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Molecular and Cellular Biology, October 1998, p. 6044-6051, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Haploinsufficiency of MSX1: a Mechanism for Selective
Tooth Agenesis
Gezhi
Hu,1,2
Heleni
Vastardis,3,4,5,
Andrew J.
Bendall,1,2
Zhaoqing
Wang,1,2
Malcolm
Logan,4
Hailan
Zhang,1,2,
Craig
Nelson,4,§
Stacey
Stein,1,2
Norma
Greenfield,2
Christine E.
Seidman,3,6
J. G.
Seidman,3,4 and
Cory
Abate-Shen1,2,*
Center for Advanced Biotechnology and
Medicine1 and
Department of Neuroscience
and Cell Biology, UMDNJ-Robert Wood Johnson School of
Medicine,2 Piscataway, New Jersey 08854, and
Howard Hughes Medical Institute3 and
Department of Genetics,4 Harvard Medical
School,
Department of Orthodontics, Harvard School of
Dental Medicine,5 and
Howard Hughes
Medical Institute, Division of Cardiology, Brigham and Women's
Hospital,6 Boston, Massachusetts 02115
Received 27 May 1998/Accepted 16 July 1998
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ABSTRACT |
Previously, we found that the cause of autosomal dominant selective
tooth agenesis in one family is a missense mutation
resulting in an arginine-to-proline substitution in the homeodomain of
MSX1. To determine whether the tooth agenesis phenotype may result from haploinsufficiency or a dominant-negative mechanism, we have performed biochemical and functional analyses of the mutant protein
Msx1(R31P). We show that Msx1(R31P) has perturbed structure and
reduced thermostability compared with wild-type Msx1. As a
consequence, the biochemical activities of Msx1(R31P) are severely
impaired, since it exhibits little or no ability to interact with DNA
or other protein factors or to function in transcriptional repression.
We also show that Msx1(R31P) is inactive in vivo, since
it does not display the activities of wild-type Msx1 in
assays of ectopic expression in the limb. Furthermore, Msx1(R31P)
does not antagonize the activity of wild-type Msx1 in any of these
assays. Because Msx1(R31P) appears to be inactive and does not
affect the action of wild-type Msx1, we propose that the phenotype of
affected individuals with selective tooth agenesis is due to
haploinsufficiency.
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INTRODUCTION |
Tooth agenesis, or missing teeth, is
one of the most common developmental anomalies in humans
(12). Agenesis of one or more teeth is reported to
occur in as many as 9% of the population, excluding third molar
(wisdom tooth) agenesis, which is more prevalent (12).
Inherited tooth agenesis is likely to be caused by an impairment of one
or more of the molecular processes that regulate tooth formation. As
with many other organs, tooth development involves sequential and
reciprocal signaling processes between epithelial and mesenchymal cell
layers that are orchestrated by a hierarchy of genes encoding
secreted growth factors, extracellular matrix components,
and transcriptional regulators (26-29). Because the
regulatory genes required for tooth formation are common components of
signaling cascades involved in development of other embryonic structures and because of its relative simplicity, the tooth is an
excellent model for studying the molecular processes that underlie organogenesis.
Among the transcriptional regulatory genes required for tooth
formation, the Msx1 homeobox gene is highly expressed in the dental mesenchyme (17, 19, 20) and is essential for tooth development, since targeted gene disruption results in arrested tooth
formation at an early stage in Msx1(
/) mice
(6, 23). In addition to its expression in the tooth primordia, Msx1 expression is prominent in regions of
epithelial-mesenchymal interactions in several other embryonic
structures, including other craniofacial structures and the limb
(reviewed in reference 7). These findings have led
to the hypothesis that Msx1 is an important component in the
signaling events that occur between epithelial and mesenchymal tissues.
Previously, we reported that a missense mutation in the human
MSX1 gene causes selective tooth agenesis of secondary
dentition in one family (30). This mutation results in a
protein, MSX1(R31P), that contains an arginine-to-proline
substitution at position 31 within the homeodomain. Since this trait
is autosomal dominant, the resulting phenotype may be due to
haploinsufficiency, a dominant-negative activity, or a novel activity
of MSX1(R31P). To distinguish among these possibilities, we have
now investigated the consequences of the R31P substitution on the
structure of the resulting protein, as well as on its biochemical and
biological activities. We present evidence that the missense mutation
in MSX1 is likely to cause selective tooth agenesis through
haploinsufficiency. Our findings highlight the importance of
dosage for mediating the biological actions of
MSX1, as well as the significance of a detailed
understanding of the consequences of missense mutations for
interpreting the molecular bases of genetic disease.
