**4. Manipulating the heart and genome of a fly**

Parallel pathways and downstream signaling cascades are thought to intersect with a number of cellular effector proteins, such as small GTPases, which in turn may influence cell migration (e.g. changes in the filopodia or lamellipodia dynamics), cell adhesion (e.g. changes in endocytosis of E-Cadherin) or cell contractions (e.g. via Rho-associated kinase activities). The activity of these genes has been studied in great detail in cell-based assays, but experimental evidence on their *in vivo* function is relatively sparse. The lack of available mutants in vertebrate model organisms often prevents such analysis, as has the shortage of tools for tissue-specific manipulations and imaging of single cells in whole animals. Therefore, very few examples of the function these proteins in the context of an entire organ or organ system exist to date (e.g. RhoDF, see Christiaen et al., 2008). In *Drosophila*, small GTPases involved in the above cellular processes have been studied by genetic manipulations during the formation of tissues other than the heart, e.g. during dorsal closure (Jacinto et al., 2002) or wound healing (Stramer et al., 2005). The embryonic *Drosophila* heart is well suited to similar experiments since it is localized just underneath the transparent cell layer of the dorsal epidermis, which allows capture of high quality

*Drosophila* Model of Congenital Heart Diseases 145

over the activity of QF, just like Gal80 and Gal80TS for Gal4. A combination of both the Gal4 and QF systems therefore would allow distinct expression of multiple transgenes in a precise tissue-specific and temporal-specific manner in an otherwise unchanged genetic

Recent advances in RNA interference (RNAi) technology, combined with the spatiotemporal control of the Gal4/UAS system, have allowed tissue-specific studies of gene function during almost any developmental stage. RNAi has therefore been useful to analyze genes that when mutated would cause early lethality or pleiotropic effects, but it is also the only method currently available to study gene function when no mutant alleles for a

Systematic analysis to determine optimal hairpin formation and careful analysis of insertion sites have greatly increased the efficacy of RNAi (Ni et al., 2008; 2009; 2011). The Transgenic RNAi project (TRiP) is currently generating these optimized RNAi lines for all *Drosophila* genes, which complements other RNAi resources (like VDRC, Dietzl et al., 2007). As an alternative reverse genetic approach, the directed mutation or knockout of a particular gene of interest by homologous recombination (reviewed in Maggert et al., 2008) has also been developed further. In addition to generating a knockout allele, Huang et al. (2009) have added a recombinase-based feature that allows modification of the deleted locus by inserting virtually any sequence ("genomic engineering"). Of note, this permits modification of gene function in an otherwise unaltered genetic background. Furthermore, efforts to create genomic duplications for regions of the X chromosome have resulted in the creation of two independent sets of fly lines, one set with a duplication located on the Y chromosome (Cook et al., 2010) and one set on the 3rd chromosome (Venken et al., 2010). This will facilitate the recovery and identification of X-linked mutations and also allow assessment of the fly's susceptibility to increased gene dosages. These improved techniques of Gal4/UAS transactivation, paired with the expression of fluorescently tagged reporters and RNAi lines as well novel forward and reverse genetic techniques and resources are likely to unravel new, previously unnoticed gene functions in different tissues and under different

**5. Exploring fly heart function to understand CHD-related cardiomyopathies**  By definition, congenital heart disease refers to the presence of structural heart and large vessel defects at the time of birth. Many of these can be repaired by surgical intervention, but depending on the severity of the defect, patients may require life-long medical followups to monitor cardiac performance and to detect signs of functional decline. Furthermore, late onset complications either due to persistent impact of structural defects or related to the applied intervention are often linked to lethal arrhythmias or progressive congestive heart failure. Such secondary complications might not be caused directly by CHD genes, but due to a maladaptive response of the cardiac tissue. In the light of these considerations it is therefore necessary to understand how cardiac tissues respond to these interventions and which other genes might contribute to arrhythmias and cardiomyopathies. The *Drosophila* model has helped to identify a number of novel genes and pathways required to maintain myofibrillar organization and overall heart structure and function. Because similarities between the *Drosophila* and murine heart are found both at the molecular and functional level it is therefore likely that new risk factors of cardiac disease can be identified using *Drosophila*. The cardiac proteasomes of the mouse and the fly have been shown to be

background.

particular gene are available.

developmental contexts.

