CARL ORFF

For the analysis of neural circuits forward genetic methods can be very useful. InDrosophila transgene expression systems have been established in the past 25 years and since then developed considerably [117, 118, 119]. These systems e.g. allow the functional dissection of a neural network by loss of function and gain of function experiments. Loss of function can be reversibly induced in classes of cells by transgene mediated blockade of neural activity. The use of such proteins to block neural activity in parallel to GECI-based imaging requires two separate systems for the expression of both transgenes in different sets of cells. Besides the classical Gal4-UAS system, I started here a second approach based on a bacterial transcription system called LexA. Next, I identified possibly interesting enhancer regions in the genome, cloned the respective genomic sequences and generated fly lines for GECI expression in visual interneurons under LexA control.

1.5.1 UAS-Gal4

Gene expression in cells and organisms is spatially and temporally well controlled. E.g., the segmentation gene fushi trarazu in Drosophila is expressed at low levels early during embryogenesis, is then upregulated and expression becomes spatially more and more re- fined until the pattern shows seven characteristic stripes around the longitudinal axis of the embryo, reflecting the even numbered parasegments 2-14 [120]. Gene expression is controlled by regulatory, non-coding DNA sequences, that are recognized by transcription factors. One type of regulatory elements is called promoter. These act on short range. An eukaryotic promoter consists of DNA motifs for RNA polymerases 50-75 bp upstream of a gene. A second class of regulatory elements are termed ’enhancers’. They act on far distances by diverse and partly unidentified mechanisms [121]. Enhancers are recognized by transcription factors and interact with the promoters of specific genes to increase the rate of transcription. Their distance from their specific promoter may be several, and even tens of kilo bases and they may reside upstream or downstream or even in exons of a gene. How enhancers exert their effect on promoters is poorly understood, but there is some evidence for the contribution of chromosomal loop structures that bring enhancer and pro- moter into physical contact or close proximity. This again may be subject to regulation by diverse elements and mechanisms. E.g. In the case offushi tarazu the interaction between different promoters and enhancers or silencers is blocked by boundaries or insulators [120]. In yeast a directional class of enhancers is called upstream activating sequence (UAS).

1.5 Expression Systems for Transgenes in Fruit Flies 27

The transcription factor Gal4 consists of a DNA binding domain, which binds to UAS and a transactivation sequence, which activates transcription of specific downstream genes. This expression system has been isolated, modified and transfered to transgenic flies to allow expression of any gene fused to the UAS sequence [118].

Enhancer-Trap

For transgene expression in flies the bipartite Gal4-UAS system is used because it allows exploitation of manifold combinations of different Gal4- and UAS-flies, and provides am- plification of transgene expression: A transgene like the cDNA for a GECI is cloned into a fly transfection vector, downstream of five UAS sequence repeats. A fly strain carrying this transgene is called the effector line and will not express the respective GECI, because it lacks the yeast transcription factor Gal4. A second fly strain is transfected with the cDNA for Gal4; this line is called the driver line. The Gal4 sequence is preceded by a weak promoter. This fusion can be randomly inserted into the genome to pick up the expression pattern of any enhancer in the genomic surround (thus enhancer-trap). The flies cellular machinery is blind for the UAS sequence and the DNA binding protein Gal4 is blind for the flies enhancer sequences. Thus, flies carrying either transgene are phenotypically wild-type - if the insertion site is not critical to some gene’s function.

When flies carrying the UAS-GECI transgene and flies carrying the Gal4 transgene are crossed, their offspring will express the GECI protein in the expression pattern of the enhancer controlling Gal4 expression. So-called libraries of driver- and effector-lines are established worldwide and can be combined for the expression of many different transgenes in many different cells. This random approach is called enhancer-trap. It requires post- transfectional screens for expression patterns of interest.

Promoter Fusions

A non-random approach to a specific expression pattern requires knowledge about a corre- sponding genomic enhancer. Often it is desirable to mimic the expression of a native gene with a transgene. Therefore several kb of non-coding DNA, mostly from the upstream regions of the native gene are cloned and fused to Gal4. The hope is that this sequence contains regulatory elements that activate expression in the desired pattern. This con- struct is inserted into the genome, in the ideal case bringing its own enhancer with it, that will eventually lead to the desired expression pattern. This expression pattern, how- ever, is hardly predictable for two reasons: First the new insertion may behave like a new

enhancer-trap. Second the genomic region fused to Gal4 may contain regulatory elements that do not produce the desired expression pattern. In the majority of cases, far distance regulatory elements show no recognized sequence characteristics and have to be sought by best guesses. Nevertheless enhancers (and other long distance regulatory elements, [121]) are often picked up in 2-10 kb upstream of a gene. E.g. 2.6 kb of genomic DNA immedi- ately upstream of the olfactory receptor gene Or83b were amplified and fused to Gal4. The Gal4 expression pattern in transgenic flies carrying this construct mimics Or83b expression in wild-type flies [88]. In the case of e.g. thefushi tarazu gene however, regulation is much more complex [120] (see 4.4).

The independent expression of two transgenes in one animal requires two separate ex- pression systems. Gal4-UAS independent expression can be achieved by direct fusion of a transgene to a presumed enhancer sequence. This system, however, lacks the amplification step provided by Gal4-UAS and often leads to insufficient transgene expression levels.

Gal80

The yeast protein Gal80 binds to the Gal4-activating domain (GAD) of Gal4, inhibiting its function. This can be used to control and refine transgene expression [122]. A temperature sensitive variant of Gal80 loses Gal4 affinity at high temperatures. This can be used as a ’temperature-switch’ for Gal4, allowing temporally controlled transgene expression [123].

1.5.2 LexA-pLOT

A second expression system, here named LexA-pLOT, has been isolated from bacteria and relies on the same logic as Gal4-UAS [124, 125]. In pLOT the bacterial operator (prokaryotic promoter) LexAop replaces UAS. The LexA protein, fused to a transcription activation domain, replaces Gal4. LexA is a bacterial DNA binding protein. It can be fused to, e.g. VP16 (the viral transcription factor VP16 acidic activation domain) or GAD (from Gal4) to function as a transcriptional activator. The GAD fusion form is susceptible to Gal80 inhibition like Gal4 [125]. The LexA-pLOT system can be applied in parallel to the Gal4-UAS system in the same animal to produce expression of two transgenes in different subsets of cells [125]. This is desired for neural network analysis by the parallel use of GECIs for calcium imaging in LPTCs and transgenes that inhibit neural activity in presynaptic partner cells.