GAL4 Driver Research Articles

Distinguishing PEX gene variant severity for mild, severe, and atypical peroxisome biogenesis disorders in Drosophila.

Peroxisomal biogenesis disorders (PBD) are autosomal recessive disorders caused by loss-of-function mutations of one of the PEX genes responsible for peroxisomal formation. Impaired peroxisome assembly causes severe multisystemic failure with patient phenotypes ranging from epilepsy, liver disease, feeding issues, biochemical abnormalities, and neurodegeneration. Variants in the same PEX gene can produce wide differences in severity, ranging from individuals with death in the first year of life to adults with milder complications. To study this strong genotype-phenotype correlation, we selected specific human PEX gene mutations and utilized Drosophila as a model organism. We generated flies replacing the coding sequence of our Pex gene of interest with a KozakGAL4 (KZ) promoter trap sequence. These cassettes simultaneously knock-out of the Pex gene and knock-in a GAL4 driver, ideal for making "humanized" flies in which the human PEX gene can replace the fly loss. We assessed Pex2 KZ and Pex16 KZ lines in lifespan, bang sensitivity, and climbing assays and confirmed that these are strong loss-of-function alleles. In parallel, we generated human reference and variant UAS-cDNA lines of PEX2 and PEX16 variants in Drosophila . We observed nearly complete phenotypic rescue of Drosophila Pex2 and Pex16 loss when human PEX2 Ref or PEX16 Ref , respectively, were expressed. We also provide evidence for an allele severity spectrum in PEX2 and PEX16 in which some missense alleles, such as PEX2 C247R , are equally severe as early truncations, such as PEX2 R119* . We also observed that alleles associated with mild PBD, such as PEX2 E55K , show variability depending on the assay but do not fully rescue. Finally, alleles associated with atypical ataxia phenotypes, such as PEX16 F332Del , can perform as well as PEX16 Ref , depending on the assay. Altogether, these Drosophila lines effectively model the range of severity of peroxisomal biogenesis disorders.

Cell type-specific driver lines targeting the Drosophila central complex and their use to investigate neuropeptide expression and sleep regulation.

The central complex (CX) plays a key role in many higher-order functions of the insect brain including navigation and activity regulation. Genetic tools for manipulating individual cell types, and knowledge of what neurotransmitters and neuromodulators they express, will be required to gain mechanistic understanding of how these functions are implemented. We generated and characterized split-GAL4 driver lines that express in individual or small subsets of about half of CX cell types. We surveyed neuropeptide and neuropeptide receptor expression in the central brain using fluorescent in situ hybridization. About half of the neuropeptides we examined were expressed in only a few cells, while the rest were expressed in dozens to hundreds of cells. Neuropeptide receptors were expressed more broadly and at lower levels. Using our GAL4 drivers to mark individual cell types, we found that 51 of the 85 CX cell types we examined expressed at least one neuropeptide and 21 expressed multiple neuropeptides. Surprisingly, all co-expressed a small neurotransmitter. Finally, we used our driver lines to identify CX cell types whose activation affects sleep, and identified other central brain cell types that link the circadian clock to the CX. The well-characterized genetic tools and information on neuropeptide and neurotransmitter expression we provide should enhance studies of the CX.

Imaging Neural Activity in Intact, Semirestrained Drosophila Larvae.

The Drosophila larval nerve cord, which is the equivalent of the vertebrate spinal cord, houses the circuits required to process somatosensory stimuli (e.g., tactile, temperature, vibration, and self-movement) and generate the patterned muscle contractions underlying movement and behavior. Within this complex structure reside many cell types and cellular processes, making it difficult to experimentally access, when compared to peripheral parts of the nervous system (i.e., primary sensory neuron dendrites, motor neuron axons and synapses, and muscles). Additionally, the neurons in the larval nerve cord have small cell bodies, precluding traditional electrophysiological approaches. As such, the function of neurons in the nerve cord is less well studied than other parts of the nervous system, severely limiting our understanding of how larvae process sensory information and generate movement. Ca2+-sensitive fluorescent proteins enable the study of neuronal activity in live, genetically tractable animals, even those with small neuronal cell bodies. In addition, live imaging of neurons within the nerve cord in whole, intact animals is possible because larvae are translucent, and the use of intact animals allows for the peripheral sensory neuron circuits to remain intact. Ca2+-sensitive fluorescent proteins increase their fluorescence when voltage-gated Ca2+ channels are opened in depolarized neurons. Here, we describe an assay where a Ca2+-sensitive fluorescent protein (GCaMP6m) is expressed under the control of a GAL4 driver in a subset of neurons that reside in a circuit for vibration sensation. External vibration (sound) stimulates sensory neurons that activate the cells expressing the Ca2+-sensitive fluorescent protein. Visualization of the calcium-induced fluorescent signal with microscopy allows for quantification of neuronal activity.