fluorescent images *in vivo* (see Figure 1B). At the same time, cardiac cells can easily be manipulated using cardiac-specific Gal4-driver lines (see below) to express GFP-tagged genes, e.g. of the actin cytoskeleton, specifically in the heart. This allows the effects of specific mutations on actin dynamics to be monitored during heart formation (Medioni et al., 2008). Furthermore, a large number of fluorescently labeled genes such as actinGFP that can be overexpressed are readily available from different laboratories and stock centers. Browsing through Flybase (Crosby et al., 2007; Tweedie et al., 2009), a *Drosophila* centered database, allows easy access to the records for any published construct. The power of the *Drosophila* model is the ability to combine mutant alleles of almost any gene with tissuespecific expression of fluorescently labeled markers. Thus, heart development can be studied in great detail at the organ or even the cellular level, permitting the role of individual genes to be examined in the context of a specific cellular function. This approach provides an experimental resolution that is unparalleled in any other model organism.

The technological advances in the *Drosophila* model are steadily growing. The Gal4/UAS system (Brand and Perrimon, 1993) had been groundbreaking for tissue-specific genetic manipulations, and continues to be further refined (Osterwalder et al., 2001; McGuire et al., 2004; Pfeiffer et al., 2010; Gohl et al., 2011). Gal4, a transcriptional activator from yeast and without endogenous binding sites in the *Drosophila* genome, is used to trans-activate genes that are engineered to contain Gal4-binding sites (upstream activating sequences, UAS). This heterologous system allows the expression of any UAS-fused gene in any tissue where Gal4 is expressed, which itself is driven by tissue-specific promoters. Gal4 and UAS-lines can be created either by random insertion of transposable elements into the fly's genome (Cooley et al., 1988) or targeted insertion at particular "landing sites" (Fish et al., 2007). The first method has been extensively used to create a vast amount of "enhancer trap" lines that express the Gal4 driver in many different, tissue-specific patterns including the heart. A recent technique by Gohl et al. (2011) has further increased the versatility of such Gal4-enhancer trap lines by developing a method to replace the Gal4 driver with any other reporter (e.g. GFP) or effector gene (e.g. the Gal4 repressor Gal80, which will inhibit the Gal4 activity of a different line in the intersecting cells). The Gal4/UAS system not only allows selective expression of marker genes in specific tissues (e.g. Figure 1B, expression in cardiac tissue using tinC∆4-Gal4, Lo and Frasch, 2001), but also permits genetic manipulations by ectopic or overexpression of genes or by reducing their expression levels using RNA interference (RNAi, see below). In combination with lines that express the Gal80 repressor in a subset of Gal4-positive cells, the Gal4 expression pattern can be further spatially refined. In addition, use of a temperature-sensitive version of Gal80 (Gal80-TS, McGuire et al., 2004) gives temporal control over Gal4 expression, which then becomes active only under the permissive temperature.

One limitation of the Gal4/UAS system is that all transgenes that carry UAS sites respond at the same time. Therefore, different tissues or cells cannot be manipulated individually, although this could be a useful approach to study their interaction. The recent invention of the Q system (Potter et al., 2010) is a novel approach to circumvent this limitation. It works in a similar manner as Gal4/UAS but uses the *Neurospora* transcriptional activator QF, which recognizes its own specific binding sequence (QUAS). Just like Gal4, a fly line that expresses QF in a certain tissue or cell will drive expression of a gene that contains the QUAS binding sites. Similar to Gal80, the activity of QF can be suppressed by expression of QS (allowing further refinement of QF expression), and feeding flies quinic acid releases this suppression in a dose-dependent manner. Thus, QS gives both, spatial and temporal control

