The Hand gene family encodes highly conserved basic helix-loop-helix (bHLH) transcription factors that play crucial roles in cardiac and vascular development in vertebrates. In Drosophila, a single Hand gene is expressed in the three major cell types that comprise the circulatory system: cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors. Drosophila Hand functions as a potent transcriptional activator, and converting it into a repressor blocks heart and lymph gland formation. Disruption of Hand function by homologous recombination also results in profound cardiac defects that include hypoplastic myocardium and a deficiency of pericardial and lymph gland hematopoietic cells, accompanied by cardiac apoptosis. Targeted expression of Hand in the heart completely rescues the lethality of Hand mutants, and cardiac expression of a human HAND gene, or the caspase inhibitor P35, partially rescues the cardiac and lymph gland phenotypes. These findings demonstrate evolutionarily conserved functions of HAND transcription factors in Drosophila and mammalian cardiogenesis, and reveal a previously unrecognized requirement of Hand genes in hematopoiesis (Han, 2006).

The initial steps in heart formation are remarkably conserved from fruit flies to mammals. In both types of organism, mesodermal progenitors become committed to a cardiac fate in response to signals from adjacent tissues and converge along the embryonic midline to form a linear cardiac tube with rhythmic contractility. In Drosophila, the myocardial cell layer of the contractile heart tube, composed of cardioblasts, is surrounded by pericardial nephrocytes, which function as secretory cells, and by lymph gland hematopoietic cells that give rise to all the major blood cells in the adult fly. Cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors -- the three major embryonic cell types that comprise the Drosophila circulatory system -- arise from the same cardiac mesoderm, specified by signaling pathways involving Decapentaplegic (DPP), Wingless (WG) and FGF. In the more complex mammalian cardiovascular system, cardiac and hematopoietic progenitors are also derived from the same mesodermal region -- the lateral mesoderm -- and are specified by conserved signaling pathways involving bone morphogenetic protein (BMP), WNT and FGF, exemplifying the conservation of developmental programs for cardiogenesis and hematopoiesis between Drosophila and mammals (Han, 2006).

NK-type homeodomain proteins and the GATA family of zinc-finger transcription factors are required for cardiac and hematopoietic development in Drosophila and mammals. The Drosophila NK family transcription factor, Tinman, and its mammalian ortholog Nkx2.5, are expressed specifically in the developing heart and are both regulated by the DPP/BMP pathway. Both Tinman and Nkx2.5 play central roles in activation of myocardial genes required for heart development. The GATA factors, Drosophila Pannier (Pnr) and its mammalian homologues GATA4, GATA5 and GATA6, are also expressed in the cardiogenic mesoderm and play crucial roles in heart development. Pannier and GATA4 function as partners of Tinman and Nkx2.5, respectively, to activate the cardiac gene program in Drosophila and mammals. Another group of GATA factors, Drosophila Serpent (Srp), and its mammalian homologues GATA1, GATA2 and GATA3 are required for hematopoiesis in Drosophila and mammals, respectively. It is likely that the functions of Tinman, Pannier and Serpent in cardiogenesis and hematopoiesis reflect the highly conserved but simplified developmental processes in Drosophila compared with mammals (Han, 2006).

The basic helix-loop-helix (bHLH) transcription factor HAND is the only transcription factor known to be specific to the three major embryonic cell types that comprise the Drosophila circulatory system (Kolsh, 2002; Han, 2005). Cardiac and hematopoietic expression of Hand is controlled by a 513 bp enhancer that integrates the activity of Tinman, Pannier and Serpent, the three central transcription factors that control cardiogenesis and hematopoiesis (Han, 2005). Hand expression is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes in the heart and by Serpent in hematopoietic progenitors in the lymph gland, through evolutionarily conserved DNA-binding sites in this enhancer. These findings place Hand at a nexus of the transcriptional networks that govern cardiogenesis and hematopoiesis, but the potential functions of Hand in these developmental processes have not been explored (Han, 2006).

By contrast, the functions of the two vertebrate Hand genes, Hand1 and Hand2, have been intensively studied. Hand1 and Hand2 are initially expressed throughout the cardiogenic region but later display complementary expression patterns in the left and right ventricular chambers (Srivastava, 1995; Srivastava, 1997; Thomas, 1998). Mice lacking Hand1 die from placental and extra-embryonic abnormalities (Firulli, 1998), whereas mice lacking Hand2 die from right ventricular hypoplasia and vascular defects (Srivastava, 1995; Srivastava, 1997; Yamagishi, 2001). Deletion of the Hand1 and Hand2 genes in the heart revealed their dose-sensitive requirement and functional redundancy for myocardial growth (McFadden, 2005), and mutation of the single hand gene in zebrafish results in a dramatic reduction in the number of cardiac cells (Yelon, 2000). In addition to its cardiac expression, Hand1 is highly expressed in the lateral plate mesoderm (Firulli, 1998) from which the intra-embryonic aorta-gonad-mesonephros (AGM), a major source of hematopoiesis, is derived. The potential functions of Hand genes in hematopoiesis have not been investigated (Han, 2006).

