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Microinjection is a widely used technique employed by biologists with applications in transgenesis, cryopreservation, mutagenesis, labeling/dye injection and in-vitro fertilization. However, microinjection is an extremely laborious manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. Here, we present a computer-guided robot that automates the targeted microinjection of Drosophila melanogaster and zebrafish (Danio rerio) embryos, two important model organisms in biological research. The robot uses a series of cameras to image an agar plate containing embryos at multiple magnifications and perspectives. This imaging is combined with machine learning and computer vision algorithms to pinpoint a location on the embryo for targeted microinjection with microscale precision. We demonstrate the utility of this microinjection robot to successfully microinject Drosophila melanogaster and zebrafish embryos. Results obtained indicate that the robotic microinjection approach can significantly increase the throughput of microinjection as compared to manual microinjection while maintaining survival rates comparable to human operators. In the future, this robotic platform can be used to perform high throughput microinjection experiments and can be extended to automatically microinject a host of organisms such as roundworms (Caenorhabditis elegans), mosquito (Culicidae) embryos, sea urchins (Echinoidea) and frog (Xenopus) oocytes.

Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. Here, we have explored the novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, we analyzed transcriptomes of embryos zygotically compromised for either clamp or zelda and found that transcript levels of many zygotically activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, we propose that CLAMP acts in concert with Zelda to regulate early zygotic transcription.

Drosophila olfactory local interneurons (LNs) in the antennal lobe are highly diverse and variable. How and when distinct types of LNs emerge, differentiate, and integrate into the olfactory circuit is unknown. Through systematic developmental analyses, we found that LNs are recruited to the adult olfactory circuit in three groups. Group 1 LNs are residual larval LNs. Group 2 are adult-specific LNs that emerge before cognate sensory and projection neurons establish synaptic specificity, and Group 3 LNs emerge after synaptic specificity is established. Group 1 larval LNs are selectively reintegrated into the adult circuit through pruning and re-extension of processes to distinct regions of the antennal lobe, while others die during metamorphosis. Precise temporal control of this pruning and cell death shapes the global organization of the adult antennal lobe. Our findings provide a road map to understand how LNs develop and contribute to constructing the olfactory circuit., Local interneurons (LNs) in the Drosophila olfactory system are highly diverse. Here, the authors labeled different LN types and described how different LN subtypes are integrated into the developing circuit.

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

The selector gene apterous (ap) plays a key role during the development of the Drosophila melanogaster wing because it governs the establishment of the dorsal-ventral (D-V) compartment boundary. The D-V compartment boundary is known to serve as an important signaling center that is essential for the growth of the wing. The role of Ap and its downstream effectors have been studied extensively. However, very little is known about the transcriptional regulation of ap during wing disc development. In this study, we present a first characterization of an essential wing-specific ap enhancer. First, we defined an 874-bp fragment about 10 kb upstream of the ap transcription start that faithfully recapitulates the expression pattern of ap in the wing imaginal disc. Analysis of deletions in the ap locus covering this element demonstrated that it is essential for proper regulation of ap and formation of the wing. Moreover, we showed that the mutations apblot and apXasta directly affect the integrity of this enhancer, leading to characteristic wing phenotypes. Furthermore, we engineered an in situ rescue system at the endogenous ap gene locus, allowing us to investigate the role of enhancer fragments in their native environment. Using this system, we were able to demonstrate that the essential wing enhancer alone is not sufficient for normal wing development. The in situ rescue system will allow us to characterize the ap regulatory sequences in great detail at the endogenous locus.

The fruit fly Drosophila melanogaster is a popular workhorse model organism that has tremendously contributed to our understanding of the nervous system across eukaryotic multicellular species. Through molecular, developmental, histochemical, anatomical, and physiological experimentation, studies that incorporate fruit flies have had immediate biomedical impact relevant to neurobiology and neuropathology. D. melanogaster is one of the most well-established eukaryotic multicellular model organisms, largely due to its sophisticated and expanding in vivo targeted neurogenetic manipulations. Here, we summarize the current status of techniques for precisely targeted spatiotemporal manipulation of the fly’s nervous system, focused on the most recent developments within the field.

Summary Background: Chemotaxis, the ability to direct movements according to chemical cues in the environment, is important for the survival of most organisms. The vinegar fly, Drosophila melanogaster, displays robust olfactory aversion and attraction, but how these behaviors are executed via changes in locomotion remains poorly understood. In particular, it is not clear whether aversion and attraction bidirectionally modulate a shared circuit or recruit distinct circuits for execution. Results:Using a quantitative behavioral assay, we determined that both aversive and attractive odorants modulate the initiation and direction of turns but display distinct kinematics. Using genetic tools to perturb these behaviors, we identified specific populations of neurons required for aversion, but not for attraction. Inactivation of these populations of cells affected the completion of aversive turns, but not their initiation. Optogenetic activation of the same populations of cells triggered a locomotion pattern resembling aversive turns. Perturbations in both the ellipsoid body and the ventral nerve cord, two regions involved in motor control, resulted in defects in aversion. Conclusions: Aversive chemotaxis in vinegar flies triggers ethologically appropriate kinematics distinct from those of attractive chemotaxis and requires specific motor-related neurons.

