The role of genes in morphological development is well understood for a variety of model organisms, but there remains a gap in our understanding of how genetics mediate behavior. Are there master genes that regulate behavior? Answering this question will lead to a better understanding of the development and function of the central nervous system, eventually allowing us to map out the pathways that regulate specific behaviors. We are using Drosophila melanogaster as a model organism and the male courtship ritual as the behavior of interest to study the relationships between genes, neural development, and behavior. Trapped in endoderm 1 (Tre1), a gene encoding an orphan G-protein coupled receptor (GPCR), is required for normal courtship behavior in fruit flies, but how this receptor regulates behavior is not yet understood. Here, we characterize the signaling cascade downstream of Tre1 by testing mutations in the Drosophila G-proteins for courtship defects similar to those seen in Tre1. Our results demonstrate that Gαi is a candidate downstream effector for Tre1, while also implicating Gγ30A in courtship behavior. Future goals include completing the characterization of the G-protein mutations and conducting experiments to explore the complex interaction between G-protein signaling and courtship initiation

Mu, Emily

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McNair Research Journal SJSUVolume 15 Spring 2019 Article 82019Gαi and GƔ30A act downstream of Tre1 inDrosophila courtshipEmily MuSan Jose State UniversityFollow this and additional works at: https://scholarworks.sjsu.edu/mcnairPart of the Molecular Biology CommonsThis Article is brought to you for free and open access by SJSU ScholarWorks. It has been accepted for inclusion in McNair Research Journal SJSU byan authorized editor of SJSU ScholarWorks. For more information, please contact scholarworks@sjsu.edu.Recommended CitationMu, Emily (2019) "Gαi and GƔ30A act downstream of Tre1 in Drosophila courtship," McNair Research Journal SJSU: Vol. 15 , Article8.Available at: https://scholarworks.sjsu.edu/mcnair/vol15/iss1/877      Emily Mu  Major: Biological Sciences, Molecular Biology  Mentor:  Dr. Rachael French, Dr. Maria E. Cruz  Co-Author: Cac Tran  Gαi and GƔ30A act downstream of Tre1in Drosophila courtship  Biography  As a first-generation American and college student, Emily’s fascination with the life sciences and passion for service drove her to work in a field that contributes to scientific and human advancement. Outside of her research in the French Laboratory and the McNair Scholars Program, she has been an active member of her campus community; having served as chapter president of Global Medical Brigades, acquiring alumni status in Alpha Omicron Pi women’s fraternity, and participating in the San José State University NSF-REU, Research by Undergraduates Using Molecular Biology Techniques (RUMBA). Emily has led one of the three medical brigades she has participated in, providing her with a worldview that has stretched her research interests beyond the scope of biology. She is interested in human genetics, biochemistry, and global health. After earning her bachelor’s degree, Emily will continue on to graduate school in pursuit of these research interests with the goal of one day mentoring, empowering, and advocating for students in academia.  1Mu: G?i and G?30A act downstream of Tre1 in Drosophila courtshipPublished by SJSU ScholarWorks, 201978  Gαi and GƔ30A act downstream of Tre1 in Drosophila courtship  ABSTRACT The role of genes in morphological development is well understood for a variety of model organisms, but there remains a gap in our understanding of how genetics mediate behavior. Are there master genes that regulate behavior? Answering this question will lead to a better understanding of the development and function of the central nervous system, eventually allowing us to map out the pathways that regulate specific behaviors. We are using Drosophila melanogaster as a model organism and the male courtship ritual as the behavior of interest to study the relationships between genes, neural development, and behavior. Trapped in endoderm 1 (Tre1), a gene encoding an orphan G-protein coupled receptor (GPCR), is required for normal courtship behavior in fruit flies, but how this receptor regulates behavior is not yet understood. Here, we characterize the signaling cascade downstream of Tre1 by testing mutations in the Drosophila G-proteins for courtship defects similar to those seen in Tre1. Our results demonstrate that Gαi is a candidate downstream effector for Tre1, while also implicating Gγ30A in courtship behavior. Future goals include completing the characterization of the G-protein mutations and conducting experiments to explore the complex interaction between G-protein signaling and courtship initiation.              