Although HAND factors are essential in vertebrate cardiogenesis, little is known about their mechanism of action. The requisite role of HAND factors in growth of the cardiac chambers during vertebrate heart development also raises interesting questions about the function of the highly conserved Drosophila Hand gene, since the Drosophila heart is thought to be a simple linear tube that does not undergo complex morphogenic changes (Han, 2006).

Drosophila Hand is shown to function as a highly potent transcriptional activator, and converting it into a transcriptional repressor blocks heart and lymph gland formation. To explore the possible roles of Hand in cardiogenesis and hematopoiesis, a null mutant was generated in the gene through homologous recombination. Hand mutant embryos and larvae display profound cardiac defects, including hypoplastic myocardium, a deficiency of pericardial nephrocytes, and abnormal cardiac morphology, suggesting an essential role of Hand during Drosophila cardiac morphogenesis. Lymph gland hematopoietic progenitors are also dramatically reduced in most Hand mutant larvae, as well as in a subset of Hand mutant embryos, indicating an important role of Hand in Drosophila hematopoiesis. These abnormalities were prevented by cardiac expression of Drosophila or human Hand genes, as well as the caspase inhibitor P35. These findings demonstrate evolutionarily conserved roles of Hand genes in Drosophila and mammalian cardiogenesis, and suggest a possible requirement of Hand genes in mammalian hematopoiesis (Han, 2006).

HAND1 and HAND2 have been shown to play essential roles the processes of cardiac remodeling and chamber specification during mammalian cardiogenesis. As the Drosophila heart has generally been considered to function as a linear tube, without a defined chamber, the function of the single highly conserved HAND factor in Drosophila has been a source of curiosity. The results show that a substantial fraction of Hand mutant larvae display cardiac morphological defects, including a thin hypoplastic heart tube and dramatically reduced pericardial nephrocytes, as well as disruption of the chamber-like structure. Hand mutant larvae also display abnormal cardiac function, reflected by their sluggish heart rate and more frequent discontinuities between continuous periods of heart beating, which could be the cause of lethality after hatching. These findings suggest that Hand plays an essential role in Drosophila heart development (Han, 2006).

HAND transcription factors are expressed during heart development in human, mouse, chick, frog, zebrafish, ciona and Drosophila embryos (Cserjesi, 1995; Srivastava, 1995; Angelo, 2000; Yelon, 2000; Davidson, 2003; Han, 2005). Mouse Hand2 and Drosophila Hand are both regulated by GATA factors during heart development (McFadden, 2000; Han, 2005). Functional studies have suggested that Hand genes are essential for cardiogenesis in mouse, chick, zebrafish and Drosophila (Srivastava, 1995; Srivastava, 1997; Yelon, 2000; McFadden, 2005). The finding that cardiac expression of human HAND2 can rescue the early larval cardiac and hematopoietic phenotype of the Drosophila Hand mutant provides strong evidence that Hand genes play evolutionarily conserved roles in cardiogenesis (Han, 2006).

Mouse embryos lacking HAND2 exhibit hypoplasia of the right ventricle and pharyngeal arches and associated apoptosis (Srivastava, 1997; Thomas, 1998; Yamagashi, 2001). Loss of the apoptosis protease-activating factor 1 (Apaf1), a downstream mediator of mitochondrial-induced apoptosis, partially rescues the ectopic apoptosis in Hand2-null embryos and delays embryonic lethality (Aiyer, 2005), suggesting that HAND2 acts, at least in part, to inhibit apoptosis (Han, 2006).

Ectopic apoptosis is observed in Hand mutant Drosophila embryos, accompanied by a dramatic reduction in pericardial nephrocytes and gaps in the cardiac tube (indicative of missing cardioblasts). Interestingly, both the ectopic apoptosis and the early cardiac and hematopoietic defects can be rescued by targeted expression the apoptosis inhibitor P35 in Hand-expressing cells, indicating that one of the important roles of Hand is to inhibit apoptosis (Han, 2006).

