1 Department of Genetics, Fordham Hall, UNC-Chapel Hill, Chapel Hill, NC
27599-3280, USA
2 Department of Biology, Fordham Hall, UNC-Chapel Hill, Chapel Hill, NC
27599-3280, USA
3 Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental
Biology and Department of Zoology, University of Cambridge, Tennis Court Road,
Cambridge CB2 1QR, UK
* Author for correspondence (e-mail: frank_conlon{at}med.unc.edu)
Accepted 23 November 2004
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SUMMARY |
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Key words: Tbx5, Tbx20, T-Box, T-domain, Cardiogenesis, Cardiac, Heart, Development, Xenopus laevis
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Introduction |
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The T-box family of transcription factors is a large family of proteins
involved in determining early cell fate decisions and controlling
differentiation and organogenesis. Two sets of clinical data have provided
direct evidence for the involvement of T-box genes in human heart development
(Packham and Brook, 2003;
Ryan and Chin, 2003
).
Deletions of Tbx1 have been found in individuals with DiGeorge
syndrome (Baldini, 2004
;
Chieffo et al., 1997
;
Yagi et al., 2003
), and
mutations in Tbx5 are associated with Holt-Oram Syndrome (HOS), a
congenital heart disease characterized by defects in heart formation and upper
limb development (Basson et al.,
1997
; Li et al.,
1997
). Clinical studies of individuals with HOS have demonstrated
a fundamental role for Tbx5 in heart development. HOS is a highly
penetrant autosomal dominant condition associated with skeletal and cardiac
malformations (Newbury-Ecob et al.,
1996
). Individuals with HOS often carry mutations within the
coding region of the T-box transcription factor Tbx5
(Basson et al., 1997
;
Basson et al., 1999
;
Benson et al., 1996
;
Li et al., 1997
). The role of
Tbx5 in heart development, and in the HOS disease state, is further
supported by recent gene-targeting experiments in mouse. These studies
demonstrate that mice heterozygous for mutations in Tbx5 display many
of the phenotypic abnormalities of individuals with HOS
(Bruneau et al., 2001
) and show
that TBX5 is required for growth and differentiation of the left ventricle and
atria as well as for proper development of the cardiac conduction system
(Moskowitz et al., 2004
).
Similar defects are seen in the zebrafish tbx5 mutant
heartstrings, suggesting that the expression and function of TBX5 is
conserved throughout vertebrate evolution
(Garrity et al., 2002
).
Previously, we have described the cloning and expression of the Xenopus
laevis (X. laevis) Tbx20 ortholog, Tbx20
(Brown et al., 2003). Studies
of Tbx20 have demonstrated that, along with Tbx5, Tbx20 is
one of the first genes expressed in the vertebrate cardiac lineage. Moreover,
Tbx20 is expressed at the same time and in many of the same regions
of the heart that also express the heart markers Tbx5, Nkx2-5 and
Gata4 (Horb and Thomsen,
1999
; Laverriere et al.,
1994
; Tonissen et al.,
1994
).
Despite our knowledge of the expression pattern of Tbx20, little
is known of Tbx20 function in heart development. In the zebrafish, it
has recently been observed that eliminating endogenous TBX20 (HrT) via
morpholinos leads to cardiac defects
(Szeto et al., 2002).
Specifically, TBX20 knockdown in zebrafish leads to dysmorphic hearts and a
loss of blood circulation. The morphological defects are not apparent until
the cardiac looping stage, despite high levels of Tbx20 during the
earlier stages of specification and development, suggesting that other T-box
genes may act redundantly with Tbx20 during early heart
development.
In this study we investigate the cellular and molecular relationship between Tbx5 and Tbx20 in X. laevis. We show that the phenotypes of knocking down TBX5 and TBX20 are highly similar, with embryos derived from either Tbx5 or Tbx20 morpholino injections displaying profound morphological defects, including pericardial edema, reduced cardiac mass and loss of circulation. In addition, we show that the morphological phenotype is not a reflection of alterations in the specification, commitment or differentiation of cardiac tissue. Thus, in addition to sharing a number of molecular properties, we show that Tbx5 and Tbx20 function in a non-redundant fashion and are essential for cardiac morphogenesis. However, despite the similarities in phenotype and shared molecular properties, Tbx5 and Tbx20 also have independent roles in heart development.
