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First published online 8 February 2006
doi: 10.1242/dev.02285
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Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd., Dallas, TX 75390, USA.
* Author for correspondence (e-mail: eric.olson{at}utsouthwestern.edu)
Accepted 16 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hand, Drosophila, Heart development, Cardiogenesis, Hematopoiesis, Lymph gland, Apoptosis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Zaffran and Frasch,
2002). 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 (Cripps and Olson,
2002). In Drosophila, the myocardial cell layer of the
contractile heart tube, composed of cardioblasts, is surrounded by pericardial
nephrocytes, which function as secretory cells
(Crossley, 1972;
Mandal et al., 2004), and by
lymph gland hematopoietic cells that give rise to all the major blood cells in
the adult fly (Evans et al.,
2003). 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
(Cripps and Olson, 2002;
Evans et al., 2003). 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
(Cripps and Olson, 2002;
Evans et al., 2003),
exemplifying the conservation of developmental programs for cardiogenesis and
hematopoiesis between Drosophila and mammals.
NK-type homeodomain proteins and the GATA family of zinc-finger
transcription factors are required for cardiac and hematopoietic development
in Drosophila and mammals
(Harvey, 1996;
Sorrentino et al., 2005). 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 (Yin and Frasch, 1998;
Xu et al., 1998;
Liberatore et al., 2002;
Lien et al., 2002). Both
Tinman and Nkx2.5 play central roles in activation of myocardial genes
required for heart development (Bodmer,
1993; Lyons et al.,
1995). 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
(Alvarez et al., 2003;
Klinedinst and Bodmer, 2003;
Molkentin et al., 1997;
Gove et al., 1997;
Reiter et al., 1999). Pannier
and GATA4 function as partners of Tinman and Nkx2.5, respectively, to activate
the cardiac gene program in Drosophila and mammals
(Gajewski et al., 1999;
Lee et al., 1998). 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
(Lebestky et al., 2000;
Mandal et al., 2004;
Tsai et al., 1994;
Ting et al., 1996;
Ferreira et al., 2005). 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.
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 and
Paululat, 2002; Han and Olson,
2005). In a recent study (Han
and Olson, 2005), we showed that 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. 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.
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 et al.,
1995; Srivastava et al.,
1997; Thomas et al.,
1998). Mice lacking Hand1 die from placental and
extra-embryonic abnormalities (Firulli et
al., 1998), whereas mice lacking Hand2 die from right
ventricular hypoplasia and vascular defects
(Srivastava et al., 1995;
Srivastava et al., 1997;
Yamagishi et al., 2001).
Deletion of the Hand1 and Hand2 genes in the heart revealed
their dose-sensitive requirement and functional redundancy for myocardial
growth (McFadden et al.,
2005), and mutation of the single hand gene in zebrafish
results in a dramatic reduction in the number of cardiac cells
(Yelon et al., 2000). In
addition to its cardiac expression, Hand1 is highly expressed in the
lateral plate mesoderm (Firulli et al.,
1998) from which the intra-embryonic aorta-gonad-mesonephros
(AGM), a major source of hematopoiesis, is derived
(Medvinsky and Dzierzak,
1996). The potential functions of Hand genes in hematopoiesis have
not been investigated.
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, as the Drosophila heart is thought to be a simple linear tube that does not undergo complex morphogenic changes.
Here, we show that Drosophila Hand functions 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, we generated a null mutant in the gene through homologous recombination. Hand mutant embryos and larvae displayed 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 were 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.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
Hand-GFP and Hand-Gal4 flies were generated using the HCH enhancer identified
in previous study (Han and Olson,
2005). The UAS-Hand, UAS-Hand-EnR and UAS-Hand-VP16 transgenic
flies were generated by cloning the corresponding DNA fragments into the pUAST
vector. Transgenic constructs were injected according to standard procedures.
Germline-transformed transgenic flies were selected by red eye color
(w+) and maintained as homozygotes. At least four
independent transgenic lines were analyzed for each construct.
