1 Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101, Japan
2 Department of Developmental Neurobiology, Institute of Development, Aging and
Cancer, Graduate School of Medicine, Tohoku University, 4-1, Seiryo, Aoba,
Sendai, 980-8575, Japan
* Author for correspondence (e-mail: ogura{at}idac.tohoku.ac.jp)
Accepted 5 May 2004
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SUMMARY |
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Key words: Dachshund, Smad1, Sin3a, Limb development, AER, Chick
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Introduction |
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Pattern formation along the AP axis is controlled by the zone of polarizing
activity (ZPA), located in the posterior margin of the limb bud. Sonic
hedgehog (Shh) is expressed in the ZPA and exerts its polarizing
activity. Expression of Shh is maintained by Fgf4 in the
AER; hence, tight communication between the ZPA and the AER is important for
pattern formation. Limb outgrowth is maintained by FGF8 in the AER, thereby
controlling morphogenesis along the PD axis
(Rubin and Saunders, 1972).
Thus, the Shh/FGF regulatory loop ensures coordinated growth and patterning
along the AP and PD axes.
Several BMP genes are also expressed in the AER and underlying mesenchyme,
playing pivotal roles in the control of proliferation, differentiation and
programmed cell death (Yokouchi et al.,
1996). When Noggin, a BMP antagonist, was misexpressed, anterior
extension of the AER and loss of its asymmetry was observed
(Pizette and Niswander, 1999
),
establishing a BMP antagonist as an apical ectodermal maintenance factor
(AEMF) (Zwilling, 1956
). When
a constitutively active BMP receptor was misexpressed, the AER and its
Fgf8 expression were lost, resulting in severe truncation
(Pizette et al., 2001
;
Ahn et al., 2001
). Both gain-
and loss-of-function approaches revealed that BMP signaling regulates pattern
formation along the DV and PD axes. However, the expression pattern of
Noggin indicates that this factor is not the bona fide AEMF. Another
BMP antagonist, Gremlin, is expressed in the limb bud, suggesting its
involvement in the AEMF pathway. As expected, misexpression of Gremlin
antagonized BMP signaling and induced hyperplasia of the AER
(Capdevila et al., 1999
;
Merino et al., 1999
;
Zuniga et al., 1999
). In
addition, Gremlin makes a tight regulatory loop between the Shh and FGF
signaling cascades, rendering this factor an excellent AEMF candidate.
Dachshund (Dac) was identified as one of the retinal
determinants in Drosophila. Null mutation of Dac results in
reduction or complete loss of compound eyes. Dac mutants have short
legs, with condensation of the femur, tibia and proximal tarsi, compatible
with the expression pattern of Dac in the intermediate domain of the
leg disc. In addition, cell death is increased
(Mardon et al., 1994).
Dac expression overlaps with that of dpp in both the
morphogenetic furrow (MF) and leg discs, making a tight regulatory loop. This
suggests that dpp and Dac are functionally related to
achieve the correct formation of legs and eyes
(Gonzalez-Crespo et al.,
1998
). Furthermore, eyeless (eye), eyes
absent (eya), sine oculis (so) and
dac are components of the pathway of compound eye formation
(Desplan, 1997
;
Wawersik and Maas, 2000
),
making a complex network (Chen et al.,
1997
; Pignoni et al.,
1997
). Recently, vertebrate homologues of eye, eya, so
and dac have been identified: Pax6, Eya1-Eya4, Six1-Six9,
and Dach1 and Dach2, respectively
(Quiring et al., 1994
;
Oliver et al., 1995
;
Hammond et al., 1998
;
Mishima and Tomarev, 1998
;
Borsani et al., 1999
;
Heanue et al., 1999
;
Kozmik et al., 1999
;
Lopez-Rios et al., 1999
).
(Expression patterns of chick Dach1 can be found at
http://dev.biologists.org/supplemental.)
We describe a novel antagonistic function of chick Dach1 on BMP signaling. We show that the nuclear events involving Dach1 and BMP signaling control pattern formation along the PD axis and maintenance of the AER.