Given the similarity in TBX5 and TBX20 morphant phenotypes, we investigated the pathways by which Tbx5 and Tbx20 function. We show that TBX5 and TBX20 do not function in a linear pathway (i.e. Tbx20 does not act downstream of Tbx5, and vice versa), but rather imply a synergistic role for these two proteins during early heart development. Consistent with this proposal, we show that TBX5 and TBX20 can physically interact, map the interaction domains, and show an interaction for the two proteins in cardiac development, therefore providing the first evidence for interaction between members of the T-box gene family.
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Materials and methods |
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Transient transfections
293T cells were plated at 1x106 cells/well in six-well
tissue culture plates 24 hours prior to transfection. Plasmids used in
transients are: the Nppa promoter-luciferase reporter
(Bruneau et al., 2001;
Hiroi et al., 2001
),
pTbx5-V5, pTbx20-V5, pCMV-LacZ and pBS/KS. The
amount of luciferase reporter plasmid DNA was kept constant at 100 ng for
Tbx5, while titering in Tbx20 (25-100 ng). Expression vector
plasmid DNA was kept constant at 100 ng total and 50 ng of lacZ
reporter plasmid was used. Total amount of DNA was kept constant at 2 µg
and transfected using Lipofectamine 2000 (Invitrogen). Plasmid DNA was diluted
in OPTI-MEM (GibcoBRL) and complexes were allowed to form for 25 minutes at
room temperature and added to each well. Forty-eight hours post-transfection,
cells were harvested using M-PER (Pierce) with gentle shaking. Luciferase
activity was normalized to ß-galactosidase activity. All assays were
carried out three independent times in triplicate. Results were plotted using
normalized Relative Luciferase Units (RLUs).
Nuclear localization
NIH/3T3 cells were seeded in chamber slides at 6x103
cells/chamber 24 hours prior to transfection. Cells were transfected with
187.5 ng pTbx20-V5 or pTbx5-V5 per chamber using 1.25 µl
Polyfect (QIAgen) transfection reagent according to manufacturer's protocol.
At 48 hours, cells were washed twice with PBS and fixed in MEMFA for 1 hour (2
ml 10xMEM, 2 ml formaldehyde, 16 ml H2O) at 4°C. Cells
were washed twice with PBST (PBS + 0.1% Triton), blocked in PBST + 10% fetal
bovine serum for 1 hour at 4°C, incubated at 4°C overnight with
anti-V5 (Invitrogen) diluted 1:1000 in PBST+Serum. Cells were washed three
times, blocked for 1 hour, then incubated for 1 hour at room temperature with
goat anti-mouse Cy2 (Jackson ImmunoResearch) diluted 1:200 in PBST+Serum. This
process was repeated using anti-phosphotyrosine (Upstate Biotechnology) as
primary antibody to visualize the cytoplasmic compartment and goat anti-mouse
Cy3 (Jackson ImmunoResearch) secondary antibody. Cells were washed three
times, coverslipped and analyzed by confocal microscopy on a Zeiss LSM
410.
Embryo injections
Preparation and injection of X. laevis embryos was carried out as
previously described (Wilson and
Hemmati-Brivanlou, 1995). Embryos were staged according to
Nieuwkoop and Faber (Nieuwkoop and Faber,
1967
). Two antisense morpholino oligonucleotides each were
designed against the Tbx5 and Tbx20 5' UTRs and start
sites. Morpholinos were obtained from Gene Tools, LLC. with the following
sequences: Tbx20-MO1, 5' AAT CCA CTT CCA AGG GCA GTT GCT T 3';
Tbx20-MO2, 5' GTT TGG GAG AAG GAG TGT ATT CCA T 3'; Tbx5-MO1,
5' TTA GGA AAG TGT CTC TGG TGT TGC C 3'; Tbx5-MO2, 5' CAT
AAG CCT CCT CTG TGT CCG CCA T 3'; and control MO, 5' CCT CTT ACC
TCA GTT ACA ATT TAT A 3'. The human ß-globin splice-mutant standard
control morpholino from Gene Tools was used as control. Equal amounts of both
Tbx5 morpholinos were used in all injections. This combination is referred to
in the text and figures as `TBX5MO'. Tbx20 morpholinos were also
injected in combination, and referred to as `TBX20MO'. TBX5MO was injected at
the optimal (40 ng) or suboptimal (20 ng) doses, and TBX20MO was injected at
the optimal (80 ng) or suboptimal (40 ng) doses. `Optimal dose' is defined as
the dose empirically found to be efficient at blocking protein translation
both in vitro and in vivo, and inducing a cardiac phenotype in nearly 100% of
injected embryos, while `suboptimal dose' refers to the dose empirically found
to be below the threshold of the full cardiac phenotype-inducing dose.
