Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA
* Author for correspondence (e-mail: eric.olson{at}utsouthwestern.edu)
Accepted 12 May 2005
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SUMMARY |
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Key words: Hand, tinman, pannier, serpent, Drosophila, Heart development, Hematopoiesis, Lymph gland, Transcription regulation
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Introduction |
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Several transcription factors have been shown to play key roles in
cardiogenesis and hematopoiesis in flies and vertebrates. The
Drosophila NK-type homeobox gene tinman (tin), the
earliest marker of the cardiac lineage, is initially expressed in the entire
mesoderm before becoming restricted to the dorsal mesoderm and later to the
cardiac mesoderm, in response to ectodermal Dpp and Wg signals. After all the
cardiac cell types are specified, tin expression is extinguished in
many cardiac cell types and maintained in only a subset of cardiac and
pericardial cells (Han et al.,
2002; Han and Bodmer,
2003
). In tin mutant embryos, the entire cardiogenic
region and the lymph gland fail to form
(Bodmer, 1993
,
Mandal et al., 2004
),
indicating the essential role of Tinman in early specification of the cardiac
and hematopoietic lineages.
There are several NK-type homeobox genes in vertebrates, which are named
Nkx2.3-Nkx2.10 (Evans,
1999). Nkx2.5 is expressed in the early cardiac crescent
and continues to be expressed throughout heart development. Mouse embryos
lacking Nkx2.5 show early cardiac defects and arrested cardiogenesis
before looping morphogenesis (Lyons et
al., 1995
). Furthermore, overexpression of a dominant-negative
form of Nkx2.5 in Xenopus blocks cardiogenesis
(Grow and Krieg, 1998
) and
mutations in Nkx2.5 cause congenital heart disease in humans
(Schott et al., 1998
). As
tin is no longer expressed in hematopoietic progenitors after stage
13, its function in hematopoiesis is limited to the early specification of the
cardiogenic mesoderm containing the progenitor cells for the lymph gland
(Mandal et al., 2004
).
Members of the GATA family of zinc-finger transcription factors play
crucial roles in both cardiogenesis and hematopoiesis in Drosophila
and vertebrates. The Drosophila GATA factor Pannier is expressed in
the cardiac mesoderm as well as the overlaying ectoderm and functions
primarily in cardiogenesis. Embryos lacking pannier (pnr)
show a dramatic reduction of cardiac progenitor cells
(Gajewski et al., 1999;
Alvarez et al., 2003
;
Klinedinst and Bodmer, 2003
).
In vertebrates, GATA4, GATA5 and GATA6 are expressed in the cardiogenic
region. Loss-of-function assays in mouse, Xenopus and zebrafish have
shown that these GATA factors are required for myocardial differentiation and
normal heart development (Molkentin et
al., 1997
; Gove et al.,
1997
; Reiter et al.,
1999
). Another Drosophila GATA factor Serpent (Srp)
functions mainly in hematopoiesis. It is expressed in all hematopoietic
progenitors formed in the head mesoderm and the lymph gland. In
serpent (srp) mutant embryos, hematopoiesis from both the
head mesoderm and the lymph gland is inhibited
(Lebestky et al., 2000
;
Mandal et al., 2004
),
indicating that Serpent plays an essential role in hematopoietic progenitor
cell specification. In vertebrates, GATA1, GATA2 and GATA3 play fundamental
roles in various aspects of hematopoietic development
(Tsai et al., 1994
;
Ting et al., 1996
;
Ferreira et al., 2005
). It is
likely that the functions of Pannier and Serpent in cardiogenesis and
hematopoiesis, respectively, reflect the highly conserved but simplified
developmental processes in Drosophila compared with vertebrates.
Several transcription factors that are directly regulated by Tinman and
Pannier have been identified, including Mef2 and
even-skipped, through enhancer mutagenesis studies
(Gajewski et al., 1997;
Gajewski et al., 1998
;
Nguyen and Xu, 1998
;
Knirr and Frasch, 2001
;
Han et al., 2002
). These
studies have begun to establish a transcriptional network that governs
Drosophila cardiogenesis. In this network, Tinman and Pannier
function in parallel as key cardiogenic factors at the top of the hierarchy.
Although several transcription factors, such as Lozenge (Lz) and
Glial-cells-missing (Gcm), appear to act `downstream' of Serpent, there is as
yet no evidence for direct activation of these genes by Serpent.