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MATERIALS AND METHODS |
Plasmid construction and protein expression.
Most studies
were performed with the murine Msx1 cDNA, which shares 94%
similarity with human MSX1 (100% identity within the homeodomain) (7). The Msx1 and
Msx1(R31A) plasmids used for in vitro transcription and
translation [pGEM7zf(+)-Msx1(1-297) and
pGEM7zf(+)-Msx1(1-297):R196A] and the expression plasmids used
for transient transfection [pCB6+-Msx1(1-297) and
pCB6+-Msx1(1-297):R196A] were described previously
(5, 33, 34). Note that Msx1(R31A) refers to
Msx1-D from a previous report (33). To construct
Msx1(R31P), we introduced, by PCR-mediated site-directed mutagenesis, a substitution to replace arginine 196 (homeodomain position 31) with proline. The product was subcloned into the BamHI and HindIII sites of plasmids
pGEM7zf(+) (Promega) and pCB6+ for use in in vitro transcription and
translation and mammalian expression, respectively. The
Msx1HD plasmid, used for production of the recombinant
homeodomain polypeptide, was described previously [pDS56-Msx1(157-233)] (3).
Msx1HD(R31P) and Msx1HD(R31A) were obtained by PCR amplification of the respective sequences encoding amino acids 157 to 233 and subcloned into the BamHI and
HindIII sites of plasmid pDS56. The recombinant
homeodomain polypeptides, made as hexahistidine fusion proteins, were
expressed in Escherichia coli and purified by nickel
affinity chromatography as described previously (3). Note
that this purification procedure renders virtually homogeneous protein
preparations (Fig. 1A). Procedures for
DNA binding, glutathione S-transferase (GST) interaction, and transient-transfection assays have been described elsewhere (3, 5, 33, 34). The complete sequences of all
Msx1, Msx1(R31P), and
Msx1(R31A) constructs were verified by using Sequenase version 2.0 (U.S. Biochemicals). Msx1(R31P) and
Msx1(R31A) are comparable to Msx1 in all
respects, except for the relevant substitutions.
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FIG. 1.
Expression of Msx1 proteins. (A) Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis demonstrates the purity of
the recombinant Msx1 homeodomain polypeptides Msx1HD, Msx1HD(R31P),
and Msx1HD(R31A). Each protein (2.5 µg) was separated on a 15%
polyacrylamide gel and visualized by staining with Coomassie brilliant
blue. (B) Western blot assay demonstrates the equivalent expression of
the indicated Msx1 proteins in infected CEFs. Cell lysates were
prepared from CEFs that were not infected (NA) or that were infected
with a retrovirus expressing Msx1, Msx1(R31P), or Msx1(R31A).
The lysates were separated on a 10% polyacrylamide gel, and the Msx1
proteins, which were Myc tagged, were detected with a monoclonal
antibody against the Myc epitope. Note that the R31P substitution
results in a more slowly migrating protein. Molecular mass standards
(shown in panel A by the marker lane and in panel B by dashes) are
phosphorylase B (100 kDa), bovine serum albumin (77 kDa), ovalbumin
(48.2 kDa), carbonic anhydrase (33.8 kDa), soybean trypsin inhibitor
(28.6 kDa), and lysozyme (20.5 kDa).
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Retroviral infection.
For construction of retroviral
expression vectors, a Myc epitope was introduced at the 5' end of the
coding region of Msx1, Msx1(R31P), and
Msx1(R31A) by PCR amplification. We have previously found that the N-terminal Myc tag, which facilitates detection, does
not affect the activity of Msx1 in various assays (data not shown). The
resulting PCR products were first cloned into the BamHI and
HindIII sites of the SLAX13 shuttle vector
(14) and then subcloned as ClaI fragments into
the corresponding site of the replication-competent avian retroviral
vectors RCASBP(A) and RCASBP(B) (see Fig. 7A) (9, 14). For
comparison, chicken Msx1(GMsx1) and the
corresponding Msx1R31P mutant (without the Myc epitope) were
subcloned into the NcoI and HindIII sites of SLAX13 and subcloned into RCASBP(A). A control retrovirus expressing human alkaline phosphatase (AP) in RCASBP(B) was described in reference
9. For production of high-titer virus
(~108 to 109 CFU/ml), supernatants from
virus-infected chicken embryo fibroblasts (CEFs) were concentrated as
described previously (9). Note that the retroviruses express
similar levels of Msx1, Msx1(R31P), and Msx1(R31A) (Fig. 1B),
and all three proteins were localized to the nucleus (data not shown).