2McNair Research Journal SJSU, Vol. 15 [2019], Art. 8https://scholarworks.sjsu.edu/mcnair/vol15/iss1/879  INTRODUCTION Genes are responsible for the development of the central nervous system (CNS), which, in turn, mediates behavior. Many of the genetic pathways underlying morphological development in animals have been identified and well-characterized. In contrast, there is still much to be understood about how a gene or genes specify behavior (Demir and Dickson 2005). Elucidating the roles of genes with regard to particular behaviors has the potential to be a powerful set of tools to map out the molecular pathways that direct the wiring of the CNS. Courtship behavior is observable and well characterized in Drosophila melanogaster, making it a practical model for the purposes of this study. In addition, the behavior is innate – it is “hard wired” into the brain. Only males perform the courtship ritual – and they are able to perform it even if they have been socially isolated since “birth.” The courtship ritual is a stereotyped set of six distinct steps that must be executed correctly, and in the proper order, to ensure reproductive success (Baker et al. 2001). Because innate behaviors such as this do not have to be learned, they are ideal models for the study of how genes pattern the nervous system to elicit specific behavioral responses. We seek to expand our knowledge on this particular trait as it is so evolutionarily favored it is programmed into the Drosophila nervous system.  We previously demonstrated that the gene Trapped in endoderm-1 (Tre1) is required for normal courtship behavior in fruit flies. Specifically, male flies in which Tre1-expressing cells are silenced, or with loss of function mutations in Tre1, initiate courtship much more rapidly than wild-type males (Luu et al. 2016). Tre1 encodes an orphan G-protein coupled receptor (GPCR). In this work, we aimed to identify components of the G-protein signaling cascade downstream of Tre1. To accomplish this goal, we tested mutations disrupting subunits of heterotrimeric G-proteins for courtship initiation defects. Here, we show that both Gαi and Gγ30A are involved in courtship initiation, and that these genes may reveal a complex role for G-protein signaling in the programming of this behavior.  METHODS Fly stocks 3Mu: G?i and G?30A act downstream of Tre1 in Drosophila courtshipPublished by SJSU ScholarWorks, 201980  Fly stocks were maintained in a 25℃ incubator and grown on standard cornmeal/molasses medium. All strains of flies were acquired from the Bloomington Drosophila Stock Center at the University of Indiana. Fly stocks used were: w;wild type Berline (our laboratory wild type strain, used as the control genetic background), GƔ30AEY11766(Bloomington Stock 20695), GƔ30Ae0084 (Bloomington Stock 17891), GαOKG01266(Bloomington Stock 13714), GαOMB00893 (Bloomington Stock 22924), GαFMB10810 (Bloomington Stock 29157), and GαIEY039776(Bloomington Stock 15698).  Courtship assays For courtship assays, virgin males were kept in isolation for 2-3 days after eclosion. Each male was then presented with a single 1-2-day-old w1118; WTB virgin female. Single male and female pairs were placed into custom plexiglass chambers 10 mm in diameter and 6 mm in height, separated by plastic transparencies. Contact between pairs was initiated by removal of the transparencies. Courtship behavior was recorded in infrared light for 20 minutes.    Statistical Analyses An  level of 0.05 was used in all experiments. Statistics were performed on log-transformed means of wing song latency. Data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test. No statistical tests were used to predetermine sample sizes, but our sample sizes are consistent with those reported in previous publications (Tran et al. 2014; Luu et al. 2015). Data in figures are back-transformed.   RESULTS Flies mutant for G⍺i mutants phenocopy Tre1 In order to identify the G-proteins that transduce the Tre1 signal, we began a systematic survey of courtship phenotypes in male flies mutant for all of the Drosophila heterotrimeric G-protein genes. The Drosophila genome encodes 8 predicted Gα subunits (concertina (cta), Gαf, Gαi, Gαo, Gαq, Gαs, CG17760, and CG30054). To date, we have tested loss-of-function alleles in cta, Gαf, Gαi, Gαo, and Gαq. We find that mutation of 4McNair Research Journal SJSU, Vol. 15 [2019], Art. 8https://scholarworks.sjsu.edu/mcnair/vol15/iss1/881  cta, Gαf, Gαo, and Gαq have no consistent effect on courtship behavior (data not shown). However, mutation of Gαi consistently phenocopies the Tre1 “rapid courtship” phenotype (Figure 1). While control animals initiated courtship in 108 seconds, on average, male flies homozygous for G⍺IEY03976b, a loss-of-function mutation in Gαi, initiate courtship approximately three times as fast, in an average time of 32 seconds (P < 0.01, student’s T test, N = 24 males). These data are consistent with Gαi functioning downstream of the Tre1 receptor in courtship initiation.   