Generating Hand null mutant by ends-out homologous recombination
Ends-out homologous recombination at the Hand locus was performed
as described (Gong and Golic,
2003). Briefly, two arms obtained from genomic PCR were subcloned
into the pw25 vector to create the construct Handko-pw25 for
generating transformants. Transgenic flies bearing this construct on
chromosome 3 were crossed to yw; 70FLP, 70I-SceI, Sco/CyO flies and
the progeny (F1) were heat-shocked at 37°C for 1 hour on days 3, 4 and 5
after egg laying. Virgins carrying Handko-pw25 and 70FLP,
70I-SceI, Sco of the F1 progeny were crossed to w1118
males. About 0.04% of the F2 progeny appeared with red-eyes (8 out of
20,000 flies). Six independent lines showed translocation of the
w+ marker from the 3rd chromosome to the 2nd chromosome,
where Hand is located. Targeted homologous recombination was verified
by genomic PCR and sequencing in four independent lines. Primer information is
available on request.
Immunohistochemistry and microscopy
Embryos from different lines were collected and stained with various
antibodies as previously described (Han et
al., 2002). The following primary antibodies were used: mouse
anti-ß-galactosidase 1:300 (Promega); rat anti-Eve 1:200 (from D.
Kosman); rabbit anti-Tinman 1:500 (from R. Bodmer); rabbit anti-Mef2 1:1000
(from B. Paterson); rabbit anti-GFP 1:2000 (Abcam); rabbit anti-Srp 1:500
(from R. Reuter). Cy2, Cy3, Cy5 or Biotin-conjugated secondary antibodies
(from Jackson Lab) were used to recognize the primary antibodies. Images were
obtained with a Zeiss LSM510-meta confocal microscope or a Leica DMRXE
compound microscope. Three-dimensional reconstruction of the
Drosophila heart was carried out using the Volocity 2.6 graphic
software from Improvision.
Transfection assays
Cell transfection and luciferase assays were performed as described
(Han et al., 2004). Reporter
plasmid (100 ng) and 100 ng of each activator plasmid were used. The
L8E6-luciferase and L8G4-luciferase were generated by cloning six tandem
copies of E-box binding sites or four tandem copies of Gal4-binding sites,
respectively, into the pGL3 vector (Promega). Luciferase activities are
expressed as mean±s.d. from three experiments.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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) (Fig. 1C). We found that the transcriptional activity of Hand depends primarily on its N-terminal region (Fig. 1C). Interestingly, mutation of the conserved basic residues in the bHLH domain increased the transcriptional activity of Gal4-Hand dramatically (Fig. 1C, Gal4-Hand-RRR), suggesting that the basic region communicates, directly or indirectly, with the transcription activation domain.
Hand functions as a transcription activator during Drosophila cardiogenesis and hematopoiesis
To determine if Hand also functions as a transcription activator in vivo,
we converted it to repressor and a super-activator by fusing it to the
Engrailed repression domain (EnR) and the VP16 transcription activation
domain, respectively. Hand-VP16 functioned as an extremely strong
transcriptional activator (Fig.
1B), whereas Hand-EnR, when co-expressed with HAND, efficiently
blocked the activity of Hand in Drosophila S2 cells
(Fig. 1D).
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To examine further the cell-autonomous requirement of Hand function within
the dorsal vessel, we overexpressed wild-type Hand, Hand-VP16 and Hand-EnR
using a Hand-Gal4 driver generated by using the Hand cardiac and
hematopoietic enhancer (HCH) identified in our previous study
(Han and Olson, 2005).
Targeted overexpression of wild-type Hand and Hand-VP16 in
Hand-expressing cells did not evoke a phenotype
(Fig. 2E,G; data not shown),
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 Hand cardiac and
hematopoietic (HCH) enhancer identified previously
(Han and Olson, 2005)
(Fig. 2F,H). The number of
cardioblasts and pericardial nephrocytes was also diminished and their
alignment was disrupted in embryos expressing Hand-EnR
(Fig. 2F,H). These data suggest
that Hand functions as an essential transcriptional activator during
cardiogenesis and hematopoiesis.
