Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Drosophila melanogaster Intestine.

The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type. We first identified 424 split-GAL4 drivers with midgut expression from ∼7300 drivers screened, and then evaluated the expression patterns of each of these 424 when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. We also evaluated a subset of the drivers expressed in progenitor cells for expression in enteroblasts using another reference driver. We show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, we show that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.

Identification and Characterization of Breakpoints and Mutations on Drosophila melanogaster Balancer Chromosomes.

Balancers are rearranged chromosomes used in Drosophila melanogaster to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in p53 causes loss of radiation-induced apoptosis and a breakpoint in Fucosyltransferase A causes loss of fucosylation in nervous and intestinal tissue-the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation. We also describe thousands of potentially harmful mutations shared among X or third chromosome balancers, or unique to specific balancers, including an Ankyrin2 mutation present on most TM3 balancers, and reiterate the risks of using balancers as experimental controls. We used long-read sequencing to confirm or refine the positions of two inversions with breakpoints lying in repetitive sequences and provide evidence that one of the inversions, In(2L)Cy, arose by ectopic recombination between foldback transposon insertions and the other, In(3R)C, cleanly separates subtelomeric and telomeric sequences and moves the subtelomeric sequences to an internal chromosome position. In addition, our characterization of In(3R)C shows that balancers may be polymorphic for terminal deletions. Finally, we present evidence that extremely distal mutations on balancers can add to the stability of stocks whose purpose is to maintain homologous chromosomes carrying mutations in distal genes. Overall, these studies add to our understanding of the structure, diversity and effectiveness of balancer chromosomes.

Mapping Second Chromosome Mutations to Defined Genomic Regions in Drosophila melanogaster.

Hundreds of Drosophila melanogaster stocks are currently maintained at the Bloomington Drosophila Stock Center with mutations that have not been associated with sequence-defined genes. They have been preserved because they have interesting loss-of-function phenotypes. The experimental value of these mutations would be increased by tying them to specific genomic intervals so that geneticists can more easily associate them with annotated genes. Here, we report the mapping of 85 second chromosome complementation groups in the Bloomington collection to specific, small clusters of contiguous genes or individual genes in the sequenced genome. This information should prove valuable to Drosophila geneticists interested in processes associated with particular phenotypes and those searching for mutations affecting specific sequence-defined genes.

Discrete Serotonin Systems Mediate Memory Enhancement and Escape Latencies after Unpredicted Aversive Experience in Drosophila Place Memory.

Feedback mechanisms in operant learning are critical for animals to increase reward or reduce punishment. However, not all conditions have a behavior that can readily resolve an event. Animals must then try out different behaviors to better their situation through outcome learning. This form of learning allows for novel solutions and with positive experience can lead to unexpected behavioral routines. Learned helplessness, as a type of outcome learning, manifests in part as increases in escape latency in the face of repeated unpredicted shocks. Little is known about the mechanisms of outcome learning. When fruit fly Drosophilamelanogaster are exposed to unpredicted high temperatures in a place learning paradigm, flies both increase escape latencies and have a higher memory when given control of a place/temperature contingency. Here we describe discrete serotonin neuronal circuits that mediate aversive reinforcement, escape latencies, and memory levels after place learning in the presence and absence of unexpected aversive events. The results show that two features of learned helplessness depend on the same modulatory system as aversive reinforcement. Moreover, changes in aversive reinforcement and escape latency depend on local neural circuit modulation, while memory enhancement requires larger modulation of multiple behavioral control circuits.

Phenotypes Associated with Second Chromosome P Element Insertions in Drosophila melanogaster.

In Drosophila melanogaster, P element transposition has been a productive means of insertional mutagenesis. Thousands of genes have been tagged with natural and engineered P element constructs. Nevertheless, chromosomes carrying P element insertions tend to have high levels of background mutations from P elements inserting and excising during transposition. Consequently, the phenotypes seen when P element-bearing chromosomes are homozygous are often not attributable to the P insertions themselves. In this study, 178 strains in the Bloomington Drosophila Stock Center collection with P insertions on the second chromosome were complementation tested against molecularly defined chromosomal deletions and previously characterized single-gene mutations to determine if recessive lethality or sterility is associated with the P insertions rather than background mutations. This information should prove valuable to geneticists using these strains for experimental studies of gene function.