Figure 1. Flies mutant for G⍺I display rapid courtship initiation. G⍺IEY03976b mutant flies initiate courtship in 32 seconds, compared to 108 seconds in wild type controls (w; WTB). (p = 8.22991E-10, student’s T-test, N = 24 males for each genotype)  GƔ30A mutants demonstrate slower courtship initiation  The Drosophila genome encodes two Gγ subunits, Gγ1 and Gγ30A. We have tested loss-of-function mutations in both of these genes, and find that, while Gγ1 does not appear to affect courtship initiation, mutation of 10832020406080100120140Wildtype GαI EY03976Wing song latency (sec)GαI Mutant Wingsong Latency5Mu: G?i and G?30A act downstream of Tre1 in Drosophila courtshipPublished by SJSU ScholarWorks, 201982  Gγ30A consistently results in a longer time to courtship initiation. Figure 2 shows the average time to courtship initiation for two alleles of Gγ30A. Male flies homozygous for either allele display an increase in courtship initiation (N = 24 males, p = 0.123). Though these results do not achieve statistical significance, the consistency of the phenotype over multiple trials in two independent alleles suggests that Gγ30A is involved in courtship initiation. Additional experiments will be needed to confirm these results.   Figure 2. GƔ30A mutant males display slower courtship initiation. GƔ30A mutant males initiate courtship in 77-81 seconds compared with 52 seconds in wild type controls (w; WTB). (p = 0.123, one-way ANOVA, N = 24 males for each genotype).   DISCUSSION We have begun a systematic analysis of the roles of all Drosophila G-protein genes in courtship initiation, in order to elucidate the signal transduction cascade downstream of Tre1. To date, we have screened 5 of 527781020406080100120140Wildtype Gγ30A EY11766Gγ30A e00834Wing song latency (sec)Gɣ30A Mutant Wingsong Latency6McNair Research Journal SJSU, Vol. 15 [2019], Art. 8https://scholarworks.sjsu.edu/mcnair/vol15/iss1/883  the 8 Gα subunits, and both Gγ subunits. We find that mutation of Gαi phenocopies the Tre1 rapid-courtship effect (Figure 1), strongly suggesting that Gαi functions downstream of the Tre1 GPCR. When activated, Gαi inhibits the activity of adenylate cyclase (reviewed in Birnbaumer, 2007). In mammals, Gαi is coupled to histamine H3 and H4 receptors (Interpro, http://www.ebi.ac.uk/interpro/entry/IPR003980). This is particularly interesting because we have evidence that Tre1 may function as a histamine receptor, and this would be the first example of a metabotropic histamine receptor in Drosophila, and only the second in any invertebrate (Zaki et al., 2017). We also show evidence that Gγ30A is involved in courtship initiation, but the phenotype of Gγ30A mutants is the opposite of Gαi mutants (Figure 2). This difference in can be explained in at least two ways. First, this result is consistent with the function of Gβγ subunits as inhibitors of Gα activity (Clapham and Neer, 1997). Second, it may be that the function of Gγ30A is independent of Gαi, and the regulation of courtship initiation by G-protein signaling is more complex. In order to distinguish between these possibilities, we will begin testing signal transduction pathways downstream of both Gγ30A and Gαi, including mutations disrupting adenylate cyclase (downstream of both Gαi and Gγ), as well as PI3K (inhibited by Gγ, so expected to phenocopy Gγ30A) and phospholipase C β (activated by Gγ, so expected to have rapid courtship).   REFERENCES Baker BS, Taylor BJ, Hall JC. 2001. Are Complex Behaviors Specified by Dedicated Regulatory Genes? Reasoning from Drosophila. Cell 105: 13-24.  Birnbaumer L. 2006. Expansion of signal transduction by G proteins. The second 15 years or so: from 3 to 16 alpha subunits plus betagamma dimers. Biochimica et biophysica acta, 1768(4): 772-93.  Clapham DE, Neer EJ. 1997. G protein beta gamma subunits. Annual Review of Pharmacology and Toxicology. 37: 167-203.    7Mu: G?i and G?30A act downstream of Tre1 in Drosophila courtshipPublished by SJSU ScholarWorks, 201984  Demir E, Dickson BJ. 2005. fruitless Splicing Specifies Male Courtship Behavior in Drosophila. Cell 121: 785-794.  Hoxha V, Lama C, Chang PL, Saurabh S, Patel N, Olate N, Dauwalder B. 2013. Sex-Specific Signaling in the Blood-Brain Barrier Is Required for Male Courtship in Drosophila. PLoS Genetics 9(1): e1003217.  Luu P, Zaki SA, Tran DH, French RL. 2016. A Novel Gene Controlling the Timing of Courtship Initiation in Drosophila melanogaster. Genetics 202: 1043-1053.  Tran DH, Meissner GW, French RL, Baker BS. 2014. A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila. PLOSO 9(4): 1-10.  Zaki SA, Sandhu J, French RL. 2017. Histamine signaling via the metabotropic receptor Trapped in endoderm 1 regulates courtship initiation in Drosophila melanogaster. bioRxiv. Preprint.  8McNair Research Journal SJSU, Vol. 15 [2019], Art. 8https://scholarworks.sjsu.edu/mcnair/vol15/iss1/8

Gαi and GƔ30A act downstream of Tre1 in Drosophila courtship

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Gαi and GƔ30A act downstream of Tre1 in Drosophila courtship

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