Cell-type-specific Labeling of Endogenous Proteins Using the Split GFP System in Drosophila.

i. Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo . A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP 1-10/11 ) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP 1-10/11 system in fruit flies. This approach depends on the reconstitution of the GFP 1-10 and GFP 11 fragments, creating a fluorescence signal. We insert the GFP 11 fragment into a specific genomic locus while expressing its counterpart, GFP 1-10 , through an available Gal4 driver line. The unique aspect of this system is that neither GFP 1-10 nor GFP 11 alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP 11 tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m ( Ten-m ) as a model, highlighting the generation and validation of GFP 11 protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

Pyriproxyfen enhances germline stem cell proliferation and reduces reproduction in Drosophila by up-regulating juvenile hormone signaling.

Pyriproxyfen is an insect growth regulator (IGR) that is effective against various types of insect pests. However, the molecular mechanism underlying pyriproxyfen effects on insect reproduction remains unclear. Thus, in this study, we attempted to uncover the mechanisms underlying the impact of pyriproxyfen on the reproductive system of the model organism Drosophila melanogaster. A significant decrease in Drosophila reproduction was observed after pyriproxyfen treatment. The juvenile hormone (JH) titer was significantly increased (120.4%) in the ovary samples of pyriproxyfen-treated flies. Likewise, the concentrations of key enzymes and the expression of key genes related to the JH signaling pathway were also increased in the pyriproxyfen-treated group compared with the control group. Furthermore, pyriproxyfen treatment significantly increased (15.6%) the number of germline stem cells (GSCs) and significantly decreased (17%) the number of cystoblasts (CBs). However, no significant differences were observed in the number of somatic cells. We performed RNA interference (RNAi) on five key genes (Met, Tai, gce, ftz-f1, and hairy) related to the JH signaling pathway in germ cells using the germ cell-specific Gal4 driver. Interestingly, RNAi of the selected genes significantly decreased the number of both GSCs and CBs in pyriproxyfen-treated transgenic flies. These results further validate that pyriproxyfen enhances GSC proliferation by up-regulating JH signaling. Our results indicate that pyriproxyfen significantly decreases reproduction by affecting germ cells in female adult ovaries. The effect of pyriproxyfen on germ cell proliferation and differentiation is mediated by an increase in JH signaling. This study has significant implications for optimizing pest control strategies, developing sustainable agriculture practices, and understanding the mechanism of insecticide action. © 2024 Society of Chemical Industry.

The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster

Intestinal stem cells (ISCs) of the fruit fly, Drosophila melanogaster, offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the escargot (esg)-GAL4 driver, expressing the yeast transcription factor gene, GAL4, under control of the esg gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of genes of interest in proximity to a GAL4-binding element, Upstream Activating Sequence, in ISCs and progenitor enteroblasts (EBs). While esg-GAL4 has been considered an ISC/EB-specific driver, recent studies have shown that esg-GAL4 is also active in other tissues, such as neurons and ovaries. Therefore, the ISC/EB specificity of esg-GAL4 is questionable. In this study, we reveal esg-GAL4 expression in the corpus allatum (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene, RasV12, esg-GAL4 induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. Our data show that esg-GAL4-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a “specific” ISC/EB driver to examine ISC/EB-mediated animal physiology.

The NFκB Dif is required for behavioral and molecular correlates of sleep homeostasis in Drosophila.