Place memory retention in Drosophila.

Some memories last longer than others, with some lasting a lifetime. Using several approaches memory phases have been identified. How are these different phases encoded, and do these different phases have similar temporal properties across learning situations? Place memory in Drosophila using the heat-box provides an excellent opportunity to examine the commonalities of genetically-defined memory phases across learning contexts. Here we determine optimal conditions to test place memories that last up to three hours. An aversive temperature of 41°C was identified as critical for establishing a long-lasting place memory. Interestingly, adding an intermittent-training protocol only slightly increased place memory when intermediate aversive temperatures were used, and slightly extended the stability of a memory. Genetic analysis of this memory identified four genes as critical for place memory within minutes of training. The role of the rutabaga type I adenylyl cyclase was confirmed, and the latheo Orc3 origin of recognition complex component, the novel gene encoded by pastrel, and the small GTPase rac were all identified as essential for normal place memory. Examination of the dopamine and ecdysone receptor (DopEcR) did not reveal a function for this gene in place memory. When compared to the role of these genes in other memory types, these results suggest that there are genes that have both common and specific roles in memory formation across learning contexts. Importantly, contrasting the timing for the function of these four genes, plus a previously described role of the radish gene, in place memory with the temporal requirement of these genes in classical olfactory conditioning reveals variability in the timing of genetically-defined memory phases depending on the type of learning.

Visual working memory: now you see it, now you don't.

Flies can form a visually-guided working memory. A new study shows that the gene termed ellipsoid body open influences multiple signals to regulate a competence factor in the ellipsoid body to support normal working memory.

Short neuropeptide F acts as a functional neuromodulator for olfactory memory in Kenyon cells of Drosophila mushroom bodies.

In insects, many complex behaviors, including olfactory memory, are controlled by a paired brain structure, the so-called mushroom bodies (MB). In Drosophila, the development, neuroanatomy, and function of intrinsic neurons of the MB, the Kenyon cells, have been well characterized. Until now, several potential neurotransmitters or neuromodulators of Kenyon cells have been anatomically identified. However, whether these neuroactive substances of the Kenyon cells are functional has not been clarified yet. Here we show that a neuropeptide precursor gene encoding four types of short neuropeptide F (sNPF) is required in the Kenyon cells for appetitive olfactory memory. We found that activation of Kenyon cells by expressing a thermosensitive cation channel (dTrpA1) leads to a decrease in sNPF immunoreactivity in the MB lobes. Targeted expression of RNA interference against the sNPF precursor in Kenyon cells results in a highly significant knockdown of sNPF levels. This knockdown of sNPF in the Kenyon cells impairs sugar-rewarded olfactory memory. This impairment is not due to a defect in the reflexive sugar preference or odor response. Consistently, knockdown of sNPF receptors outside the MB causes deficits in appetitive memory. Altogether, these results suggest that sNPF is a functional neuromodulator released by Kenyon cells.

Learning and memory in Drosophila: behavior, genetics, and neural systems.

The rich behavioral repertoire that Drosophila use to navigate in their natural environment suggests that flies can use memories to inform decisions. Development of paradigms to examine memories that restrict behavioral choice was essential in furthering our understanding of the genetics and neural systems of memory formation in the fly. Olfactory, visual, and place memory paradigms have proven influential in determining principles for the mechanisms of memory formation. Several parts of the nervous system have been shown to be important for different types of memories, including the mushroom bodies and the central complex. Thus far, about 40 genes have been linked to normal olfactory short-term memory. A subset of these genes have also been tested for a role in visual and place memory. Some genes have a common function in memory formation, specificity of action comes from where in the nervous system these genes act. Alternatively, some genes have a more restricted role in different types of memories.

Chemical neuroanatomy of the Drosophila central complex: distribution of multiple neuropeptides in relation to neurotransmitters.