Generation of a Hand null mutant by homologous recombination
To examine the functions of Hand in vivo, we generated a null
mutant of the gene by replacing it with a mini-white gene using the
ends-out homologous recombination technology
(Fig. 3A)
(Gong and Golic, 2003). We
obtained five independent homozygous lethal lines with a trans-location of the
mini-white gene from the 3rd chromosome where it was originally
located to the 2nd chromosome where the Hand gene resides. Four out
of these five lines failed to complement a deficiency line that deletes the
Hand locus (BL-7819). RT-PCR from homozygous mutant larvae from these
four independent lines, identified by the absence of a GFP-positive balancer
chromosome, showed a loss of Hand transcripts
(Fig. 3B). Hand
transcripts were also undetectable by in situ hybridization of homozygous
Hand mutant embryos, identified by the absence of a
ß-Gal-positive balancer chromosome
(Fig. 3C), further
demonstrating that the Hand mutation resulted in a null allele.
Sequencing of genomic PCR products demonstrated that expected homologous
recombination occurred identically in these four independent mutant lines.
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3%) survived
for a few days after hatching, but they were sluggish and remained as small as
1st-instar larvae.
Cardiac and hematopoietic defects in Hand mutant embryos
Approximately 20% of Hand mutant embryos showed a range of cardiac
morphological defects that included discontinuities and irregularities in the
architecture of the heart tube, shown by the misalignment of Mef2-expressing
cardioblasts (Fig. 4D,F),
reduced numbers of pericardial nephrocytes, shown by Odd-skipped (Odd)
expression, and random gaps in expression of the secreted extracellular matrix
protein Pericardin (Fig. 4E,F).
A small subset of mutant embryos (3%) showed more severe cardiac defects
characterized by a significant reduction of Mef2-expressing cardioblasts,
Odd-expressing pericardial cells and Pericardin expression
(Fig. 4G-I). In addition, the
number of lymph gland hematopoietic cells was reduced in more than half of
Hand mutant embryos. In many of these mutants, the lymph gland cell
clusters labeled by Odd antibody were completely absent, whereas the ring
gland, which is located anterior to the lymph gland and is labeled by the
Pericardin antibody, was intact (Fig.
4D-F).
Cardiac and hematopoietic defects in Hand mutant larvae
About 80% of Hand mutant embryos showed normal embryonic heart
development and 60% of Hand mutants hatched to become 1st-instar
larvae. In order to examine for possible abnormalities in larval cardiac
morphology, we crossed the Hand-GFP transgene into the Hand mutant
background. Recent work has shown that the Drosophila heart undergoes
dramatic cardiac remodeling during late larva and early pupa development
(Monier et al., 2005).
However, little is known about the cardiac morphological changes during the
early larval stages because of the lack of markers of the living heart and the
inaccessibility of antibodies at larval stages. The Hand-GFP transgene
strongly labels the entire heart from embryos to adults, providing an
opportunity to examine the cardiac morphological changes during the late
embryo and early larva transition by confocal microscopy. At 18 hours after
egg laying (AEL), cardioblasts and pericardial cells were well aligned at the
dorsal midline in wild type and a majority of Hand mutants
(Fig. 5A,D). The number of
lymph gland hematopoietic cells flanking the anterior aorta was largely
reduced in most Hand mutants (Fig.