The nuclear factor binding the κ light chain in B-cells (NFκB) is involved in a wide range of cellular processes including development, growth, innate immunity, and sleep. However, genetic studies of the role of specific NFκB transcription factors in sleep have been limited. Drosophila fruit flies carry three genes encoding NFκB transcription factors, Dorsal, Dorsal Immunity Factor (Dif), and Relish. We previously found that loss of the Relish gene from fat body suppressed daily nighttime sleep, and abolished infection-induced sleep. Here we show that Dif regulates daily sleep and recovery sleep following prolonged wakefulness. Mutants of Dif showed reduced daily sleep and suppressed recovery in response to sleep deprivation. Pan-neuronal knockdown of Dif strongly suppressed daily sleep, indicating that in contrast to Relish, Dif functions from the central nervous system to regulate sleep. Based on the unique expression pattern of a Dif- GAL4 driver, we hypothesized that its effects on sleep were mediated by the pars intercerebralis (PI). While RNAi knock-down of Dif in the PI reduced daily sleep, it had no effect on the recovery response to sleep deprivation. However, recovery sleep was suppressed when RNAi knock-down of Dif was distributed across a wider range of neurons. Induction of the nemuri (nur) antimicrobial peptide by sleep deprivation was reduced in Dif mutants and pan-neuronal overexpression of nur also suppressed the Dif mutant phenotype by significantly increasing sleep and reducing nighttime arousability. Together, these findings indicate that Dif functions from brain to target nemuri and to promote deep sleep.

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.

Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

A drug stabilizable GAL80ds for conditional control of gene expression via GAL4-UAS and CRISPR-Cas9 systems in Drosophila

The binary GAL4-UAS expression system has been widely used in Drosophila to achieve tissue-specific expression of genes. To further allow for simultaneous spatial and conditional control of gene expression in existing GAL4 expression lines backgrounds, temperature and chemical controllable GAL80 variants have been engineered. Here we add a new drug stabilizable GAL80ds variant, by fusing it to a low-background DHFR-22-DD. We first quantify both single (DD-GAL80) and double (DD-GAL80-DD) architectures and show varied background and activation levels. Next, we demonstrate the utility of GAL80dsDrosophila line to regulate a cell death gene ectopically, in a drug-dependent manner, by utilizing an existing tissue-specific GAL4 driver that regulates the expression of a cell death gene under a UAS. Finally, we showcase the usefulness of GAL80ds in tight drug-mediated regulation of a target gene, from an endogenous locus, by utilizing an existing tissue-specific GAL4 to drive the expression of a dead Cas9 variant fused to the transcriptional coactivator nejire, under a UAS and in gRNA lines. Overall, these new GAL80ds lines expand the use of the wide variety of existing tissue-specific GAL4 and gene-specific gRNA lines. This enables conditional control of genes, both ectopically and endogenously, for a broad array of gene expression control applications.

New genetic tools for mushroom body output neurons in Drosophila

How memories of past events influence behavior is a key question in neuroscience. The major associative learning center in Drosophila, the mushroom body (MB), communicates to the rest of the brain through mushroom body output neurons (MBONs). While 21 MBON cell types have their dendrites confined to small compartments of the MB lobes, analysis of EM connectomes revealed the presence of an additional 14 MBON cell types that are atypical in having dendritic input both within the MB lobes and in adjacent brain regions. Genetic reagents for manipulating atypical MBONs and experimental data on their functions have been lacking. In this report we describe new cell-type-specific GAL4 drivers for many MBONs, including the majority of atypical MBONs that extend the collection of MBON driver lines we have previously generated (Aso et al., 2014a; Aso et al., 2016; Aso et al., <named-content content-type="page-number">20</named-content>19). Using these genetic reagents, we conducted optogenetic activation screening to examine their ability to drive behaviors and learning. These reagents provide important new tools for the study of complex behaviors in Drosophila.

New genetic tools for mushroom body output neurons in Drosophila.

