InteractiveFly: GeneBrief

Hand: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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).

REGULATION

Promoter Structure

The existence of hemangioblasts, which serve as common progenitors for hematopoietic cells and cardioblasts, has suggested a molecular link between cardiogenesis and hematopoiesis in Drosophila. However, the molecular mediators that might link hematopoiesis and cardiogenesis remain unknown. This study shows that the highly conserved bHLH transcription factor Hand is expressed in cardioblasts, pericardial nephrocytes and hematopoietic progenitors. The homeodomain protein Tinman and the GATA factors Pannier and Serpent directly activate Hand in these cell types through a minimal enhancer, which is necessary and sufficient to drive Hand expression in these different cell types. Hand is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes, and by Serpent in hematopoietic progenitors in the lymph gland. These findings place Hand at a nexus of the transcriptional networks that govern cardiogenesis and hematopoiesis, and indicate that the transcriptional pathways involved in development of the cardiovascular, excretory and hematopoietic systems may be more closely related than previously appreciated (Han, 2005).

To search for cis-regulatory elements capable of conferring the specific expression pattern of Hand in cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors, a series of reporter genes were generated containing lacZ and the hsp70 basal promoter linked to genomic fragments within a 13 kb genomic region encompassing the gene, and reporter gene expression was examined in transgenic embryos. A 513 bp minimal enhancer was identified referred to as Hand cardiac and hematopoietic (HCH) enhancer, between exons 3 and 4 of the Hand gene. HCH is both necessary and sufficient to direct lacZ expression in the entire embryonic heart and lymph gland in a pattern identical to that of the endogenous Hand gene. Further deletions of this enhancer caused either a partial or complete loss of activity. The 513 bp HCH enhancer showed the same expression pattern in the heart and lymph gland as larger genomic fragments that were positive for enhancer activity. It is concluded that this enhancer fully recapitulates the temporal and spatial expression pattern of Hand transcription in the distinct cell types derived from the cardiogenic region (Han, 2005).

The homeobox protein Tinman is essential for the formation of the cardiac mesoderm, from which the heart and blood progenitors arise. However, its potential late functions remain unknown. It is believed that Tinman is not required for the entirety of heart development in flies, because it is not maintained in all the cardiac cells at late stages. The data reveal at least one function for the late-embryonic Tinman expression, which is to maintain Hand expression. The fact that ectopic Tinman can turn on Hand expression dramatically in the somatic muscles is striking and suggests the existence of a Tinman-co-factor in muscle cells that can cooperate with Tinman to activate Hand expression; this co-factor would not be expected to be expressed in pericardial cells or the lymph gland. This co-factor should also be expressed in Drosophila S2 cells, since transfected Tinman can increase activity of the HCH enhancer in S2 cells by more than 100-fold. The generally reduced activity of the HCH enhancer that results from mutation of the Tinman-binding sites also suggests that Tinman activity is required to fully activate the Hand enhancer (Han, 2005).

Although Pannier and Serpent bind to the same consensus sites, these GATA factors produce distinct phenotypes when overexpressed in the mesoderm. Ectopic Pannier induces cardiogenesis, shown by the extra number of cardioblasts and pericardial nephrocytes, but does not affect the lymph gland hematopoietic progenitors. Ectopic Serpent, however, induces ectopic lymph gland hematopoietic progenitors, but reduces the number of cardioblasts and pericardial cells. Interestingly, pericardial cells with ectopic Serpent expression have a tendency to form cell clusters such as the lymph gland progenitors, suggesting a partial cell fate transformation. These results suggest that Pannier functions as a cardiogenic factor, whereas Serpent functions as a hematopoietic factor. Although both can activate Hand expression, Pannier and Serpent activate the HCH enhancer in different cell types. This assumption is also supported by the specific expression pattern of Serpent and Pannier in late embryos. Serpent is detected specifically in the lymph gland hematopoietic progenitors but not in any cardiac cells. Pannier expression in the cardiogenic region of late embryos is not clear because of the interference by the high level Pannier expression from the overlaying ectoderm. However, the lymph gland was examined in late stage embryos and no Pannier expression was detected in these cells. Together with the evidence from loss-of-function and gain-of-function experiments with Serpent, it is concluded that the HCH-5G-GFP transgene is not expressed in the lymph gland because Serpent could not bind to the mutant enhancer in the lymph gland cells; whereas the lack of HCH-5G-GFP expression in cardiac cells is due to the inability of Pannier to bind the mutant enhancer in these cardiac cells (Han, 2005).

Since tin and pnr are not expressed in all the cardiac cells of late stage embryos but the Hand-GFP transgene is expressed in these cells, it is likely that additional factors control Hand expression in the heart. One group of candidates is the T-box family. Since Doc1, Doc2 and Doc3 genes (Drosophila orthologs to vertebrate Tbx5) are expressed in the Svp-positive cardioblasts where tin is not expressed, but H15 and midline (Drosophila orthologs to vertebrate Tbx-11) are expressed in most of the cardiac cells in late embryos, it is likely that the T-box genes activate Hand expression in cells that do not express tin and pannier. However, the enhancer lacking GATA and Tinman sites has no activity, indicating that the additional factors that may activate Hand expression in the heart and lymph gland also requires these crucial Tinman and GATA sites, probably through protein interaction between Tinman and the GATA factors (Han, 2005).

Putative Hand enhancers were detected from divergent Drosophila species. In most of these species, the entire 513 bp Hand enhancer region is highly conserved. However, the D. virilis HCH enhancer does not exhibit highly conserved sequence between the consensus binding sites, even though it has a similar number of consensus binding sites for both Tinman and Pannier. The fact that this D. virilis enhancer can also drive reporter gene expression in the heart indicates that these Tinman and GATA-binding sites are the crucial elements for enhancer activity. Besides the enhancers with all Tinman or all GATA binding sites mutated, transgenic flies were also generated carrying one or two mutations of the Tinman or GATA-binding sites. None of these transgenic lines shows significant changes in enhancer activity, indicating that this enhancer is robustly activated by Tinman, Pannier and Serpent through functionally redundant binding sites. These data also explain why the Hand enhancers from different Drosophila species have different numbers of Tinman or GATA-binding sites (Han, 2005).

Interestingly, Hand expression is also dependent on GATA factors in vertebrates. An enhancer has been described that is necessary and sufficient to direct cardiac expression of the mouse Hand2 gene, which contains two essential GATA-binding sites. Thus, it is proposed that the Hand genes are directly regulated by GATA factors in an evolutionarily conserved developmental pathway in both Drosophila and mice. Although no functional NK binding sites were identified in the mouse Hand2 enhancer, there are perfectly matched NK consensus sites in the Hand2 locus that may function in a redundant or refined way to regulate Hand2 expression (Han, 2005).

In mammals, the adult hematopoietic system originates from the yolk sac and the intra-embryonic aorta-gonad-mesonephros (AGM) region. The AGM region is derived from the mesodermal germ layer of the embryo in close association with the vasculature. Indeed, the idea of the hemangioblast, a common mesodermal precursor cell for the hematopoietic and endothelial lineages, was proposed nearly 100 years ago without clear in vivo evidence. Recently, this idea was substantiated by the identification of a single progenitor cell that can divide into a hematopoietic progenitor cell in the lymph gland and a cardioblast cell in the dorsal vessel in Drosophila (Mandal, 2004). In addition to providing the first evidence for the existence of the hemangioblast, this finding also suggested a close relationship between the Drosophila cardiac mesoderm, which gives rise to cardioblasts, pericardial nephrocytes and pre-hemocytes, and the mammalian cardiogenic and AGM region, which gives rise to the vasculature (including cardiomyocytes), the excretory systems (including nephrocytes) as well as adult hematopoietic stem cells. In fact, in both Drosophila and mammals, the specification of the cardiogenic and AGM region requires the input of Bmp, Wnt and Fgf signaling. In addition to the conserved role of the NK and GATA factors, GATA co-factors (U-shaped in Drosophila and Fog in mice) also play important roles in cardiogenesis and hematopoiesis in both Drosophila and mammals. Recent studies have shown that the Notch pathway is required for both cardiogenic and hematopoietic progenitor specification in Drosophila. It is likely that Notch also plays an important role in mammalian hematopoiesis (Han, 2005).

This study found that Drosophila Hand is expressed in cardioblasts, pericardial nephrocytes and pre-hemocytes, and is directly regulated by conserved transcription factors (NK and GATA factors) that control both cardiogenesis and hematopoiesis. The bHLH transcription factor Hand is highly conserved in both protein sequence and expression pattern in almost all organisms that have a cardiovascular system. In mammals, Hand1 is expressed at high levels in the lateral plate mesoderm, from which the cardiogenic region and the AGM region arise, in E9.5 mouse embryos. Functional studies of Hand1 and Hand2 using knockout mice have demonstrated the essential role of Hand genes during cardiogenesis, whereas the functional analysis of Hand genes during vertebrate hematopoiesis has not yet been explored. It will be interesting to determine whether mammalian Hand genes are also regulated in the AGM region by GATA1, GATA2 and GAT3 (vertebrate orthologs to Drosophila Serpent), and whether they play a role in mammalian hematopoiesis (Han, 2005).

In summary, this study places Hand at a pivotal point to link the transcriptional networks that govern cardiogenesis and hematopoiesis. Since the Hand gene family encodes highly conserved bHLH transcription factors expressed in the cardiogenic region of widely divergent vertebrates and probably in the AGM region in mouse, these findings open an avenue for further exploration of the conserved transcriptional networks that govern both cardiogenesis and hematopoiesis, by studying the regulation and functions of Hand genes in vertebrate model systems (Han, 2005).

Requirement of the LIM homeodomain transcription factor tailup for normal heart and hematopoietic organ formation in Drosophila; Tup regulates a dorsal vessel enhancer of Hand

Dorsal vessel morphogenesis in Drosophila melanogaster serves as a superb system with which to study the cellular and genetic bases of heart tube formation. A cardioblast-expressed Toll-GFP transgene was used to screen for additional genes involved in heart development and tailup was identified as a locus essential for normal dorsal vessel formation. tailup, related to vertebrate islet1, encodes a LIM homeodomain transcription factor expressed in all cardioblasts and pericardial cells of the heart tube as well as in associated lymph gland hematopoietic organs and alary muscles that attach the dorsal vessel to the epidermis. A transcriptional enhancer regulating expression in these four cell types was identified and used as a tailup-GFP transgene with additional markers to characterize dorsal vessel defects resulting from gene mutations. Two reproducible phenotypes were observed in mutant embryos: hypoplastic heart tubes with misaligned cardioblasts and the absence of most lymph gland and pericardial cells. Conversely, a significant expansion of the lymph glands and abnormal morphology of the heart were observed when tailup was overexpressed in the mesoderm. Tailup was shown to bind to two DNA recognition sequences in the dorsal vessel enhancer of the Hand basic helix-loop-helix transcription factor gene, with one site proven to be essential for the lymph gland, pericardial cell, and Svp/Doc cardioblast expression of Hand. Together, these results establish Tailup as being a critical new transcription factor in dorsal vessel morphogenesis and lymph gland formation and place this regulator directly upstream of Hand in these developmental processes (Tao, 2007).