Whole-mount RNA in situ hybridization
Whole-mount in situ hybridization was performed as previously described
(Harland, 1991). Embryos were
cleared using 2:1 benzyl benzoate/benzyl alcohol (Sigma) (Figs
5 and
9).
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Translation inhibition by morpholinos
In vitro translations were performed using TNT Coupled Reticulocyte Lysate
System (Promega) following the manufacturer's protocol. We have recently
demonstrated that X. laevis SHP-2 is uniformly expressed throughout
early development (Y. Langdon and F.L.C., unpublished) and anti-PTP1D/SHP2
primary antibody was used at 1:2500 (Transduction Laboratories) as a loading
control with peroxidase-conjugated AffiniPure donkey anti-mouse (H+L) 2°
antibody (1:10,000). V5-tagged proteins were probed with anti-V5 primary
antibody (Invitrogen) at 1:5000 dilution, and peroxidase-conjugated AffiniPure
Donkey anti-mouse (H+L) secondary antibody (Jackson ImmunoResearch
Laboratories) at 1:10,000 dilution. For in vivo translation analyses, embryos
were injected with MOs and mRNA at the one-cell stage and animal caps cut at
stage 8. At sibling stage 10, 10 animal caps per treatment were collected and
lysed in 100 µl of lysis buffer: 200 mM NaCl, 20 mM NaF, 50 mM Tris (pH
7.5), 5 mM EDTA, 1% IGEPAL, 1% Triton X-100 (Sigma), Complete EDTA-free
Protease Inhibitor (Roche). Lysates were resolved on 12% SDS-PAGE gels, and
visualization was carried out using Western Lightning Chemiluminescence
Reagent Plus (PerkinElmer Life Sciences).
Glutathione-S-transferase pull-down assays
GST pull-down assays were performed using the MicroSpin GST Purification
Module (Amersham Biosciences) according to the manufacturer's protocol. GST
constructs were transformed into BL21-Gold (DE3) cells (Stratagene) for
protein induction. Transformed cells were grown at 37°C to
ODA600=0.8 and GST proteins were induced for 1.5 hours at
20-27°C with 1 mM IPTG (Amersham Biosciences). Hemagglutinin (HA)-tagged
putative interacting proteins were produced in 293T cells. Lysates were
sonicated three times for 10 seconds prior to centrifugation at 16,000
g at 4°C for 10 minutes, and the supernatant was
collected. GST-fusion protein lysates and putative interacting protein lysates
were loaded on GST columns, incubated for 1.5 hours at 25°C, eluted,
electrophoresed on a 12% SDS-PAGE gel and transferred to PolyScreen PVDF
Transfer Membranes. HA-tagged proteins were detected with mouse HA.11 primary
antibody (1:1,000, Covance Research Products) and with peroxidase-conjugated
AffiniPure donkey anti-mouse (H+L) secondary antibody (1:10,000, Jackson
ImmunoResearch Laboratories). GST-fusion proteins were detected with rabbit
anti-GST primary antibody (1:25,000, Sigma-Aldrich) and with
peroxidase-conjugated AffiniPure donkey anti-rabbit (H+L) secondary antibody
(1:10,000, Jackson ImmunoResearch Laboratories).
