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Central and Peripheral Regulation of Appetite and Food Intake in Drosophila
Published in Ruth B.S. Harris, Appetite and Food Intake, 2017
Perhaps the most valuable aspect of the fly as a model for behavioral neurobiology is the astounding array of genetic tools available for targeting, manipulating, and monitoring neural circuit activity. One particularly important tool set is based on the upstream activation sequence (UAS)/GAL4 system for genetically targeted gene expression, which provides a high level of spatial and temporal control over cell targeting and transgene expression. This system exploits the activity of a yeast transcription factor, GAL4, and it’s identified cis-regulatory binding sites (UASs) that, when present upstream of a gene, result in its transcriptional activation by GAL4 (Brand and Perrimon 1993). The GAL4 transcriptional activator can be expressed randomly or in cell types or tissues of interest by placing it under the control of unique gene promotors (Gordon and Scott 2009; Marella et al. 2012). Alternately, GAL4 expression can be controlled by heat- or drug-inducible promoters for temporal control over transgene expression (Duffy 2002). Variations of this system include the LexA/LexAop and QF/QUAS systems, which can be used alone or in tandem with the UAS/GAL4 system to further refine targeting or to express multiple transgenes (Pfeiffer et al. 2010; del Valle Rodriguez et al. 2012; Riabinina et al. 2015). A wide variety of UAS- and LexAop-based transgenes are available for labeling neurons as well as their nuclear, axonal, and dendritic compartments (Rolls et al. 2007; Nicolai et al. 2010), for monitoring activity (calcium [Akerboom et al. 2013], 3′,5′-cyclic adenosine monophosphate (cAMP) [Shafer et al. 2008], or voltage [Cao et al. 2013]), and for manipulating activity in a temperature- (Pool et al. 2014) or light- (Claridge-Chang et al. 2009; Perisse et al. 2013) dependent fashion. In addition to their use for monitoring the activity of select neuronal populations, these tools can be used to perform exquisitely targeted lesions in individual or small groups of neurons by administration of a targeted laser, allowing for precise loss of function analysis. The body wall of fly larvae is largely transparent, allowing focused laser beams to penetrate the brain and generate lesions in soma and neurites of identifiable central and peripheral neurons labeled with compartment specific markers such as nucleus- or axon-specific GFP (Xu et al. 2010; Wang et al. 2013; Zhang et al. 2013a). This technique has been particularly useful for testing the roles of defined neurons and circuits in feeding regulation in behaving animals. In tandem with the behavioral assays described previously, these genetic tools allow fly researchers a high level of temporal and spatial control and facilitate the characterization of the underlying biology regulating complex behavioral circuits.
Cystathionine β-synthase Deficiency Impairs Vision in the Fruit Fly, Drosophila melanogaster
Published in Current Eye Research, 2021
Marycruz Flores-Flores, Leonardo Moreno-García, Felipe Castro-Martínez, Marcos Nahmad
One of these genetic tools is the Drosophila Gal4-UAS system23,24 that allows overexpressing and/or downregulate gene expression in a spatiotemporal manner. It is composed by two transgenes inserted into the fly genome: the first one contains a tissue-specific or ubiquitous driver upstream the Gal4 gene, that encodes for a yeast transcription factor; the second one is a specific DNA sequence known as the Upstream Activation Sequence (UAS). When both transgenes are put together in a single fly, a gene of choice is ectopically expressed in the place and time determined by the Gal4 driver. A particular use of the Gal4-UAS system is gene silencing: the UAS sequence is placed upstream of an interference RNA (RNAi), allowing the system to knock-down a specific gene.25 Here, we used ubiquitous and eye-specific Gal4 drivers to express an RNAi of cbs (cbsRNAi), in order to investigate homocystinuria vision phenotypes. We conducted behavioral studies to ask if vision is affected in cbsRNAi animals. Particularly, we investigated one of the best-documented behaviors in Drosophila, its attraction to a light stimulus, or phototaxis,26 in flies expressing cbsRNAi ubiquitously or in the eyes, with respect to a control group with normal CBS expression. We found that ubiquitous down-regulation of CBS leads to abnormal phototaxis. Furthermore, we confirmed that the behavior is not due to motor impairment, suggesting that vision is affected in these mutants. Since, vision problems are a very common manifestation in classical homocystinuria patients, we propose to establish Drosophila melanogaster as an animal model of the disease.