To determine if Hand can generally inhibit apoptosis, tests were performed to see whether overexpression of Hand in transfected Drosophila S2 cells could block apoptosis induced by genes that induce apoptosis, such as Reaper and HID, or with drugs that induce apoptosis, such as Etoposide and Taxol. However, Hand failed to inhibit apoptosis in response to these stimuli, suggesting that it does not function as a general inhibitor of apoptosis. The fact that targeted overexpression of P35 could not completely rescue the cardiac morphological defects in Hand mutant larvae also suggests that Hand performs functions in addition to inhibiting apoptosis. It is possible that Hand could control differentiation of the cardiac and lymph gland cells and the absence of Hand would lead to apoptosis indirectly as a result of its role in some differentiation event (Han, 2006).

Although Hand family genes have been identified for a long time, their mechanism of action has not been fully elucidated. The results of this study demonstrate Drosophila Hand to be a potent transcriptional activator in vitro and during heart and lymph gland development in vivo. Converting Hand into a transcription repressor evokes more severe cardiac and hematopoietic defects than simply removing it, suggesting that its function depends on the activation of its downstream target genes. Based on the phenotypes resulting from Hand mutants and from overexpression of Hand-EnR, it is predicted that these target genes participate in cell growth and survival and in maintaining cardiac and hematopoietic cell fates. Given the functional redundancy among Hand genes in mammals, Drosophila offers a powerful system with which to uncover conserved functions and mechanisms of action of this gene family in both cardiogenesis and hematopoiesis (Han, 2006).

In Drosophila, adult blood cells originate from the lymph gland hematopoietic progenitors, which are derived from cardiac mesoderm. The lymph gland dissociates at the pupal stages to release all the adult blood cells. Hand is the only transcription factor identified to date that is expressed in all hematopoietic progenitors and the entire heart. The dramatic reduction of lymph gland hematopoietic progenitors in Hand mutants suggests that Hand is essential for Drosophila hematopoiesis (Han, 2006).

In mammals, the adult hematopoietic system originates from the yolk sac and the intra-embryonic aorta-gonad-mesonephros (AGM) region. Previous studies have suggested a close relationship between the Drosophila cardiac mesoderm and the mammalian cardiogenic and AGM region. In both Drosophila and mammals, the specification of these regions requires the input of BMP, WNT and FGF signaling from the neighboring germ layer and function of NK and GATA factors in the mesoderm. Although the possible role of HAND factors in mammalian hematopoiesis has not been explored, mouse Hand1 is expressed at high levels in the lateral plate mesoderm, from which the cardiogenic region and the AGM region arise (Firulli, 1998). This study provides the first evidence for the requirement of Hand in Drosophila hematopoiesis, suggesting similar functions for its mammalian orthologs (Han, 2006).

GENE STRUCTURE

Exons - 4

PROTEIN STRUCTURE

Amino Acids - 171

Structural Domains

A 521-bp hand cDNA was isolated by RT-PCR with gene-specific primers that allowed the amplification of the complete open reading frame of hand. The isolated cDNA was sequenced and the deduced amino acid sequence showed highest similarities to the vertebrate dHand and eHand proteins. The most conserved regions are the bHLH-domain, responsible for DNA-binding and dimerization, and a C-terminal stretch of about 16 amino acid residues (Hand-domain), which is characteristic for this subtype of class A bHLH proteins. The high conservation of the Hand-domain throughout all Hand proteins identified so far implies a functional but still undetermined role for this motif (Kolsch, 2002).

Similar to the observations in Drosophila and Caenorhabditis, only a single hand gene was found in fish, whereas two related hand genes (dHand and eHand) are found in all higher vertebrates. Phylogenetic analysis placed the fish Hand into the same branch as the dHand proteins, suggesting that dHand might represent the ancestral form. A typical feature of fish, frog and other dHand proteins is a polyalanine tract N-terminal of the bHLH domain. Polyalanine stretches were found in several transcriptional repressors and are thought to be essential for their repression activities. Drosophila Hand lacks such a polyalanine stretch, indicating that the vertebrate dHand proteins might have evolved additional features. It should be noted that the basic region of the bHLH domain of Drosophila Hand is more similar to the corresponding region in vertebrate dHand proteins. Phylogenetic analysis was performed with the full-length Drosophila Hand protein sequence, as well as the bHLH domain only, and various vertebrate Hand proteins to determine the phylogenetic position of Drosophila Hand. This analysis does not indicate a closer relationship of Drosophila Hand to dHand or eHand proteins, but clearly shows that the Drosophila gene represents a real orthologue of the vertebrate hand genes. Whether dHand genes indeed represent the ancestral form from which eHand genes have been derived by a gene duplication event remains to be elucidated. To answer this question, the identification of additional Hand orthologues, e.g. from urochordates and others, would be helpful (Kolsch, 2002).

date revised: 5 August 2006

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