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Results |
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To determine the requirement of TBX5 and TBX20 in heart development, we
injected TBX5MO, TBX20MO, or ControlMO into one-cell stage embryos. No
significant differences are seen between TBX5 morphants, TBX20 morphants,
control morphants, or uninjected siblings throughout gastrulation and
neurulation stages. However, a slight delay in developmental stage is evident
in TBX5 and TBX20 morphants relative to control morphants and uninjected
embryos by neurulation stages (stage 16). By cardiac looping stages
(
stage 38) (Kolker et al.,
2000
; Mohun and Leong,
1999
; Mohun, 2000
;
Newman and Krieg, 1999
), a
reduction in cardiac mass is evident in the morphants, and by stage 38 both
morphants display grossly abnormal heart morphology
(Fig. 2A-F). At this stage, 82%
of TBX5 morphants and 100% of TBX20 morphants display prominent cardiac
defects, as scored by the presence of an unlooped heart tube, a reduction in
cardiac mass and the presence of a pericardial edema
(Fig. 2G). After terminal
cardiomyocyte differentiation has begun (
stage 45)
(Kolker et al., 2000
;
Mohun and Leong, 1999
;
Mohun et al., 2000
;
Newman and Krieg, 1999
), TBX5
and TBX20 morphants display dramatically smaller hearts and in many embryos
cardiac tissue is barely detectable (Fig.
2E,F). However, the remaining cardiac tissue still retains some
degree of contractility, although it is confined to a small patch of
contractile tissue in the dorsal-most aspect of the cardiac cavity. Both TBX5
and TBX20 morphants also display abnormal eyes, which is consistent with
studies showing the involvement of both genes in eye development
(Fig. 2) (Carson et al., 2004
;
Koshiba-Takeuchi et al., 2000
;
Leconte et al., 2004
). Embryos
derived from injection of Tbx20 morpholinos directed against the
antisense transcript, Tbx5 morpholinos containing mismatches, MOs
directed against zebrafish Tbx5 and Gene Tools LLC's MO control,
produced no observable phenotype at any concentration (data not shown). These
observations, and the findings that the TBX5 and TBX20 protein levels can be
reduced or eliminated both in vitro and in vivo, suggest that the phenotypes
we observe are specific for knocking down TBX5 and TBX20.
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Analysis of hearts derived from TBX5MO and TBX20MO embryos shows a significant decrease in cardiac cell number. To determine if this is due to alterations in cardiac cell commitment, we performed whole-mount in situ hybridization with the early heart marker, Nkx2.5 (Fig. 4). This analysis was carried out on staged-matched embryos derived from TBX5MO, TBX20MO and ControlMO embryos over the period of cardiac cell commitment, migration and differentiation (stages 16-36). We could not detect any obvious difference in the number or spatial distribution of Nkx2.5-expressing cells prior to stage 24 (Fig. 4). Consistent with our initial analysis, after stage 24, the hearts from TBX5MO and TBX20MO embryos are morphologically abnormal and smaller in size, and therefore show a reduced domain of Nkx2.5 expression.
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We next tested whether TBX5 and TBX20 can function to regulate the levels
of transcription of the TBX5 target gene Nppa/ANF. To test for
DNA-specific binding and transcriptional activities, we transfected in
full-length versions of Tbx5 and Tbx20, either alone or in
combination, with the putative Tbx5 target Nppa/ANF reporter
construct into 293T cells. Consistent with studies using the mouse
Tbx5 ortholog (Bruneau et al.,
2001; Hiroi et al.,
2001
), TBX5 can weakly activate the rat Nppa/ANF
reporter. By contrast, Tbx20 alone can activate Nppa/ANF in
a dose dependent fashion. However, in the presence of TBX5, TBX20 can have the
converse effect on the Nppa/ANF reporter. In the presence of TBX5, at
high and low doses of TBX20 there is increased activation of the reporter
construct, while at moderate doses there is a repressive effect
(Fig. 6I). Thus, the presence
of TBX5 appears to alter TBX20 transcriptional activity.
TBX5 and TBX20 physically interact with one another
Given the similarity in phenotypes of TBX5 and TBX20 morphant embryos, and
the observation that Tbx5 and Tbx20 are not dependent on the
expression of one another, we next assessed whether TBX5 and TBX20 can
physically interact. TBX5 fused to Glutathione-S-Transferase (GST) was
incubated with HA-tagged TBX20 or NKX2-5. Pull-down experiments were then
performed to assess whether TBX20 can bind to TBX5. NKX2-5 has been shown to
interact with TBX5 and thus serves as a positive control
(Bruneau et al., 2001;
Hiroi et al., 2001
). As shown
in Fig. 8A, bacterially
translated GST-TBX5 is able to bind HA-TBX20 and HA-NKX2-5 produced from 293T
cells, in contrast to GST alone, which does not bind either protein. These
results reveal that TBX5 and TBX20 can interact in vitro. This is the first
report of physical interaction between T-box proteins.
