Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7530, USA
* Author for correspondence (e-mail: kimelman{at}u.washington.edu)
Accepted 30 July 2002
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SUMMARY |
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Key words: Zebrafish, hrT, Cardiogenesis, Dorsal aorta, tbx5, fli1, floating head
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INTRODUCTION |
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The family of T-box transcription factors shares a phylogenetically
conserved DNA-binding domain, which is required for specific DNA sequence
recognition. The functional activity of these genes are mediated by binding to
their cognate regulatory sites within the promoter and enhancer regions of
genes, leading to the activation or repression of gene expression depending
upon the type of T-box gene and the context within the promoter
(Carreira et al., 1998;
He et al., 1999
;
Sinha et al., 2000
;
Tada and Smith, 2001
). Members
of this gene family have been shown to have selective patterns of expression
and play many key roles in patterning and specifying the development of a
variety of tissues (Basson et al.,
1999
; Merscher et al.,
2001
; Lamolet et al.,
2001
; Bruneau et al.,
2001
; Tada and Smith,
2001
).
We recently described a novel zebrafish T-box gene, hrT
(tbx20 Zebrafish Information Network), which is expressed in
the developing heart and dorsal aorta
(Griffin et al., 2000;
Ahn et al., 2000
). hrT
is expressed in the cardiogenic mesoderm of the anterior lateral plate from
the beginning of segmentation, and continues to be expressed in the heart
field until at least 72 hours (Griffin et
al., 2000
; Ahn et al.,
2000
). The onset of hrT expression in the lateral plate
is earlier than the first expression of nkx2.5 or tbx5, and
coincident with the start of nkx2.7 expression
(Lee et al., 1996
;
Begemann and Ingham, 2000
).
hrT is thus expressed during all the key stages of cardiac
development, which include cardiac cell fate specification, morphogenesis,
looping of the heart tube and chamber formation (Srivastavia and Olson, 2000;
Yelon et al., 1999
;
Stainier, 2001
). In addition,
hrT begins to be expressed in the dorsal aorta at the 15-somite
stage, in addition to sites of expression in the hindbrain, eye and at the
anal opening (Griffin et al.,
2000
; Ahn et al.,
2000
).
To investigate the developmental role of hrT, we used hrT-specific morpholino antisense oligonucleotides to produce zebrafish with a reduced amount of HrT. We find that hrT plays an important role in the later development of the heart, including normal cardiac looping and the division of the cardiac chambers. Specifically, we show that hrT is required to regulate tbx5 expression levels correctly during the stages of cardiac looping. In addition, we find that hrT is required for the morphogenesis of the dorsal aorta, resulting in embryos lacking blood circulation. Interestingly, the vascular defects in hrT morphant embryos are similar to midline mutants such as floating head (flh), which we show lack expression of hrT in vascular progenitors. Taken together, these data indicate that hrT helps to mediate essential functions in vascular morphogenesis downstream of the midline mesoderm. This study provides the first demonstration of the crucial role for hrT in cardiovascular development and sheds new insight into the mechanism regulating the expression of tbx5 during cardiogenesis.
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MATERIALS AND METHODS |
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In situ hybridization
Whole-mount in situ hybridization was performed using digoxigeninlabeled
antisense RNA probe and visualized using anti-digoxigenin Fab fragments
conjugated with alkaline phosphatase (Roche Molecular Biochemicals) as
described (Griffin et al.,
1998). Riboprobes were made from DNA templates, which were
linearized and transcribed with either SP6 or T7 RNA polymerase. Embryos were
processed and hybridized as described
(Griffin et al., 1998
), except
that 10 µg/ml of proteinase K in PBS/0.1% Tween-20 was used for 10 to 30
minutes depending the age of the collected embryos.
Whole-mount immunostaining
Whole-mount immunostaining was performed using monoclonal antibodies MF20
(generous gift of Dr Stephen Hauschka) and S46 (generous gift of Dr Frank
Stockdale). Embryos were processed as previously described
(Westerfield, 1995), except
that the pericardium of each embryo was punctured before treatment with
proteinase K using a fine gauge syringe needle. The color reaction was
visualized with goat anti-mouse antibodies conjugated to horseradish
peroxidase and 3,3'-Diaminobenzidine tetrahydrochloride
(Polysciences).