Virus was injected into stage 10 or stage 17 chicken embryos in the
area fated to become the right wing as described previously
(11). Embryos were staged according to the method of
Hamburger and Hamilton (13). Wings were dissected at stages
36 to 39, stained with Alcian blue or green, and cleared with
KOH-glycerol as described previously (11). Dissected wings
were imaged by video capture; the bone lengths were measured, and other
parameters of the phenotype (feather germ formation and altered
morphology) were scored. The ratios of infected (right wing) to
uninfected (left wing) bone lengths from the same embryos were
calculated for the humerus, radius, ulna, and the longest digit (digit
III). The mean bone length indices of Msx1-infected embryos
were compared to the 99% confidence interval for the mean bone length
indices of control [AP- and Msx1(R31P)-infected]
embryos.
CD.
Circular dichroism (CD) measurements were performed with
an Aviv model 62 DS spectropolarimeter fitted with a thermally
regulated cell holder in 0.1-cm (far-UV spectra) or 1.0-cm (near-UV
spectra) rectangular cuvettes. The midpoint temperatures
(Tms) of the unfolding transitions were
determined by fitting the change in ellipticity at 208 nm to the
equations k = exp{[
H/(RT)] [(T/Tm) 
1]}, y = k/(1 + k),
and f = [(u l)y] + 1, where k is the equilibrium constant of folding at any temperature (T),
Tm is the midpoint temperature of the folding
transition, and f is the fraction folded at any temperature.
T and Tm are in degrees kelvin,
converted to degrees celsius in Fig. 3. R is the gas
constant, and H is the enthalpy of folding, u
represents the ellipticity values where the protein is completely
folded, and l is the ellipticity value where the protein is
completely unfolded. To calculate the Tm values,
initial values of H, u, l, and
Tm are estimated. The CD data are then fit to
f by using the Levenberg-Marquardt algorithm (21)
implemented in SigmaPlot. The percentage of 
-helicity was calculated
from the ellipticity at 222 nm with the equation %helix = 100[
(Observed) (Coil)]/[(Helix) (Coil)], where (Helix) = 40,000 (1 2.5/n) + 100T
and (Coil) = 640 45T. (Helix) and (Coil)
are the values for 100% -helix and 100% random coil respectively,
expressed in degrees times centimeter squared per decimole,
T is the temperature in degrees celsius, and n is
the number of residues in the chain (24).
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RESULTS AND DISCUSSION |
Functional domains of Msx1.
The murine Msx1 gene
encodes a highly conserved DNA binding protein that functions as a
transcriptional repressor through its interactions with general
transcription factors, such as the TATA binding protein (TBP), and
other homeoproteins, including members of the Dlx family (4, 5,
31, 33, 34). Of several conserved functional domains of Msx1, the
homeodomain in particular is essential for DNA binding, transcriptional
repression, interactions with TBP and Dlx, and protein stability (Fig.
2) (3, 4, 8, 25, 33, 34).
Therefore, the R31P substitution may affect any, or all, of these
biochemical activities of Msx1.
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FIG. 2.
Functional domains of Msx1. Schematic diagram of Msx1
showing the regions conserved among Msx proteins: the Msx homology
regions I to III (MHRI to -III), the extended homeodomain (EHD), and
the homeodomain (7). MHRI and MHRII contribute to
transcriptional repression, whereas MHRIII promotes protein stability
(4). Contributions made by the homeodomain subdivisions (the
N-terminal arm [NT Arm] and helices I, II, and III) to protein
stability, DNA binding specificity, transcriptional repression, and
protein interactions are indicated by bars (8, 25, 33, 34).
Note that the R31P substitution occurs in helix II, which is primarily
important for protein stability.
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In considering the possible consequences of the R31P substitution, we
noted that this mutation occurs within helix II of the
homeodomain,
which makes an important contribution to protein
stability (Fig.