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Materials and methods |
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Cell culture, transfections and luciferase assay
C2C12 and Cos7 cells were maintained in high-glucose DMEM supplemented with
10% FCS. Transient transfection was performed using lipofectamine (Invitrogen)
or polyethylenimine (Polysciences). In all transfection experiments, the total
amount of transfected DNA was kept constant by adding an appropriate amount of
empty vector. C2C12 cells (1x104 cells/well in 24-well tissue
culture plates) were transfected with various combinations of plasmids: 200 ng
of reporter constructs (Xvent-2-Luc or 4R-UAS-Luc plasmid),
200 ng of ß-galactosidase expression plasmids, 25-500 ng of various
expression constructs containing Dach1, Gal4-DBD fused Dach1, BMP4, Smad1,
Gal4-DBD-fused Smad1 (a kind gift from Dr Miyazono), mouse Sin3a, HDAC1 and
N-CoR (a kind gift from Dr Ishii). Forty hours after transfection, cells were
lysed, and lysates were subjected to luciferase assay. Luciferase activities
were measured by Luminescencer-JNR (ATTO). ß-Galactosidase activities
were measured to standardize the transfection efficiency.
Immunoprecipitation and western blotting
Cos7 cells were transfected with expression plasmids containing
hemagglutinin (HA)-tagged Dach1 or the DD2 domain of Dach1 along with
Flag-tagged Smad1 or Flag-tagged mouse Sin3a. Transfected cells were harvested
40 hours after transfection, then lysed in lysis buffer [50 mM HEPES (pH 7.5),
250 mM NaCl, 0.2 mM EDTA, 10 µM NaF, 0.5% NP-40]. Lysates were
immunoprecipitated using an anti-HA antibody (Covance). Immunoprecipitants
were subjected to SDS-PAGE, and western blot analysis was performed using an
anti-Flag antibody (Sigma) and ECL detection reagents (Amersham).
In ovo electroporation and bead implantation
For electroporation, a BTX T-820 electroporator (BTX, San Diego) and pulse
monitor (Meiwa Shoji, Japan) were used. Fertilized eggs were purchased from
Takeuchi and Yamagishi poultry farms (Nara, Japan). Platinum electrodes
(Muramatsu, Japan) and a sharpened tungsten needle (Nilaco) were used as an
anode and a cathode, respectively. An anode was inserted beneath the endoderm,
and a cathode was placed on the surface of the embryos. For ectodermal
expression, DNA solution was poured over the ectoderm with a sharp glass
pipette, and electric pulses were applied. Mesodermal expression was described
previously (Takeuchi et al.,
2003). Heparin-acrylic beads (diameter 100-160 µm; Sigma)
soaked for 1 hour at room temperature in recombinant BMP4 (0.5 mg/ml) were
implanted into the limb buds.
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Results |
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We further investigated whether Dach1 modulates BMP signaling. We
transfected an Xvent2 promoter-luciferase reporter plasmid
(Xvent2-Luc) (Hata et al.,
2000) into C2C12 cells, together with expression plasmids of
BMP4, Smad1 and Smad4. When a full-length Dach1 was
co-expressed, luciferase activities were repressed
(Fig. 1F). We constructed a
transcriptionally active Dach1 by fusing the VP16 transactivation domain at
the N-terminal end of Dach1 (VP=Dach1). As Dach1 makes a repressor complex,
VP=Dach1 should form a transcriptional active complex. When VP=Dach1 was
co-expressed, the Xvent2 promoter was activated strongly, even in the
presence of Dach1 (Fig. 1F).
This suggests that VP=Dach1 acts as a transcriptionally active form and
abrogates the Dach1-mediated transcriptional repression.