Thus, Tup is a newly discovered player in the regulatory network controlling dorsal vessel morphogenesis and hematopoietic organ formation. Tup is expressed in all cardioblast and pericardial cells of the heart tube, prohemocytes of the lymph glands, and alary muscles needed to secure the dorsal vessel to the epidermis. Phenotypic studies demonstrate a requirement for tup function in three of these cells types. tup mutant embryos exhibit a hypoplastic dorsal vessel, with a variable number of cardioblasts that fail to organize into a heart tube structure. It appears that correct numbers of cardioblasts are not specified in mutant embryos, since gaps were observed in the bilateral cardioblast rows early in the process of dorsal vessel formation. Missing cardioblasts included cells of both the Tin- and Svp/Doc-positive subclasses. The late cardioblast misalignment phenotype is likely due to the dorsal closure and germ band retraction defects known to occur in tup embryos (Tao, 2007).

While the degree of cardioblast hypoplasia is variable in mutant embryos, the severe reduction in prohemocytes of the lymph glands and pericardial cells surrounding the contractile tube is fully penetrant. The Collier (Col) protein serves as an excellent marker for lymph gland primordia and the posterior signaling centers of lymph glands associated with the mature dorsal vessel. Since Col expression is normal in tup mutants, Tup function is not required for the early specification of lymph gland primordia within the dorsal mesoderm. However, the severe reduction of several mature lymph gland markers such as tup-GFP, Hand-GFP, Srp, and Odd suggests that either prohemocytes are present within lymph glands with Tup activity essential for expression of all four of these indicator genes or the cells are absent due to defects in prohemocyte proliferation and/or programmed cell death. The latter is an attractive possibility since Hand knockout embryos show ectopic apoptosis among lymph gland progenitor cells (Tao, 2007).

A function for the Hand basic helix-loop-helix transcription factor has been reported for cardioblast, pericardial, and lymph gland cells. This is the same set of dorsal vessel and hematopoietic cells that require Tup function. Through analysis of the Hand cardiac and hematopoietic enhancer, Tup was demonstrated to be a direct transcriptional regulator of Hand in these cell types. Specifically, mutation of the single Tup-2 element in the Hand cardiac and hematopoietic regulatory module resulted in a dramatic loss or reduction of Hand enhancer activity in prohemocytes, pericardial cells, and the Svp/Doc cardioblast subtype. These findings invoke two possibilities. (1) tup phenotypes may be due to the lack of Hand expression and function in cardioblasts, pericardial cells, and lymph gland progenitors. However, Tup function is likely to be even more critical for cardiogenic and hematopoietic events; forced expression of tup results in the production of excess prohemocytes, while the ectopic expression of Hand does not. Thus, Tup can be considered to be a seminal upstream regulator of genetic and cellular events controlling lymph gland formation. (2) Tin and GATA factors have been shown to regulate the Hand cardiac and hematopoietic enhancer. Thus, it is possible the Hand cardiac and hematopoietic transcription occurs due to combinatorial control, specifically via Tup and Doc cofunction in Svp/Doc-expressing cardioblasts and Tup and Srp coactivity in lymph gland progenitors. Ample evidence exists for the function of multiple interacting transcription factors in the regulation of heart and blood cell gene expression in Drosophila. To summarize regulatory aspects of its function, the data showing that Tup is a direct transcriptional activator of Hand expression in lymph glands, pericardial cells, and Svp/Doc-positive cardioblasts through the HCH enhancer module are compelling. Likewise, Tup serves as either a direct or indirect regulator of srp expression in lymph gland cells and odd expression in lymph gland and pericardial cells (Tao, 2007).

Hand is a direct target of the forkhead transcription factor Biniou during Drosophila visceral mesoderm differentiation

The visceral trunk mesoderm in Drosophila develops under inductive signals from the ectoderm. This leads to the activation of the key regulators Tinman, Bagpipe and Biniou that are crucial for specification of the circular visceral muscles. How further differentiation is regulated is widely unknown, therefore it seems to be essential to identify downstream target genes of the early key regulators. This study focuses on the analysis of the transcriptional control of the highly conserved transcription factor Hand in circular visceral muscle cells, providing evidence that the hand gene is a direct target of Biniou. A regulatory region has been identified in the hand gene that is essential and sufficient for the expression in the visceral mesoderm during embryogenesis. hand expression in the circular visceral mesoderm is abolished in embryos mutant for the FoxF domain containing transcription factor Biniou. Furthermore it is demonstrated that Biniou regulates hand expression by direct binding to a 300 bp sequence element, located within the 3rd intron of the hand gene, and marked by the presence of four putative motifs with homology to the HFH-8 consensus binding site A/G C/T A A A C/T A, recognized by Biniou. This regulatory element is highly conserved in different Drosophila species. In addition, evidence is provided that Hand is dispensable for the initial differentiation of the embryonic visceral mesoderm. This study shows that cross species sequence comparison of non-coding sequences between orthologous genes is a powerful tool to identify conserved regulatory elements. Combining functional dissection experiments in vivo and protein/DNA binding studies hand was identified as a direct target of Biniou in the circular visceral muscles (Popichenko, 2007; full text of article).

Hand functions as a potent transcriptional activator

To begin to understand the mechanism of action of HAND, structure/function studies of the HAND protein were performed in Drosophila S2 cells. Members of the HAND family transcription factors share homology in a bHLH domain and a 15 amino acid peptide at their C termini, referred to as the HAND domain, which is unique to this subfamily of bHLH proteins. bHLH transcription factors bind to a conserved DNA-binding site called an E-box (CANNTG). The transcriptional activity of a series of Hand deletion mutants was tested using a luciferase reporter linked to six copies of the E-box sequence (L8E6-luc). Drosophila Hand was a remarkably effective transcriptional activator. Mutation of the conserved residues in the basic domain (RRR), or deletion of either the N-terminal region or the C-terminal HAND domain, abolished transcriptional activity, indicating the central bHLH domain cooperates with the latter domains to activate transcription (Han, 2006).

The transcription activation domain of Hand was mapped by fusing regions of the protein to the Gal4 DNA binding domain and assaying activity with a luciferase reporter linked to four copies of the Gal4-binding site (L8G4-luc). It was found that the transcriptional activity of Hand depends primarily on its N-terminal region. Interestingly, mutation of the conserved basic residues in the bHLH domain increased the transcriptional activity of Gal4-Hand dramatically (Gal4-Hand-RRR), suggesting that the basic region communicates, directly or indirectly, with the transcription activation domain (Han, 2006).

To determine if Hand also functions as a transcription activator in vivo, it was converted into a repressor and a super-activator by fusing it to the Engrailed repression domain (EnR) and the VP16 transcription activation domain, respectively. Hand-VP16 functions as an extremely strong transcriptional activator, whereas Hand-EnR, when co-expressed with HAND, efficiently blocks the activity of Hand in Drosophila S2 cells (Han, 2006).

Using the UAS-Gal4 system, wild-type HAND, Hand-VP16 and Hand-EnR were overexpressed in Drosophila embryos. Pan-mesodermal overexpression of Hand has no effect on embryonic heart or muscle development, although it results in lethality at the late larval stage for reasons that are unclear. By contrast, pan-mesodermal over-expression of Hand-EnR dramatically disrupts embryonic heart and lymph gland formation. The number of cardioblasts (labeled by Mef2 antibody) and pericardial cells (labeled by Even-skipped antibody) is significantly reduced in embryos with ectopic Hand-EnR. The residual cardiac cells in Hand-EnR-expressing embryos were able to migrate to the dorsal midline at the end of embryogenesis, but their alignment was disrupted. Formation of the lymph gland hematopoietic progenitors, labeled by Odd-skipped antibody, is also completely blocked by ectopic Hand-EnR (Han, 2006).

To examine further the cell-autonomous requirement of Hand function within the dorsal vessel, wild-type Hand, Hand-VP16 and Hand-EnR were overexpressed using a Hand-Gal4 driver generated by using the Hand cardiac and hematopoietic enhancer (HCH; Han, 2005). Targeted overexpression of wild-type Hand and Hand-VP16 in Hand-expressing cells did not evoke a phenotype, whereas targeted overexpression of Hand-EnR in Hand-expressing cells abolished the formation of lymph gland hematopoietic progenitors, labeled by antibody against the hematopoietic GATA factor Serpent and Hand-GFP, which is a transgene carrying a GFP reporter driven by the HCH enhancer. The number of cardioblasts and pericardial nephrocytes was also diminished and their alignment was disrupted in embryos expressing Hand-EnR. These data suggest that Hand functions as an essential transcriptional activator during cardiogenesis and hematopoiesis (Han, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

The highly conserved basic helix-loop-helix transcription factor Hand plays a crucial role in cardiogenesis, limb formation and other developmental processes of vertebrates. Humans, mice and other higher vertebrates have two related genes, dHand (also known as Hand2, Hed, Thing2) and eHand (also known as Hand1, Hxt, Thing1), whereas fish and Drosophila have only a single hand gene. Drosophila hand has been cloned and its embryonic expression examined in detail by using various tissue-specific markers that allowed the identity of hand-expressing cells to be analyzed. hand was found to be expressed in the entire heart, including all cardioblasts and pericardial cells, in the progenitors of the circular visceral muscles, the lymph gland and garland cells, and in a few cells in the CNS. The expression of Drosophila hand starts after the inductive activity of the early regulators in these tissues, e.g., Tinman and Bagpipe, suggesting a role for Hand in differentiation rather than in tissue determination. In many aspects the expression pattern of Drosophila hand resembles the patterns of its vertebrates orthologues, for instance in cardiac tissues. It is assumed that Hand proteins might play a highly conserved role throughout evolution (Kolsch, 2002).