The central complex of the insect brain is an integration center, receiving inputs from many parts of the brain. In Drosophila it has been associated with the control of both locomotor and visually correlated behaviors. The central complex can be divided into several substructures and is comprised of a large number of neuronal types. These neurons produce classical neurotransmitters, biogenic amines, and different neuropeptides. However, the distribution of neurotransmitters and neuromodulators in central-complex circuits of Drosophila is poorly known. By immunolabeling and GAL4-directed expression of marker proteins, we analyzed the distribution of acetylcholine, glutamate, GABA, monoamines, and eight different neuropeptides; Drosophila tachykinin, short neuropeptide F, myoinhibitory peptide, allatostatin A, proctolin, SIFamide, neuropeptide F, and FMRFamide. All eight neuropeptides were localized to the fan-shaped body, the largest substructure of the central complex, and were mapped to different layers within this structure. Several populations of peptide-immunoreactive tangential and columnar neurons were identified, of which some colocalized acetylcholine. Fewer peptides were found to be expressed in the other substructures: the ellipsoid body, the protocerebral bridge, and the noduli. The ellipsoid body and the protocerebral bridge were innervated by extrinsic peptide expressing neurons. Our findings reveal that numerous neuropeptides are expressed in the central complex and that each peptide has a distinct distribution pattern, suggesting important roles for neuropeptides as neuromediators and cotransmitters in this brain area.

Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides.

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.

Neuropeptides in the Drosophila central complex in modulation of locomotor behavior.

The central complex is one of the most prominent neuropils in the insect brain. It has been implicated in the control of locomotor activity and is considered as a pre-motor center. Several neuropeptides are expressed in circuits of the central complex, and thus may be modulators of locomotor behavior. Here we have investigated the roles of two different neuropeptides, Drosophila tachykinin (DTK) and short neuropeptide F (sNPF), in aspects of locomotor behavior. In the Drosophila brain, DTK and sNPF are expressed in interneurons innervating the central complex. We have directed RNA interference (RNAi) towards DTK and sNPF specifically in different central complex neurons. We also expressed a temperature-sensitive dominant negative allele of the fly ortholog of dynamin called shibire(ts1), essential in membrane vesicle recycling and endocytosis, to disrupt synaptic transmission in central complex neurons. The spontaneous walking activity of the RNAi- or shibire(ts1)-expressing flies was quantified by video tracking. DTK-deficient flies displayed drastically increased center zone avoidance, suggesting that DTK is involved in the regulation of spatial orientation. In addition, DTK deficiency in other central complex neurons resulted in flies with an increased number of activity-rest bouts. Perturbations in the sNPF circuit indicated that this peptide is involved in the fine regulation of locomotor activity levels. Our findings suggest that the contribution of DTK and sNPF to locomotor behavior is circuit dependent and associated with particular neuronal substrates. Thus, peptidergic pathways in the central complex have specific roles in the fine tuning of locomotor activity of adult Drosophila.

Insulin signaling, lifespan and stress resistance are modulated by metabotropic GABA receptors on insulin producing cells in the brain of Drosophila.

Insulin-like peptides (ILPs) regulate growth, reproduction, metabolic homeostasis, life span and stress resistance in worms, flies and mammals. A set of insulin producing cells (IPCs) in the Drosophila brain that express three ILPs (DILP2, 3 and 5) have been the main focus of interest in hormonal DILP signaling. Little is, however, known about factors that regulate DILP production and release by these IPCs. Here we show that the IPCs express the metabotropic GABA(B) receptor (GBR), but not the ionotropic GABA(A) receptor subunit RDL. Diminishing the GBR expression on these cells by targeted RNA interference abbreviates life span, decreases metabolic stress resistance and alters carbohydrate and lipid metabolism at stress, but not growth in Drosophila. A direct effect of diminishing GBR on IPCs is an increase in DILP immunofluorescence in these cells, an effect that is accentuated at starvation. Knockdown of irk3, possibly part of a G protein-activated inwardly rectifying K(+) channel that may link to GBRs, phenocopies GBR knockdown in starvation experiments. Our experiments suggest that the GBR is involved in inhibitory control of DILP production and release in adult flies at metabolic stress and that this receptor mediates a GABA signal from brain interneurons that may convey nutritional signals. This is the first demonstration of a neurotransmitter that inhibits insulin signaling in its regulation of metabolism, stress and life span in an invertebrate brain.