Comparison of Drosophila Dac and vertebrate Dach1 identified two
highly conserved domains in the amino and C termini (DD1/DS and DD2/EYA
domains, respectively) (Li et al.,
2002; Wu et al.,
2004
). Our data indicate that mouse Sin3a binds directly to DD2
(data not shown), as previously reported
(Li et al., 2002
). As HDAC and
NCoR bind to DD1 (Li et al.,
2002
), a synergistic interaction between DD1 and DD2 might be
necessary for the formation of the functional co-repressor complex. In
addition, DD2 binds to Smad1 (data not shown) and Smad4
(Wu et al., 2004
). Based upon
these observations, we speculated that DD2 per se blocks the synergistic
action between Smad1 and Dach1. To confirm this hypothesis, we assessed the
function of the DD2 construct in the Xvent2-luciferase context
(Fig. 1G). When the full-length
Dach1 was co-expressed with BMP4, Smad1 and Smad4, repression of this promoter
became evident. Interestingly, co-transfection of increasing amounts of the
DD2 expression construct abrogated this Dach1-mediated repression. This
suggests that DD2 per se acts as a dominant-negative in the
BMP/Smad1/Sin3a-mediated transcriptional control. Nonetheless, in contrast to
VP=Dach1 (Fig. 1F), DD2 did not
activate the Xvent2 promoter, but instead de-repressed the
Dach1-mediated repression. From these observations, we speculate that using
these two different constructs, we can investigate the functional roles of
Dach1 with two different approaches. Namely, VP=Dach1 activates the
BMP/Smad1-mediated transcription in a manner opposite to the normal Dach1, and
DD2 de-represses its action, acting as a dominant-negative form.
As reported previously, the extracellular molecule Gremlin acts as a BMP
antagonist (Capdevila et al.,
1999). To confirm whether this extracellular antagonism sets up a
`BMP-zero status' in the limb buds, we stained sections of embryos with an
anti-phosphorylated Smad1/5/8 antibody and found weak but distinct signals in
the ectoderm and mesoderm of the limb (data not shown). These observations
suggest that BMP signaling is weak but active in both the ectoderm and the
mesoderm of the limb buds, despite the expression of Gremlin. This also
suggests that Dach1 exerts its action with the BMP signal, which is attenuated
by Gremlin. Hence, we could alter the BMP/Smad1/Dach1-mediated transcriptional
control by the transcriptionally active VP=Dach1 and the dominant-negative
DD2.
VP=Dach1 in the limb buds
Next, we misexpressed VP=Dach1 in the surface ectoderm at stages 8-11 (top
insets in Fig. 2). At stage 19,
expression of Fgf8 was faint and blurred
(Fig. 2A,B). At stage 24,
severe deformities were seen in the distal ectoderm where strong enhanced
green fluorescent protein (EGFP) signals derived from co-electroporated
pCAGGS-EGFP were observed (Fig.
2C,D). Compatible with this, expression of Fgf-8 was
distorted and lost in the central part
(Fig. 2E). A gap was also found
in expression of Bmp7 in the AER
(Fig. 2F), and mesenchymal
Fgf10 expression was repressed
(Fig. 2G). By contrast,
Shh expression in the posterior part was not affected, although
severe deformity was evident in the distal end
(Fig. 2H).
|
DD2 in the limb buds
Next, we misexpressed DD2 in the limb mesenchyme at stages 13-15 (top
insets in Fig. 3).
Electroporation was monitored by the EGFP signals derived from the co-injected
pCAGGS-EGFP (Fig. 3A). At stage
19, 24 hours after electroporation, repression of Fgf10 first became
evident in the anterior side (Fig.
3B). At this stage, repression of Wnt5a was also observed
in the anterior domain with deformation of the AER
(Fig. 3C).
|
In severe cases, invagination of the thick epithelium was observed at stage 20 (red arrowhead in Fig. 4A). In such limbs, Fgf8 expression was detected in this invaginated part (Fig. 4B), suggesting that the AER was formed, but invaginated in the underlying mesenchyme. Cyclin D1 was expressed normally in the mesenchyme, suggesting that this invagination was not caused by mesenchymal growth arrest.
|
When these limbs were allowed to develop further, polydactylous digits developed (Fig. 4L,M) and were arranged in a double row, with digits a, b and c formed in a line, and digits a' and b' in another (Fig. 4M). When stained with Alcian Blue, this double row appearance was evident (Fig. 4N-P). As misexpression of DD2 induced the split AER (Fig. 3G) and/or the invagination of the ventral epithelium, we speculated that digits were formed in a double row arrangement in the split AER and/or the separate two limb protrusions (Fig. 4J,K).