Using the isolated hand cDNA as probe for whole-mount in situ hybridization experiments, the expression of Drosophila hand was examined in embryos with emphasis on cardiogenesis and visceral mesoderm development. Expression starts at stage 11 and is restricted to 11 bilateral segmentally arranged cell clusters in the dorsal mesoderm. Shortly after these cells form a continuous one- to two-cell-wide stripe that will give rise to part of the primordium of the circular visceral muscles. The circular and longitudinal visceral muscles consist of syncytia. Two different cell populations contribute to the formation of gut muscles: muscle progenitors and fusion-competent cells that form binucleated circular muscle fibers at stage 12. At this time, hand is expressed exclusively in the visceral progenitor cells but not in the fusion-competent myoblasts of the circular visceral mesoderm. When fusion in the circular visceral muscle tissue starts at stage 12, hand expression appears broader and more diffuse, indicating that hand transcripts are present in the syncytial visceral myofibers (Kolsch, 2002).

At stage 12 hand expression is initiated in the heart primordium in segmentally arranged groups of two to five cells. Shortly thereafter they form a continuous row that gives rise to the embryonic heart. At the same time a small group of cells located anterior of the trunk mesoderm starts to express hand. At later stages these cells are associated with the proventriculus and give rise to the garland cells. At stage 13, hand expression is also detectable in the lymph gland cells. These cells are already associated with the developing heart and, as development proceeds, are arranged bilaterally near the anterior end of the heart tube. Expression of hand in the lymph gland cells continues until the end of embryogenesis. Beyond stage 12 hand is also expressed in yet unidentified cells within the CNS. At the end of embryogenesis, hand expression in the visceral mesoderm becomes most prominent in myofibers located at the midgut constrictions. The strongest expression is seen in the heart, the lymph glands and the garland cells (Kolsch, 2002).

The spatial and temporal dynamics of hand expression in the developing heart prompted an analysis to identify hand-expressing cells in more detail. In situ hybridization of whole-mount embryos with cDNA as probe indicates that hand is expressed in the majority of heart cells. Since different cell types contribute to the formation of the embryonic heart, it was of interest to know in detail the identity of hand-expressing cells. To verify when during cardiogenesis hand expression starts, Mef2 and Eve were used as markers. At stage 11, Mef2 expression in the dorsal mesoderm becomes prominent in heart precursors. At this stage hand was not detected in heart cells. A short time later during development, Mef2 and hand are coexpressed in the developing heart. Similar results were obtained with Eve as marker. At early stage 11 Eve is expressed in one somatic muscle progenitor (which gives rise to dorsal muscle 1) and in one pericardial cell that divides into two daughter cells, both of which express Eve and give rise to two pericardial cells (EPC cells). Double staining for hand transcripts and Eve protein reveals that hand is neither coexpressed with Eve in the EPC cells at this stage, nor is there a detectable level of hand expression in neighboring cells (other progenitors of pericardial cells or cardioblasts). From stage 12 onwards, Eve and hand are coexpressed in the developing heart. These results indicate that hand expression starts after the early determination and specification of cardioblasts and pericardioblasts. To identify individual subsets of heart cells at later stages, a set of specific antibodies was used for double-staining experiments and confocal microscopic analysis. Expression of hand was found in all cardioblasts per hemisegment until the end of embryogenesis as indicated by colocalization with Mef2 (all cardioblasts) and Tinman (four out of six cardioblasts). Furthermore, confocal microscopic analysis revealed that hand is expressed in all pericardial cells, as indicated by colocalization with Mab3, Zfh1, Tin and Eve (Kolsch, 2002).

Recently it was shown that the visceral mesoderm originates from at least two different cell types: progenitor cells and fusion-competent myoblasts. Both cell types contribute to the formation of syncytial visceral myofibers and are distinguishable by the expression of specific marker genes. Advantage was taken of a reporter line carrying a lacZ gene under the control of a bagpipe enhancer and an antibody against Tinman that stains a subset of visceral cells. Thus, the spatial and temporal expression of hand was analyzed during visceral mesoderm differentiation. At early stage 11, when bagpipe/lacZ-positive cells of the visceral mesoderm are arranged in segmental groups, hand transcripts are not present at a detectable level, neither in the primordium of the circular visceral muscles nor in the caudal mesoderm. At mid stage 11, when bagpipe/lacZ-expressing progenitors of the circular visceral muscles have formed a continuous stripe, hand is strongly expressed in the trunk mesoderm. Coexpression with bagpipe/lacZ is exclusively observed in the distally located visceral progenitors (lower level of bagpipe/lacZ expression), but not in the fusion-competent cells. In the caudal mesoderm, giving rise to the progenitors of the longitudinal visceral muscles, hand is not expressed, neither at stage 11 nor later. hand and bagpipe/lacZ are coexpressed in all circular visceral muscle progenitors at mid stage 11. At this stage, tinman shows a transient and segmentally interrupted expression in a subset of visceral cells. Recently it was shown that the segmented expression pattern of connectin in the visceral mesoderm is dependent on the intersecting influence of Wingless and Decapentaplegic. The circular visceral muscle progenitor population consists of two distinct cell types at this time. All progenitor cells coexpress bagpipe and hand, but only a subset of cells coexpresses bagpipe, tinman and hand, suggesting specific functional properties of both cell types. When syncytial circular muscles start to form at stage 12, hand expression is still strong. At stage 13 and 14 hand is found in all circular myofibers, as shown by colocalization with bagpipe-lacZ. At stage 16 hand transcripts are still detectable in circular visceral myofibers with highest concentration in fibers near the midgut constrictions (Kolsch, 2002).

In the Drosophila larvae, hematopoiesis takes place in the lymph glands that consist of five pairs of lobes associated with the heart. Precursors of the larval lymph gland are first seen during embryogenesis where they are located in close proximity to the heart in two clusters of about 20 cells in the second thoracic segment. The lymph gland cells start to express hand after their association with the developing heart. As development proceeds, the number of hand-expressing lymph gland cells increases to about 20 on each side of the heart. The garland cells have a so far unidentified function in the fly. They appear during embryogenesis in close connection to the foregut-midgut transition and later during development form a U-shaped cluster around the proventriculus. Pericardial cells and garlands cells show a morphologically and ultrastructurally similar appearance and it was speculated that both cell types might function as nephrocytes. Expression of hand is clearly detectable in garland cells at stage 11/12. The expression continuous until the end of embryogenesis when hand-positive cells are clustered around the proventriculus (Kolsch, 2002).

Drosophila Hand is expressed in a specific pattern in the cardiogenic mesoderm. Hand expression is initiated in the cardiogenic region at late stage 12, immediately following the differentiation of Even-skipped (Eve)-positive mesodermal progenitors into segmentally repeated Eve pericardial cells (EPCs) and DA1 muscles; this differentiaion marks the completion of progenitor cell divisions that give rise to the cardioblasts and pericardial nephrocytes (Han, 2003). Cardiac expression of Hand is initially weak and segmental, but soon becomes strong in most cardioblasts and pericardial cells from stage 13. At the end of embryogenesis, when the heart is completely formed, Hand is expressed in all the cardioblasts that also express Dmef2 and in all the pericardial nephrocytes that express even-skipped (eve) (Han, 2005).

At stage 15, tin is expressed in four of the six cardioblasts in each hemisegment from segment A1 to A5, and all the Eve-positive pericardial cells, as well as all cardioblasts from segment T2 to T3, but not in the lymph gland. Hand expression is detected in all the Tinman-positive cardiac cells. Hand is likely to be expressed in all the pericardial nephrocytes since all Zfh-1-positive pericardial cells express Hand. odd-skipped (odd) is expressed in both the lymph gland hematopoietic progenitor cells and a subset of pericardial nephrocytes. Hand expression is also detected in all the Odd-skipped-positive hematopoietic progenitors and pericardial nephrocytes. In addition, Hand is co-expressed with Serpent in all the lymph gland progenitors. The secreted extracellular protein Pericardin (Prc) labels the ring gland and the extracellular matrix surrounding the pericardial nephrocytes. Hand expression is not detected in the ring gland, but Hand-expressing cells are surrounded by Prc from segment T2-A6. Hand expression also appears in the visceral mesoderm, the garland cells and in a subset of central nervous system cells (Han, 2005).

The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation

In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. This paper presents the first developmental study on these organs and reports that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality (Tögel, 2008).

Unlike in vertebrates, where an elaborate closed blood vessel system extends throughout the whole body, insects possess only one vessel, the tubular heart, in their otherwise open circulatory system. Once the hemolymph has left the heart, it moves freely between the internal organs and can not be directed into narrow body appendages such as antennae, legs or wings. To ensure sufficient hemolymph supply of these appendages additional circulatory organs evolved (Pass, 2000; Pass, 2006). In Drosophila, circulation in the wings is maintained by the so-called wing hearts (Krenn, 1995), a pair of autonomous muscular pumps located bilaterally in the scutellum, the dorsal elevation of the second thoracic segment. Due to this location, they are also referred to as scutellar pulsatile organs. Although known for many years, no developmental studies on the origin or morphogenesis of these organs have been performed. Probably, this was due to the lack of available methods to track their differentiation. However, studies on the origin of the thoracic somatic muscles in Drosophila and comparative anatomical investigations in insects suggested that the wing hearts originate from the cardiac mesoderm or from the heart itself (Tögel, 2008).

A previous study identified an enhancer region of the Drosophila hand gene that is able to drive reporter gene activity in the wing hearts (Sellin, 2006). In the present work, this reporter was used to identify the embryonic anlagen of the wing hearts and to elucidate the dynamics of their postembryonic development with in vivo time lapse imaging. It was found that the anlagen of the Drosophila wing hearts indeed derive from the cardiac mesoderm but, astonishingly, not from the muscular cardioblast lineage. Instead, they represent a so far undescribed subpopulation of the well-known Even-skipped (Eve) positive pericardial cells (EPCs) (Tögel, 2008).

In addition to their unknown origin, little is known about the contribution of wing hearts to wing morphogenesis and functionality. After eclosion, wings are unfolded by a sudden influx of hemolymph and subsequently undergo maturation. During this process, the epidermal cells that until then bonded the dorsal and ventral wing surfaces enter programmed cell death, delaminate from the cuticle, and disappear into the thorax (Kimura, 2004). Subsequently, the cuticles of the intervein regions become tightly bonded to form a flexible wing blade, while the cuticles of the vein regions form tubes, lined by living cells, through which hemolymph circulates in mature adult insects. Measurements of hemolymph flow in adult butterflies showed that wing hearts function as suction pumps that draw hemolymph out of the wings starting shortly after wing unfolding. Whether wing hearts might play a role in wing maturation was tested by generating flies lacking wing hearts. The findings demonstrate that the delaminated epidermal cells are removed from the wings by the hemolymph flow generated by the wing hearts. Loss of wing heart function leads to remains of epidermal cells resting between the unbonded dorsal and ventral wing surfaces which results in malformation of the wing blade and flightlessness. It is concluded that wing hearts are essential for wing maturation and, thus, for acquiring flight ability in Drosophila (Tögel, 2008).

