**2. Comparison between** *Drosophila* **and vertebrate cardiogenesis**

The early development of the *Drosophila* heart shows remarkable similarities with its vertebrate counterparts, both morphologically and genetically (for review, see Bodmer, 1995; Bier and Bodmer, 2004). Our understanding of the regulation of cardiac development by a core cardiac transcription factor network (Venkatesh et al., 2000; Cripps and Olson, 2002; Olson, 2006; Bodmer and Frasch, 2010) began with the identification of the *Drosophila Nkx2.5* homologue *tinman* twenty years ago (Bodmer et al., 1990; Azpiazu and Frasch, 1993; Bodmer, 1993). One decade later, the completion of the sequencing of the *Drosophila*, mouse and human genomes has led to the identification of fly homologues of most cardiac transcription factors. The *Drosophila* model allowed the extensive genetic screening and functional analysis of Tinman (NKX2.5 Yin et al., 1997; Akasaka et al., 2006; Zaffran et al., 2006; Qian et al., 2011; Ryu et al., 2011), Hand (dHAND, eHAND, Han and Olson, 2005; Han

*Drosophila* Model of Congenital Heart Diseases 143

turn is likely to prevent further lumen formation (Santiago-Martínez et al., 2008). Both, Slit and Robo are expressed during mouse heart development and expression of Slit3 and Robo2 depend on Tbx20 and Nkx2-5, respectively (Medioni et al., 2010). Functional analysis in zebrafish done by Fish et al. (2011) indicates a role for Slit/Robo signaling during zebrafish heart development. Slit/robo mutant fish hearts show a number of developmental defects, indicating a conserved requirement for this pathway during vertebrate cardiogenesis and thus a role in CHD. Again, the analysis of Slit/Robo in *Drosophila* has paved the way for the

Recent work on fly heart development suggests several possible future research directions. Firstly, genetic screens in *Drosophila* should reveal additional genes involved in cell-cell signaling during development. Among them is the Netrin/Unc-5 pathway that, similar to Slit/Robo, was found to be involved in axon guidance and cell migration. In the heart, the UNC-5 ligand Netrin is also required for heart lumen formation (Albrecht et al., 2011), although with a lesser penetrance than Slit/Robo. This indicates that in fact multiple pathways are required during the formation of the heart, and it would be interesting to see if these two pathways genetically interact, which would indicate a potential cross talk between them. The *Drosophila* model therefore helps pinpoint which pathways may also interact in CHD in humans. Secondly, a more complete and detailed understanding of the signaling pathways themselves during heart development and establishment of cardiac function will be essential for understanding CHD initiation and progression. For example, co-receptors might play an important role in defining pathway sensitivity and downstream activity. In *Drosophila*, cardiac Slit/Robo signaling has been shown to require the activity of the heparan sulfate proteoglycan Syndecan (Knox et al., 2011). Since Syndecan is involved in angiogenesis (through VEGF, Chen et al., 2004) and is also upregulated during cardiac remodeling after myocardial infarction, the *Drosophila* model might help identify important components of Syndecan signaling in these disease-relevant contexts. Thirdly, the cellular machineries through which signaling pathways exert their specific function are largely unknown. Thus, we currently have no clear understanding of the intracellular mechanisms

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

subsequent experiments done in vertebrates.

that give rise to the *slit* mutant phenotype.

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

et al., 2006; Lo et al., 2007), tailup/isl-1 (Islet, Tao et al., 2007; Mann et al., 2009), Pannier (GATA4, Alvarez et al., 2003; Fromental-Ramain et al., 2008; Qian et al., 2008; Qian and Bodmer, 2009), Neuromancer-1/-2 (TBX20, Miskolczi-McCallum et al., 2005; Qian et al., 2005a; Reim et al., 2005; Leal et al., 2009), and Dorsocross-1/-2/-3 (TBX5, Reim and Frasch, 2005) and revealed a conserved cardiac transcription factor network responsible for heart specification (Olson, 2006). *Drosophila* and vertebrates also share the same inductive and instructive signaling pathways (Wnt, FGF, BMPs, for review see Frasch, 1999; Cripps and Olson, 2002) during early heart development. This further underscores that the *Drosophila* heart, despite its evolutionary distance from vertebrates, is specified by similar, fundamental mechanisms. This remarkable degree of genetic conservation is paralleled by morphological similarities during early development: the heart originates from a lateral portion in the early mesoderm, and two bilateral regions will eventually fuse and undergo lumen formation. Such genetic and morphological similarities across phyla have led to the conclusion that the cardiovascular system of the fly and vertebrates share true homologies (Hartenstein and Mandal, 2006). Lessons learned from *Drosophila* are likely to translate into a greater understanding of vertebrate heart development and function, and thus will help to understand the pathology and improve the treatment of cardiovascular diseases, as exemplified by Neely (2010) and Qian (2011).

