A genetic resource for complex cell-cell interaction studies in Drosophila

To study the processes involved in development, tissue maintenance, or higher order interactions of complex tissues such as the brain, researchers need tools that allow for the manipulation of specific cells in time and space. This can be achieved by combining a regulatory element (enhancer) with a gene of interest. The enhancer, in this new context, will then control when and where the gene of interest is expressed. Since there are thousands of different genes and even more cells in an organism, it would be very difficult to generate a specific enhancer/gene of interest combination for each particular manipulation.
An elegant solution to this problem, is the use of binary expression systems which separate the enhancer from the gene of interest. The enhancer is now part of a control unit called "driver" which regulates the expression of a DNA binding protein while the gene of interest is combined with binding sites for the DNA binding protein into a functional unit termed "responder". Upon activation of the enhancer, the DNA binding protein is made and binds to its recognition sites next to the gene of interest and triggers its expression. Due to this architecture, driver and responder elements can be completely separated in two different animal lines and only when crossed together will the offspring express the gene of interest in the desired pattern. This allows for the creation of vast driver and responder collections which can be freely combined to generate thousands of different combinations without the need of establishing permanent transgenic animals.
However, complex cell-cell interaction studies may require several different manipulations at the same time, e.g. the up-regulation of a gene in one cell and the down-regulation of another gene in the neighbouring cell. To achieve this, several different binary expression systems need to be employed simultaneously. To date, only three binary systems are available, which limits the maximum number of parallel manipulations to three. To overcome this limitation, we have developed a new binary expression system based on a DNA binding protein (TALE) that can be altered to recognise different binding sites (VAS). This new TALE-VAS system has the potential to generate an almost unlimited number of driver/responder pairs. However, to be readily applicable for other researchers, a basic resource of drivers and responders is needed.
In this project, we propose to generate this basic resource and to further enhance and modify the TALE-VAS system in order to provide a valuable and versatile genetic tool for the scientific community. In particular, we will ensure that all TALE-VAS driver-responder pairs are completely background-free. Additionally, we will modify the TALE drivers so that they can be turned off by a repressor, have a faster turnover rate, or work with the existing split technology where the driver can be separated into two components to achieve better spatial resolution. We will also provide a new split system that will allow for the simultaneous employment of many split drivers simultaneously. In parallel, we will generate donor flies that can be used to convert existing drivers from the most widely used binary expression system, the GAL4-UAS system, to the TALE-VAS system. Finally, we will further extend the range of applications of the TALE-VAS system by generating logic gates ("switches") that will only activate the gene of interest if several conditions are true. For example, activation occurs only if gene-1 AND gene-2 AND gene-3 are expressed, or if gene-1 AND gene-2 but NOT gene-3 are expressed. Using this system, researchers will be able to manipulate cell populations that are characterised by complex gene expression patterns and are difficult to address otherwise. Taken together, the basic TALE-VAS toolkit will greatly benefit the scientific community and will enable researchers to perform cell-cell interaction studies of unprecedented complexity.

Cell-cell interactions are a crucial component of many biological processes such as development or neuronal activity. The ability to study these events relies on tools capable of manipulating cells with precise spatial and temporal resolution. Binary expression systems are very powerful tools especially designed for this task. However, complex interactions like the ones found in the brain, require even more sophisticated technologies. We have therefore developed a new binary expression system, the TALE-VAS system, which is capable of multiple parallel manipulations. Here, we propose to further enhance this system and to turn it into a versatile genetic resource that can easily be applied by other researchers. To this end, we will generate five background-free TALE-VAS pairs, a version of each TALE that is repressible by GAL80, split TALEs fully compatible with existing split libraries, new orthogonal split TALEs based on the coiled-coil technology, and degradation tagged TALEs suitable for studies requiring faster turnover rates. Furthermore, we will provide donor fly libraries for easy and convenient in vivo GAL4 to TALE conversion. This will either be based on the Cas9 mediated HACK strategy or the integrase and recombinase driven InSITE system. Both will make two large resources accessible and enable researchers to convert almost any GAL4 line using genetics. Finally, we will add a unique logic gate component which will significantly expand the spectrum of possible applications. In this technology, the TALEs will be used as input to drive the expression of recombinases which in turn will act on logic gates by removing regulatory elements. This will enable researchers to manipulate cell populations that are defined by a combinatorial gene expression pattern and cannot be addressed otherwise. We anticipate that the proposed new resource will be a valuable addition to the existing repertoire of genetic tools and will greatly benefit the scientific community.