A hand-C-GFP reporter was generated (Sellin, 2006) that reflects the described hand expression pattern and was found to be active in wing hearts. To confirm that the hand-C-GFP reporter is expressed in all cells of mature wing hearts, their morphology was examined based on the signal from the reporter in conjunction with histological sections. In the adult fly, wing hearts are located at the lateral angles of the scutellum, which are joined to the posterior wing veins by cuticular tubes. Each organ is curved in anterior–posterior direction as well as dorso-ventrally. It consists of about 7-8 horizontally arranged rows of prominent muscle cells, which are attached at their proximal side to a thin layer of cells that has a greater dorsal extension than the muscle cells. Both cell types are labeled by the reporter. The fine acellular strands that hold the wing hearts to the adjacent epidermal cells were not observed to be marked by the reporter. Movies are provided to demonstrate the location and the beating of wing hearts (Tögel, 2008).

The hand-C-GFP reporter was tested for expression in earlier stages of wing heart development and it was found to be active throughout the entire organogenesis. This enabled identification of the embryonic anlagen of the wing hearts, which consist of eight progenitor cells located dorsally and anterior to the heart, in two pairs in the second and third thoracic segment from stage 16/17 onward. The progenitors exhibit a flattened triangular shape and are interconnected by thin cytoplasmic extensions. In addition, the second and the fourth pair of the progenitors are closely associated with the dorsal tracheal branches at their interconnection in the second and third thoracic segments. The characteristic pairwise arrangement and the connection to the tracheae are retained during the subsequent three larval stages. Proliferation starts at about the transition from the second to the third larval instar, leading to eight clusters of cells that remain arranged in four pairs in the anterior region until 1h after puparium formation (APF). Between 1 and 10h APF, the cell number increases significantly and the anterior three pairs of cell clusters are retracted to join the last pair of clusters, eventually forming one large median cluster. Between 13 and 50h APF, the single large cluster splits along its anterior-posterior axis into two groups of cells that migrate laterally in the forming scutellum, thereby adopting the characteristic arched appearance of the adult wing hearts. During this process some of the cells on either side form the underlying thin layer while the remaining cells arrange in horizontal rows along that layer. First contractions of the mature organs were observed at about 45-50h APF (Tögel, 2008).

The expression of the bHLH transcription factor Hand in the wing heart progenitors, which serves as a general marker for all classes of heart cells in Drosophila, prompted a to screen for the expression of genes known to be active in cardiac lineages. Analysis of Even-skipped (Eve) expression revealed that the embryonic wing heart progenitors arise through the same lineage as the well described Eve expressing pericardial cells (EPCs). At stage 10 in embryogenesis, 12 Eve clusters are present on either side of the embryo, located in parasegments (PS) 2 to 12. Each cluster gives rise to a pair of EPCs, except for the most posterior cluster in PS 14, which generates only one EPC. During subsequent development, the first and the second pair of EPCs, located in parasegment 2 and 3, turn toward the midline of the embryo to accompany the tip of the heart, which later bends ventrally into the embryo. The third and the fourth pair of EPCs in PS 4 and 5 are shifted anteriorly in relation to the heart. This step is not based on migration but on the remodeling of the embryo during head involution, since the cells remain in their PS close to the likewise Eve positive anlagen of the DA1 muscle. The EPCs in PS 4 and 5 subsequently differentiate into the later wing heart progenitors, while all others become the classical EPCs and accompany the heart in a loosely associated fashion. At least from PS 4 to 12, all pairs of Eve positive cells (wing heart progenitors and classical EPCs) are interconnected by cytoplasmic extensions forming a rope ladder-like strand above the heart after dorsal closure at stage 16/17. This mode of contact between the cells persists in the wing heart progenitors in postembryonic stages and might be essential for proper relocation in the prepupae (Tögel, 2008).

Although the Drosophila wing hearts have been known for many years, their origin and development have remained unknown. This study provides the first developmental approach on these organs using in vivo time lapse imaging as well as genetic and immunohistochemical methods. It was found that the wing hearts develop from embryonic anlagen that consist of eight progenitor cells located anterior to the heart. Analysis of gene expression in these progenitors confirmed the hypothesis that the wing hearts originate from the cardiac mesoderm, but not from the contractile cardioblast lineage, as has been suggested based on anatomical data. Surprisingly, the embryonic anlagen derive from a particular subset of the well-known EPCs. EPCs arise in pairs in PS 2 to 12 from the dorsal progenitor P2, which divides asymmetrically into the founder of the dorsal oblique muscle 2 and the founder of the EPCs in a numb-dependent lineage decision. Additionally, a single EPC arises in PS 14. The subsequent differentiation of the founders into EPCs requires the activity of the transcription factors Zfh1 and Eve. This study shows that the EPCs located in PS 4 and 5 are relocated in relation to the heart during head involution at stage 14/15 of embryogenesis and subsequently differentiate into the wing heart progenitors. Until this step, no difference to the EPCs in the anterior and posterior PS could be detected. Like the classical EPCs, which remain close to the heart, the EPCs that give rise to the wing heart progenitors depend on factors involved in asymmetric cell division, e.g. Insc or Numb, and fail to differentiate in embryos mutant for zfh1 as well as in animals lacking mesodermal Eve. Loss of tinman expression is the only event that could be identified that discriminates between a classical EPC fate and the specification of wing heart progenitors. Consistently, ectopic expression of Tinman in the wing heart progenitors effectively represses their specification, probably by committing them to a classical EPC fate, indicating that Tinman plays a crucial role in the involved regulatory pathway (Tögel, 2008).

So far, the biological role of pericardial cells (PCs), and EPCs in particular, is not well understood. In the embryo, three populations of PCs arise in each segment, which are characterized by the expression of different combinations of genes (Odd positive PCs, Eve positive PCs, and Tinman positive PCs). During postembryonic stages, the number of PCs decreases, raising the question which population contributes to the final set of PCs in the adult and whether all PCs have the same function throughout development. Recent studies have shown that postembryonic PCs express Odd and Eve, a combination which is not observed in the embryo, and are dispensable for cardiac function. Genetic ablation of all larval PCs had no effect on heart rate, but increased sensitivity to toxic stress. In contrast, the specification of the correct number of embryonic PCs is crucial for normal heart function. Loss of mesodermal Eve during embryogenesis results in fewer larval pericardial cells, which causes a reduction in heart rate and lifespan. Conversely, hyperplasia of embryonic PCs has no effect on heart rate but causes decreased cardiac output. This was explained by an excess of Pericardin secreted by the PCs into the extracellular matrix enveloping the heart (Johnson, 2007). Taken together, embryonic PCs seem to influence cardiac development by e.g., secreting substances whereas postembryonic PCs function as nephrocytes. However, in this study, functional data is provided on a subset of embryonic EPCs, which differentiate into adult progenitors giving rise to a myogenic lineage. This represents a completely new function of PCs, raising the question whether EPCs might in general have myogenic potential and rather represent a population of adult progenitors, than PCs in a functional sense (Tögel, 2008).

The organogenesis of the wing hearts is a highly dynamic process, which includes distinct cellular interactions. At first, adjacent EPCs (including the wing heart progenitors) on either side of the embryo establish contact via cytoplasmic extensions. After dorsal closure of the embryo, interconnections are also formed between opposing EPCs resulting in a rope ladder-like strand above the heart. These interconnections are assumed to be needed to retain contact between the wing heart progenitors during the subsequent development. During larval stages, some of the wing heart progenitors establish a second contact to specific tracheal branches and proliferation starts. In the prepupa, a relocation event joins all wing heart progenitors in one large cluster. During this step, the progenitors are probably passively relocated in conjunction with the tracheal branches to which they are connected. Finally, the wing heart progenitors initiate active migration and form the mature wing hearts in the pupa. Considering the complexity of their development, it is proposed that wing hearts provide an ideal model for studying organogenesis on several different levels such as signaling, cell polarity, or path finding (Tögel, 2008).

Elimination of the embryonic progenitors by ectopic expression of tinman or by laser ablation causes the loss of wing hearts, which results in a specific wing phenotype in conjunction with flightlessness. In the identified phenotype, the delaminated epidermal cells are not cleared from the wings during wing maturation and bonding of the dorsal and ventral wing surfaces is omitted. Recently, it was reported that the epidermal cells transform into mobile fibroblasts and actively migrate out of the wings. However, in in vivo time-lapse studies migration of epidermal cells could not be observed during wing clearance. Conversely, their movements correlated with the periods of wing heart beating, indicating that they are passively transported by the hemolymph flow. One-sided ablation of mature wing hearts in pupae, confirms that wing hearts play a crucial physiological role in wing maturation, since the wing phenotype occurs only on the treated side, but in the same genetic background. In contrast, mutations in genes coding for proteins involved in cell adhesion, e.g. integrins, or in adhesion to the extra cellular matrix, cause a blistered wing phenotype. In the latter phenotype, the epidermal cells of the immature wings are not attached to their opposing cells or to the cuticle and the wing surfaces are separated during unfolding by the sudden influx of hemolymph. In contrast, in animals lacking wing hearts the wings resemble those of the wild-type shortly after unfolding. The epidermal cells also delaminate later from the cuticle, as indicated by their disarrayed pattern, but are not removed from the wings due to the missing hemolymph circulation and probably impede spatially the bonding of the dorsal and the ventral cuticle. Thus, the wings remain in their immature state and do not acquire aerodynamic properties, which accounts for the flightlessness. It is concluded that wing hearts are crucial for establishing proper wing morphology and functionality in Drosophila (Tögel, 2008).

Wing hearts occur in all winged insects, but differ considerably in their morphology. However, their function is highly conserved, since they all function as suction pumps that draw hemolymph from the wings. In the basal condition, the heart itself is directly connected to the scutellum and constitutes the pump. This connection was lost several times during evolution and other muscles, e.g. the separate wing hearts in Drosophila, were recruited to retain the function indicating a high selection pressure on wing circulation. It is suggested that this is due to the crucial role of wing hearts during wing maturation. Since proper wing morphogenesis is essential for flight ability, insect flight might not have been possible before the evolution of wing hearts (Tögel, 2008).