5D). At around 20 hours AEL, cardioblasts and pericardial cells in
wild-type larvae no longer aligned in perfect rows, as the cardioblasts
started to form the heart tube and the pericardial nephrocytes started to
migrate to their final positions around the heart tube
(Fig. 5B). A subset of
Hand mutants started to show defects around this time with a reduced
number of pericardial cells and thinner heart tube
(Fig. 5E). The cardiac
morphological defects of Hand mutants became more significant around
24 hours AEL, when 1st-instar larvae hatched from the cuticle. In wild-type
1st-instar larvae, a chamber-like structure was seen in the posterior heart
and the size of the pericardial nephrocytes was significantly enlarged
(Fig. 5C). By contrast, most
newly hatched Hand mutant 1st-instar larvae displayed a hypoplastic
heart with an abnormally thin heart tube and further reduced numbers of
pericardial cells, as well as gaps in the posterior heart tube
(Fig. 5F). Higher magnitude
confocal scans showed the lymph gland cell clusters flanking the anterior
opening of the aorta (Fig.
5G,I), and the three-dimensional structures of the posterior heart
(Fig. 5H,J). In wild-type
1st-instar larvae, the posterior heart tube formed two chamber-like structures
flanked by two pairs of ostias and the highly organized posterior heart tip
(Fig. 5H). By contrast, the
lymph gland was completely absent or largely reduced in most Hand
mutant 1st-instar larvae (Fig.
5I). The three-dimensional chamber-like structure of the posterior
heart was also dramatically disrupted in Hand mutant larvae
(Fig. 5J). Most pericardial
nephrocytes were also missing at 26 hours AEL
(Fig. 5J).
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Targeted expression of P35 rescues the cardiac and lymph gland phenotypes due to ectopic apoptosis
To test whether inhibiting apoptosis in the lymph gland and hearts of
Hand mutants might rescue the Hand mutant phenotypes, we
overexpressed the apoptosis inhibitor P35
(Clem et al., 1991), which
prevents cell death by inactivating effector caspases (reviewed by
Goyal, 2001), in the heart
using Hand-Gal4. P35 has been shown to be an efficient caspase suppressor in
Drosophila cells (Hay et al.,
1994). Targeted expression of P35 in Hand-expressing
cells alone did not evoke any phenotypes, whereas targeted expression of P35
in Hand mutant embryos prevented ectopic apoptosis
(Fig. 6E), as well as the
phenotype of reduced lymph gland hematopoietic progenitors and pericardial
nephrocytes in late stage embryos (Fig.
6F). Targeted overexpression of P35 also delayed but did not
prevent the larval lethality in Hand mutants. At 18 hours AEL,
Hand mutant larvae with targeted P35 expression started to display an
abnormal appearance (Fig. 6G).
At 24 hours AEL, these larvae developed thin hypoplastic heart and reduced
lymph gland hematopoietic progenitors similar to, but less severe than, that
of Hand mutant larvae (Fig.
6H, compare with Fig.
5F).
Rescue of the Hand mutant phenotype with Drosophila Hand and human HAND2
To confirm that the phenotypes of the Hand null mutant were due
solely to the absence of Hand, we specifically overexpressed
wild-type Hand in Hand mutants using Hand-Gal4, and were
able to completely rescue the phenotype and lethality of Hand mutants
(data not shown). We also expressed human HAND2 in Drosophila
Hand mutants using Hand-Gal4. Control experiments showed that transgenic
expression of human HAND2 in wild-type flies caused no abnormalities.
Remarkably, expression of human HAND2 in the Hand mutant
background effectively rescued the cardiac and lymph gland defects, such that
almost all mutant embryos hatched and developed to 1st-instar larvae with
nearly normal hearts and lymph glands (Fig.
6I). Hand mutant larvae rescued by targeted expression of
human HAND2 survived up to 6 days and developed a fairly normal heart
and lymph gland at 24 hours AEL (Fig.
6J), suggesting an evolutionary conserved role of HAND factors in
cardiogenesis and hematopoiesis.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hand is required for heart development in Drosophila
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.
Our 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 displayed 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 (data not shown). These findings suggest
that Hand plays an essential role in Drosophila heart
development.
