Academic journal article Genetics

Spatial and Temporal Control of Gene Expression in Drosophila Using the Inducible GeneSwitch GAL4 System. I. Screen for Larval Nervous System Drivers

Academic journal article Genetics

Spatial and Temporal Control of Gene Expression in Drosophila Using the Inducible GeneSwitch GAL4 System. I. Screen for Larval Nervous System Drivers

Article excerpt

ABSTRACT

There is a critical need for genetic methods for the inducible expression of transgenes in specific cells during development. A promising approach for this is the GeneSwitch GAL4 system of Drosophila. With GeneSwitch GAL4 the expression of upstream activating sequence (UAS) effector lines is controlled by a chimeric GAL4 protein that becomes active in the presence of the steroid RU486 (mifepristone). To improve the utility of this expression system, we performed a large-scale enhancer-trap screen for insertions that yielded nervous system expression. A total of 204 GeneSwitch GAL4 lines with various larval expression patterns in neurons, glia, and/or muscle fibers were identified for chromosomes I-III. All of the retained lines show increased activity when induced with RU486. Many of the lines reveal novel patterns of sensory neurons, interneurons, and glia. There were some tissue-specific differences in background expression, with muscles and glia being more likely to show activity in the absence of the inducing agent. However, >90% of the neuron-specific driver lines showed little or no background activity, making them particularly useful for inducible expression studies.

TARGETED gene expression is a powerful technique for analyzing neural development and function in Drosophila. For example, the selective expression of constructs such as modified ion channels, which alter the electrical properties of neurons, can be used to control neural activity (PARADIS et al. 2001; WHITE et al. 2001; NITABACH et al. 2002; MOSCA et al. 2005; L UAN et al. 2006). However, these constructs often have potent and systemic effects when expressed constitutively, and their effective use requires fine spatial and temporal control of expression. The aim of this study is to developmolecular tools for confining transgene expression to well-defined subsets of neurons, muscle fibers, or glia at specific times during development. We have focused on the larval nervous system of Drosophila as it provides a favorable model system for studying neuronal circuitry and synaptic plasticity.

In Drosophila, several techniques exist that give both temporal and spatial control over gene expression (MCGUIRE et al. 2004). Many of these approaches are derived from the widely used bipartite GAL4-upstream activating sequence (UAS) system (BRAND and P ERRIMON 1993). In this system, the transcriptional activator GAL4 is expressed in a spatially restricted pattern using a tissue-specific promoter. GAL4 then activates the expression of transgenes under UAS control in tissues that express GAL4. While this system provides no independent control over the timing of transgene expression, a number of methods that add temporal control to the GAL4-UAS expression system have now been developed (HAN et al. 2000; OSTERWALDER et al. 2001; ROMAN et al. 2001; MCGUIRE et al. 2003). These either use modified steroid-activated versions of GAL4 to achieve inducible expression or introduce a temperature-sensitive GAL4 repressor (GAL80ts) to regulate GAL4 expression. Both of these approaches show significant promise for controlling gene expression in both time and space. Alternative tetracycline-inducible systems have also been developed (BELLO et al. 1998; BIESCHKE et al. 1998; STEBBINS and YIN 2001; STEBBINS et al. 2001), but are limited by the small number of tetO-dependent transgenes currently available.

Steroid-activated chimeric GAL4 proteins have been independently developed by three different groups (HAN et al. 2000; OSTERWALDER et al. 2001; R OMAN et al. 2001). Each protein combines the GAL4 DNA-binding domain with a steroid hormone receptor transcriptional activation domain, which requires ligand binding to become transcriptionally active. The GAL4 estrogen-receptor fusion protein (GAL4-ER) becomes active in the presence of estrogen (HAN et al. 2000), while GAL4-progesterone-receptor fusion proteins (Gene Switch, here referred to as GeneSwitch GAL4; Figure 1) are activated by RU486 (mifepristone) (O STERWALDER et al. …

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