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Tbx5 and Tbx20 cooperate to regulate heart morphogenesis
Given that TBX5 and TBX20 physically interact with one another, we
hypothesized that Tbx5 and Tbx20 may function cooperatively
to control cardiogenesis. To test this hypothesis, we co-injected
concentrations of TBX5MO and TBX20MO below the threshold at which cardiac
phenotypes are efficiently induced when injected individually. At a
concentration of 40 ng per embryo for Tbx5 morpholinos and 80 ng per
embryo for Tbx20 morpholinos, injections yield consistent heart
phenotypes in 82% of TBX5MO-injected embryos and in 100% of TBX20MO-injected
embryos (Fig. 2). We refer to
this dose as the `optimal' dose, because it is the dose that efficiently
blocks translation of Tbx5 and Tbx20 in vivo
(Fig. 1D,E) and the dose that
gives efficient and penetrant cardiac phenotypes. At half doses, 20 ng per
embryo for TBX5MO and 40 ng per embryo for TBX20MO, each morpholino yields
significantly fewer and weaker heart phenotypes compared with the full dose
(Fig. 9M, data not shown). We
refer to this concentration as the `suboptimal' dose for inducing cardiac
defects. The terms `optimal' and `suboptimal' are only used to refer to the
concentrations that yield fully penetrant or partially penetrant cardiac
phenotypes, respectively.
To address the question of whether Tbx5 and Tbx20 cooperate in cardiogenesis, we injected TBX5MO and TBX20MO individually at suboptimal doses in combination with ControlMO to keep total morpholino concentrations equal in all injections. TBX5MO was then co-injected with TBX20MO, each at the suboptimal dose. ControlMO injected at 80 ng/embryo served as control. As shown in Fig. 9, only 4% of embryos injected with suboptimal TBX5MO/ControlMO displayed a pericardial edema, unlooped heart tubes and a reduction in cardiac mass. Suboptimal TBX20MO/ControlMO yields only 13% cardiac defects. In suboptimal injections, the majority of embryos appeared normal, while the few cardiac phenotypes produced were much less severe than at optimal doses (e.g. barely detectable reduction in cardiac mass, slight perturbation of looping and little or no pericardial edema). When co-injected at suboptimal doses, 74% of TBX5MO/TBX20MO co-injected embryos display dramatic cardiac defects compared with 0% of ControlMO-injected embryos (Fig. 9A-L). The observation that the percentage of heart defects in double morphants is more than additive suggests that Tbx5 and Tbx20 synergistically act to control heart morphogenesis.
If Tbx5 and Tbx20 cooperate to regulate cardiogenesis, one might expect a more severe alteration in cardiac morphology and marker expression when the levels of both proteins are reduced. To address this issue, we performed in situ hybridization on stage 36 embryos from the above double injection experiment using Nkx2-5, XANF and XTnIc probes. As shown in Fig. 9, all three markers are expressed normally in embryos injected with suboptimal doses of TBX5MO and TBX20MO when compared with ControlMO. However, heart marker expression in the double morphant embryos is markedly reduced, particularly XANF. Both Nkx2-5 and XTnIc are still detectable in the heart region, albeit in fewer cells. Thus, the synergistic cooperation of TBX5 and TBX20 are required for proper heart development.
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Discussion |
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Functions of Tbx5 and Tbx20 in cardiac morphogenesis
Our studies show that Tbx5 and Tbx20 are required for
similar cellular processes in the developing heart. These data demonstrate a
non-redundant function for TBX5 and TBX20 during cardiac morphogenesis;
neither protein can compensate for the other in heart morphogenesis. The lack
of redundancy at the molecular level is corroborated by the observation that
the putative TBX5 target gene XANF either is not expressed or is
expressed very weakly in TBX5 morphant embryos, while being expressed at the
proper time, place and levels in TBX20 morphant embryos. Together, these data
suggest that TBX5 and TBX20 act in a non-redundant fashion to control
morphogenetic movements of early heart tissue.