HAND transcription factors are expressed during heart development in human,
mouse, chick, frog, zebrafish, ciona and Drosophila embryos (Cserjesi
et al., 1995; Srivastava et al.,
1995; Angelo et al.,
2000; Yelon et al.,
2000; Davidson and Levine,
2003; Han and Olson,
2005). Mouse Hand2 and Drosophila Hand are both
regulated by GATA factors during heart development
(McFadden et al., 2000;
Han and Olson, 2005).
Functional studies have suggested that Hand genes are essential for
cardiogenesis in mouse, chick, zebrafish and Drosophila
(Srivastava et al., 1995;
Srivastava et al., 1997;
Yelon et al., 2000;
McFadden et al., 2005) (this
study). 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.
Inhibition of apoptosis by HAND transcription factors
Mouse embryos lacking HAND2 exhibit hypoplasia of the right ventricle and
pharyngeal arches and associated apoptosis
(Srivastava et al., 1997;
Thomas et al., 1998; Yamagashi
et al., 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 et al., 2005),
suggesting that HAND2 acts, at least in part, to inhibit apoptosis.
We also observed ectopic apoptosis 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 could 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.
To determine if Hand can generally inhibit apoptosis, we tested if
overexpression of Hand in transfected Drosophila S2 cells
could block apoptosis induced by genes that induce apoptosis, such as Reaper
and HID (Vucic et al., 1998),
or with drugs that induce apoptosis, such as Etoposide and Taxol
(Fang et al., 1998). However,
Hand failed to inhibit apoptosis in response to these stimuli (data not
shown), 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.
Possible functional mechanism of Hand
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, we predict
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.
Hand function in hematopoiesis
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.
In mammals, the adult hematopoietic system originates from the yolk sac and
the intra-embryonic aorta-gonad-mesonephros (AGM) region
(Medvinsky and Dzierzak,
1996). Previous studies have suggested a close relationship
between the Drosophila cardiac mesoderm and the mammalian cardiogenic
and AGM region (Evans et al.,
2003; Mandal et al.,
2004). 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
(Cripps and Olson, 2002;
Evans et al., 2003). 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 et al., 1998). Our
study provides the first evidence for the requirement of Hand in
Drosophila hematopoiesis, suggesting similar functions for its
mammalian orthologs.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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.[CrossRef][Medline]
Alvarez, A. D., Shi, W., Wilson, B. A. and Skeath, J. B.
(2003). Pannier and pointedP2 act sequentially to regulate
Drosophila heart development. Development
130,3015
-3026.
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.
Bodmer, R. (1993). The gene tinman is required for specification of the heart and visceral muscles in Drosophila.Development 118,719 -729.[Abstract]
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Clem, R. J., Fechneimer, M. and Miller, L. K.
(1991). Prevention of apoptosis by a baculovirus gene during
infection of insect cells. Science
254,1388
-1390.
Cripps, R. M. and Olson, E. N. (2002). Control of cardiac development by an evolutionarily conserved transcriptional network. Dev. Biol. 246,14 -28.[CrossRef][Medline]
Crossley, A. C. (1972). The ultrastructure and function of pericardial cells and other nephrocytes in an insect: Calliphora erythrocephala. Tissue Cell 4, 529-560.[Medline]
Davidson, B. and Levine, M. (2003).
Evolutionary origins of the vertebrate heart: Specification of the cardiac
lineage in Ciona intestinalis. Proc. Natl. Acad. Sci.
USA 100,11469
-11473.
Evans, C. J., Hartenstein, V. and Banerjee, U. (2003). Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev. Cell 5, 673-690.[CrossRef][Medline]
Fang, G., Chang, B. S., Kim, C. N., Perkins, C., Thompson, C. B.
and Bhalla, K. N. (1998). "Loop" domain is
necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as
well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c
and apoptosis. Cancer Res.
58,3202
-3208.
Ferreira, R., Ohneda, K., Yamamoto, M. and Philipsen, S.
(2005). GATA1 function, a paradigm for transcription factors in
hematopoiesis. Mol. Cell. Biol.
25,1215
-1227.