How memories of past events influence behavior is a key question in neuroscience. The major associative learning center in Drosophila, the mushroom body (MB), communicates to the rest of the brain through mushroom body output neurons (MBONs). While 21 MBON cell types have their dendrites confined to small compartments of the MB lobes, analysis of EM connectomes revealed the presence of an additional 14 MBON cell types that are atypical in having dendritic input both within the MB lobes and in adjacent brain regions. Genetic reagents for manipulating atypical MBONs and experimental data on their functions have been lacking. In this report we describe new cell-type-specific GAL4 drivers for many MBONs, including the majority of atypical MBONs that extend the collection of MBON driver lines we have previously generated (Aso et al., 2014a; Aso et al., 2016; Aso et al., <named-content content-type="page-number">20</named-content>19). Using these genetic reagents, we conducted optogenetic activation screening to examine their ability to drive behaviors and learning. These reagents provide important new tools for the study of complex behaviors in Drosophila.

The insc-GAL4 driver marks distinct cell types in Drosophila midgut

Drosophila geneticists frequently employ the binary GAL4-UAS system of conditional gene expression to direct expression of the desired transgene in tissues of interest. The inscuteable -GAL4 driver (insc-GAL4) expresses in the type 1 and type 2 neuroblasts of Drosophila larval brain, a frequent target tissue in many investigations. This GAL4 line additionally displayed its expression in the midgut. In this study, we examined the expression of the UAS-mCD8GFP reporter under the command of the insc-GAL4 driver and observed that this driver expresses exclusively to intestinal stem cells (ISCs) of the Drosophila adult midgut as well as adult midgut precursors (AMPs) of the larval midgut besides its expression in larval brain. Additionally, using the G-TRACE method, it was observed that AMPs in the larval midgut consistently expressed insc-GAL4 in real-time, and the lineage expression of this GAL4 was observed in the enterocyte cells. This study reveals for the first time that insc-GAL4 is specific to larval AMPs and adult ISCs of the midgut.

Peripheral Neuropathy and Decreased Locomotion of a RAB40B Mutation in Human and Model Animals.

Rab40 proteins are an atypical subgroup of Rab GTPases containing a unique suppressor of the cytokine signaling (SOCS) domain that is recruited to assemble the CRL5 E3 ligase complex for proteolytic regulation in various biological processes. A nonsense mutation deleting the C-terminal SOCS box in the RAB40B gene was identified in a family with axonal peripheral neuropathy (Charcot-Marie-Tooth disease type 2), and pathogenicity of the mutation was assessed in model organisms of zebrafish and Drosophila. Compared to control fish, zebrafish larvae transformed by the human mutant hRAB40B-Y83X showed a defective swimming pattern of stalling with restricted localization and slower motility. We were consistently able to observe reduced labeling of synaptic markers along neuromuscular junctions of the transformed larvae. In addition to the neurodevelopmental phenotypes, compared to normal hRAB40B expression, we further examined ectopic expression of hRAB40B-Y83X in Drosophila to show a progressive decline of locomotion ability. Decreased ability of locomotion by ubiquitous expression of the human mutation was reproduced not with GAL4 drivers for neuron-specific expression but only when a pan-glial GAL4 driver was applied. Using the ectopic expression model of Drosophila, we identified a genetic interaction in which Cul5 down regulation exacerbated the defective motor performance, showing a consistent loss of SOCS box of the pathogenic RAB40B. Taken together, we could assess the possible gain-of-function of the human RAB40B mutation by comparing behavioral phenotypes in animal models; our results suggest that the mutant phenotypes may be associated with CRL5-mediated proteolytic regulation.

Dissecting neuron-specific functions of circadian genes using modified cell-specific CRISPR approaches

Circadian behavioral rhythms in Drosophila melanogaster are regulated by about 75 pairs of brain neurons. They all express the core clock genes but have distinct functions and gene expression profiles. To understand the importance of these distinct molecular programs, neuron-specific gene manipulations are essential. Although RNAi based methods are standard to manipulate gene expression in a cell-specific manner, they are often ineffective, especially in assays involving smaller numbers of neurons or weaker Gal4 drivers. We and others recently exploited a neuron-specific CRISPR-based method to mutagenize genes within circadian neurons. Here, we further explore this approach to mutagenize three well-studied clock genes: the transcription factor gene vrille, the photoreceptor gene Cryptochrome (cry), and the neuropeptide gene Pdf (pigment dispersing factor). The CRISPR-based strategy not only reproduced their known phenotypes but also assigned cry function for different light-mediated phenotypes to discrete, different subsets of clock neurons. We further tested two recently published methods for temporal regulation in adult neurons, inducible Cas9 and the auxin-inducible gene expression system. The results were not identical, but both approaches successfully showed that the adult-specific knockout of the neuropeptide Pdf reproduces the canonical loss-of-function mutant phenotypes. In summary, a CRISPR-based strategy is a highly effective, reliable, and general method to temporally manipulate gene function in specific adult neurons.