#### **3. Lessons learned from studying** *Drosophila* **heart morphogenesis**

To gain new insights into the role of the cardiac transcriptional network, different groups have begun to analyze the mechanisms underlying heart morphogenesis and heart lumen formation during *Drosophila* embryonic development. After specification, cardiomyocyte precursors (called cardioblasts) migrate towards the dorsal midline of the embryo. These cells will extend filopodia towards their contralateral counterparts to establish a dorsal cell-cell contact. They then undergo cell shape changes, thereby bending around to form a second, ventral contact and enclosing a luminal space (see Figure 1 and Rugendorff et al., 1994). By the end of embryogenesis, these cells will have differentiated into a tubular dorsal vessel, providing the circulation of hemolymph during larval and adult stages. The mechanism by which this migratory behavior is orchestrated is still poorly understood, but recent studies have established a framework of genes involved in heart morphogenesis. An important participating signal transduction pathway is the Slit/Robo pathway, which was originally identified and characterized for its role in axon guidance and in regulation of midline crossing of growing neurons (Dickson and Gilestro, 2006). Slit, an EGF-like ligand, and the Slit-receptor Roundabout (Robo) are both expressed by cardioblasts during morphogenesis and lumen formation, and ChIP data suggest that cardiac genes, such as *tinman* (Liu et al., 2009), directly regulate their expression. Mutants for *slit* or *robo* together with its paralogue *robo2* have distinct defects during these processes (Qian et al., 2005b; MacMullin and Jacobs, 2006; Santiago-Martínez et al., 2006; Medioni et al., 2008; Santiago-Martínez et al., 2008): impaired cardioblasts cell-cell adhesion, which disrupts subsequent heart morphogenesis, and impairment of cell shape changes and lumen formation. In *slit* mutants, polar (or polarly distributed) markers, including the *Drosophila* MAGUK protein Discs-large, are incorrectly localized indicating a loss of overall cardioblast polarity (Qian et al., 2005b). In addition, the cardioblasts fail to correctly change their cell shape in order to enclose a heart lumen (Medioni et al., 2008). This is accompanied by upregulation of the cell adhesion molecule Shotgun/E-Cadherin at the presumptive luminal domain, leading to increased adhesion at the luminal surfaces, which in

et al., 2006; Lo et al., 2007), tailup/isl-1 (Islet, Tao et al., 2007; Mann et al., 2009), Pannier (GATA4, Alvarez et al., 2003; Fromental-Ramain et al., 2008; Qian et al., 2008; Qian and Bodmer, 2009), Neuromancer-1/-2 (TBX20, Miskolczi-McCallum et al., 2005; Qian et al., 2005a; Reim et al., 2005; Leal et al., 2009), and Dorsocross-1/-2/-3 (TBX5, Reim and Frasch, 2005) and revealed a conserved cardiac transcription factor network responsible for heart specification (Olson, 2006). *Drosophila* and vertebrates also share the same inductive and instructive signaling pathways (Wnt, FGF, BMPs, for review see Frasch, 1999; Cripps and Olson, 2002) during early heart development. This further underscores that the *Drosophila* heart, despite its evolutionary distance from vertebrates, is specified by similar, fundamental mechanisms. This remarkable degree of genetic conservation is paralleled by morphological similarities during early development: the heart originates from a lateral portion in the early mesoderm, and two bilateral regions will eventually fuse and undergo lumen formation. Such genetic and morphological similarities across phyla have led to the conclusion that the cardiovascular system of the fly and vertebrates share true homologies (Hartenstein and Mandal, 2006). Lessons learned from *Drosophila* are likely to translate into a greater understanding of vertebrate heart development and function, and thus will help to understand the pathology and improve the treatment of cardiovascular diseases, as