Firulli, A. B., McFadden, D. G., Lin, Q., Srivastava, D. and Olson, E. N. (1998). Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1. Nat. Genet. 18,266 -270.[CrossRef][Medline]
Gajewski, K., Fossett, N., Molkentin, J. D. and Schulz, R. A. (1999). The zinc finger proteins Pannier and GATA4 function as cardiogenic factors in Drosophila. Development 126,5679 -5688.[Abstract]
Gong, W. J. and Golic, K. G. (2003). Ends-out,
or replacement, gene targeting in Drosophila. Proc. Natl. Acad.
Sci. USA 100,2556
-2561.
Gove, C., Walmsley, M., Nijjar, S., Bertwistle, D., Guille, M., Partington, G., Bomford, A. and Patient, R. (1997). Over-expression of GATA-6 in Xenopus embryos blocks differentiation of heart precursors. EMBO J. 16,355 -368.[CrossRef][Medline]
Goyal, L. (2001). Cell death inhibition: keeping caspases in check. Cell 104,805 -808.[CrossRef][Medline]
Han, Z. and Olson, E. N. (2005). Hand is a
direct target of Tinman and GATA factors during Drosophila
cardiogenesis and hematopoiesis. Development
132,3525
-3536.
Han, Z., Fujioka, M., Su, M., Liu, M., Jaynes, J. B. and Bodmer, R. (2002). Transcriptional integration of competence modulated by mutual repression generates cell-type specificity within the cardiogenic mesoderm. Dev. Biol. 252,225 -240.[CrossRef][Medline]
Han, Z., Li, X., Wu, J. and Olson, E. N.
(2004). A myocardin-related transcription factor regulates
activity of serum response factor in Drosophila. Proc. Natl. Acad.
Sci. USA 101,12567
-12572.
Harvey, R. P. (1996). NK-2 homeobox genes and heart development. Dev. Biol. 178,203 -216.[CrossRef][Medline]
Hay, B. A., Wolff, T. and Rubin, G. (1994). Expression of baculovirus P35 prevents cell death in Drosophila.Development 120,2121 -2129.[Abstract]
Klinedinst, S. L. and Bodmer, R. (2003). Gata
factor Pannier is required to establish competence for heart progenitor
formation. Development
130,3027
-3038.
Kolsch, V. and Paululat, A. (2002). The highly conserved cardiogenic bHLH factor Hand is specifically expressed in circular visceral muscle progenitor cells and in all cell types of the dorsal vessel during Drosophila embryogenesis. Dev. Genes Evol. 212,473 -485.[CrossRef][Medline]
Lee, Y., Shioi, T., Kasahara, H., Jobe, S. M., Wiese, R. J.,
Markham, B. E. and Izumo, S. (1998). The cardiac
tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the
zinc finger protein GATA4 and cooperatively activates atrial natriuretic
factor gene expression. Mol. Cell. Biol.
18,3120
-3129.
Lebestky, T., Chang, T., Hartenstein, V. and Banerjee, U.
(2000). Specification of Drosophila hematopoietic lineage by
conserved transcription factors. Science
288,146
-149.
Liberatore, C. M., Searcy-Schrick, R. D., Vincent, E. B. and Yutzey, K. E. (2002). Nkx-2.5 gene induction in mice is mediated by a Smad consensus regulatory region. Dev. Biol. 244,243 -256.[CrossRef][Medline]
Lien, C. L., McAnally, J., Richardson, J. A. and Olson, E. N. (2002). Cardiac-specific activity of an Nkx2-5 enhancer requires an evolutionarily conserved Smad binding site. Dev. Biol. 244,257 -266.[CrossRef][Medline]
Lyons, I., Parsons, L. M., Hartley, L., Li, R., Andrews, J. E.,
Robb, L. and Harvey, R. P. (1995). Myogenic and morphogenetic
defects in the heart tubes of murine embryos lacking the homeo box gene
Nkx2-5. Genes Dev. 9,1654
-1666.