fluorescent images *in vivo* (see Figure 1B). At the same time, cardiac cells can easily be manipulated using cardiac-specific Gal4-driver lines (see below) to express GFP-tagged genes, e.g. of the actin cytoskeleton, specifically in the heart. This allows the effects of specific mutations on actin dynamics to be monitored during heart formation (Medioni et al., 2008). Furthermore, a large number of fluorescently labeled genes such as actinGFP that can be overexpressed are readily available from different laboratories and stock centers. Browsing through Flybase (Crosby et al., 2007; Tweedie et al., 2009), a *Drosophila* centered database, allows easy access to the records for any published construct. The power of the *Drosophila* model is the ability to combine mutant alleles of almost any gene with tissuespecific expression of fluorescently labeled markers. Thus, heart development can be studied in great detail at the organ or even the cellular level, permitting the role of individual genes to be examined in the context of a specific cellular function. This approach provides an experimental resolution that is unparalleled in any other model organism. The technological advances in the *Drosophila* model are steadily growing. The Gal4/UAS system (Brand and Perrimon, 1993) had been groundbreaking for tissue-specific genetic manipulations, and continues to be further refined (Osterwalder et al., 2001; McGuire et al., 2004; Pfeiffer et al., 2010; Gohl et al., 2011). Gal4, a transcriptional activator from yeast and without endogenous binding sites in the *Drosophila* genome, is used to trans-activate genes that are engineered to contain Gal4-binding sites (upstream activating sequences, UAS). This heterologous system allows the expression of any UAS-fused gene in any tissue where Gal4 is expressed, which itself is driven by tissue-specific promoters. Gal4 and UAS-lines can be created either by random insertion of transposable elements into the fly's genome (Cooley et al., 1988) or targeted insertion at particular "landing sites" (Fish et al., 2007). The first method has been extensively used to create a vast amount of "enhancer trap" lines that express the Gal4 driver in many different, tissue-specific patterns including the heart. A recent technique by Gohl et al. (2011) has further increased the versatility of such Gal4-enhancer trap lines by developing a method to replace the Gal4 driver with any other reporter (e.g. GFP) or effector gene (e.g. the Gal4 repressor Gal80, which will inhibit the Gal4 activity of a different line in the intersecting cells). The Gal4/UAS system not only allows selective expression of marker genes in specific tissues (e.g. Figure 1B, expression in cardiac tissue using tinC∆4-Gal4, Lo and Frasch, 2001), but also permits genetic manipulations by ectopic or overexpression of genes or by reducing their expression levels using RNA interference (RNAi, see below). In combination with lines that express the Gal80 repressor in a subset of Gal4-positive cells, the Gal4 expression pattern can be further spatially refined. In addition, use of a temperature-sensitive version of Gal80 (Gal80-TS, McGuire et al., 2004) gives temporal control over Gal4 expression,

which then becomes active only under the permissive temperature.

One limitation of the Gal4/UAS system is that all transgenes that carry UAS sites respond at the same time. Therefore, different tissues or cells cannot be manipulated individually, although this could be a useful approach to study their interaction. The recent invention of the Q system (Potter et al., 2010) is a novel approach to circumvent this limitation. It works in a similar manner as Gal4/UAS but uses the *Neurospora* transcriptional activator QF, which recognizes its own specific binding sequence (QUAS). Just like Gal4, a fly line that expresses QF in a certain tissue or cell will drive expression of a gene that contains the QUAS binding sites. Similar to Gal80, the activity of QF can be suppressed by expression of QS (allowing further refinement of QF expression), and feeding flies quinic acid releases this suppression in a dose-dependent manner. Thus, QS gives both, spatial and temporal control over the activity of QF, just like Gal80 and Gal80TS for Gal4. A combination of both the Gal4 and QF systems therefore would allow distinct expression of multiple transgenes in a precise tissue-specific and temporal-specific manner in an otherwise unchanged genetic background.

Recent advances in RNA interference (RNAi) technology, combined with the spatiotemporal control of the Gal4/UAS system, have allowed tissue-specific studies of gene function during almost any developmental stage. RNAi has therefore been useful to analyze genes that when mutated would cause early lethality or pleiotropic effects, but it is also the only method currently available to study gene function when no mutant alleles for a particular gene are available.

Systematic analysis to determine optimal hairpin formation and careful analysis of insertion sites have greatly increased the efficacy of RNAi (Ni et al., 2008; 2009; 2011). The Transgenic RNAi project (TRiP) is currently generating these optimized RNAi lines for all *Drosophila* genes, which complements other RNAi resources (like VDRC, Dietzl et al., 2007). As an alternative reverse genetic approach, the directed mutation or knockout of a particular gene of interest by homologous recombination (reviewed in Maggert et al., 2008) has also been developed further. In addition to generating a knockout allele, Huang et al. (2009) have added a recombinase-based feature that allows modification of the deleted locus by inserting virtually any sequence ("genomic engineering"). Of note, this permits modification of gene function in an otherwise unaltered genetic background. Furthermore, efforts to create genomic duplications for regions of the X chromosome have resulted in the creation of two independent sets of fly lines, one set with a duplication located on the Y chromosome (Cook et al., 2010) and one set on the 3rd chromosome (Venken et al., 2010). This will facilitate the recovery and identification of X-linked mutations and also allow assessment of the fly's susceptibility to increased gene dosages. These improved techniques of Gal4/UAS transactivation, paired with the expression of fluorescently tagged reporters and RNAi lines as well novel forward and reverse genetic techniques and resources are likely to unravel new, previously unnoticed gene functions in different tissues and under different developmental contexts.