Insights into the mechanism of histamine synthesis and recycling disruption induced by exposure to CdO NPs in the fruit fly (Drosophila melanogaster).

Exposure to a sublethal concentration of CdO nanoparticles impairs the vision of the fruit fly (Drosophila melanogaster) by disrupting histamine (HA) synthesis and recycling mechanisms. To gain more insights, we measured HA titer using HPLC in CdO NP-treated vs. non-treated adults in the current study and found that CdO NPs caused an increase in the level of HA in the head and the decapitated body. We asked whether HA accumulation (increase) is a response of photoreceptors or CNS histaminergic neurons, and whether there is any difference in the expression levels of HA recycling and transport encoding genes (Lovit, CarT, Ebony, Tan, BalaT) between the adult fly head and decapitated body that could explain this HA accumulation. We used GAL4/UAS system tool with three GAL4 drivers: ubiquitous tubP-GAL4, nervous system driver (elav Gal4), and compound eye drivers (sev Gal4 and GMR Gal4) to silence HA synthesis in site specific manner followed by detecting the expression level of genes involved in HA recycling and transport in both the heads and the decapitated bodies of CdO treated and non-treated flies. We found an increase in Lovit expression in the heads of treated adults, which is responsible for HA loading into synaptic vesicles and release from photoreceptors, as well as a decrease in catalytic enzymes involved in HA recycling, which leads to HA accumulation without increasing the real signal. To conclude, both photoreceptors and CNS histaminergic neurons are responsible for the increase in HA in CdO NP-treated flies, but through different mechanisms. Our results provide more insights on the underlying molecular mechanism of vision impairment because of nano-sized cadmium particles exposure.

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" invivo 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.

Downregulation of Plasma Membrane Ca2+ ATPase driven by tyrosine hydroxylase-Gal4 reduces Drosophila lifespan independently of effects in neurons

ABSTRACTIn Drosophila melanogaster, several Gal4 drivers are used to direct gene/RNAi expression to different dopaminergic neuronal clusters. We previously developed a fly model of Parkinson’s disease, in which dopaminergic neurons had elevated cytosolic Ca2+ due to the expression of a Plasma Membrane Ca2+ ATPase (PMCA) RNAi under the thyroxine hydroxylase (TH)-Gal4 driver. Surprisingly, TH-Gal4>PMCARNAi flies died earlier compared to controls and showed swelling in the abdominal area. Flies expressing the PMCARNAi under other TH drivers also showed such swelling and shorter lifespan. Considering that TH-Gal4 is also expressed in the gut, we proposed to suppress the expression specifically in the nervous system, while maintaining the activation in the gut. Therefore, we expressed Gal80 under the direction of the panneuronal synaptobrevin (nSyb) promoter in the context of TH-Gal4. nSyb-Gal80; TH-Gal4>PMCARNAi flies showed the same reduction of survival as TH-Gal4>PMCARNAi flies, meaning that the phenotype of abdomen swelling and reduced survival could be due to the expression of the PMCARNAi in the gut. In perimortem stages TH-Gal4>PMCARNAi guts had alteration in the proventriculi and crops. The proventriculi appeared to lose cells and collapse on itself, and the crop increased its size several times with the appearance of cellular accumulations at its entrance. No altered expression or phenotype was observed in flies expressing PMCARNAi in the dopaminergic PAM cluster (PAM-Gal4>PMCARNAi). In this work we show the importance of checking the global expression of each promoter and the relevance of the inhibition of PMCA expression in the gut.

A searchable image resource of Drosophila GAL4 driver expression patterns with single neuron resolution.

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

Cell-Surface Targeting of Fluorophores in Drosophila for Rapid Neuroanatomy Visualization.

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. Front. Neurosci. 2021, 15, 754027) and SNAPf-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines, which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAPf-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.