EVOLUTIONARY HOMOLOGS

Hand and muscle development in C. elegans

Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in C. elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. Two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naive blastomeres to muscle when overproduced ectopically in the embryo. In addition, genetic null mutants were used to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type (Fukushige, 2006).

Cardiac development and Hand genes of ascidians

The extensive cell lineage information and streamlined genome of the ascidian, Ciona intestinalis has been exploited to investigate heart development in a basal chordate. Several cardiac genes were analyzed, including the sole Ciona ortholog of the Drosophila tinman gene, and tissue-specific enhancers were isolated for some of the genes. Conserved sequence motifs within these enhancers facilitated the isolation of a heart enhancer for the Ciona Hand-like gene. Altogether, these studies provide a regulatory framework for the differentiation of the cardiac mesoderm, beginning at the 110-cell stage, and extending through the fusion of cardiac progenitors during tail elongation. The cardiac lineage shares a common origin with the germ line, and zygotic transcription is first detected in the heart progenitors only after its separation from the germ line at the 64-cell stage. It is proposed that germ-line determinants influence the specification of the cardiac mesoderm, both by inhibiting inductive signals required for the development of noncardiac mesoderm lineages, and by providing a localized source of Wnt-5 and other signals required for heart development. The possiblility is discussed that the germ line also influences the specification of the vertebrate heart (Davidson, 2003).

Hand genes in fish and frogs

dHAND and eHAND are related basic helix-loop-helix transcription factors that are expressed in the cardiac mesoderm and in numerous neural crest-derived cell types in chick and mouse. To better understand the evolutionary development of overlapping expression and function of the HAND genes during embryogenesis, the zebrafish and Xenopus orthologues were cloned. Comparison of dHAND sequences in zebrafish, Xenopus, chick, mouse and human demonstrated conservation throughout the protein. Expression of dHAND in zebrafish is seen in the earliest precursors of all lateral mesoderm at early gastrulation stages. At neurula and later stages, dHAND expression is observed in lateral precardiac mesoderm, branchial arch neural crest derivatives and posterior lateral mesoderm. At looping heart stages, cardiac dHAND expression remains generalized with no apparent regionalization. Interestingly, no eHAND orthologue was found in zebrafish. In Xenopus, dHAND and eHAND are co-expressed in the cardiac mesoderm without the segmental restriction seen in mice. Xenopus dHAND and eHAND are also expressed bilaterally in the lateral mesoderm without any left-right asymmetry. Within the branchial arches, XdHAND is expressed in a broader domain than XeHAND, similar to their mouse counterparts. Together, these data demonstrate conservation of HAND structure and expression across species (Angelo, 2000).

The precursors of several organs reside within the lateral plate mesoderm of vertebrate embryos. The zebrafish hands off locus is essential for the development of two structures derived from the lateral plate mesoderm -- the heart and the pectoral fin. hands off mutant embryos have defects in myocardial development from an early stage: they produce a reduced number of myocardial precursors, and the myocardial tissue that does form is improperly patterned and fails to maintain tbx5 expression. A similar array of defects is observed in the differentiation of the pectoral fin mesenchyme: small fin buds form in a delayed fashion, anteroposterior patterning of the fin mesenchyme is absent and tbx5 expression is poorly maintained. Defects in these mesodermal structures are preceded by the aberrant morphogenesis of both the cardiogenic and forelimb-forming regions of the lateral plate mesoderm. Molecular analysis of two hands off alleles indicates that the hands off locus encodes the bHLH transcription factor Hand2, which is expressed in the lateral plate mesoderm starting at the completion of gastrulation. Thus, these studies reveal early functions for Hand2 in several cellular processes and highlight a genetic parallel between heart and forelimb development (Yelon, 2000).

The ventrally expressed secreted polypeptide endothelin1 (Edn1) patterns the skeleton derived from the first two pharyngeal arches into dorsal, intermediate and ventral domains. Edn1 activates expression of many genes, including hand2 and Dlx genes. It was of interest to know how hand2/Dlx genes might generate distinct domain identities. Differential expression of hand2 and Dlx genes was shown to delineate domain boundaries before and during cartilage morphogenesis. Knockdown of the broadly expressed genes dlx1a and dlx2a results in both dorsal and intermediate defects, whereas knockdown of three intermediate-domain restricted genes dlx3b, dlx4b and dlx5a results in intermediate-domain-specific defects. The ventrally expressed gene hand2 patterns ventral identity, in part by repressing dlx3b/4b/5a. The jaw joint is an intermediate-domain structure that expresses nkx3.2 and a more general joint marker, trps1. The jaw joint expression of trps1 and nkx3.2 requires dlx3b/4b/5a function, and expands in hand2 mutants. Both hand2 and dlx3b/4b/5a repress dorsal patterning markers. Collectively, this work indicates that the expression and function of hand2 and Dlx genes specify major patterning domains along the dorsoventral axis of zebrafish pharyngeal arches (Talbot, 2010).

Extracellular matrix (ECM) remodeling is critical for organogenesis, yet its molecular regulation is poorly understood. In zebrafish, asymmetric migration of the epithelial lateral plate mesoderm (LPM) displaces the gut leftward, allowing correct placement of the liver and pancreas. To observe LPM migration at cellular resolution, EGFP was transgenically expressed under the control of the regulatory sequences of the bHLH transcription factor gene hand2. Laminin was found to be distributed along the LPM/gut boundary during gut looping, and it appears to become diminished by the migrating hand2-expressing cells. Laminin diminishment is necessary for LPM migration and is dependent on matrix metalloproteinase (MMP) activity. Loss of Hand2 function causes reduced MMP activity and prolonged laminin deposition at the LPM/gut boundary, leading to failed asymmetric LPM migration and gut looping. This study reveals an unexpected role for Hand2, a key regulator of cell specification and differentiation, in modulating ECM remodeling during organogenesis (Yin, 2010).

Chicken Hand genes

Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors. Two bHLH genes, dHAND and eHAND, have now been isolated that are expressed in the bilateral heart primordia and subsequently throughout the primitive tubular heart and its derivatives during chick and mouse embryogenesis. Incubation of stage 8 chick embryos with dHAND and eHAND antisense oligonucleotides revealed that either oligonucleotide alone has no effect on embryonic development, whereas together they arrested development at the looping heart tube stage. Thus, dHAND and eHAND may play redundant roles in the regulation of the morphogenetic events of vertebrate heart development (Srivastava, 1995).

During embryonic development in amniotes, the extraembryonic mesoderm, where the earliest hematopoiesis and vasculogenesis take place, also generates smooth muscle cells (SMCs). It is not well understood how the differentiation of SMCs is linked to that of blood (BCs) and endothelial (ECs) cells. This study shows that, in the chick embryo, the SMC lineage is marked by the expression of a bHLH transcription factor, dHand. Notch activity in nascent ventral mesoderm cells promotes SMC progenitor formation and mediates the separation of SMC and BC/EC common progenitors marked by another bHLH factor, Scl. This is achieved by crosstalk with the BMP and Wnt pathways, which are involved in mesoderm ventralization and SMC lineage induction, respectively. These findings reveal a novel role of the Notch pathway in early ventral mesoderm differentiation, and suggest a stepwise separation among its three main lineages, first between SMC progenitors and BC/EC common progenitors, and then between BCs and ECs (Shin, 2009).

The precise function of the Notch pathway in the process of muscle and BC/EC lineage separation remains to be elucidated. The data suggest that, during chick ventral mesoderm differentiation, the Notch pathway acts together with the BMP and Wnt pathways, and that it plays a 'permissive', rather than an 'instructive', role in mediating the separation of SMCs and BC/ECs. The Notch pathway does not control the induction of but rather the balance between these two populations. Evidence is provided that the induction of these lineages is controlled by the activities of both the BMP pathway, as a general ventral mesoderm inducer, and the canonical Wnt pathway, as a strong SMC lineage inducer. Ectopic activation of the BMP pathway can induce both SMC and BC/EC lineages, with the balance of SMCs and BC/ECs being regulated by Notch activity. It is not clear whether the induction of SMCs by the BMP pathway is a direct or indirect process, or whether it requires an active Wnt pathway. In this analysis, a stronger and wider ectopic dHand induction was observed by CA-β-Catenin than by CA-ALK6 around the anterior primitive streak where BMP antagonists are highly expressed, suggesting that the induction of SMCs by the Wnt pathway does not require active BMP signaling. A recent in vitro study suggested that Notch activity promotes the degradation of Scl by facilitating its ubiquitination, and that this process requires the transcriptional regulation of Notch pathway activity through Suppressor of Hairless. Although there is no direct evidence in support of a similar phenomenon in the current system, it could in principle act as a possible mechanism for the Notch activity-mediated segregation of SMCs and BC/ECs. Furthermore, Nrarp (an ankyrin-repeat protein that is transcriptionally regulated by the Notch signaling pathway), in addition to serving as a Notch-activity readout and a feedback regulator of the Notch pathway, has also been shown to positively regulate the canonical Wnt pathway by blocking the ubiquitination and increasing the stability of Lef1 in zebrafish. This might also serve as a possible mechanism for the Notch and Wnt pathway-mediated SMC specification observed in this system (Shin, 2009).

Expression of mammalian Hand genes

The yeast two-hybrid technique was employed to screen a mouse embryo cDNA library for novel tissue-specific Class B basic helix-loop-helix (bHLH) transcription factors that heterodimerize with the ubiquitously expressed Class A bHLH protein E12. From this screen, a novel bHLH protein was cloned, which was named eHAND. Its low sequence identity with other bHLH family members and unique expression pattern during development suggest that eHAND defines a new subclass of Class B bHLH proteins. eHAND was expressed at high levels in trophoblast cells and extraembryonic membranes throughout development. The first site of eHAND expression in embryos is the heart, where it is expressed at high levels between 8.5 and 10.5 days post coitum, after which transcript levels declined abruptly. By 13.5 d.p.c., eHAND expression in the heart is localized to regions of valve formation. Expression in other regions of the embryo is confined to tissues with a substantial neural crest component. eHAND is expressed in the first branchial arch and its derivatives, in the sympathoadrenal lineage, and in the enteric systems. The expression pattern of eHAND during development is distinct from that of other bHLH genes and suggests that it has a role in formation of extraembryonic tissues, heart, and neural crest derivatives (Cserjesi, 1995).