2)
(
8,
25). Moreover, proline residues are
rarely found at
position 31 in homeodomain sequences (
2), which
is not
surprising given the known propensity of prolines to disrupt
-helices (
22). Therefore, the R31P substitution may
affect
the activity of Msx1 as a consequence of introducing a proline
residue within helix II, which may be distinct from effects due
to the
loss of the basic arginine side chain. To distinguish between
these
possibilities, we have compared the activities of Msx1(R31P)
to
that of Msx1 and also to that of an Msx1 polypeptide containing
a
substitution of arginine 31 by alanine [Msx1(R31A)], since
alanine
is a neutral amino acid that is known to promote, rather than
destabilize,
-helix formation (
22).
Msx1HD(R31P) has altered structure and reduced
stability relative to Msx1HD and Msx1HD(R31A).
To
determine whether the R31P substitution affects the structure
of Msx1, we performed CD analysis using homeodomain polypeptides corresponding to the wild-type sequence [Msx1HD], the R31P
substitution [Msx1HD(R31P)], or the R31A substitution
[Msx1HD(R31A)] (Fig. 1A). CD analysis in the far-UV range
provides a quantitative measurement of the -helical content of
proteins (1), which is particularly useful for homeodomains,
since they are primarily -helical in structure (10). CD
analysis in the near-UV range measures the aromatic amino acid side
chain conformations and provides an indirect measurement of
protein conformation (1). As shown by their far-UV CD
spectra, all three homeodomain polypeptides form -helices at 20°C
(Fig. 3A). However, Msx1HD(R31P) has
reduced -helical content (56%) relative to Msx1HD (65%),
whereas Msx1HD(R31A) has increased -helical content (71%). The
near-UV CD spectra show that the protein conformation of
Msx1HD(R31P) is altered relative to those of Msx1HD and
Msx1HD(R31A) (Fig. 3B). In particular, the characteristic
tryptophan peak (at 290 nm) is similar for all three polypeptides,
whereas the tyrosine (280 nm) and phenylalanine (260 nm) peaks are
reduced for Msx1HD(R31P) relative to those for Msx1HD and
Msx1HD(R31A) (Fig. 3B). The single tryptophan residue in the Msx1
homeodomain is located in helix III, whereas tyrosine and phenylalanine
residues are found in helices I and II, indicating a local unfolding in
the vicinity of the proline substitution.
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FIG. 3.
CD analysis demonstrates altered structure
and reduced stability of Msx1HD(R31P) relative to Msx1HD and
Msx1HD(R31A). CD spectra were collected by using the purified
Msx1 homeodomain polypeptides (Fig. 1A) Msx1HD ( ), Msx1HD(R31P)
( ), and Msx1HD(R31A) ( ). (A) Far-UV CD spectra (200 to 250 nm) show that the -helical content of Msx1HD(R31P) (56%) is
less than that of Msx1HD (65%), whereas Msx1HD(R31A) (71%) has
greater -helical content. This is consistent with the known
helix-disrupting propensity of proline and the helix-promoting
propensity of alanine (22). (B) Near-UV CD spectra (250 to
320 nm) show that Msx1HD(R31P) has reduced absorbance in the
characteristic tyrosine and phenylalanine regions (260 and 280 nm,
respectively), whereas the tryptophan peak (290 nm) is similar for all
three proteins. (C) Tm curves show that
Msx1HD(R31P) has a lower Tm (33°C) than
Msx1HD (53°C) or Msx1HD(R31A) (58°C). The fraction of folded
protein was calculated from the far-UV CD spectra at 208 nm, taken
between 0 and 80°C; a similar profile was obtained at 222 nm (data
not shown). In panels A, B, and C, protein concentrations were
determined by A280 ( 280 = 7,000 cm1/M1) in the presence of 6 M
guanidine-HCl (8) and were adjusted to 0.06 mg/ml (A and C)
or 0.6 mg/ml (B). All data were collected in 10 mM potassium phosphate
buffer (pH 7.0) in triplicate with a step size of 0.25 nm.
In panels A and B, spectra were recorded at 20°C; for clarity,
only five data points are shown. CD analysis was performed three times
with two independent protein preparations; representative data are
shown.