**3. Lessons learned from studying** *Drosophila* **heart morphogenesis** 

To gain new insights into the role of the cardiac transcriptional network, different groups have begun to analyze the mechanisms underlying heart morphogenesis and heart lumen formation during *Drosophila* embryonic development. After specification, cardiomyocyte precursors (called cardioblasts) migrate towards the dorsal midline of the embryo. These cells will extend filopodia towards their contralateral counterparts to establish a dorsal cell-cell contact. They then undergo cell shape changes, thereby bending around to form a second, ventral contact and enclosing a luminal space (see Figure 1 and Rugendorff et al., 1994). By the end of embryogenesis, these cells will have differentiated into a tubular dorsal vessel, providing the circulation of hemolymph during larval and adult stages. The mechanism by which this migratory behavior is orchestrated is still poorly understood, but recent studies have established a framework of genes involved in heart morphogenesis. An important participating signal transduction pathway is the Slit/Robo pathway, which was originally identified and characterized for its role in axon guidance and in regulation of midline crossing of growing neurons (Dickson and Gilestro, 2006). Slit, an EGF-like ligand, and the Slit-receptor Roundabout (Robo) are both expressed by cardioblasts during morphogenesis and lumen formation, and ChIP data suggest that cardiac genes, such as *tinman* (Liu et al., 2009), directly regulate their expression. Mutants for *slit* or *robo* together with its paralogue *robo2* have distinct defects during these processes (Qian et al., 2005b; MacMullin and Jacobs, 2006; Santiago-Martínez et al., 2006; Medioni et al., 2008; Santiago-Martínez et al., 2008): impaired cardioblasts cell-cell adhesion, which disrupts subsequent heart morphogenesis, and impairment of cell shape changes and lumen formation. In *slit* mutants, polar (or polarly distributed) markers, including the *Drosophila* MAGUK protein Discs-large, are incorrectly localized indicating a loss of overall cardioblast polarity (Qian et al., 2005b). In addition, the cardioblasts fail to correctly change their cell shape in order to enclose a heart lumen (Medioni et al., 2008). This is accompanied by upregulation of the cell adhesion molecule Shotgun/E-Cadherin at the presumptive luminal domain, leading to increased adhesion at the luminal surfaces, which in

exemplified by Neely (2010) and Qian (2011).

turn is likely to prevent further lumen formation (Santiago-Martínez et al., 2008). Both, Slit and Robo are expressed during mouse heart development and expression of Slit3 and Robo2 depend on Tbx20 and Nkx2-5, respectively (Medioni et al., 2010). Functional analysis in zebrafish done by Fish et al. (2011) indicates a role for Slit/Robo signaling during zebrafish heart development. Slit/robo mutant fish hearts show a number of developmental defects, indicating a conserved requirement for this pathway during vertebrate cardiogenesis and thus a role in CHD. Again, the analysis of Slit/Robo in *Drosophila* has paved the way for the subsequent experiments done in vertebrates.

Recent work on fly heart development suggests several possible future research directions. Firstly, genetic screens in *Drosophila* should reveal additional genes involved in cell-cell signaling during development. Among them is the Netrin/Unc-5 pathway that, similar to Slit/Robo, was found to be involved in axon guidance and cell migration. In the heart, the UNC-5 ligand Netrin is also required for heart lumen formation (Albrecht et al., 2011), although with a lesser penetrance than Slit/Robo. This indicates that in fact multiple pathways are required during the formation of the heart, and it would be interesting to see if these two pathways genetically interact, which would indicate a potential cross talk between them. The *Drosophila* model therefore helps pinpoint which pathways may also interact in CHD in humans. Secondly, a more complete and detailed understanding of the signaling pathways themselves during heart development and establishment of cardiac function will be essential for understanding CHD initiation and progression. For example, co-receptors might play an important role in defining pathway sensitivity and downstream activity. In *Drosophila*, cardiac Slit/Robo signaling has been shown to require the activity of the heparan sulfate proteoglycan Syndecan (Knox et al., 2011). Since Syndecan is involved in angiogenesis (through VEGF, Chen et al., 2004) and is also upregulated during cardiac remodeling after myocardial infarction, the *Drosophila* model might help identify important components of Syndecan signaling in these disease-relevant contexts. Thirdly, the cellular machineries through which signaling pathways exert their specific function are largely unknown. Thus, we currently have no clear understanding of the intracellular mechanisms that give rise to the *slit* mutant phenotype.
