Faculty Bibliography

INTRODUCTIONThe GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.

INTRODUCTIONThe generation of gain-of-function phenotypes by ectopic expression of known genes provides a powerful complement to the genetic approach, in which genes are studied or identified through mutations that generally reduce or eliminate gene function. The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. A key advantage of the system is the separation of the GAL4 protein from its target gene in distinct transgenic lines, which ensures that the target gene is silent until the introduction of GAL4. Recent modifications and adaptations of the GAL4 system to make the system inducible have further expanded its scope, enabling greater temporal control over the activity of GAL4. There are now large resources for the community, including thousands of GAL4 lines and a wide selection of reporter lines. Here we present an overview of the GAL4 system, highlighting recent developments and discussing methods for generating and analyzing transgenic flies for GAL4-mediated ectopic expression.

Asymmetric cell division is an important and conserved strategy in the generation of cellular diversity during animal development. Many of our insights into the underlying mechanisms of asymmetric cell division have been gained from Drosophila, including the establishment of polarity, orientation of mitotic spindles and segregation of cell fate determinants. Recent studies are also beginning to reveal the connection between the misregulation of asymmetric cell division and cancer. What we are learning from Drosophila as a model system has implication both for stem cell biology and also cancer research.

During development, many neural stem cells "age" as they sequentially generate distinct neuronal or glial cell types. In this issue, Maurange et al. (2008) now identify the temporal control factors in Drosophila neural stem cells (neuroblasts) that regulate the fate of stem cell progeny and signal the end of stem cell proliferation.

Drosophila neuroblasts are similar to mammalian neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. In the past two decades, great advances have been made in understanding the molecular mechanisms underlying embryonic neuroblast formation, the establishment of cell polarity and the temporal regulation of cell fate. It is now a challenge to connect, at the molecular level, the different cell biological events underlying the transition from neural stem cell maintenance to differentiation. Progress has also been made in understanding the later stages of development, when neuroblasts become mitotically inactive, or quiescent, and are then reactivated postembryonically to generate the neurons that make up the adult nervous system. The ability to manipulate the steps leading from quiescence to proliferation and from proliferation to differentiation will have a major impact on the treatment of neurological injury and neurodegenerative disease.

Over the past decade the adoption and refinement of the GAL4 system by the Drosophila field has resulted in a wide array of tools with which the researcher can drive transgene expression in a precise spatiotemporal pattern. The GAL4 system relies on two components: (1) GAL4, a transcriptional activator from yeast, which is expressed in a tissue-specific manner and (2) a transgene under the control of the upstream activation sequence that is bound by GAL4 (UASG). The two components are brought together in a simple genetic cross. In the progeny of the cross, the transgene is only transcribed in those cells or tissues expressing the GAL4 protein. Recent modifications of the GAL4 system have improved the control of both the initiation and the spatial restriction of transgene expression. Here we describe the GAL4 system highlighting the properties that make it a powerful tool for the analysis of gene function in Drosophila and higher organisms.

Stem cells can divide symmetrically to generate two similar daughter cells and expand the stem cell pool or asymmetrically to self-renew and generate differentiating daughter cells. The proper balance between symmetric and asymmetric division is critical for the generation and subsequent repair of tissues. Furthermore, unregulated stem cell division has been shown to result in tumorous overgrowth. The Drosophila nervous system has proved to be a fruitful model system for studying the biology of neural stem cell division and uncovering the molecular mechanisms that, when disrupted, can lead to tumor formation. We are using the Drosophila embryonic and larval nervous systems as models to study the regulation of symmetric and asymmetric stem cell division.

The correct control of gene expression is essential for the proper development of organisms. Abnormal expression of genes can lead to cancerous growth and certain diseases. To understand how gene expression is controlled on a genome-wide scale, methods for assaying transcription factor binding sites are required. There are two prevailing techniques for mapping protein-chromatin interactions, ChIP (chromatin immunoprecipitation) and DamID (DNA adenine methyltransferase identification). Both of these methods, when combined with microarray technology, can provide powerful insights into transcription factor function, higher order chromatin structure and gene regulatory networks. In vivo chromatin profiling studies are now being performed on model organisms, targeting specific tissues to help generate more accurate maps of protein-DNA interactions.