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To examine directly whether the R31P substitution affects protein
stability, we determined the
Tms of Msx1HD,
Msx1HD(R31P),
and Msx1HD(R31A) (Fig.
3C). The
Tm refers to the temperature at
which 50% of
the protein is folded and is calculated from the
far-UV CD spectra
taken between 0 and 80°C. This analysis revealed
that Msx1(R31P)
is significantly less stable (
Tm = 33°C) than
Msx1HD and Msx1HD(R31A) (
Tm = 53 and 58°C,
respectively) (Fig.
3C). We note that the
Tm of
Msx1HD(R31P) is lower than the physiological
temperature,
suggesting that a considerable fraction of the Msx1HD(R31P)
protein
may be in a partially unfolded state in vivo.
Msx1R31P has reduced activity in biochemical functions that require
the homeodomain.
We next examined the consequences of the R31P
substitution on biochemical activities of Msx1 mediated by the
homeodomain. To compare the DNA binding activities of Msx1,
Msx1(R31P), and Msx1(R31A), we performed gel retardation assays
using full-length proteins obtained by in vitro translation (Fig.
4A). As shown in Fig. 4A and as
previously described (3), Msx1 interacts with its consensus
DNA site (e.g., CTAATTGG) and certain variations of this
site (e.g., CTAATTAG), but not with sites that
have substitutions of critical nucleotides within the consensus site
(e.g., CTAATGGA and CTACTTGG)
(Fig. 4A). Whereas the binding profile of Msx1(R31A) is
essentially identical to that of Msx1, Msx1(R31P) does not interact
significantly with any of these DNA sites (Fig. 4A) or with several
other DNA sites tested (data not shown).
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FIG. 4.
Msx1R31P has reduced DNA binding activity,
compared with that of Msx1 or Msx1(R31A), and is temperature
sensitive. (A) A gel retardation assay was performed at 20°C with
proteins obtained by in vitro transcription and translation (1 or 2 µl, indicated by the triangle) and with the DNA sites shown; these
sites were described in reference 3. The lanes
labeled NA contain the unprogrammed reticulocyte lysate (2 µl). The
upper arrow indicates the position of the specific Msx1-DNA complex;
the lower arrow indicates a nonspecific complex present in the
unprogrammed lysate. (B) The gel retardation assay was performed with
recombinant protein (5, 10, or 20 ng, indicated by the triangle) and
the Msx1 consensus DNA site (CTAATTGG) (3).
Protein-DNA complexes were formed at 20 or at 37°C, as shown. The
lanes labeled NA contain no added protein; the arrow indicates the
position of the Msx1HD-DNA complex. Note that Msx1HD(R31A) binds to
DNA more avidly than Msx1HD, which is presumably a consequence of its
enhanced stability (Fig. 3). For panels A and B, assays were performed
a minimum of three times; representative data are shown.
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To examine the relationship between loss of DNA binding and reduced
stability of Msx1(R31P), we compared the binding activities
of
Msx1HD, Msx1HD(R31P), and Msx1HD(R31A) at temperatures
below
(20°C) and above (37°C) the
Tm
of Msx1HD(R31P) (Fig.
4B). We used
the recombinant
homeodomain polypeptides, since Msx1HD(R31P) interacts
better
with DNA than full-length Msx1(R31P) (compare Fig.
4A and
B).
We found that the DNA binding activities of Msx1HD and Msx1HD(R31A)
were not altered at the temperatures examined (Fig.
4B). In
contrast,
the DNA binding activity of Msx1HD(R31P) was
significantly reduced
at 37°C compared to that at 20°C (Fig.
4B).
Taken together, these
findings indicate that the impaired DNA binding
activity of Msx1(R31P)
is a consequence of its reduced stability
rather than the loss
of the arginine side chain.
The Msx1 homeodomain also mediates functional interactions with various
protein factors, including Dlx2 and TBP (Fig.
2) (
33,
34). Therefore, we tested the ability of Msx1(R31P) to
associate
with these proteins in GST interaction assays (Fig.
5). In contrast
to the strong interaction
observed with Msx1 and Msx1(R31A), Msx1(R31P)
exhibited a weak
interaction with Dlx2 and TBP (Fig.
5). Since
residues in helix II do
not contribute directly to these protein-protein
interactions (Fig.
2)
(
33,
4), we infer that the reduced
ability of Msx1(R31P)
to bind to Dlx2 and TBP is also due to its
structural perturbation
and/or reduced stability.