Physical interactions of mammalian Hand proteins

dHAND and eHAND are basic helix-loop-helix (bHLH) transcription factors expressed during embryogenesis and are required for the proper development of cardiac and extraembryonic tissues. HAND genes, like the myogenic bHLH genes, are classified as class B bHLH genes, which are expressed in a tissue-restricted pattern and function by forming heterodimers with class A bHLH proteins. Myogenic bHLH genes are shown not to form homodimers efficiently, suggesting that their activity is dependent on their E-protein partners. To identify HIPs (HAND-interacting proteins) that regulate the activity of the HAND genes, a 9.5-10.5-day-old mouse embryonic yeast two-hybrid library was screened with eHAND. Several HIPs held high sequence identity to eHAND, indicating that eHAND could form and function as a homodimer. Based on the high degree of amino acid identity between eHAND and dHAND, it is possible that dHAND can also form homodimers and heterodimers with eHAND. Yeast and mammalian two-hybrid assays as well as biochemical pull-down assays showed that eHAND and dHAND are capable of forming both HAND homo- and hetero-dimers in vivo. To investigate whether HAND genes form heterodimers with other biologically relevant bHLH proteins, HAND heterodimerization with the recently identified Hairy-related transcription factors, HRT1-3, was demonstrated. This finding is exciting, because both HRT and HAND genes are coexpressed in the developing heart and limb and both have been implicated in establishing tissue boundaries and pattern formation. Moreover, competition gel shift analysis demonstrates that dHAND and eHAND can negatively regulate the DNA binding of MyoD/E12 heterodimers in a manner similar to MISTI and Id proteins, suggesting a possible transcriptional inhibitory role for HAND genes. Taken together, these results show that dHAND and eHAND can form homo- and hetero-dimer combinations with multiple bHLH partners and that this broad dimerization profile reflects the mechanisms by which HAND genes regulate transcription (Firulli, 2000).

The basic helix-loop-helix (bHLH) factor Hand1 plays an essential role in cardiac morphogenesis, and yet its precise function remains unknown. Protein-protein interactions, involving Hand1, provide a means of determining how Hand1-induced gene expression in the developing heart might be regulated. Hand1 is known to form either heterodimers with near-ubiquitous E-factors and other lineage-restricted class B bHLH proteins or homodimers with itself in vitro. To date, there have been no reported Hand1 protein interactions involving non-bHLH proteins. Heterodimer-versus-homodimer choice is mediated by the phosphorylation status of Hand1; however, little is known about the in vivo function of these dimers or, importantly, how they are regulated. In an effort to understand how Hand1 activity in the heart might be regulated postdimerization, tertiary Hand1-protein interactions with non-bHLH factors was studied. A novel interaction of Hand1 with the LIM domain protein FHL2, a known transcriptional coactivator and corepressor expressed in the developing cardiovascular system, is described. FHL2 interacts with Hand1 via the bHLH domain and is able to repress Hand1/E12 heterodimer-induced transcription but has no effect on Hand1/Hand1 homodimer activity. This effect of FHL2 is not mediated either at the level of dimerization or via an effect of Hand1/E12 DNA binding. In summary, these data describe a novel differential regulation of Hand1 heterodimers versus homodimers by association of the cofactor FHL2 and provide insight into the potential for a tertiary level of control of Hand1 activity in the developing heart (Hill, 2004).

dHAND is a transcription factor belonging to the class B basic helix-loop-helix protein family and is expressed during embryogenesis in the heart, branchial arches, limb buds, and neural crest derivatives. Despite much study, the molecular mechanisms involved in the regulation of dHAND activity are not well understood. Yeast two-hybrid screening was performed using full-length dHAND as bait; this led to identification of several dHAND-binding proteins, including three E-proteins: E2A, ME2, and ALF1. Subsequent analysis revealed that although their heterodimerization and transcriptional activities were similar, dHAND/E-protein heterodimers bind to an E-box element with differing affinities, suggesting they have distinct DNA binding specificities. Moreover, in situ hybridization showed that E-protein genes are expressed fairly ubiquitously among embryonic tissues, including the branchial arches and limb buds. By contrast, little signal was detected in the heart, suggesting that dHAND complexes with partners other than E-proteins in cardiac tissue (Murakami, 2004b).

An intricate array of cell-specific multiprotein complexes participate in programs of cell-specific gene expression through combinatorial interaction with different transcription factors and cofactors. The dHAND basic helix-loop-helix (bHLH) transcription factor, which is essential for heart development and extra embryonic structures, is thought to regulate cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted transcription factors such as GATA4 and NKX2.5. dHAND also interacts with the myocyte enhancer binding factor-2c (MEF2C) protein, a MADS-box transcription factor that is essential for heart development. dHAND and MEF2C synergistically activate expression of the atrial naturetic peptide gene (ANP) in transfected HeLa cells. GST-pulldown and immunoprecipitation assay demonstrate that full-length MEF2C protein is able to interact with dHAND in vitro and in vivo, just like MEF2A and bHLH transcription factors MyoD in skeletal muscle cells. In addition, electrophoretic mobility shift assays (EMSAs) demonstrate that MEF2C and dHAND do not influence each other's DNA binding activity. Using chromatin immunoprecipitation (ChIP) analysis in H9c2 cells it has been shown that dHAND interacts with MEF2C to form protein complex and binds A/T sequence in the promoter of ANP. These results suggest the existence of large multiprotein transcriptional complex with core DNA binding proteins that physically interact with other transcriptional factors to form favorable conformation to potentiate transcription (Zang, 2004).

Phorphorylation of mammalian Hand proteins

HAND2/dHAND is a basic helix-loop-helix transcription factor expressed in the heart and neural crest derivatives during embryogenesis. Although dHAND is essential for branchial arch, cardiovascular and limb development, its target genes have not been identified. The regulatory mechanisms of dHAND function also remain relatively unknown. Akt/PKB, a serine/threonine protein kinase involved in cell survival, growth and differentiation, phosphorylates dHAND and inhibits dHAND-mediated transcription. AU5-dHAND expressed in 293T cells becomes phosphorylated, possibly at its Akt phosphorylation motif, in the absence of kinase inhibitors, whereas the phosphatidylinositol 3-kinase inhibitor wortmannin and the Akt inhibitor NL-71-101, but not the p70 S6 kinase inhibitor rapamycin, significantly reduce dHAND phosphorylation. Coexpression of HA-Akt augments dHAND phosphorylation at multiple serine and threonine residues mainly located in the bHLH domain and, as a result, decreases the transcriptional activity of dHAND. Consistently, alanine mutation mimicking the nonphosphorylation state abolishes the inhibitory effect of Akt on dHAND, whereas aspartate mutation mimicking the phosphorylation state results in a loss of dHAND transcriptional activity. These changes in dHAND transcriptional activity are in parallel with changes in the DNA binding activity rather than in dimerization activity. These results suggest that Akt-mediated signaling may regulate dHAND transcriptional activity through the modulation of its DNA binding activity during embryogenesis (Murakami, 2004a).

Mutation of mammalian Hand genes

dHAND and eHAND are related basic helix-loop-helix (bHLH) transcription factors that are expressed in mesodermal and neural crest-derived structures of the developing heart. In contrast to their homogeneous expression during avian cardiogenesis, during mouse heart development dHAND and eHAND are expressed in a complementary fashion and are restricted to segments of the heart tube fated to form the right and left ventricles, respectively. dHAND and eHAND represent the earliest cardiac chamber-specific transcription factors yet identified. Targeted gene deletion of dHAND in mouse embryos resulted in embryonic lethality at embryonic day 10.5 from heart failure. This description of the cardiac phenotype of dHAND mutant embryos is the first demonstration of a single gene controlling the formation of the mesodermally derived right ventricle and the neural crest-derived aortic arches and reveals a novel cardiogenic subprogramme for right ventricular development (Srivastava, 1997).

The basic helix-loop-helix (bHLH) transcription factors, Hand1 and Hand2, also called eHand/Hxt/Thing1 and dHand/Hed/Thing2, respectively, are expressed in the heart and certain neural-crest derivatives during embryogenesis. In addition, Hand1 is expressed in extraembryonic membranes, whereas Hand2 is expressed in the deciduum. Previous studies have demonstrated that Hand2 is required for formation of the right ventricle of the heart and the aortic arch arteries. A germline mutation was generated in the mouse Hand1 gene by replacing the first coding exon with a beta-galactosidase reporter gene. Embryos homozygous for the Hand1 null allele died between embryonic days 8.5 and 9.5 and exhibited yolk sac abnormalities due to a deficiency in extraembryonic mesoderm. Heart development was also perturbed and did not progress beyond the cardiac-looping stage. These results demonstrate important roles for Hand1 in extraembryonic mesodermal and heart development (Firulli, 1998).

dHAND and eHAND are basic helix-loop-helix transcription factors that play critical roles in cardiac development. The HAND genes have a complementary left-right cardiac asymmetry of expression with dHAND predominantly on the right side and eHAND on the left side of the looped heart tube. Although eHAND is asymmetrically expressed along the anterior-posterior and dorsal-ventral embryonic axes, it is symmetrically expressed along the left-right axis at early stages of embryonic and cardiac development. After cardiac looping, dHAND and eHAND are expressed in the right (pulmonary) and left (systemic) ventricles, respectively. The left-right (LR) sidedness of dHAND and eHAND expression is demonstrated to be anatomically reversed in situs inversus (inv/inv) mouse embryos; however, dHAND expression persists in the pulmonary ventricle and eHAND in the systemic ventricle regardless of anatomic position, indicating chamber specificity of expression. dHAND-null mice fail to form a right-sided pulmonary ventricle. Mice homozygous for the dHAND and inv mutations are demonstrated to have only a right-sided ventricle [which is morphologically a left (systemic) ventricle]. These data suggest that the HAND genes are involved in development of segments of the heart tube that give rise to specific chambers of the heart during cardiogenesis, rather than controlling the direction of cardiac looping by interpreting the cascade of LR embryonic signals (Thomas, 1998).

Nkx2.5/Csx and dHAND/Hand2 are conserved transcription factors that are coexpressed in the precardiac mesoderm and early heart tube and control distinct developmental events during cardiogenesis. To understand whether Nkx2.5 and dHAND may function in overlapping genetic pathways, mouse embryos lacking both Nkx2.5 and dHAND were generated. Mice heterozygous for mutant alleles of Nkx2.5 and dHAND are viable. Although single Nkx2.5 or dHAND mutants have a morphological atrial and single ventricular chamber, Nkx2.5;dHAND double mutants have only a single cardiac chamber which was molecularly defined as the atrium. Complete ventricular dysgenesis was observed in Nkx2.5;dHAND double mutants; however, a precursor pool of ventricular cardiomyocytes was identified on the ventral surface of the heart tube. Because Nkx2.5 mutants failed to activate eHAND expression even in the early precardiac mesoderm, the double mutant phenotype appears to reflect an effectively null state of dHAND and eHAND. Cell fate analysis in dHAND mutants suggests a role of HAND genes in survival and expansion of the ventricular segment, but not in specification of ventricular cardiomyocytes. These molecular analyses also reveal the cooperative regulation of the homeodomain protein, Irx4, by Nkx2.5 and dHAND. These studies provide the first demonstration of gene mutations that result in ablation of the entire ventricular segment of the mammalian heart, and reveal essential transcriptional pathways for ventricular formation (Yamagishi, 2001).