Histology
Whole-mount in situ hybridized embryos were washed in water several times,
and placed in a series of washes in 25%, 50%, 75% and 100% ethanol. The
completely dehydrated embryos were cleared in acetone and then embedded in
Paraplast II (Tissue Tek). Sections 7 µm thick were cut and mounted on
glass with the same embedding plastic. The mounted slides were covered with
coverslips and incubated at 60°C to dry.
RNA injection of hrT-GFP and GFP in the presence of
hrTMO(1)
RNAs were synthesized from Asp718 linearized CS2-hrT-GFP
(details available on request) and CS2-GFP (generous gift of Dr Jeff
Miller) templates using the mMessage Machine kit (Ambion) and dissolved in
RNase-free sterile water. RNA (0.1 ng) was injected in the presence or absence
of 1.5 ng hrTMO(1) into one cell zebrafish embryos. The expression of
hrT-GFP and GFP were analyzed at the shield stage using
green fluorescent microscopy.
Overexpression of an inducible hrT expression plasmid
Synthetic capped mRNA transcripts were synthesized by SP6 in vitro
transcription (mMessage machine; Ambion) of an Asp718 linearized CS2
template containing an insert of the coding region of hrT fused to
the glucocorticoid receptor ligand binding domain (GR-hrT; details
available on request). GR-hrT mRNA was dissolved in RNase-free
sterile water and 1 nl volume of RNA at a concentration of 0.2 mg/ml was
injected into one cell zebrafish embryos. The GR-hrT protein was activated by
adding 0.1% volume of 100 mM dexamethasone in 100% ethanol to give a final
concentration of 100 µM dexamethasone. Control treated embryos were put
into 0.1% ethanol at the same time.
Photography and image processing
For photography, whole-mount in situ hybridized embryos were post-fixed in
4% paraformaldehyde, washed three times with PBS, dehydrated with methanol,
cleared in methyl salicylate and mounted onto a glass slide with Permount as
described (Melby et al.,
1997). Plastic tissue sections and whole-mount in situ hybridized
embryos were photographed on an Axioplan microscope (Zeiss) using a digital
camera (Dage). Photo images were cropped and assembled using the Photoshop
program version 5.5 (Adobe).
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RESULTS |
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The abnormal cardiac development was visible in hrTMO(1)-injected zebrafish embryos at 24 hours post fertilization (hpf) (Fig. 1B,C). By 48 hpf, the heart tube of uninjected zebrafish embryos developed into clearly separate chambers (Fig. 1F,H), whereas the heart tube in hrT morphants remained in a tubular structure with no obvious morphological distinction between the chambers (Fig. 1G,I). This morphological alteration was also associated with abnormal contractility of the heart (see Movies at http://dev.biologists.org/supplemental/). The hearts of hrT morphants show slower cardiac rhythm than those of their uninjected siblings.
To confirm that these morphant phenotypes were specifically due to the inactivation of hrT function, we first investigated the efficacy of hrTMO(1) in blocking the translation of the hrT gene. To do this, we constructed a fusion between the coding region of hrT and the GFP reporter gene, hrT-GFP, which includes the binding region of hrTMO(1). Injection of 0.1 ng hrT-GFP RNA in the presence of 1.5 ng hrTMO(1) resulted in the absence of GFP protein expression (Fig. 1J-M; 100%, n=55). By contrast, there was no effect on the expression of GFP in embryos co-injected with 0.1 ng GFP RNA and 1.5 ng hrTMO(1) (Fig. 1N-Q; 90%, n=45). These experiments demonstrate that hrTMO(1) is able to specifically block the translation of the hrT gene via the binding sequence for hrTMO(1). To demonstrate further the morphant phenotypes were the result of specific loss of the hrT gene function, we designed a second morpholino oligonucleotides, hrTMO(2), which binds to the hrT transcript at a different region than hrTMO(1) (Fig. 1A). At a range of 6.0 to 12.5 ng, the hrTMO(2)-injected zebrafish embryos exhibited the same phenotype as that observed with hrTMO(1) (Fig. 1D,E), and the resulting phenotypes were dose dependent (Table 1). At higher doses, both morpholino oligonucleotides caused nonspecific effects (data not shown). Thus, we conclude that the specific morphant phenotypes produced by hrTMO(1) result from the specific inhibition of HrT.