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FIG. 5.
Msx1(R31P) interacts inefficiently with Dlx2 and
TBP. The GST interaction assay was performed with 5 µg of GST,
GST-Dlx2, or GST-TBP and 35S-labeled Msx1, Msx1(R31P),
or Msx1(R31A), as shown. Immobilized proteins were resolved by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
visualized by autoradiography (arrow). The input lane contains 20% (1 µl) of the total 35S-labeled protein (5 µl) used in the
interaction assays. The dashes show the positions of the molecular mass
standards (bovine serum albumin, 77 kDa; carbonic anhydrase, 33.8 kDa).
Assays were performed a minimum of three times; representative data are
shown.
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The biological actions of Msx1 are presumed to be mediated through its
function as a transcriptional repressor, and the homeodomain
is known
to be essential for this activity (
4,
5,
31,
33). Therefore,
we examined whether Msx1(R31P) is capable of
functioning as a
transcriptional repressor in transient-transfection
assays (Fig.
6). As we have found previously (
15,
33), Msx1
and Msx1(R31A) exhibited potent repressor activity
on an Msx1-responsive
reporter in transfection assays (Fig.
6A).
In contrast, Msx1(R31P)
had no transcriptional repressor
activity through this Msx1-responsive
reporter (Fig.
6A) or
through various other reporters (data not
shown).
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FIG. 6.
Msx1(R31P) lacks transcriptional repressor activity
and does not influence the transcriptional repressor activity of Msx1.
Transient-transfection assays were performed with C2C12 cells by using
expression plasmids encoding the indicated proteins and a luciferase
reporter plasmid containing an Msx1-responsive element (the WIP element
[15]). In panel A, the amounts of each expression
plasmid were 62.5, 125, or 250 ng (indicated by triangles). In panel B,
each sample contained 125 ng of the Msx1 expression plasmid (indicated
by the bar) alone or in combination with 125 or 250 ng of the Msx1,
Msx1(R31P), and Msx1(R31A) expression plasmids (indicated by
triangles). Data are represented as fold luciferase activity; a
representative assay is shown with error bars indicating the difference
between duplicates. Assays were performed a minimum of three times;
representative data are shown.
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Although Msx1(R31P) is not transcriptionally active, it may
influence the repressor activity of Msx1. To test this possibility,
we
performed mixing experiments by using a constant amount of
the Msx1
expression plasmid and increasing amounts of additional
Msx1,
Msx1(R31P), or Msx1(R31A) (Fig.
6B). A comparable level
of
repression was observed with equivalent amounts of additional
Msx1 or
Msx1(R31A) (Fig.
6B). In contrast, Msx1(R31P) did not
modify
the repressor activity of Msx1 significantly (Fig.
6B).
In other mixing
experiments, we have observed that Msx1(R31P)
neither
increased nor decreased the DNA binding activity of wild-type
Msx1 in
gel retardation assays (data not shown). Taken together,
these biochemical studies demonstrate that Msx1(R31P) (i)
has
little or no activity on its own, (ii) does not perturb the actions
of wild-type Msx1, and (iii) has no apparent novel activities.
Ectopic expression of Msx1, but not Msx1(R31P), alters
chicken embryonic limb morphology.
To address whether
these in vitro studies may also reflect the biological properties of
Msx1(R31P), we misexpressed Msx1, Msx1(R31P), or Msx1(R31A)
in embryonic chicken limb buds by using retroviral infection (Fig.
7). Clearly, there are
differences between tooth and limb development that are exemplified by
the expression pattern and function of Msx1 in these
tissues. In particular, Msx1 expression is more dynamic in
the developing tooth than in the limb, and the functional consequences
of targeted gene disruption of Msx1 and the R31P mutation
are evident in the tooth, but not in the limb (7, 23, 30).
On the other hand, limb development is similar to tooth formation in
that both require sequential and reciprocal signaling processes between
epithelial and mesenchymal cell layers, and Msx1 expression
in the limb and tooth mesenchyme is an important component in these
signaling events (7). Therefore, the chicken limb bud assay
provides a means of comparing the biological activities of
Msx1 and Msx1(R31P), whose functions in the
tooth may be more specialized.
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FIG. 7.
