Jonathan M. Blagburn - US grants

DESCRIPTION (provided by applicant): Engrailed is a ubiquitous transcriptional regulator that is potentially of great significance to human health. It has recently been linked to fate determination and survival of midbrain dopaminergic neurons, with En knockout mice showing Parkinson-like symptoms, and has also been linked to autism spectrum disorder. It is therefore essential to understand the role En plays in regulating neuronal connectivity. The long term objectives of this research are to use simple model systems, that have identifiable neurons, to investigate how Engrailed controls synaptic target recognition, and what are the downstream effector genes that it regulates. My previous work has used the cockroach cereal system to study this;however, there are substantial barriers to further progress in this system. The aims of this pilot project will enable me to develop a similar system of identifiable Engrailed-expressing neurons in the genetically tractable Drosophila melanogaster. The first aim is to use Gal4-UAS to express green fluorescent protein in neurons that normally express Engrailed. Confocal microscopy will then be used to characterize these neurons in a range of sensory systems, such as the olfactory, auditory and touch cells. The second aim is to focus on one of these sensory systems using the electrophysiological and anatomical techniques that have been developed in the lab, and the expertise of the mentor, Dr. Rod Murphey, in order to be able to test the role of Engrailed in controlling the synaptic connectivity of this system. The third aim will involve the co-expression of ectopic Engrailed along with GFP, enabling a direct test of the idea that Engrailed controls connectivity in the circuit. In the final aim, with the help of Dr. Rod Murphey, I will use genetic screens to begin a search for other genes downstream of Engrailed that control synaptic specificity. An alternative strategy will be to test En binding targets previously identified by other groups. Drosophila models are particularly useful for the discovery of molecular pathways that are directly relevant to human health, because most of these pathways have been conserved during evolution. All animals have Engrailed protein, so it is very likely that any molecules that are regulated by it during the process of synapse formation in Drosophila have their counterparts in humans. These molecules may be of great potential importance in neurological diseases such as Parkinson's or autism.

DESCRIPTION (provided by applicant): Engrailed (En) is a transcription factor first discovered in Drosophila but later found to be present in all animals, playing an important role in controllin neuronal development. Little is known about the cell surface molecules that it regulates. The long-term goal of this research is to find out how En regulates synaptic connectivity in the CNS, with a particular focus on identifying and characterizing its target genes of cell surface effector molecules. Preliminary data show that overexpression of En in Drosophila olfactory neurons alters their axonal path finding to their targets, the olfactory glomeruli. Additionally, ectopic E expression in a normally En- negative subset of auditory neurons allows them to form synaptic connections with the Giant Fiber (GF) escape neuron. The first aim is to selectively knock out En in olfactory neurons using fly lines in which RNAi is driven by the Gal4-UAS system. I will determine the effects on axonal guidance by assaying changes in the morphology of GFP-labeled olfactory axons, and alterations in the positions of immunolabeled olfactory glomeruli. The second aim will drive En RNAi in the auditory neurons and measuring their synaptic input to the GF. The third aim is to test whether increasing or knocking down expression of the En target Connectin, a member of the conserved LRR superfamily of adhesion molecules, alters axon guidance or glomerulus positioning. I will also test whether En knockout results in overexpression of this protein, as would be expected if it is a target of En repression. In the finl aim, I will ectopically express or knock down the En-binding target gene Neuroglian, a cell surface adhesion molecule homologous to vertebrate L1-CAM, then use the auditory synapse assays of connection to the GF. I will also test whether its immunostaining is altered by overexpression or knockout of En, as would be expected if it is negatively regulated by En. Relevance: En has been shown to control the survival of midbrain dopaminergic neurons, with En knockout mice showing Parkinson-like symptoms, and it has also been linked to autism spectrum disorder. Drosophila models are particularly useful for the discovery of molecular pathways that are directly relevant to human health, because most of these pathways have been conserved during evolution. All animals have En protein, so it is very likely that any molecules that are regulated by it during the process of synapse formation in Drosophila have their counterparts in humans, playing similar roles. These molecules may be of great potential importance in neurological diseases such as Parkinson's or autism. In addition, the basic knowledge gained from this project about the development of dipteran olfactory and auditory systems could be of use in designing ways to disrupt the feeding and mating patterns of mosquitoes, which are vectors of diseases such as malaria and dengue fever. PUBLIC HEALTH RELEVANCE: This research will help our understanding of the molecular pathways involved in the formation of synaptic connections in the central nervous system, using the fruit fly, Drosophila melanogaster, as a model system. The long-term goal of this research is to find out how a protein that is present in all animals' brains regulates the accuracy of the formation of synaptic connections, by controlling the presence of other cell surface molecules. The results of this study may have relevance to diseases of humans such as Parkinson's disease and autism, and may help in the control of disease-transmitting insects such as mosquitoes.

Abstract Engrailed (En) is a transcription factor; a protein that binds to DNA and that switches on or off other genes. It was first discovered in Drosophila but later found to be present in all animals, where it plays an important role in controlling the development of neurons, or nerve cells. The long-term goal of this research is to find out how En controls the way that nerve cells connect to each other (form synapses) within the brain, with a particular focus on identifying and characterizing the network of genes and molecules that it regulates. This proposal uses the Drosophila auditory neuron ? to ? giant fiber (GF) synapse as a model system with which to investigate the role of En in the control of synaptic connections. The first specific aim will be to use electrophysiology and anatomical staining techniques to measure how auditory neuron action potentials and output synapses to the GF change as the animal ages, and to determine whether there are sex-specific differences. The second specific aim will be to study the effects of changing the amounts of En present in the neurons in the adult animal. As in mammalian neurons, En expression in Drosophila auditory neurons persists through adult life, but its functions during this period are not understood. It could perhaps maintain their electrical properties or the patterns of their synaptic connections. To test this, genetic methods will be used to add or take away En from the sensory neurons at different times. This work is relevant to public health because human En has been linked to several brain disorders, such as Parkinson?s disease and autism. Drosophila models are particularly useful for the discovery of molecular pathways that are directly relevant to humans, because most of these pathways have been conserved during evolution. All animals have En protein, so it is very likely that any molecules that are regulated by it during the process of synapse formation in Drosophila have their counterparts in humans, playing similar roles in brain development.