Defective cardiac looping in hrT morphants
hrT is expressed throughout all stages of heart formation
including cardiac cell fate specification, morphogenesis, looping of the heart
tube and chamber formation (Griffin et
al., 2000; Ahn et al.,
2000
). These different key stages have been well characterized
with molecular markers, including nkx2.5, tbx5, cardiac myosin light chain
2 and ventricle myosin heavy chain
(Lee et al., 1996
;
Serbedzija et al., 1998
;
Begemann and Ingham, 2000
;
Yelon et al., 1999
). These
markers were used as in situ hybridization probes to assess gene expression
changes during cardiac development in embryos injected with hrTMO(1) to
determine when defects in hrT morphants occurred.
We first examined the pattern of nkx2.5 expression during early
cardiac development, starting from the specification of cardiac progenitors to
the formation of a linear heart tube. One of the initial steps in
cardiogenesis is the establishment of the heart field in the anterior region
of the lateral plate mesoderm. These cardiac precursors are characterized by
the bilateral expression of nkx2.5
(Lee et al., 1996;
Serbedzija et al., 1998
). In
hrT-morphant embryos, nkx2.5 expression at the 12-somite
stage in the anterior lateral plate mesoderm was unaffected by the depletion
of hrT, indicating that hrT is not required for
establishment of bilateral sets of cardiac precursors
(Fig. 2A,B). Similarly, using
nkx2.5 expression to visualize the cardiac primordium, heart
development appeared normal in hrT-morphant embryos through the
stages of fusion of the bilateral heart fields and leftward jogging of the
heart (Fig. 2C,D). We also
compared the relative distributions of cardiac-myosin light chain-2
(cmlc2), a pan-cardiac marker and ventricular myosin heavy
chain (vmhc), to examine the formation of atrial and ventricular
precursors (Yelon et al.,
1999
). At the 17-somite stage and at 24 hpf, expression of
cmlc2 and vmhc in injected embryos was indistinguishable
from uninjected embryos, indicating that the specification of ventricular and
atrial precursors proceeded normally in hrT-morphant embryos (data
not shown). We conclude therefore that the early expression of hrT in
cardiac precursors (up to 24 hpf) is not essential for the early
differentiation or morphogenesis of the heart.
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At 36 hpf, we used cmlc2 to visualize the process of cardiac looping in hrT morphants. cmlc2 is expressed in the atrial and ventricular regions, outlining the development of the whole heart. In uninjected embryos, the process of cardiac looping is marked by a rightward bending in the ventricular region (Fig. 2E). This morphological signature of cardiac looping was not observed in hrT morphants (Fig. 2F). Thus, HrT plays an essential role in cardiac morphogenesis that is not evident until the cardiac looping stage, significantly later than the onset of hrT expression in cardiac precursors.
Abnormal cardiac chamber formation in hrT morphants
In addition to the absence of looping, the distribution of cmlc2
expression at 36 hpf also revealed a defect in chamber morphology. In
uninjected embryos, the dense cmlc2 expression observed in the
ventricle contrasts with the relatively diffuse appearance of the atrium
(Fig. 2E). In injected embryos,
however, atrial expression of cmlc2 appeared similar to ventricular
expression (Fig. 2F). This
defect could either be due to collapse of the atrium, so that there was an
apparent increase in the density of cmlc2 expression there, or
because atrial chamber identity was defective. Consistent with the latter
possibility, we observed that vmhc expression in hrT
morphants was no longer specific to the ventricle and was now also detected in
the atrium (Fig. 2G compare
with 2H), suggesting that hrT may play a role in maintaining
chamber-specific patterns of gene expression.