The Drosophila Hand gene encodes a highly conserved basic
helix-loop-helix (bHLH) transcription factor. Interestingly, Hand is
the only gene identified so far that is expressed in a specific pattern in all
the cardioblasts, pericardial nephrocytes and hematopoietic progenitors in the
lymph gland (Kolsh and Paululat, 2002). The vertebrate Hand genes have been
shown to play essential roles during heart development
(Srivastava et al., 1995;
Srivastava et al., 1997
;
Yamagishi et al., 2001
;
McFadden et al., 2005
). Hand
genes have also been shown to be expressed during heart development in
Xenopus, zebrafish and Ciona
(Sparrow et al., 1998
;
Yelon et al., 2000
;
Davidson and Levine, 2003
). The
conserved cardiac expression patterns of Hand genes across vast evolutionary
distances suggest that these genes play conserved roles during cardiogenesis
and may be regulated by conserved genetic pathways.
In an effort to understand the position of Hand in the genetic networks that govern cardiogenesis and hematopoiesis, we searched for and identified the cis-regulatory region of the Drosophila Hand gene. We describe a minimal Hand enhancer that completely recapitulates endogenous Hand expression in cardioblasts, pericardial nephrocytes and lymph gland prehemocytes. This enhancer contains consensus binding sites for the NK factor Tinman and the GATA factors Pannier and Serpent, which are conserved across evolutionarily divergent Drosophila species. Mutagenesis of these consensus binding sites shows that Hand is directly activated by Tinman and Pannier in the heart, and by Serpent in the lymph gland. Overexpression of Tinman, Pannier or Serpent induces ectopic Hand in muscle progenitors, dorsal vessel and hematopoietic progenitors, respectively, indicating that Hand is activated separately by Tinman, Pannier and Serpent in distinct cell types. These findings place Hand at a central position to link the transcriptional networks that govern cardiogenesis and hematopoiesis.
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Materials and methods |
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Generation of transgenic fly lines
The various Hand enhancer fragments
(Fig. 2A) were PCR amplified
and subcloned into pC4LZ (containing the lacZ reporter gene) or
pPelican (containing the GFP reporter gene)
(Barolo et al., 2000), using
SphI/XhoI or KpnI/NotI sites,
respectively. The 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.
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-Dmef2 1:1000
(from B. Peterson); rabbit anti-GFP 1:2000 (Abcam); and 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.
Electrophoretic mobility shift assays
GST-Tin and GST-Pnr fusion proteins were prepared according to standard
procedures. Complimentary oligonucleotides containing Tin or GATA consensus
site were radiolabeled using Klenow fill-in reaction as probes. Complimentary
oligonucleotides containing wild-type consensus binding sites or binding-site
mutations were used as non-labeled competitors to compete for the binding of
GST fusion proteins in the presence of the radio-labeled probe. After 30
minutes incubation of the protein, probe and competitor oligonucleotides at
4°C, the products were electrophoresed in 7.5% non-denaturing
polyacrylamide gels at 4°C. The sense strand DNA sequences of the
oligonucleotides used are shown as follows with consensus binding sites in
parentheses and mutated nucleotides underlined: Tin1, TTT CCA AAA AGG
(CACTTAA) TTA ATC AAA CCC; Tin2: TTT CTG AAG CAC (CACTTAG) ACA CTT GTC TCT;
Tin3, CTT TTT ATA AAG (TCAAGTG) CTT TTG TTT CTT; Tin4/G5: ATA ATA AAC AAA
(CAATTGA) (GATA) TCT ACG CCC CAG; G1, CTC TTG TGT TCA (TATC) TAA AAC CAG ATT;
G2, GCG TCT GCG GTT (TATC) ACT TCC GAA ATT; G3, CCA TTA GGA ATA (TATC) TAC AAT
CAA TCG; G4: CAA TCG AGT TTT (TATC) TGC GGA TTA CAA; Tin1m, TTT CCA AAA AGG
(CATCCAA) TTA ATC AAA CCC; Tin2m, TTT CTG AAG CAC
(CATCCAG) ACA CTT GTC TCT; Tin3m, CTT TTT ATA AAG
(TCGGATG) CTT TTG TTT CTT; Tin4m, ATA ATA AAC AAA
(CATCCGA) (GATA) TCT ACG CCC CAG; G1m, CTC TTG TGT TCA
(TCCC) TAA AAC CAG ATT; G2m, GCG TCT GCG GTT (TCCC) ACT
TCC GAA ATT; G3m, CCA TTA GGA ATA (TCCC) TAC AAT CAA TCG; G4m, CAA
TCG AGT TTT (TCCC) TGC GGA TTA CAA; G5m, ATA ATA AAC AAA (CAATTGA)
(GGGA) TCT ACG CCC CAG.
