**7. Conclusions**

To understand the complex etiology and genetics of congenital heart disease, synergistic efforts from all fields of medical and biological sciences are required. For many decades, the invertebrate model organism *Drosophila* has provided exciting new insights into the genetics, development and function of multi-cellular organisms. In this review, we have highlighted some of the recent advances and findings gained from a *Drosophila* model for CHD. Despite its evolutionary distance from vertebrates there is a remarkable conservation of genetics and function. The development of technologies such as time-lapse analysis of heart formation and optical techniques to study function suggest that further studies using this system will provide insights into fundamental cellular mechanisms underlying heart function and disease. The fly has been shown to be a useful model that is able to complement the shortcomings of other model systems. Its simpler genetic architecture allows researchers to dissect the basic networks involved in organ formation and by extension to gain insights into the genetics underlying CHD and cardiac diseases in the same way that the *Drosophila* model has advanced our understanding of human genetics and embryonic development.

*Drosophila* Model of Congenital Heart Diseases 151

Fig. 2. Morphological features of the adult *Drosophila* heart and determination of structural and functional parameters for phenotypic analysis. Overview: The adult *Drosophila* heart is a contractile tube located at the dorsal midline of the abdomen. Along the heart, specialized cells and structures can be identified: 5 pairs of inflow valves (ostia, red) and 3 pairs of valves inside

Fig. 1. **A.** Morphology of the late embryonic heart of *Drosophila*. After 17 hours of development, the cardiac precursor cells have completed migration and heart assembly. The heart is located underneath the epidermis along the dorsal midline. It consists of two morphologically different portions, the anterior aorta (spanning segments T3-A4) and the posterior heart proper (segment A4-A7), which is characterized by a much wider lumen. The two major cell types are cardioblasts (CBs), which will differentiate into cardiomyocytes, and pericardial cells (PCs), which will become nephrocyte-like cardiac support cells. The heart is also connected to specialized lateral body wall muscles, named alary muscles. The cardioblast nuclei can be specifically labeled (e.g. by anti-Nmr1 antibody, green) to assess CB alignment. Cell surfaces are stained using Dystroglycan antibody (red). The aorta and heart contain a central (HL). **B.** Heart assembly, visualized by a time-lapse movie of cardiac cells expressing actinGFP. Before alignment, two lateral rows of CBs and PCs migrate towards the dorsal midline (indicated as hatched line). The CBs elongate at the dorsal side and extend filopodia towards the contralateral side to form the dorsal contact. Following this contact, the cells change in shape to contact ventrally, thereby enclosing a luminal space.

Fig. 1. **A.** Morphology of the late embryonic heart of *Drosophila*. After 17 hours of

change in shape to contact ventrally, thereby enclosing a luminal space.

development, the cardiac precursor cells have completed migration and heart assembly. The heart is located underneath the epidermis along the dorsal midline. It consists of two morphologically different portions, the anterior aorta (spanning segments T3-A4) and the posterior heart proper (segment A4-A7), which is characterized by a much wider lumen. The two major cell types are cardioblasts (CBs), which will differentiate into cardiomyocytes, and pericardial cells (PCs), which will become nephrocyte-like cardiac support cells. The heart is also connected to specialized lateral body wall muscles, named alary muscles. The cardioblast nuclei can be specifically labeled (e.g. by anti-Nmr1 antibody, green) to assess CB alignment. Cell surfaces are stained using Dystroglycan antibody (red). The aorta and heart contain a central (HL). **B.** Heart assembly, visualized by a time-lapse movie of cardiac cells expressing actinGFP. Before alignment, two lateral rows of CBs and PCs migrate towards the dorsal midline (indicated as hatched line). The CBs elongate at the dorsal side and extend filopodia towards the contralateral side to form the dorsal contact. Following this contact, the cells

Fig. 2. Morphological features of the adult *Drosophila* heart and determination of structural and functional parameters for phenotypic analysis. Overview: The adult *Drosophila* heart is a contractile tube located at the dorsal midline of the abdomen. Along the heart, specialized cells and structures can be identified: 5 pairs of inflow valves (ostia, red) and 3 pairs of valves inside

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the tube. The anterior portion of the heart shows a prominent specialization (the conical chamber, which is larger in size) and anastomoses into the aorta that runs from the posterior end of the thorax into the head capsule. Several pairs of alary muscles are attached to the heart tube, which are likely to help to maintain heart position. The pericardial cells are found alongside the heart and have a nephrocyte-like as well as other cardiac support functions. The ventral longitudinal layer consists of several multi-nucleated muscle cells that ensheath the heart from A1 to about mid-A5 (indicated as VLL in transverse section of the conical chamber). Anatomical features: The conical chamber is the largest chamber of the adult fly heart (compare transverse sections 1+2). The VLL that ventrally and laterally covers the heart can be seen in transverse section 1. Myofibrillar structure: The cardiomyocytes of the conical chamber are much larger in size and have a higher acto-myosin content compared to other regions of the heart. The cardiomyocytes of the valves show a very dense packaging of myofibrils compared to regular cardiomyocytes of the heart. Cardiac physiology: High-speed image capturing from semi-dissected fly hearts allows determination of several parameters, which are indicative for fly heart morphology and function: the diameters of the heart during diastole (DD) and systole (SD) are determined from the original movies. M-modes are generated by aligning a 1-pixel wide strip from each frame showing the location of the heart walls (Y axis) over time (X-axis) (Ocorr et al., 2007). Heart rate, durations of diastoles and systoles (DI and SI) and rhythmicity are determined by semi-automated analysis using MATlab-based software (Fink et al. 2009).