Dach1 and the PD axis of limb buds
As reported previously, BMP signaling regulates pattern formation of limb
buds along its PD axis (Capdevila et al.,
1999). This suggests that Dach1 might be involved in this process,
acting along with the BMP signaling pathway. To examine the effects of DD2 on
the PD axis, we misexpressed DD2 in the entire limb mesoderm at stage 15
(Fig. 5A-F). In this case,
expression of Meis2 was induced in broader domains at stages 17 and
19 (Fig. 5B,E), compared with
the normal expression on the control sides
(Fig. 5C,F).
|
We isolated a 700 bp putative regulatory region of the human MEIS1
gene near its first exon. When this region was compared with the human
MEIS2 gene, it was highly conserved between these two genes,
compatible with the similar expression patterns of these two Meis genes
(Mercader et al., 2000). These
results indicate that the Meis1 and Meis2 genes share
similar regulatory elements crucial for their expression along the PD
axis.
To investigate the roles of this region, we constructed a luciferase reporter by inserting the 5' 700 bp sequence of the human MEIS1 gene upstream of the herpes simplex virus thymidine kinase (TK) promoter (Fig. 5J). This construct was transfected into HepG2 cells along with various combinations of effector plasmids (Fig. 5J). Addition of the BMP4 expression plasmid repressed this reporter weakly. Co-transfection of Dach1 and Sin3A super-repressed it, indicating that BMP signaling represses expression of Meis1 acting with Smads, Dach1 and Sin3a. By contrast, co-expression of VP=Dach1 activated this reporter (Fig. 6J). These results suggest that Dach1 is involved in pattern formation along the PD axis, controlling the expression of Meis genes through the BMP signaling cascade.
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Discussion |
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Recent studies have revealed that vertebrate Dach proteins interact with
other transcription factors. In the limb bud, Dach2 is expressed in
migrating myoblast precursors (Heanue et
al., 1999). Dach2 interacts with Eya2, and Eya2 interacts with
Six1; therefore, a synergism among the Dach2, Eya2 and Six1 proteins regulates
myogenic differentiation. In retinogenesis and pituitary development, Dach1
and Dach2 interact with Six6, HDAC, N-CoR and Sin3a to make a repressor
complex, which then represses cyclin-dependent kinase inhibitors, such as
p27Kip1 (Li et al.,
2002
). In this report, Dach1/2 were shown to possess multiple
interfaces; the DD1 domain interacts with Six6, HDAC and N-CoR, and the DD2
domain with Sin3a. In addition, the DD2 of Dach2 interacts with Eya2
(Heanue et al., 1999
). In both
cases, the Dach protein functions in the context of the DNA-binding protein
Six, highlighting the Dach protein as a potent modulator of multiple
transcriptional controls.
Dach1 was not expressed in the early stages of limb development
(stages 13-17) (Heanue et al.,
2002) (see Figs S1, S2 at
http://dev.biologists.org/supplemental).
Dach1 expression began at stage 20 in the AER. This suggests that
Dach1 is not involved in the initiation of the AER or the DV axis
determination; rather, it plays a role in its maintenance. Both in mouse and
chick limbs, expression of Dach1 was induced by implantation of beads
soaked in several FGFs (Horner et al.,
2002
). This is compatible with our data showing that expression of
Fgf8 begins earlier than that of Dach1 and supports the
roles of Dach1 in maintenance. When a constitutively active VP=Dach1 was
misexpressed in the pre-AER ectoderm, disruption of the AER and distal
truncation were observed (Fig.