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
Hand-luciferase was generated by cloning the minimal Hand enhancer
identified in this study into the pGL3 vector (Promega). Tin-pAc5.1,
Pnr-pAc5.1 or SrpNC-pAc5.1 were generated by cloning the full length tin,
pnr or srp cDNAs into the pAc5.1-HisA vector (Invitrogen),
respectively. Luciferase activities are expressed as mean±s.d. from
three experiments.
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Results |
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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 in from segment T2 to T3, but not in the lymph gland (Fig. 1G). Hand expression is detected in all the Tinman-positive cardiac cells (Fig. 1H). Hand is likely to be expressed in all the pericardial nephrocytes as all Zfh-1-positive pericardial cells express Hand (Fig. 1I). odd-skipped (odd) is expressed in both the lymph gland hematopoietic progenitor cells and a subset of pericardial nephrocytes (Fig. 1J). Hand expression is also detected in all the Odd-skipped-positive hematopoietic progenitors and pericardial nephrocytes (Fig. 1K). In addition, Hand is co-expressed with Serpent in all the lymph gland progenitors (Fig. 1L). The secreted extracellular protein Pericardin (Prc) labels the ring gland and the extracellular matrix surrounding the pericardial nephrocytes (Fig. 1M). Hand expression is not detected in the ring gland, but Hand-expressing cells are surrounded by Prc from segment T2-A6 (Fig. 1N-O). Hand expression also appears in the visceral mesoderm (Fig. 1A-C, data not shown), the garland cells (data not shown) and in a subset of central nervous system cells (data not shown).
|
Replacing the lacZ reporter gene with a GFP reporter made it possible to examine HCH enhancer activity after embryogenesis. The HCH-GFP is expressed in embryos in the same pattern as Hand transcripts (Fig. 2C, part d). After embryogenesis, the enhancer activity remains strong in the lymph gland, cardioblasts and pericardial nephrocytes in larvae (Fig. 2C, part e), and GFP expression persists in the heart throughout the Drosophila life cycle (data not shown).
|
|
Tinman, Pannier and Serpent bind directly to the consensus sites in the HCH enhancer
To test for binding of Tinman protein to the Tinman consensus-binding sites
in the HCH enhancer, we performed gel mobility shift assays with GST-Tinman
fusion protein and a radiolabeled probe corresponding to first Tinman
consensus site (Tin1). GST-Tinman bound avidly to this site, and binding could
be competed by unlabeled oligonucleotides corresponding to any of the Tinman
consensus-binding sites in the HCH enhancer
(Fig. 4A). We then tested
whether the GATA factor Pannier could bind to the GATA consensus sites. By
using a radiolabeled probe containing the second GATA consensus site (G2), we
found that GST-Pannier fusion protein could bind this probe, and binding could
be competed by unlabeled oligonucleotides corresponding to any of the GATA
consensus-sites in the HCH enhancer (Fig.
4B). Next, we tested whether the same GATA consensus sites could
be bound by the hematopoietic GATA factor Serpent. As expected, both forms of
the Serpent protein (SrpNC and SrpC) could bind to the radiolabeled probe
containing the second GATA consensus site (G2), and the binding could be
competed by any of the unlabeled GATA consensus sites in the HCH enhancer
(Fig. 4C). Mutation of the
Tinman and GATA consensus-sites severely diminished their ability to compete
for binding of the corresponding proteins to the labeled probes
(Fig. 4A-C). As not all of
these consensus-binding sites are conserved in all the Drosophila
species, but they were all bound by the corresponding proteins, it is likely
that some of the NK and GATA consensus-binding sites are functionally
redundant.