HAND2 is an essential transcription factor for cardiac, pharyngeal arch, and limb development. Apoptosis in the HAND2-null embryo causes hypoplasia of the right ventricle and pharyngeal arches leading to lethality by embryonic day (E)10.0 from heart failure. In order to investigate the role of apoptosis in inducing the HAND2-null phenotype, mouse embryos were generated lacking both HAND2 and Apaf-1, a central downstream mediator of mitochondrial damage-induced apoptosis. In contrast to HAND2-/- embryos, HAND2-/-Apaf-1-/- embryos at E10.5-11.0 had well-developed pharyngeal arches, aortic arch arteries, and no signs of cardiac failure. TUNEL analysis through pharyngeal arches of HAND2-/-Apaf-1-/- embryos revealed decreased apoptosis and the embryos had clearly patent aortic arch arteries. However, ventricular hypoplasia and cell death were unchanged in these animals compared to HAND2-/- embryos, resulting in growth arrest at E11.0. This study suggests that loss of HAND2 in the pharyngeal arch mesenchyme leads to apoptosis in an Apaf-1-dependent fashion and that, while loss of aortic arch integrity contributes to the early lethality, the ventricular defects are independent of arch development (Aiyer, 2005).

The basic helix-loop-helix transcription factors Hand1 and Hand2 display dynamic and spatially restricted expression patterns in the developing heart. Mice that lack Hand2 die at embryonic day 10.5 from right ventricular hypoplasia and vascular defects, whereas mice that lack Hand1 die at embryonic day 8.5 from placental and extra-embryonic abnormalities that preclude analysis of its potential role in later stages of heart development. To determine the cardiac functions of Hand1, mice were generated harboring a conditional Hand1-null allele and the gene was excised by cardiac-specific expression of Cre recombinase. Embryos homozygous for the cardiac Hand1 gene deletion displayed defects in the left ventricle and endocardial cushions, and exhibited dysregulated ventricular gene expression. However, these embryos survived until the perinatal period when they died from a spectrum of cardiac abnormalities. Creation of Hand1/2 double mutant mice revealed gene dose-sensitive functions of Hand transcription factors in the control of cardiac morphogenesis and ventricular gene expression. These findings demonstrate that Hand factors play pivotal and partially redundant roles in cardiac morphogenesis, cardiomyocyte differentiation and cardiac-specific transcription (McFadden, 2005).

The basic helix-loop-helix (bHLH) transcription factor Hand2 is required for growth and development of the heart, branchial arches and limb buds. To determine whether DNA binding is required for Hand2 to regulate the growth and development of these different embryonic tissues, mutant mice were generated in which the Hand2 locus was modified by a mutation (referred to as Hand2^EDE) that abolished the DNA-binding activity of Hand2, leaving the remainder of the protein intact. In contrast to Hand2 null embryos, which display right ventricular hypoplasia and vascular abnormalities, causing severe growth retardation by E9.5 and death by E10.5, early development of the heart appeared remarkably normal in homozygous Hand2^EDE mutant embryos. These mutant embryos also lacked the early defects in growth of the branchial arches seen in Hand2 null embryos and survived up to 2 to 3 days longer than did Hand2 null embryos. However, Hand2^EDE mutant embryos exhibited growth defects in the limb buds similar to those of Hand2 null embryos. These findings suggest that Hand2 regulates tissue growth and development in vivo through DNA binding-dependent and -independent mechanisms (Liu, 2009).

Targeted deletion of the bHLH DNA-binding protein Hand2 in the neural crest, impacts development of the enteric nervous system (ENS), possibly by regulating the transition from neural precursor cell to neuron. This hypothesis was tested by targeting Hand2 deletion in nestin-expressing neural precursor (NEP) cells. The mutant mice showed abnormal ENS development, resulting in lethal neurogenic pseudo-obstruction. Neurogenesis of neurons derived from NEP cells identified a second nestin non-expressing neural precursor (NNEP) cell in the ENS. There was substantial compensation for the loss of neurons derived from the NEP pool by the NNEP pool but this was insufficient to abrogate the negative impact of Hand2 deletion. Hand2-mediated regulation of proliferation affected both neural precursor and neuron numbers. Differentiation of glial cells derived from the NEP cells was significantly decreased with no compensation from the NNEP pool of cells. These data indicate differential developmental potential of NEPs and NNEPs; NNEPs preferentially differentiate as neurons, whereas NEPs give rise to both neurons and glial cells. Deletion of Hand2 also resulted in complete loss of NOS and VIP and a significant decrease in expression of choline acetyltransferase and calretinin, demonstrating a role for Hand2 in neurotransmitter specification and/or expression. Loss of Hand2 resulted in a marked disruption of the developing neural network, exemplified by lack of a myenteric plexus and extensive overgrowth of fibers. Thus, Hand2 is essential for neurogenesis, neurotransmitter specification and neural network patterning in the developing ENS (Lei, 2011).

The basic helix-loop-helix transcription factor Twist1 is essential for normal limb development. Twist1^-/- embryos die at midgestation. However, studies on early limb buds found that Twist1^-/- mutant limb mesenchyme has an impaired response to FGF signaling from the apical ectodermal ridge, which disrupts the feedback loop between the mesenchyme and AER, and reduces and shifts anteriorly Shh expression in the zone of polarizing activity. This study combined Twist1 null, hypomorph and conditional alleles to generate a Twist1 allelic series that survives to birth. As Twist1 activity is reduced, limb skeletal defects progress from preaxial polydactyly to girdle reduction combined with hypoplasia, aplasia or mirror symmetry of all limb segments. With reduced Twist1 activity there is striking and progressive upregulation of ectopic Shh expression in the anterior of the limb, combined with an anterior shift in the posterior Shh domain, which is expressed at normal intensity, and loss of the posterior AER. Consequently limb outgrowth is initially impaired, before an ectopic anterior Shh domain expands the AER, promoting additional growth and repatterning. Reducing the dosage of FGF targets of the Etv gene family, which are known repressors of Shh expression in anterior limb mesenchyme, strongly enhances the anterior skeletal phenotype. Conversely this and other phenotypes are suppressed by reducing the dosage of the Twist1 antagonist Hand2. These data support a model whereby multiple Twist1 activity thresholds contribute to early limb bud patterning, and suggest how particular combinations of skeletal defects result from differing amounts of Twist1 activity (Krawchuk, 2011).

Transcriptional regulation of mammalian Hand genes

One of the first morphological manifestations of left/right (L/R) asymmetry in mammalian embryos is a pronounced rightward looping of the linear heart tube. The direction of looping is thought to be controlled by signals from an embryonic L/R axial system. Morphological L/R asymmetry in the murine heart first becomes apparent at the linear tube stage as a leftward displacement of its caudal aspect. Beginning at the same stage, the basic helix-loop-helix (bHLH) factor gene eHand is expressed in a strikingly left-dominant pattern in myocardium, reflecting an intrinsic molecular asymmetry. In embryo hearts lacking the homeobox gene Nkx2-5, which does not loop, left-sided eHand expression is abolished. The data predict that eHand expression is enhanced in descendants of the left heart progenitor pool as one response to inductive signaling from the L/R axial system, and that eHand controls intrinsic morphogenetic pathways essential for looping (Biben, 1997).

Basic helix-loop-helix (bHLH) transcription factors control developmental decisions in a wide range of embryonic cell types. The HLH motif mediates homo- and hetero-dimerization; this juxtaposes the basic regions within the dimeric complex to form a bipartite DNA binding domain that recognizes a DNA consensus sequence known as an E-box. eHAND and dHAND (also known as HAND1 and HAND2) are closely related bHLH proteins that control cardiac, craniofacial and limb development. Within the developing limb, dHAND expression encompasses the zone of polarizing activity in the posterior region, where it has been shown to be necessary and sufficient to induce the expression of the morphogen sonic hedgehog. Misexpression of dHAND in the anterior compartment of the limb bud induces ectopic expression of sonic hedgehog, with resulting preaxial polydactyly and mirror image duplications of posterior digits. To investigate the potential transcriptional mechanisms involved in limb patterning by dHAND, a structure-function analysis was performed of the protein in cultured cells and dHAND mutant proteins were ectopically expressed in the developing limbs of transgenic mice. An N-terminal transcriptional activation domain, and the bHLH region, are required for E-box-dependent transcription in vitro. Remarkably, however, digit duplication by dHAND requires neither the transcriptional activation domain nor the basic region, but only the HLH motif. eHAND has a similar limb patterning activity to dHAND in these misexpression experiments, indicating a conserved function of the HLH regions of these proteins. These findings suggest that dHAND may act via novel transcriptional mechanisms mediated by protein-protein interactions independent of direct DNA binding (McFadden, 2000).

Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. This study characterized the Gli-dependent cis-regulatory network through a combination of whole-genome chromatin immunoprecipitation (ChIP)-on-chip and transcriptional profiling of the developing mouse limb. These analyses identified approximately 5000 high-quality Gli3-binding sites, including all known Gli-dependent enhancers. Discrete binding regions exhibit a higher-order clustering, highlighting the complexity of cis-regulatory interactions. Further, Gli3 binds inertly to previously identified neural-specific Gli enhancers, demonstrating the accessibility of their cis-regulatory elements. Intersection of DNA binding data with gene expression profiles predicted 205 putative limb target genes. A subset of putative cis-regulatory regions were analyzed in transgenic embryos, establishing Blimp1 (see Drosophila Blimp-1) as a direct Gli target and identifying Gli activator signaling in a direct, long-range regulation of the BMP antagonist Gremlin. In contrast, a long-range silencer cassette downstream from Hand2 likely mediates Gli3 repression in the anterior limb. These studies provide the first comprehensive characterization of the transcriptional output of a Shh-patterning process in the mammalian embryo and a framework for elaborating regulatory networks in the developing limb (Vokes, 2008).