Understanding neurobehavioral genetics of zebrafish
Published in Journal of Neurogenetics, 2020
Sergey V. Cheresiz, Andrey D. Volgin, Alexandra Kokorina Evsyukova, Alim A.O. Bashirzade, Konstantin A. Demin, Murilo S. de Abreu, Tamara G. Amstislavskaya, Allan V. Kalueff
Until mid-2000s, using transgenic zebrafish in neuroscience has been rather limited (Akerberg, Stewart, & Stankunas, 2014). However, the problem of inefficient conventional (e.g. plasmid-based) transgenesis has been later resolved by germline retroviral transduction (Gaiano, Allende, Amsterdam, Kawakami, & Hopkins, 1996) or the germline insertion of Tol2 transposon (Asakawa & Kawakami, 2009; Emelyanov & Parinov, 2008). The advent of high-efficiency transgenesis in zebrafish has developed gene- and enhancer-trapping constructs on transposon (Choo et al.,2006) or retroviral backbone (Ellingsen et al.,2005) and identified numerous cis-acting sequences in zebrafish genome that drive the transgene expression in a spatially-restricted manner. An adaptation of Gal4/UAS (Galactose-induced genes transcription factor/Upstream Activation Sequence) two-component transcriptional activation switch from yeast to zebrafish has generated various Gal4 zebrafish lines able to drive the expression of UAS-controlled transgenes of interest in discrete cell types and/or anatomical regions of the brain with only background expression in non-neural tissues (Scott et al.,2007). The original Gal4/UAS system relies on the expression of Gal4 DNA-binding domain (DBD) and its activation of synthetic promoters consisting of tandem repeats of UAS upstream of the basal promoter (Köster & Fraser, 2001). The design of the gene- and enhancer-trapping transgenic constructs expressing the chimeric Gal4-VP16 protein or transcriptional activator Gal4FF enables screening for transgenic zebrafish with spatially restricted Gal4 expression in neural cells (Davison et al.,2007; Scott, 2009; Scott et al.,2007), to modulate the expression of genes in relevant neural structures by crossing fish expressing Gal4 in the region of interest with the transgenes of interest under the control of UAS promoter.
The use of Drosophila melanogaster as a model organism to study immune-nanotoxicity
Published in Nanotoxicology, 2019
Cheng Teng Ng, Liya E Yu, Choon Nam Ong, Boon Huat Bay, Gyeong Hun Baeg
Numerous genetic tools allow the study of gene expression in Drosophila. The yeast transcription activator protein GAL4/Upstream Activation Sequence (UAS) system is an extremely versatile system that allows researchers to knockdown and overexpress gene of interest in a cell or tissue specific manner (Koon and Chan, 2017; Duffy, 2002). In particular, Drosophila transgenic RNA interference (RNAi) project (www.flyrnai.org) is a platform for accessing numerous transgenic animal models with an RNAi hairpin under the UAS/Gal4 system. In addition to the GAL4/UAS system, a large number of in vivo reporter lines that can reflex the activity of signaling cascades has been established in Drosophila. For example, tissues or organs of interest can be stained with lacZ antibody to measure reporter activities for the Wnt (naked cuticle-lacZ), EGFR (pointed-lacZ), Hippo (diap1-lacZ), TGF-β (dad-lacZ), JAK/STAT (10xStat92e-lacZ), Hedgehog (patched-lacZ), Notch (GBE-Su(H)m8-lacZ), and Insulin/PI3K signaling pathways (PH-lacZ). In case of measuring intracellular ROS levels in vivo, one can also use transgenic lines harboring an oxidative stress reporter gene such as gstD1-GFP (Sykiotis and Bohmann, 2008), which allows to monitor specific tissues and organs that are susceptible to oxidative stress induced by NMs through quantifying the intensity of green fluorescent protein (GFP) (Koon and Chan, 2017), as shown in Figure 9. Furthermore, under the Berkeley Drosophila Genome Project (http://www.fruitfly.org; gene disruption project), tens of thousands of loss-of-function mutant lines have been generated by using single transposable element to disrupt annotated Drosophila genes (Spradling et al., 1999). The majority of classical fly mutants from forward genetic screens are typically loss-of-function alleles. On the other hand, gain-of-function alleles are modeled by overexpressing hyper-activated form of mutant protein using the GAL4/UAS system (St Johnston, 2002). For example, Toll trans-heterozygotes can be generated through crossing flies carrying different loss-of-function alleles of Toll to study the role of Toll and host-pathogen interactions in the event of infection by Candida albicans. (Alarco et al., 2004). Recently, there are burgeoning research studies using the genome editing system in Drosophila (http://flycrispr.molbio.wisc.edu; flyCRISPR), the clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) system for selectively creating highly efficient mutagenesis (∼88%) in specific regions of the genome (Bassett and Liu, 2014). This method is highly applicable to almost any gene in a time efficient manner (∼1 month), thereby generating more isogenic mutant lines and allowing us to combine mutations with preexisting stocks. This system provides an opportunity for up-scale genetic screening in Drosophila (Bassett et al., 2013).