2); a similar change is induced by excess BMP signaling
(Macias et al., 1997
;
Zou et al., 1997
). When DD2
was misexpressed in the mesenchyme, repression of marker genes and deformation
of the AER were also obtained (Fig.
3). This suggests that Dach1 in the mesenchyme regulates the
formation of the AER.
The actions of BMP molecules are antagonized by extracellular Gremlin.
Nonetheless, BMP signaling is still weakly active in both the mesoderm and the
ectoderm, as revealed by staining with an anti-phosphorylated Smad protein
antibody (data not shown). This suggests that the attenuated BMP signals play
a repressive role with Smads, Sin3a and Dach1. In contrast to Gremlin, Dach1
regulates the transcription of putative target genes, leaving other signaling
cascades, such as p38 MAP kinase, intact
(Kozawa et al., 2002). This
might create different signaling contexts in the limb bud. When BMP signaling
is shut off by forced expression of Gremlin, Noggin or dominant-negative BMP
receptors, both the Smad/Dach1 pathway and the MAP kinase pathway are
affected. By contrast, DD2 and VP=Dach1 affect only Smad-mediated
transcription. In addition, Dach1 was reported to contain a DNA-binding motif
(Kim et al., 2002
), suggesting
that complex formation of Dach1 and Smads might be influenced by sequences
near the Smad-binding motif. Consequently, genes that contain only the
Smad-binding motif might be activated by the Smad1/Smad4 complex. Hence, the
transcriptional control of target genes might be dependent on the target
sequences, although Smads play an essential role in both cases. The difference
between extracellular and nuclear BMP antagonism might be related to the
elongation of the ventral ectoderm, which was never observed with
extracellular BMP antagonism. In addition, a balance between Dach1 and the
phosphorylated Smad proteins might be important, as excess BMP signaling
induces nuclear accumulation of Smad proteins, which might change the
stoichiometric ratio of phosphorylated Smad and Dach1 proteins, thereby
affecting the transcriptional levels of the target genes.
Misexpression of VP=Dach1 induced expansion of Meis2 expression in the distal domain, with intense expression in the anteroproximal area (see Figs S1, S2 at http://dev.biologists.org/supplemental). This suggests that Dach1 is involved in pattern formation along the PD axis of the limb bud. Consistent with this, implantation of BMP4-soaked beads repressed Meis2 expression (Fig. 5G,H), suggesting that the Smads/Dach1 complex represses Meis2. When DD2 was misexpressed, repression of Meis2 was cancelled (Fig. 5I), probably because DD2 abrogated the formation of the repressor complex. Drosophila data indicate that Dachshund is involved in pattern formation of the leg along its PD axis. Hence, the functional link between BMP/Dpp and Dach1/Dachshund is highly conserved in both invertebrates and vertebrates, placing Smad/Mad as a junction of signaling.
Recently, the mouse Dach1 gene was successfully knocked out,
showing no overt morphological changes
(Davis et al., 2001). This
suggests that there might be genetic redundancy, because expression of mouse
Dach2 overlaps with that of Dach1
(Davis et al., 2001
;
Heanue et al., 1999
). In
addition, chick Ski, which also binds to Smad proteins to repress BMP-mediated
transcription (Wang et al.,
2000
), is expressed in the developing limb buds
(Dai et al., 2002
). This
suggests that multiple mechanisms of transcriptional control operate in the
limb buds.
Our data have shown that Dach1 plays pivotal roles as an intracellular BMP antagonist and contributes to pattern formation along the PD axis and maintenance of the AER. In addition, our data have revealed that a direct interaction among Dach1, Smads and co-repressors is essential. Hence, our data uncover a novel set of interactions, introducing a new paradigm in the regulation of limb outgrowth. Further molecular dissection of Dach1 should provide novel insight into the highly conserved genetic program operating in both invertebrate and vertebrate appendages.
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ACKNOWLEDGMENTS |
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Footnotes |
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