dm8→Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ∼16 R7 signals for transfer to the lobula, a higher visual center.
Reproduction induces increased food intake across females of many animal species
, 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.
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
.
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
, 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.
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.
To grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Trigger Hormone (ETH) and has been extensively studied as a ...model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone's neuronal targets. An alternatively spliced gene encoding a G-protein-coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. Here we use the recently introduced "Trojan exon" technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. We show that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA- but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. We also identify several functionally important subsets of ETHR-expressing neurons including one that coexpresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy presented here of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks.
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.
Display omitted
•More than 40 transgenic lines allow for restricted expression in dopamine (DA) neurons•TH-based FLP recombinase permits isolation of DA neurons from any GAL4 driver•microRNA transgenic lines enable selective manipulation of DA signaling•HACK reagents can be used to replace any GAL4 with GAL80 or Split GAL4
The rapid analysis of how dopaminergic circuits regulate behavior is limited by the genetic tools available to target and manipulate small numbers of these neurons. Xie et al. present genetic tools in Drosophila that allow rational targeting of sparse dopaminergic neuronal subsets and selective knockdown of dopamine signaling.
PDF