Activation of the HCH enhancer by Tinman, Pannier and Serpent in Drosophila S2 cells
To examine if the HCH enhancer could be activated by Tinman and GATA
factors in vitro, we generated a luciferase reporter construct using the HCH
enhancer and tested it in Drosophila S2 cells. Remarkably, Tinman was
able to activate this enhancer over 100-fold, whereas Pannier and Serpent
activated the enhancer approximately sixfold
(Fig. 4D). Although previous
studies suggested that Tinman and Pannier function synergistically to activate
cardiac gene expression (Gajewski et al.,
1998), we did not detect significant synergy between these factors
on the HCH enhancer when transfected simultaneously
(Fig. 4D).
In order to show that the activation occurred specifically through binding of the three transcription factors to their consensus-binding sites, we mutated the Tinman and GATA-binding sites in the HCH enhancer. Tinman could still activate the HCH enhancer with all the GATA-binding sites mutated, but could not activate the enhancer with all the Tinman-binding sites mutated, whereas Pannier or Serpent could activate the enhancer with the Tinman-binding sites mutated, but not with the GATA-binding sites mutated (Fig. 4D). An enhancer with both Tinman- and GATA-binding sites mutated could not be activated by either Tinman, Pannier or Serpent (Fig. 4D). These results further support the conclusion that the HCH enhancer is a direct transcriptional target of Tinman, Pannier and Serpent.
|
In contrast to Tinman, Pannier overexpression in the mesoderm using the
same Gal4 driver induced the formation of ectopic Mef2-positive cardioblasts
(indicated by arrows in Fig.
5H,I), as shown in a previous study
(Klinedinst and Bodmer, 2003).
Ectopic expression of HCH-GFP was also detected in all the extra cardioblasts
(Fig. 5G-I). The expanded
HCH-GFP pattern also showed that more pericardial nephrocytes were induced by
ectopic Pannier (indicated by the arrowhead in
Fig. 5G-I). We did not detect
supernumerary Eve-positive pericardial cells
(Fig. 5I), but Odd-positive
pericardial cells were significantly increased (data not shown). Although
ectopic expression of HCH-GFP was detected randomly in a few muscle cells,
this effect was insignificant compared with the ectopic HCH-GFP expression
induced by Tinman. We did not detect an expansion of the lymph gland when
Pannier was overexpressed in the mesoderm.
Unlike Tinman or Pannier, ectopic Serpent driven by twi-Gal4; 24B-Gal4 did
not induce any cardioblasts or pericardial nephrocytes, but instead repressed
their formation (indicated by arrows in
Fig. 5J-L). By contrast, cell
clusters forming the lymph gland (identified by their position, shape and
Hand-GFP expression) were significantly expanded by ectopic Serpent (indicated
by arrowheads in Fig. 5J-L). Furthermore, in embryos with ectopic mesodermal Serpent, pericardial
nephrocytes around the aorta and heart often failed to align along the dorsal
vessel, but formed cell clusters like hematopoietic progenitors in the lymph
gland (Fig. 5J,L), suggesting a
cell fate transformation from pericardial nephrocytes to hematopoietic
progenitors. A gain-of-function study of Srp using a mef2-Gal4 driver showed
similar results with Odd as a marker
(Mandal et al., 2004).
|
As the dependence of the HCH enhancer on Tinman and Pannier could result
secondarily from defects in tin or pannier mutant embryos,
we established a system to test the requirement of Tinman and Pannier for
activation of Hand expression specifically in the cells that express
Hand. Using the HCH enhancer, we generated HCH-Gal4 transgenic flies,
which could drive a UAS-GFP reporter in a pattern identical to that of the
endogenous Hand gene (data not shown). We then overexpressed
dominant-negative forms of Tinman or Pannier specifically in the
Hand-expressing cardiac and hematopoietic cells using the HCH-Gal4
driver. The dominant-negative forms of Tinman (Tin-EnR) or Pannier (Pnr-EnR)
were made by fusing the Engrailed repression domain (EnR) to the Tinman or
Pannier DNA-binding domain (Han et al.,
2002; Klinedinst and Bodmer,
2003
). Overexpression of Tin-EnR in the Hand-expressing
cells nearly abolished HCH-GFP expression in cardioblasts and pericardial
nephrocytes, and also reduced HCH-GFP in the lymph gland but less dramatically
(Fig. 6F), indicating that
dominant-negative Tinman can suppress HCH activity more efficiently in cardiac
cells than in hematopoietic cells. Overexpression of Pnr-EnR in the
Hand-expressing cells using HCH-Gal4 abolished most of the HCH
activity in the heart and lymph gland (Fig.