At 48 hpf, when morphologically distinct cardiac chambers begin to form in
untreated embryos, hrT morphants did not exhibit clearly distinct
physical boundaries between the chambers. To examine the chamber identity in
hrT morphants, we analyzed cardiac chamber formation using the
monoclonal antibodies MF20 to detect a myosin chain common to both chambers
(Fig. 2K), and S46 which
detects an atrial-specific myosin epitope
(Fig. 2M)
(Yelon et al., 1999;
Evans et al., 1988
). Staining
of the hrT morphants with MF20 indicated the presence of two distinct
chambers (Fig. 2L). This result
was confirmed with the atrium-specific antibody S46, which revealed a clear
atrioventricular boundary (Fig.
2N). Despite the absence of morphologically distinct cardiac
chambers in the hrT morphants, the heart was characteristically
divided into an atrium and ventricle at this stage. Thus, the depletion of
hrT function does not prevent the acquisition of anteroposterior
fates within the heart tube, although there are clear alterations to gene
expression within the atrium.
Regulation of tbx5 by HrT
Our initial study of hrT suggested that it might be genetically
upstream of tbx5 as the expression of hrT precedes the
expression of tbx5 in the heart field
(Griffin et al., 2000).
Consistent with this idea, mice lacking the tbx5 gene have normal
expression of tbx20, the mouse ortholog of hrT
(Bruneau et al., 2001
). Because
tbx5 is a key transcription factor that regulates cardiac
morphogenesis and gene expression within the heart field
(Basson et al., 1999
;
Horb and Thomsen, 1999
;
Bruneau et al., 2001
), we
wished to determine the relationship between HrT and tbx5 expression.
In hrT morphants at 33 hpf, we observed a dramatic upregulation of
tbx5 expression in the embryonic heart
(Fig. 3E,F; 44%,
n=34). However, there was no change in tbx5 expression at
earlier times (Fig. 3A-D), or
in the developing pectoral fin buds where tbx5 is expressed but
hrT is not (Fig.
3G,H).
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As depletion of HrT increased the expression levels of tbx5 in the
heart, we predicted that overexpression of hrT would decrease the
expression of tbx5. In order to regulate the expression of
hrT, we constructed a fusion between the coding region of
hrT and the ligand-binding domain of the glucocorticoid
receptor (GR-hrT; gr Zebrafish Information Network). As
shown previously, these fusion proteins are inactive until the hormone
dexamethasone is added (Kolm and Sive,
1995; Tada et al.,
1997
). For the experiments shown here, dexamethasone was added at
the 12-somite stage (Fig.
3I-L), but the same results were observed when the hormone was
added at earlier stages (data not shown). We also determined that the same
concentration of dexamethasone does not cause any apparent developmental
defect in the developing uninjected embryos (data not shown). Induction of
GR-HrT had no effect on the morphological appearance of the heart field or on
the expression of tbx5 before the stages of cardiac looping (data not
shown). However, at 30 hpf, we observed a significant downregulation of
tbx5 expression in the embryos after induction of GR-HrT
(Fig. 3I,J; 55%,
n=40), as well as morphological alterations in the heart at later
stages. While the hearts of control embryos underwent the normal process of
heart looping, the hearts in GR-HrT-induced embryos did not loop (data not
shown). The effects of hrT overexpression on tbx5 were
specific to the heart, as the levels of tbx5 expression in the fin
buds of both GR-HrT-induced and control embryos were the same
(Fig. 3K,L). Both the
overexpression and the morpholino antisense experiments demonstrate that HrT
acts to regulate the levels of tbx5 expression during the stages of
cardiac looping. As normal cardiac morphogenesis and gene expression requires
a precise regulation of the levels of tbx5 (reviewed by Hatcher and
Beeson, 2001), our results suggest that a major role for HrT is to modulate
the levels of tbx5 during cardiac looping.