DNA binding of mammalian Hand genes

HAND2 (dHAND) is a basic helix-loop-helix (bHLH) transcription factor expressed in numerous tissues during development including the heart, limbs, and a subset of neural crest derivatives. Functional analysis has shown that HAND2 is involved in development of the branchial arches, heart, limb, vasculature, and nervous system. Although it is essential for development of numerous tissues, little is known about its mode of action. To this end, HAND2 transcriptional regulatory mechanisms have been characterized. Using mammalian one-hybrid analysis it has been shown that HAND2 contains a strong transcriptional activation domain in the amino-terminal third of the protein. Like most tissue-restricted bHLH factors, HAND2 heterodimerizes with the broadly expressed bHLH factors, the E-proteins. The consensus DNA binding site of HAND2 was determined; HAND2 binds a subset of E-boxes as a heterodimer with E12. Yeast two-hybrid screening of a neuroblastoma cDNA library for HAND2-interacting proteins selected HAND2 and numerous additional members of the E-protein family. Although HAND2 homodimer formation was confirmed by in vitro analysis, HAND2 fails to homodimerize in a mammalian two-hybrid assay but demonstrates robust HAND2/E12 interaction. It is concluded that HAND2 functions as a transcription activator by binding a subset of E-boxes as a heterodimer with E-proteins (Dai, 2002).

Transcriptional targets of mammalian Hand genes

The bHLH protein eHAND plays an important role in the development of extraembryonic, mesodermal, and cardiac cell lineages, presumably through heterodimerization with other HLH proteins and DNA binding. A novel transcriptional activity of eHAND has been identified. In transient transfection assays, eHAND is a potent inhibitor of activation by some but not all bHLH proteins. eHAND can prevent E-box DNA binding by these bHLH proteins. Interestingly, eHAND can also strongly inhibit transactivation activity by a MyoD approximately E47 tethered dimer, which suggests a distinct mechanism of action. eHAND also inhibits MyoD-dependent skeletal muscle cell differentiation and expression of the muscle-specific myosin heavy chain protein. In addition, eHAND can repress activity of the natural p75LNGFR promoter, whose expression overlaps that of eHAND and dHAND. The inhibitory activity of eHAND may be attributed to multiple mechanisms, such as the ability to act as a corepressor, the presence of a repression domain, and its ability to sequester E proteins in an inactive complex. Based upon its inhibitory effect on bHLH proteins and cellular differentiation, it is proposed that eHAND may function by several mechanisms to promote placental giant cell proliferation by negatively regulating the activities of the bHLH protein MASH-2 (Bounpheng, 2000).

Reported here is the isolation and characterization of murine and human cDNAs encoded for by Irx4 (Iroquois homeobox gene 4). Mouse and human Irx4 proteins are highly conserved (83%) and their 63-aa homeodomains are more than 93% identical to those of the Drosophila Iroquois patterning genes. Human IRX4 maps to chromosome 5p15.3, which is syntenic to murine chromosome 13. Irx4 transcripts are present in the developing central nervous system, skin, and vibrissae, but are predominantly expressed in the cardiac ventricles. In mice at embryonic day (E) 7.5, Irx4 transcripts are found in the chorion and at low levels in a discrete anterior domain of the cardiac primordia. During the formation of the linear heart tube and its subsequent looping (E8.0 -8.5), Irx4 expression is restricted to the ventricular segment and is absent from both the posterior (eventual atrial) and the anterior (eventual outflow tract) segments of the heart. Throughout all subsequent stages in which the chambers of the heart become morphologically distinct (E8.5-11) and into adulthood, cardiac Irx4 expression is found exclusively in the ventricular myocardium. Irx4 gene expression has also been assessed in embryos with aberrant cardiac development: mice lacking RXRalpha or MEF2c have normal Irx4 expression, but mice lacking the homeobox transcription factor Nkx2-5 (Csx) have markedly reduced levels of Irx4 transcripts. dHand-null embryos initiate Irx4 expression, but cannot maintain normal levels. These data indicate that the homeobox gene Irx4 is likely to be an important mediator of ventricular differentiation during cardiac development downstream of Nkx2-5 and dHand (Bruneau, 2000).

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate the specification and differentiation of numerous cell types during embryonic development. Hand1 and Hand2 are expressed by a subset of neural crest cells in the anterior branchial arches and are involved in craniofacial development. However, the precise mechanisms by which Hand proteins mediate biological actions and regulate downstream target genes in branchial arches is largely unknown. This study reports that Hand2 negatively regulates intramembranous ossification of the mandible by directly inhibiting the transcription factor Runx2, a master regulator of osteoblast differentiation. Hand proteins physically interact with Runx2, suppressing its DNA binding and transcriptional activity. This interaction is mediated by the N-terminal domain of the Hand protein and requires neither dimerization with other bHLH proteins nor DNA binding. Partial colocalization of Hand2 and Runx2 was observed in the mandibular primordium of the branchial arch, and downregulation of Hand2 precedes Runx2-driven osteoblast differentiation. Hand2 hypomorphic mutant mice display insufficient mineralization and ectopic bone formation in the mandible due to accelerated osteoblast differentiation, which is associated with the upregulation and ectopic expression of Runx2 in the mandibular arch. This study shows that Hand2 acts as a novel inhibitor of the Runx2-DNA interaction and thereby regulates osteoblast differentiation in branchial arch development (Funato, 2009).

Lower jaw development is a complex process in which multiple signaling cascades establish a proximal-distal organization. These cascades are regulated both spatially and temporally and are constantly refined through both induction of normal signals and inhibition of inappropriate signals. The connective tissue of the tongue arises from cranial neural crest cell-derived ectomesenchyme within the mandibular portion of the first pharyngeal arch and is likely to be impacted by this signaling. Although the developmental mechanisms behind later aspects of tongue development, including innervation and taste acquisition, have been elucidated, the early patterning signals driving ectomesenchyme into a tongue lineage are largely unknown. This study shows that the basic helix-loop-helix transcription factor Hand2 plays key roles in establishing the proximal-distal patterning of the mouse lower jaw, in part through establishing a negative-feedback loop in which Hand2 represses Dlx5 and Dlx6 expression in the distal arch ectomesenchyme following Dlx5- and Dlx6-mediated induction of Hand2 expression in the same region. Failure to repress distal Dlx5 and Dlx6 expression results in upregulation of Runx2 expression in the mandibular arch and the subsequent formation of aberrant bone in the lower jaw along with proximal-distal duplications. In addition, there is an absence of lateral lingual swelling expansion, from which the tongue arises, resulting in aglossia. Hand2 thus appears to establish a distal mandibular arch domain that is conducive for lower jaw development, including the initiation of tongue mesenchyme morphogenesis (Barron, 2011).

Hand and neural crest derived cells

Hand genes encode basic helix-loop-helix transcription factors that are expressed in the developing gut, where their function is unknown. Enteric Hand2 expression is limited to crest-derived cells, whereas Hand1 expression is restricted to muscle and interstitial cells of Cajal. Hand2 is developmentally regulated and is intranuclear in precursors but cytoplasmic in neurons. Neurons develop in explants from wild-type but not Hand2^-/- bowel, although, in both, crest-derived cells are present and glia arise. Similarly, small interfering RNA (siRNA) silencing of Hand2 in enteric crest-derived cells prevents neuronal development. Terminally differentiated enteric neurons do not develop after conditional inactivation of Hand2 in migrating crest-derived cells; nevertheless, conditional Hand2 inactivation does not prevent precursors from expressing early neural markers. It is suggested that enteric neuronal development occurs in stages and that Hand2 expression is required for terminal differentiation but not for precursors to enter the neuronal lineage (D'Autreaux, 2007).

Defective Hand1 phosphoregulation uncovers essential roles for Hand1 in limb morphogenesis

The morphogenesis of the vertebrate limbs is a complex process where cell signaling and transcriptional regulation coordinate diverse structural adaptations across species. This study examined the consequences of altering Hand1 (see Drosophila Hand) dimer choice regulation within the developing vertebrate limbs. Although Hand1 deletion via the limb-specific Prx1-Cre reveals a non-essential role for Hand1 in limb morphogenesis, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of anterior-proximal limb elements. Molecular analysis reveals a non-cell autonomous mechanism that causes widespread cell death within embryonic limb bud. In addition, changes were observed in proximal anterior gene regulation including a reduction in the expression of Irx3&5 (see Drosophila Araucan), Gli3 (see Drosophila Ci), and Alx4 (see Drosophila Aristaless), all of which are upregulated in Hand2 limb conditional knockouts. A reduction of Hand2 and Shh (see Drosophila Hedgehog) gene dosage improves the integrity of anterior limb structures validating this proposed mechanism (Firulli, 2017).

REFERENCES

Search PubMed for articles about Drosophila Hand

Aiyer, A. R., Honarpour, N., Herz, J. and Srivastava, D. (2005). Loss of Apaf-1 leads to partial rescue of the HAND2-null phenotype. Dev. Biol. 278: 155-162. 15649468

Angelo, S., Lohr, J., Lee, K. H., Ticho, B. S., Breitbart, R. E., Hill, S., Yost, H. J. and Srivastava, D. (2000). Conservation of sequence and expression of Xenopus and zebrafish dHand during cardiac, branchial arch and lateral mesoderm development. Mech. Dev. 95: 231-237. 10906469

Barron, F., et al. (2011). Downregulation of Dlx5 and Dlx6 expression by Hand2 is essential for initiation of tongue morphogenesis. Development 138(11): 2249-59. PubMed Citation: 21558373

Biben, C., et al. (1997). Homeodomain factor Nkx2-5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development. Genes Dev. 11(11): 1357-1369. PubMed Citation: 9192865

Bounpheng, M. A., Morrish, T. A., Dodds, S. G. and Christy. B. A. (2000). Negative regulation of selected bHLH proteins by eHAND. Exp. Cell Res. 257(2): 320-31. 10837146

Bruneau, B. G., et al. (2000). Cardiac expression of the ventricle-specific homeobox gene Irx4 is modulated by Nkx2-5 and dHand. Dev. Biol. 217: 266-277. 10625552

Cserjesi, P., Brown, D., Lyons, G. E. and Olson, E. N. (1995). Expression of the novel basic helix-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development. Dev. Biol. 170(2): 664-78. 7649392

Dai, Y. S. and Cserjesi. P. (2002). The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer. J. Biol. Chem. 277(15): 12604-12. 11812799

D'Autreaux, F., Morikawa, Y., Cserjesi, P. and Gershon, M. D. (2007). Hand2 is necessary for terminal differentiation of enteric neurons from crest-derived precursors but not for their migration into the gut or for formation of glia. Development 134(12): 2237-49. Medline abstract: 17507395

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date revised: 1 January 2024

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