Drosophila, genetic screens, and cardiac function

Abstract

The fruit fly, Drosophila melanogaster, has been used to study genetics, development, and signaling for nearly a century, but only over the past few decades has this tremendous resource been the focus of cardiovascular research. Fly genetics offers sophisticated transgenic systems, molecularly defined genomic deficiencies, genome-wide transgenic RNAi lines, and numerous curated mutants to perform genetic screens. As a genetically tractable model, the fly facilitates gene discovery and can complement mammalian models of disease. The circulatory system in the fly comprises well-defined sets of cardiomyocytes, and methodological advances have permitted accurate characterization of cardiac morphology and function. Thus, fly genetics and genomics offer new approaches for gene discovery of adult cardiac phenotypes to identify evolutionarily conserved molecular signals that drive cardiovascular disease.

Figure 1. The Drosophila melanogaster genome

(A) The karyotyping of D. melanogaster prepared from the salivary gland showing chromosomes X, 2L/R, and 3L/R. The Y and fourth chromosomes are not depicted. (B) An enlarged view of the 3L chromosome with cytological map showing bands 61 through 80 (above) and molecularly-defined deficiencies (below). (C) An enlarged view of the 61D through 62A cytological map showing predicted genes encoded within the region and corresponding molecularly-defined genomic deficiencies from the Exelixis and DrosDel collections.^– Figures adapted from http://flystocks.bio.indiana.edu.

Figure 2. Transgenic-Expression Systems in Drosophila

(A) The Gal4-UAS bipartite transgene expression approach relies on breeding transgenic flies that harbor either a tissue-specific promoter that drives Gal4 production or UAS-transgene constructs. The progeny possess one copy of the Gal4 and UAS constructs and express the transgene of interest under the control of the promoter of interest. This system allows versatility by using different promoters to control transgene expression. (B) The ubiquitous expression of a temperature-sensitive Gal80 (Gal80^ts) is incorporated into the Gal4-UAS system to add temporal control of transgene expression using a shift from a restrictive to permissive temperature. Gal80^ts reversibly suppresses Gal4 activity at 18°C and permits Gal4 binding to UAS at 29°C.

Figure 3. Targeted gene knockout and gene replacement in Drosophila

(A) The “ends-out” approach for gene knockout. A fly harboring a transgenic construct containing the mini-white gene within the genomic sequence of a gene targeted for replacement is bred with a fly that harbors an inducible FLP recombinase and I-SceI homing enzyme. In a subset of progeny, the targeting construct is excised by the FLP recombinase, linearized by I-SceI, and undergoes homologous recombination with the endogenous gene. The endogenous gene is disrupted by replaced with the mini-white gene. (B) The “ends-out” approach for gene replacement. A fly harboring a transgenic construct that contains an engineered mutation (asterisk) and the mini-white gene within the genomic sequence of a gene targeted for replacement is bred with a fly that harbors an inducible FLP recombinase and I-SceI homing enzyme. In a subset of the first generation, the targeting construct is excised by FLP recombinase, linearized by I-SceI, and undergoes homologous recombination with the endogenous gene locus to produce tandem gene duplication. Then, the fly containing the gene duplication is bred with a fly that harbors an inducible Cre recombinase that recognizes the I-CreI site that is engineered in the targeting construct. In a subset of the progeny, the tandem genes undergo homologous recombination and reduction of gene copy number thereby producing flies that harbor either a wild-type gene or the mutant gene. The figure is based on Maggert, K.A. et al

Figure 4. The embryonic and adult Drosophila circulatory system

(A) The developing embryonic circulatory system arises from cardial precursor cells that migrate to form the dorsal vessel at Stage 16. Stages 12, 13, and 17 are shown. The figure is adapted from Fly Embryo RNAi Project (http://flyembryo.nhlbi.nih.gov). (B) The adult fly circulatory system consists of an open system with the main conical chamber, heart, located along the dorsal aspect of the A1 abdominal segment. Suspensory muscles including the Alary and Ventral Longitudinal Muscle also referred to as the Dorsal Diaphragm. Pericardial cells are closely juxtaposed along the length of the abdominal portion of the circulatory system. The figure is adapted from Miller. A 1 mm scale bars are shown for comparison.

Figure 5. Strategies to assess adult Drosophila cardiac morphology and function

(A) A dissected adult fly heart perfused in artificial hemolymph is shown. Abdominal segments are denoted as A1, A2, A3, and A4. This specimen preparation can be used to assess heart rate and rhythm, myocardial calcium handling, cardiac morphology, and cardiac function. (B) An example of cardiac morphology in a tinC-GFP transgenic fly depicting the cardiac tube (green) and actin stained with phalloidin (red) using confocal microscopy with Z-stack reconstruction as previously described.(C) Examples of heart rate and rhythm obtained using a Leica 165FC steromicroscope equipped with an Andor iXon high-speed camera. Heart rate was measured by examining movement of the lateral wall as described by Wessells and Bodmer.^, An example of the effects of Diltiazem, a calcium-channel blocker, is shown. (D) Representative m-mode of cardiac function was obtained using high-speed brightfield imaging similar to methods as described. M-modes showing w¹¹¹⁸ (control) and a dilated cardiomyopathic mutant are shown for comparison. (E) An example of myocardial calcium transients measured in adult transgenic flies that had cardiac-specific expression of GCaMP2 in a w1118 background (control) or in the presence of hdp2, a mutation in Troponin-I, that has a dilated cardiomyopathy phenotype are shown.(F) Longitudinal B-mode OCT image during diastole superimposed on a live adult fly (left) and transverse B-mode OCT during diastole and systole with representative M-mode OCT showing cardiac chamber size and function in w¹¹¹⁸ (control) and a mutant that has dilated cardiomyopathy as described by Wolf et. al.(G) Example of transverse sectioning and hemotylin/eosin staining of a fixed adult fly. The cardiac chamber (CC) and surrounding tissues including the ventral longitudinal muscle (VML) are shown. The image is adapted from Yu et. al.