The cardiac defects, in response to a reduction of either TBX5 or TBX20, appear to represent a block in an early morphological step in heart formation. As the spatial distribution of Nkx2-5 is unaltered throughout early development in TBX5MO-, TBX20MO- and ControlMO-injected embryos, and as Nkx2.5, Tbx5 and Tbx20 continue to be expressed until the later stages of heart development, and TBX5 and TBX20 morphants express markers of terminal muscle differentiation, neither Tbx5 nor Tbx20 appears to be required for commitment, migration or terminal differentiation of cardiac tissue. Thus, both Tbx5 and Tbx20 appear to be required to direct the coordinated events that occur during the early steps of heart morphogenesis.
Consistent with this hypothesis, both TBX5 and TBX20 morphant-derived hearts are greatly extended along the anteroposterior axis, and the heart tube fails to correctly loop and undergo chamber formation. As a result, embryos display pericardial edemas, have impaired blood flow (see Figs S2 and S3 in the supplementary material), an irregular heartbeat (data not shown) and ultimately die. Thus, the alteration in heart morphology appears to be the primary outcome of perturbing TBX5 or TBX20 function.
Past attempts to interfere with Tbx5 function in X.
laevis were carried out by the misexpression of a putative interfering
form of Tbx5 that leads to either the absence or severe malformations
of the heart (Horb and Thomsen,
1999). In instances in which the heart does form, there is a
reduction or block in myocardial tissue formation and a failure of the heart
to undergo looping. Our results with Tbx5-specific morpholinos show a
less severe heart phenotype than those reported with the dominant interfering
Tbx5 but bear a close resemblance to those reported for the zebrafish
Tbx5 mutant, heartstrings
(Garrity et al., 2002
). This
may be due to the dominant-interfering form of Tbx5 used in the
X. laevis studies interfering with the function of both Tbx5
and Tbx20 or possibly other T-box family members expressed in the
developing heart, e.g. Tbx1 and Tbx2
(Chapman et al., 1996
), as has
been shown for other Engrailed fusions
(Horb and Thomsen, 1997
).
However, in the absence of a TBX5-specific antibody, we cannot rule out the
possibility that some residual TBX5 protein is present in morphant embryos
leading to a less severe phenotype in our studies.
Tbx5 and Tbx20 are not dependent on the function of one another
The phenotypes of TBX5 and TBX20 morphant embryos do not appear to act in a
linear pathway as the spatial and temporal expression of Tbx5 appears
unaltered in TBX20 morphants, and vice versa. These findings are in agreement
with studies showing normal expression of Tbx20 in Tbx5
mutant mice (Bruneau et al.,
2001) but in apparent conflict with a second study reporting the
downregulation of Tbx5 in zebrafish embryos injected with a
Tbx20 morpholino (Szeto et al.,
2002
). Although the zebrafish and X. laevis orthologs of
Tbx20 share a very high degree of identity at the protein level
(86%), the differences between the two orthologs may reflect a species
difference as, for example, has been reported for the endodermal-inducing
activities of the T-box-containing gene Brachyury
(Marcellini et al., 2003
).
Although no alterations in Tbx5 or Tbx20 RNA levels were
observed in morphant embryos, we did observe a downregulation of TBX5 protein
in response to Tbx20 morpholinos in vivo, and vice versa, but not in
vitro (Fig. 1), raising the
interesting possibility that cross-regulation may be occurring between TBX5
and TBX20 at the level of translation. As similar studies have not been
conducted in zebrafish, it is not possible at this time to know the mechanisms
of crossregulation or whether this is a conserved response to interfering with
TBX5 or TBX20.
TBX5 and TBX20 heterodimerization
Although Tbx5 and Tbx20 are co-expressed and both
function in early heart development, the genes appear to be regulated through
separate pathways. For example, Tbx20 but not Tbx5 can be
induced in response to BMP2 signaling
(Plageman and Yutzey, 2004).
Taken together with our results demonstrating a physical interaction between
TBX5 and TBX20, these data would suggest that TBX5 and TBX20 function in
parallel pathways that converge upon TBX5:TBX20 heterodimerization. This model
is also supported by our results showing a functional interaction between TBX5
and TBX20: embryos derived from injections of suboptimal doses of
Tbx5 and Tbx20 morpholinos have only minor effects on heart
development in a small proportion of the embryos. However, when injected in
combination, 74% of all embryos examined displayed grossly abnormal heart
formation.