The role of hrT in hematopoiesis
In addition to the defective heart, hrT morphants do not have
circulating blood. To determine the cause of this defect, we examined the
process of hematopoiesis and vasculogenesis in embryos depleted of
hrT function. Hematopoiesis occurs in several waves and in distinct
locations, giving rise to the terminal differentiation of various
hematopoietic progenitor cells, including early macrophages, erythrocytes,
megakaryocytes and leucocytes (Zon,
1995; Herbomel et al.,
1999
; Detrich et al.,
1995
; Thompson et al.,
1998
). From the onset of gastrulation to 24 hpf in the
hrT morphants, we observed no apparent change in the expression of
genes that are known to denote the development of different hematopoietic cell
lineages (data not shown), indicating that the different hematopoietic cell
lineages form in the absence of hrT function. Thus, the absence of
circulating blood is not a result of an absence of blood, but is most probably
due to a problem with vasculogenesis. This conclusion is supported by the
observed blood pooling in the peri-anal region of the hrT morphants
at 36 hpf (Fig. 4F). Although
the hematopoiesis in the hrT morphants was essentially normal, we did
observe an interesting alteration in the pattern of gata1 and
gata2 expression. At the 2- to 12-somite stage, gata1
expression resides in two stripes flanking the posterior paraxial mesoderm in
the developing zebrafish embryos, marking the erythrocyte and megakaryocyte
lineages (Detrich et al.,
1995
). At the 8- to 10-somite stage, we found a `U'-shaped pattern
of gata1 expression in hrT morphants, rather than the
bilateral stripes of expression in the uninjected sibling embryos, because of
ectopic expression of gata1 in the most posterior lateral plate
mesoderm (Fig. 4B; 61%,
n=81). We observed a similar result for gata2 expression,
which marks all of the definitive hematopoietic lineages (data not shown).
Interestingly, hrT is expressed in the most posterior region of the
lateral plate where the normal bilateral stripes join to form the `U'-shaped
pattern in the hrT morphants (Fig.
4C,D), suggesting that HrT normally prevents the hematopoietic
fate in this domain.
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The role of hrT in vasculogenesis
hrT is expressed in the dorsal aorta
(Griffin et al., 2000;
Ahn et al., 2000
), and thus the
blood circulation defects could be due to aberrant formation of the dorsal
aorta. To determine if there is a vascular defect when hrT function
is impaired, we investigated the formation of the major blood vessels in the
hrT morphants. After gastrulation, vasculogenesis begins with the
formation of two stripes of endothelial precursors at the lateral edges of the
mesoderm, and these cells express fli1, a member of the ETS-domain
family of transcription factors (Brown et
al., 2000
). At 14 hpf, the expression of fli1 in the
lateral mesoderm extends along the entire axis during segmentation in two
continuous bands, forming a `U'-shaped surrounding the axial and paraxial
mesoderm (Fig. 4G). At this
stage, we found that the pattern of fli1 expression was normal in
hrT morphants when compared with that in the uninjected siblings
(Fig. 4G,H). This result
suggests that the early specification of endothelial precursors is unaffected
when hrT function is disrupted. At approximately 20 hpf, the
`U'-shaped pattern of fli1 expression in trunk and tail coalesces in
the midline (Fig. 4I).
Subsequently, the fli1 expression is found in the walls of major
vessels, including the dorsal aorta, axial vein and intersegmental vessels
(Fig. 4K). At 20 hpf, the
expression of fli1 was detected in a broad distribution pattern
around the midline of hrT morphants
(Fig. 4J), suggesting that the
fusion of endothelial cells to form the dorsal aorta in the midline was
disrupted. By 24 hpf, the abnormal expression pattern of fli1 in
hrT morphants was even more apparent. In uninjected embryos, the
fli1 expression was detected in the dorsal aorta, axial vein and
intersegmental vessels (Fig.
4M), whereas in hrT morphants, fli1 was
expressed in a single domain running along the midline ventral to the
notochord (Fig. 4N; 53%,
n=30). In addition, the sprouting of intersegmental vessels was not
detected in hrT morphants (Fig.
4N). However, the pattern of fli1 expression in the
pharyngeal primordium appeared to be normal
(Fig. 4K,L), suggesting that
the vasculogenic requirement for hrT is localized to the trunk of the
embryos.