Rhythmic motor behaviors such as feeding are driven by neural networks that can be modulated by external stimuli and internal states. In Drosophila , ingestion is accomplished by a pump that draws fluid into the esophagus. Here we examine how pumping is regulated and characterize motor neurons innervating the pump. Frequency of pumping is not affected by sucrose concentration or hunger but is altered by fluid viscosity. Inactivating motor neurons disrupts pumping and ingestion, whereas activating them elicits arrhythmic pumping. These motor neurons respond to taste stimuli and show prolonged activity to palatable substances. This work describes an important component of the neural circuit for feeding in Drosophila and is a step toward understanding the rhythmic activity producing ingestion.

Boundary elements or insulators subdivide eukaryotic chromosomes into a series of structurally and functionally autonomous domains. They ensure that the action of enhancers and silencers is restricted to the domain in which these regulatory elements reside. Three models, the roadblock, sink/decoy, and topological loop, have been proposed to explain the insulating activity of boundary elements. Strong predictions about how boundaries will function in different experimental contexts can be drawn from these models. In the studies reported here, we have designed assays that test these predictions. The results of our assays are inconsistent with the expectations of the roadblock and sink models. Instead, they support the topological loop model.

The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. In this study, we analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, we genetically and molecularly analyzed the trans-factors that regulate these CRMs. Our results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs., MINECO to CE (No. BFU2012-34353) and grants from the Kantons Basel-Stadt and Basel-Land, and the Swiss National Science Foundation to MA.

In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like.

Visual motion cues provide animals with critical information about their environment and guide a diverse array of behaviors. The neural circuits that carry out motion estimation provide a well-constrained model system for studying the logic of neural computation. Through a confluence of behavioral, physiological, and anatomical experiments, taking advantage of the powerful genetic tools available in the fruit fly Drosophila melanogaster, an outline of the neural pathways that compute visual motion has emerged. Here we describe these pathways, the evidence supporting them, and the challenges that remain in understanding the circuits and computations that link sensory inputs to behavior. Studies in flies and vertebrates have revealed a number of functional similarities between motion-processing pathways in different animals, despite profound differences in circuit anatomy and structure. The fact that different circuit mechanisms are used to achieve convergent computational outcomes sheds light on the evolution of the nervous system.

Determining the genomic locations of transposable elements is a common experimental goal. When mapping large collections of transposon insertions, individualized amplification and sequencing is both time consuming and costly. We describe an approach in which large numbers of insertion lines can be simultaneously mapped in a single DNA sequencing reaction by using digital error-correcting codes to encode line identity in a unique set of barcoded pools.

Dopamine is synonymous with reward and motivation in mammals. However, only recently has dopamine been linked to motivated behaviour and rewarding reinforcement in fruitflies. Instead, octopamine has historically been considered to be the signal for reward in insects. Here we show, using temporal control of neural function in Drosophila, that only short-term appetitive memory is reinforced by octopamine. Moreover, octopamine-dependent memory formation requires signalling through dopamine neurons. Part of the octopamine signal requires the α-adrenergic-like OAMB receptor in an identified subset of mushroom-body-targeted dopamine neurons. Octopamine triggers an increase in intracellular calcium in these dopamine neurons, and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking octopamine. Analysis of the β-adrenergic-like OCTβ2R receptor reveals that octopamine-dependent reinforcement also requires an interaction with dopamine neurons that control appetitive motivation. These data indicate that sweet taste engages a distributed octopamine signal that reinforces memory through discrete subsets of mushroom-body-targeted dopamine neurons. In addition, they reconcile previous findings with octopamine and dopamine and suggest that reinforcement systems in flies are more similar to mammals than previously thought. © 2012 Macmillan Publishers Limited. All rights reserved.

Tissue-specific gene expression using the UAS/GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we have developed a convertible genetic platform, called the Integrase Swappable In vivo Targeting Element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient, in vivo manipulations, making it possible to generate many swaps in parallel. Furthermore, this system is entirely modular, allowing future genetic tools to be easily incorporated into the existing framework.

Intra- and interchromosomal interactions have been implicated in a number of genetic phenomena in diverse organisms, suggesting that the higher-order structural organization of chromosomes in the nucleus can have a profound impact on gene regulation. In Drosophila, homologous chromosomes remain paired in somatic tissues, allowing for trans interactions between genes and regulatory elements on the two homologs. One consequence of homolog pairing is the phenomenon of transvection, in which regulatory elements on one homolog can affect the expression of a gene in trans. We report a new instance of transvection at the Drosophila apterous (ap) locus. Two different insertions of boundary elements in the ap regulatory region were identified. The boundaries are inserted between the ap wing enhancer and the ap promoter and have highly penetrant wing defects typical of mutants in ap. When crossed to an ap promoter deletion, both boundary inserts exhibit the interallelic complementation characteristic of transvection. To confirm that transvection occurs at ap, we generated a deletion of the ap wing enhancer by FRT-mediated recombination. When the wing-enhancer deletion is crossed to the ap promoter deletion, strong transvection is observed. Interestingly, the two boundary elements, which are inserted ∼10 kb apart, fail to block enhancer action when they are present in trans to one another. We demonstrate that this is unlikely to be due to insulator bypass. The transvection effects described here may provide insight into the role that boundary element pairing plays in enhancer blocking both in cis and in trans.