What are the possible cellular functions of TBX5 and TBX20 in heart
development? Past studies of T-box genes have shown a direct link between
members of the T-box gene family and cell adhesion. For example, embryos
homozygous for mutations in Brachyury, the founding member of the
T-box gene family, show an inability of the mesoderm to migrate properly along
the extracellular matrix leading to an inability of the mesodermal germ layer
to complete the morphogenetic movements normally associated with gastrulation
(reviewed by Showell et al.,
2004). We propose an analogous model for TBX5 and TBX20 function
in regulating cell polarity or adhesion events associated with heart
morphogenesis. We propose that TBX5 and TBX20 function to control polarity or
adhesive properties of cardiac tissue once the two heart fields merge along
the anterior midline, and that target specificity is regulated through TBX5
and TBX20 protein-protein interactions. In agreement with this proposal, we
have recently shown that alterations in cardiac cell numbers, survival and
proliferation in TBX5MO-derived embryos are a secondary consequence of
disrupting TBX5 function (S. Goetz and F.L.C., unpublished). This observation,
taken together with our findings that cardiac gene expression patterns are not
disrupted in TBX5MO- or TBX20MO-derived embryos, suggests that the primary
role for TBX5 and TBX20 is to control cardiac cell polarity or adhesion.
It is worth noting that neither TBX5 nor TBX20 have strong transcriptional
activation or repression activity by themselves
(Fig. 6)
(Bruneau et al., 2001;
Hiroi et al., 2001
;
Plageman and Yutzey, 2004
;
Stennard et al., 2003
). Thus,
transcriptional activity appears to be governed by protein-protein
interactions. Past studies have identified several other interacting partners
for both TBX5 and TBX20. For example, both TBX5 and TBX20 have been shown to
interact with the homeobox-containing transcription factor NKX2-5
(Bruneau et al., 2001
;
Hiroi et al., 2001
;
Stennard et al., 2003
),
consistent with clinical studies showing that HOS patients and humans
heterozygous for NKX2-5 display many of the same cardiac defects
(Elliott et al., 2003
;
Goldmuntz et al., 2001
;
Prall et al., 2002
).
How might TBX5:TBX20 heterodimerization affect target choice? It is
possible that the role of TBX5:TBX20 dimerization is to sequester TBX5 and
thereby block its interaction with other proteins such as NKX2.5, thereby
indirectly inhibiting the induction of cardiac specific genes such as
XANF. However, several lines of evidence argue against such a
proposal. For example, at low and high concentrations TBX20 can increase
transcription of the Nppa/ANF reporter in the presence of TBX5, while
showing a repressive activity at intermediate concentrations, suggesting that
in certain contexts TBX20 can cooperate with TBX5 to activate transcription,
while antagonizing TBX5 activity in others. An alternative possibility is that
TBX20 target choice and ability to function as a transcriptional activator or
repressor is governed by its choice of interacting partners. Consistent with
this hypothesis, Stennard et al. (Stennard
et al., 2003) have shown that NKX2.5, GATA4 and GATA5 interact
with TBX20, and the interactions occur through the same domain of TBX20 that
we have shown interacts with TBX5, at least in the cases of NKX2.5 and GATA4.
Furthermore, the authors have demonstrated that TBX20 can repress synergistic
activation of a connexin 40 reporter by NKX2.5 and GATA4, while
synergistically activating the same reporter with NKX2.5 and GATA5. Thus,
TBX20 may be able to function both as a transcriptional activator or
repressor, and this decision is based on its choice of protein partners. In
addition, TBX5 and TBX20 have been shown to display different binding
affinities for different T-box-binding sites
(Stennard et al., 2003
). For
example, TBX20, unlike TBX5 can bind to the Brachyury target site
while TBX5 has a higher affinity than TBX20 for the T-box binding site in the
Nppa/ANF promoter. Thus, downstream target selection may be
dictated by homodimerization versus heterodimerization. This is supported by
the recent findings that several genes involved in heart development are found
to contain multiple T-box binding sites (R. Schwartz, personal communication;
F.L.C., unpublished). Our model suggests that TBX5 and TBX20 target selection
and transcriptional activity is based on partner choice in a specific tissue
at a specific time. However, it still remains to be established which protein
interactions take place in the developing heart and in turn, what governs the
choice of partners for TBX5 or TBX20. These are presently areas under active
investigation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/3/553/DC1
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ACKNOWLEDGMENTS |
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