In sections of fli1-stained, uninjected embryos, the dorsal aorta is seen ventral to the notochord and the axial vein is below the aorta (Fig. 4O). In the hrT morphants, the fli1-expressing cells have not organized into two clear vessels, although we typically saw one lumen above the gut tube (Fig. 4P). As hrT is expressed in the dorsal aorta but not the axial vein, we expect that the major defect is due to the formation of this vessel. Thus, we conclude that the failure of hrT morphants to circulate blood is primarily due to a defect in the formation of the dorsal aorta.
HrT is a potential downstream effector of flh
We noticed a strong resemblance between the trunk vascular defects in the
hrT morphants and floating head (flh) mutant
embryos (Sumoy et al., 1997;
Brown et al., 2000
;
Fouquet et al., 1997
).
flh is a homeodomain transcription factor expressed in the notochord
precursors, and the flh mutation causes an absence of notochord
(Talbot et al., 1995
). In
flh mutants as in the hrT morphants, blood circulation does
not occur, and the blood accumulates in the peri-anal region because of a
failure to form the dorsal aorta (Fouquet
et al., 1997
; Brown et al.,
2000
; Sumoy et al.,
1997
). Similarly in the hrT morphants and flh
mutants, fli1 is expressed in a single stripe in the midline with no
apparent intersegmental vessels.
The similarity between the vascular defects in flh mutant embryos
and hrT morphants, prompted us to examine hrT expression in
flh embryos. In flh homozygotes, which were identified
morphologically and confirmed by the absence of flh expression
(Talbot et al., 1995), we
observed a complete absence of hrT expression in the vascular
progenitors whereas other domains of hrT expression were unaffected
(Fig. 5). This is consistent
with the possibility that hrT expression in vascular progenitors
depends, directly or indirectly, upon a signal from the midline mesoderm
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DISCUSSION |
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Although hrT is expressed very early in the heart field, depletion
of its function did not have an apparent effect until 33 hpf. It is possible
that this is due to incomplete inhibition of hrT function using the
antisense oligonucleotides or due to the presence of a gene that compensates
at earlier times for a loss of hrT. However, it is intriguing that a
similar effect has been observed with murine tbx5; while
tbx5 is also expressed very early in the heart field
(Liberatore et al., 2000),
elimination of the tbx5 gene did not have an effect until later times
of development (Brunneau et al., 2001). This finding suggests that the T-box
genes may not function by themselves in the regulation of heart development,
but may need to interact with other factors that are expressed at later times
of development.
Regulatory role of hrT during cardiogenesis
A key finding in this study is that HrT acts as a regulator of
tbx5 expression. Depletion of HrT function causes the cardiac
expression of tbx5 to be upregulated, whereas overexpression of
hrT leads to the downregulation of tbx5 expression in the
developing heart. Importantly, in both cases, the regulation of tbx5
was specific to the heart and did not affect the expression of tbx5
in the limb buds. Although we do not yet know whether or not Tbx5 regulates
hrT expression in zebrafish, mice lacking the tbx5 gene do
not show alterations in the expression of the murine hrT ortholog
tbx20 (Brunneau et al., 2001), indicating that hrT may
function solely upstream of tbx5.
tbx5 has emerged as a key gene regulating heart development from
amphibians to mammals (Horb and Thomsen,
1999; Liberatore et al.,
2000
; Bruneau et al.,
2001
). Patients with Holt-Oram syndrome, a disorder caused by
haploinsufficiency of the tbx5 gene, have a high penetrance of
cardiac defects (Li et al.,
1997
; Basson et al.,
1999
). Similar defects have been found in mice lacking one copy of
the tbx5 gene (Bruneau et al.,
2001
). Mice lacking both copies of tbx5 have more severe
defects, including an absence of heart looping and alterations in a subset of
cardiac-specific genes, although the early steps in cardiac development appear
normal (Bruneau et al., 2001
).
Curiously, mice overexpressing tbx5 also have cardiac looping defects
as well as alterations in the expression of some cardiac genes
(Liberatore et al., 2000
).
