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).
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).
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).
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).
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