A novel approach to identifying aggression genes in Drosophila

Aggression is a universally expressed highly conserved trait that represents humanity's greatest affliction. Over the past few years, the fruitfly has become an excellent model system for studying aggression. A number of studies have reported that many hundreds, even thousands of genes change their expression patterns when flies are selected for high or low levels of aggression. We have used a novel approach where we take genetically identical pairs of males and let them fight for 30 minutes. Some pairs fight a lot (the Rambos) and some do not (the Gandhis). We compared the genes that were expressed differently between the two groups and found a small number that were either upregulated or downregulated in Rambos. Some of these genes encoded properties of neurotransmitters and some encoded proteins important for olfaction, and all of these could rationally be expected to play a role in aggression. We have focused on one of these, Dat which encodes a protein that destroys a number of neurotransmitters called monoamines at the synapse. There are two isoforms of this protein encoded by the gene, one produces Rambos, the other produces Gandhis, and they appear to be expressed in different parts of the fly brain. In humans, a mutation in a gene called MAOA, which does the same job as Dat in the fly, causes hyperaggression in the boys in which it is expressed, and most of the boys in this partiucular Dutch family which carry the mutant gene are imprisoned. Consequently, using our unusual approach, we have discovered an aggression gene in the fly that has a functional parallel in humans. In this proposal we will use sophisticated genetic tools to discover how the two Dat isoforms can generate Rambo and Gandhi behaviour. We will also initiate studies of some of the other aggression genes that appear to be involved in aggression, including two other genes that are implicated in neurotransmitter function, DopR2 which encodes a receptor for the neurotransmitter dopamine, and a gene called CG13794, which nobody what it does, but by its protein sequence may be implicated in the transport of the neurotranmsmitter serotonin. We shall also investigate the roles of two odourant binding protein genes, Obp99b and Obp56d, both of which are expressed highly in Rambo flies. Our results will enlighten the molecular basis of aggression in the fly, and, given our findings so far, our results may be very relevant for understanding the nature of human aggression.

Previous transcriptome studies of Drosophila aggression have used selection studies or various types of inbred or outbred lines to generate complex putative gene pathways involving hundreds if not thousands of genes. The conclusions from this work is that gene pleiotropy and epistasis provides the overwhelming major component of the aggression phenotype, indeed, investigating single gene effects on aggression may be futile. We took a different approach and investigated genetically identical males that were allowed to fight (or not) for 30 minutes. Some pairs fought very aggressively (Rambos), other were more peaceful (Gandhis). We took the two extremes of the distribution and performed a differential microarray analysis on head RNA. Not surprisingly, we found a relatively small number of genes that differentiated between the two groups. Oor most prominent hit was Dat (dopamine-N-acetyltransferase) which catabolises monoamine neurotransmitters. Validation by qPCR and RNAi was successful, and we observed that one isoform DatA reduces aggression, while teh otehr DatB, enhances aggression, particularly in a DatA mutant background. The two isoforms appear to be expressed in different parts of the brain, suggesting they interact at the systems level to generate adaptive levels of aggression. Remarkably, a mutation in the human MAOA gene that performs a similar function to Dat, generates hyperaggression in the boys that carry the null mutation in a Dutch kindred. We have validates a number of other hits by RNAi including two odourant binding protein geens Obp99b and 56d and the gene encoding DopR2. Another gene, CG13794, which appears in all four fly aggression transcriptome experiments, including ours, encodes a putative 5-HT transporter. Our approach thus avoids epistasis and pleiotropy from the genetic background and our proposal seeks to consolidate our successful neurogenetic approach to study further the genetic basis of this important phenotype.

The main beneficiaries of our work in the narrow sense will be neurobiologists, geneticists and evolutionary biologists that are interested in aggression. As aggression is a highly topical subject as judged by the number of high impact papers published in this field, our studies, once published, might find themselves in textbooks with obvious impact for biology education. Our remarkable discovery of the Dat gene as a major determinant of fly aggression which mirrors its human functional equivalent, the MAOA gene, which when mutated in males, generates hyperaggression, shows just how relevant our work is to human studies. This consequently makes our work interesting to the general public who are worried about the seemingly increasing levels of aggression in our society. Consequently, our work, through GENIE (see our impact plan), will have a certain resonance with the general public and we shall consolidate this at every opportunity. On a more pragmatic note, as we are working with aggression that is mediated by neurotransmitter-related genes, our studies will also be of interst perhaps to phanrmaceutical companies. Indeed, some of our candidate genes may already be targets for anti-depressants. Thus we will liase with our Business Development Office should any commercial opportunities arise (see Impact plan)

Briefly, GENIE (Genetics, Education, Networking, Innovation and Excellence) is a national CETL (Centre of Excellence in Teaching and Learning) run by Prof Annette Cashmore of our Genetics department. A significant activity of GENIE is therefore outreach, and its website attract >22,000 hits per month (http://www.le.ac.uk/genetics/genie/?searchterm=cetl). Within this website is a Virtual Genetics Education Centre (VGEC) for schools and colleges, higher education centres, the general public, as well as health professionals and policymakers. GENIE conducts about 35 meetings/workshops per year, and CPK and ET have contributed to GENIE functions on an ad hoc basis regularly. Consequently, we shall,

1. Work with the GENIE lecturers to add a section on aggression and Drosophila neurogenetics on our research group's website on the VGEC which we have already created as part of other grant related impact activities.
2. Use the opportunities offered to us organised by GENIE, to present our work to various groups, both local and national. We shall be particularly keen to present our aggression studies to health professionals, social workers schools, women's groups etc. However CETL also has regular forums with policymakers (eg House of Lords presentations) and commercial groups (Rotary Clubs) and so we shall deliver lectures to these influential groups whenever possible.
3. We will propose a summer science exhibition to the Royal Society in 2016