Mandal, L., Banerjee, U. and Hartenstein, V. (2004). Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm. Nat. Genet. 36,1019 -1023.[CrossRef][Medline]
McFadden, D. G., Charite, J., Richardson, J. A., Srivastava, D., Firulli, A. B. and Olson, E. N. (2000). A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart. Development 127,5331 -5341.[Abstract]
McFadden, D. G., Barbosa, A. C., Richardson, J. A., Schneider,
M. D., Srivastava, D. and Olson, E. N. (2005). The Hand1 and
Hand2 transcription factors regulate expansion of the embryonic cardiac
ventricles in a gene dosage-dependent manner.
Development 132,189
-201.
Medvinsky, A. and Dzierzak, E. (1996). Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86,897 -906.[CrossRef][Medline]
Molkentin, J. D., Lin, Q., Duncan, S. A. and Olson, E. N.
(1997). Requirement of the transcription factor GATA4 for heart
tube formation and ventral morphogenesis. Genes Dev.
11,1061
-1072.
Monier, B., Astier, M., Semeriva, M. and Perrin, L.
(2005). Steroid-dependent modification of Hox function drives
myocyte reprogramming in the Drosophila heart.
Development 132,5283
-5293.
Reiter, J. F., Alexander, J., Rodaway, A., Yelon, D., Patient,
R., Holder, N. and Stainier, D. Y. (1999). Gata5 is required
for the development of the heart and endoderm in zebrafish. Genes
Dev. 13,2983
-2995.
Sorrentino, R. P., Gajewski, K. M. and Schulz, R. A. (2005). GATA factors in Drosophila heart and blood cell development. Semin. Cell. Dev. Biol. 16,107 -116.[CrossRef][Medline]
Srivastava, D., Cserjesi, P. and Olson, E. N.
(1995). A subclass of bHLH proteins required for cardiac
morphogenesis. Science
270,1995
-1999.
Srivastava, D., Thomas, T., Lin, Q., Kirby, M. L., Brown, D. and Olson, E. N. (1997). Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nat. Genet. 16,154 -160.[CrossRef][Medline]
Thomas, T., Yamagishi, H., Overbeek, P. A., Olson, E. N. and Srivastava, D. (1998). The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev. Biol. 196,228 -236.[CrossRef][Medline]
Ting, C. N., Olson, M. C., Barton, K. P. and Leiden, J. M. (1996). Transcription factor GATA-3 is required for development of the T-cell lineage. Nature 384,474 -478.[CrossRef][Medline]
Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W. and Orkin, S. H. (1994). An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371,221 -226.[CrossRef][Medline]
Vucic, D., Kaiser, W. J. and Miller, L. K.
(1998). Inhibitor of apoptosis proteins physically interact with
and block apoptosis induced by Drosophila proteins HID and GRIM.
Mol. Cell. Biol. 18,3300
-3309.
Xu, X., Yin, Z., Hudson, J. B., Ferguson, E. L. and Frasch,
M. (1998). Smad proteins act in combination with synergistic
and antagonistic regulators to target Dpp responses to the Drosophila
mesoderm. Genes Dev. 12,2354
-2370.
Yamagishi, H., Yamagishi, C., Nakagawa, O., Harvey, R. P., Olson, E. N. and Srivastava, D. (2001). The combinatorial activities of Nkx2.5 and dHAND are essential for cardiac ventricle formation. Dev. Biol. 239,190 -203.[CrossRef][Medline]
Yelon, D., Ticho, B., Halpern, M. E., Ruvinsky, I., Ho, R. K., Silver, L. M. and Stainier, D. Y. (2000). The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development. Development 127,2573 -2582.[Abstract]
Yin, Z., Xu, X. L. and Frasch, M. (1997). Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development. Development 124,4971 -4982.[Abstract]
Zaffran, S. and Frasch, M. (2002). Early
signals in cardiac development. Circ. Res.
91,457
-469.
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