6G), indicating that dominant-negative Pannier is able to suppress
HCH activity efficiently in both heart and lymph gland, probably by competing
with both endogenous Pannier and Serpent for binding to the HCH enhancer.
Ectopic expression of Tin-EnR or Pnr-EnR in the Hand-expressing cells
did not ablate these cells in the embryos but rather appeared to induce some
kind of cell fate changes that we are currently investigating (data not
shown).
|
To examine the in vivo function of the GATA consensus-binding sites, we generated transgenic flies carrying the HCH enhancer with all five GATA sites mutated (HCH-5G). Interestingly, this mutant enhancer activated GFP expression only in Tinman-positive cardiac cells (Fig. 7G-I). The expression pattern of HCH-5G-GFP was almost identical to that of Tinman (Fig. 7G). The level of GFP expression in these Tinman-positive cardioblasts and pericardial nephrocytes (Fig. 7H,I) was the same as that of the wild-type HCH enhancer (compare Fig. 7H with Fig. 7B). The absence of HCH-5G-GFP activity in the Tinman-negative cardioblasts and pericardial nephrocytes indicates that the binding of Pannier to the consensus GATA sites is necessary to activate Hand expression in Tinman-negative cardioblasts and pericardial cells. However, the absence of the HCH-5G-GFP in the lymph gland hematopoietic progenitors (Fig. 7G) suggests that the binding of Serpent to the consensus GATA-binding sites is required for Hand expression in the lymph gland hematopoietic progenitors.
In order to test whether the Tinman and GATA sites are necessary for all the expression of Hand in the cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors, we created a mutant HCH enhancer with all the four Tinman-binding sites and five GATA-binding sites mutated. This enhancer, HCE-4T5G, was completely devoid of activity in cardioblasts, pericardial nephrocytes and the lymph gland (Fig. 7J-L), demonstrating that the activation of Hand in these three closely linked cell types is absolutely dependent on the binding of Tinman, Pannier and Serpent to the Hand cardiac and hematopoietic (HCH) enhancer.
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Discussion |
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The homeobox-containing protein Tinman is essential for the formation of
the cardiac mesoderm, from which the heart and blood progenitors arise
(Bodmer, 1993). 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. Our 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, as
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.
|
As 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. As Doc1, Doc2 and
Doc3 genes (Drosophila orthologs to vertebrate Tbx5) are
expressed in the Svp-positive cardioblasts where tin is not expressed
(Lo and Frasch, 2001), but H15
and midline (Drosophila orthologs to vertebrate Tbx-11) are expressed
in most of the cardiac cells in late embryos
(Miskolczi-McCallum et al.,
2005
; Qian et al.,
2005
), 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.
Evolution of the HCH enhancer
We have identified putative Hand enhancers 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, we also generated transgenic flies carrying one or two
mutations of the Tinman or GATA-binding sites. None of these transgenic lines
shows significant changes in enhancer activity (data not shown), 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.
Interestingly, Hand expression is also dependent on GATA factors
in vertebrates. We have previously described an enhancer necessary and
sufficient to direct cardiac expression of the mouse Hand2 gene,
which contains two essential GATA-binding sites
(McFadden et al., 2000). Thus,
we propose 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 (Z.H. and E.N.O., unpublished).
Identification of Hand as a common target of transcriptional cascades that govern cardiogenesis and 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). 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 et al.,
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
(Evans et al., 2003
). In fact,
in both Drosophila and mammals, the specification of the cardiogenic
and AGM region requires the input of Bmp, Wnt and Fgf signaling
(Cripps and Olson, 2002
;
Evans et al., 2003
). 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
(Fossett et al., 2001
;
Sorrentino et al., 2005
).
Recent studies have shown that the Notch pathway is required for both
cardiogenic and hematopoietic progenitor specification in Drosophila
(Han and Bodmer, 2003
; Mandel
et al., 2004), as well as for mammalian embryonic vascular development
(Fischer et al., 2004
). It is
likely that Notch also plays an important role in mammalian hematopoiesis.
|
In summary, this study places Hand at a pivotal point to link the transcriptional networks that govern cardiogenesis and hematopoiesis, as shown in Fig. 8. As 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.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/15/3525/DC1
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