Pergamon Progress in Neurobiology Vol. 42, pp. 299-308, 1994 Copyright© 1994Elsevier Science Ltd Printed in Great Britain.All dghts raerved 0301-0082/94/$24.00 REVERSE GENETICS OF DROSOPHILA BRAIN STRUCTURE AND FUNCTION J. W. SENTRY, S. F. GOODWIN, C. D. MILLIGAN, A. DUNCANSON, M.YANG and K. KAISER* Department of Genetics, University of Glasgow, Church Street, Glasgow GI I 5JS, U.K. Abstraet--A set of molecular genetic technologies are described, which will have far reaching consequences for the study of brain structure, function and development in Drosophila melanogaster. Site selected mutagenesis (a PCR-based screen for P-element insertion events) allows insertion mutants to he isolated for any cloned gene, and is being used in this laboratory to ask questions about the rolls of particular cellular components in learning and memory. Transposants have been isolated in genes encoding a regulatory (RI) and a catalytic (DCO) subunit of cAMP-dependent protein kinase, and in a gene encoding a Gi-like alpha subunit. The alternative use of I factors is described. The PKA RI homozygous mutants display a significant decrement in initial learning ability. Enhancer-trap strategies, for which the GAL-4 P-element system is particularly convenient, allow the identification of genes expressed in the developing fly brain. Strategies for the efficient detection of such events are described. CONTENTS 1. Introduction 2. Site-selected mutagenesis 2.1. P-elements 2.2. P-element mutagenesis 2.3. PCR-based detection of new insertions 2.4. Sib-selection 3. Reverse genetics of learning and memory 4. Brain specific gene expression: an enhancer trap approach 5. Future prospects 5.1. Alternative transposons for site-selected mutagenesis 5.2. Alternative PCR detection strategies 5.2. Targeted genome modification 6. Summary References 299 299 300 300 301 301 301 303 304 304 307 307 307 307 1. INTRODUCTION Over 100 billion neurons, specifically and intri- cately connected, constitute the human brain. This organizational feat requires neurons to be generated in correct numbers, in defined locations and at specific developmental stages. Neuronal processes must then follow appropriate pathways to their targets and finally make the correct connections. The functioning adult brain is testimony to the high degree of fidelity maintained throughout this bewil- dering construction task, very little of which appears to be left to chance. As a first step towards unravelling the complexities of brain structure and function, there is a lot to be said for the study of less complex systems. One of these is the nervous system of the fruit fly, Drosophila melanogaster. Its relative simplicity, combined with the unparalleled genetic opportunities in Drosophila, have much to recommend it as a model. This article describes our own laboratory approaches, in par- ticular the use of reverse genetics for the phenotypic *To whom correspondence should be addressed. dissection of nervous system signal transduction pathways and the generation of Drosophila lines in which specific elements of the nervous system are marked by expression of the yeast transcription factor, GAL4. 2. SITE-SELECTED MUTAGENESIS Though genetics is traditionally a discipline in which recognition of a mutant phenotype precedes characterization of the pertinent gene, a large number of fly counterparts of genes relevant to mammalian brain structure and function have been cloned solely on the basis of DNA sequence homology. Other genes expressed specifically or predominantly in the Drosophila nervous system have been cloned, by this laboratory and by others, via differential/subtractive hybridization methods. Only rarely, however, has such a gene been found to correspond to a pre-exist- ing Drosophila mutation. It is therefore clearly desir- able that reverse genetic approaches, enabling the generation of mutants corresponding to any chosen gene, be developed. 299