These results indicate that the gene dose of tbx5 is crucial for
normal cardiac morphogenesis (Bruneau et
al., 2001
; Hatcher and Basson,
2001
). These studies fit very well with our observations that
inhibition of hrT function or overexpression of hrT, which
cause an increase or decrease of tbx5 expression, respectively, leads
to defects in cardiac morphogenesis. Importantly, zebrafish tbx5
mutants also show a complete absence of heart looping (M. Fishman, personal
communication). It will be of great interest to determine if the alterations
we observed in cardiac development are due solely to changes in the level of
tbx5 expression, or whether hrT has additional
tbx5-independent functions in the heart.
The role of hrT in the hematopoiesis and vasculogenesis
Another key aspect of hrT depletion is the absence of circulating
blood. By examining the expression of a variety of different blood cell
markers at various stages in hrT morphants, we find that there is no
observable defect in hematopoiesis. In analyzing vasculogenesis, however, we
have observed abnormal trunk vascular development in the hrT
morphants. In hrT morphants, the endothelial marker fli1 is
expressed normally at 14 hpf when endothelial cells form a `U'-shape around
the axial and paraxial mesoderm, demonstrating that hrT plays no
significant role in the specification of endothelial cells. At 24 hpf, the
trunk vascular defect is evident by the altered expression of fli1,
as well as the absence of normal trunk vessel formation. In addition, no
intersegmental vessels sprout from the domain of the aberrant fli1
expression. As hrT is expressed only in the dorsal aorta
(Griffin et al., 2000;
Ahn et al., 2000
), we suggest
that hrT is required specifically in the dorsal aorta rather than the
axial vein, although we do not rule out the possibility of indirect effects on
axial vein formation due to a defective dorsal aorta. As the endothelial cells
come close to the midline in hrT morphants but do not completely fuse
to give the dorsal aorta, our results suggest that hrT plays a
critical role in assembling the endothelial cells into a vessel.
We noticed a striking similarity between the hrT morphants and
flh mutants with regard to the formation of the vascular system, and
we found that hrT expression is specifically eliminated in the dorsal
aorta of flh mutants, while hrT expression in other regions
is unaffected. As the major defect in flh mutants is an absence of
notochord, it has been suggested that formation of the endothelial cells into
the dorsal aorta requires a notochord-derived signal such as sonic
hedgehog (shh) (Brown et al.,
2000; Fouquet et al.,
1997
; Sumoy et al.,
1997
). Our results suggest that HrT is an essential component of
the response to this signal, and indeed we have observed that hrT
expression is abolished in the hedgehog signaling mutant you-too (D.
P. S., K. J. P. G. and D. K., unpublished). As the process of vessel formation
is still not well understood, it will be very interesting to determine the
targets of HrT in the dorsal aorta.
Inhibition of gene expression by hrT
We observed a very interesting alteration in the expression pattern of
gata1 and gata2 at the 8- to 10-somite stage in hrT
morphants. While gata1 and gata2 are normally expressed in
bilateral stripes, in hrT morphants, gata1 and
gata2 form a `U'-shaped pattern because of the presence of an extra
posterior domain of expression that connects the two bilateral stripes. This
extra domain of expression coincides with a region of the embryo that
expresses hrT (Griffin et al.,
2000; Ahn et al.,
2000
), indicating that one function of hrT is to prevent
the expression of gata1 and gata2 in the most posterior
lateral plate of the developing embryo. This ectopic expression of
gata1 and gata2, as well as the increased cardiac expression
of tbx5 and vmhc, suggests that an important function of HrT
is to negatively regulate gene expression, at least indirectly.
A role for hrT in human cardiovascular disease?
This study has demonstrated a clear role for hrT in zebrafish
cardiovascular development. Mutations in the human hrT ortholog,
TBX20, are candidates for producing congenital cardiovascular defects
that are among the most prevalent defects affecting live births. Since several
human T-box genes have been shown to cause birth defects when
haploinsufficient (Basson et al.,
1999; Li et al.,
1997
; Bamshad et al.,
1997
; Merscher et al.,
2001
), it will be worthwhile examining the TBX20 gene in
humans with congenital heart or vascular defects if a disease allele maps to
this genetic interval.
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ACKNOWLEDGMENTS |
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REFERENCES |
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