Ectopic expression of Msx1, but not Msx1(R31P),
alters chicken embryonic limb morphology. (A) The replication-competent
retroviral vector RCASBP (9, 14) provides a vehicle with
which to introduce Msx1, Msx1(R31P), or
Msx1(R31A) into the limb and to drive their expression.
Note that the murine genes are Myc tagged, but the chicken genes are
not (see Materials and Methods). The retroviruses that contain the
murine Msx1 sequences allow for detection of the exogenous
genes as distinguished from the endogenous chicken gene
(GMsx1) by in situ hybridization. (B) Whole-mount in situ
hybridization shows expression of endogenous GMsx1 at stage
26 in the embryonic limb buds, where it is confined primarily to the
distal mesenchyme (progress zone; arrows). At this stage,
GMsx1 is also expressed in the dorsal neural tube (white
arrowhead) and branchial arches (not shown). (C) Whole-mount in situ
hybridization shows ectopic expression of murine Msx1 in the
forelimb bud at stage 26 following retroviral infection at stage 17. Exogenous Msx1 is expressed throughout the forelimb, but not
the hindlimb, on the infected side (black arrows) and not in the
forelimb of the uninfected (control) side (white arrowhead) or in other
regions of endogenous GMsx1 expression. A similar pattern of
ectopic expression was observed for GMsx1 and
GMsx1(R31P) (data not shown). Whole-mount in situ
hybridization was performed with a chicken (B) or murine (C)
Msx1 antisense riboprobe as described in reference
34. (D to F) Ectopic expression of Msx1
(D) or Msx1(R31A) (F), but not Msx1(R31P)
(E), in the forelimb produces a smaller wing on the infected side
(upper right) relative to the wing on the uninfected side (control,
lower left). (G) Ectopic expression of Msx1 together with
Msx1(R31P) produces a smaller wing comparable to that
seen with ectopic expression of Msx1 alone. (H to I) Ectopic
expression of GMsx1 (H), but not GMsx1(R31P)
(I), in the forelimb produces a smaller wing and smaller feathers on
the infected side relative to the uninfected one (control). In panels D
to I, infection was achieved by injection of high-titer virus
(108 CFU/ml) into the region of the prospective forelimb
(right side) at stage 10 (H and I) or stage 17 (D to G); embryos were
staged according to the method of Hamburger and Hamilton
(13). Infection was generally restricted to the site of
injection (Fig. 7C); however, blood-borne virus sometimes led to
ectopic expression in the heart (not shown). Two retroviral vectors
encoding alternative viral envelope proteins were used [RCASBP(A) and
RCASBP(B)]. Ectopic expression of Msx1 by using either
retroviral vector produced equivalent results. In panel G, coinfection
of Msx1 and Msx1(R31P) was achieved with a
1:1 mixture of Msx1-RCASBP(A) and
Msx1(R31P)-RCASBP(B). Panels D to I show representative
wings (stages 36 to 39) with the ventral surface facing up. Data
analysis is provided in Table 1. R, radius; U, ulna; H, humerus; D,
digit III.
|
|
Within the developing chicken limb bud, expression of endogenous
Msx1 (
GMsx1) is restricted to the outer margin of
the mesoderm,
termed the progress zone, which is in close proximity to
the overlying
ectoderm (Fig.
7B). We infected the prospective right
forelimb
with the
Msx1-expressing retroviruses at the onset
of limb bud
outgrowth at stage 17 (
13) and examined the
consequences of
this ectopic expression at later stages of limb
development (stages
24 to 37) (Fig.
7). Ectopic expression of
Msx1 throughout the
wing (Fig.
7C) resulted in a reduction
in the size of the wing
on the infected side compared with the control,
uninfected, wing
(Fig.
7D and Table
1).
In particular, the skeletal elements (humerus,
radius, ulna, and
digits) were reduced by an average of 10 to
20% in infected wings
compared with uninfected, control wings
(Table
1). We observed a
reduction in the size and number of
the feather buds in the infected
wings compared with the control
wings (Table
1). For the
Msx1-infected wings, the mean infected/uninfected
(right/left) ratio of length of the various skeletal elements
was
statistically different from the same ratio for control wings
(
P < 0.01 for all skeletal elements measured). A
similar phenotype
was produced by infecting the prospective forelimb
field with
retroviruses expressing
GMsx1 at an earlier
developmental stage
(stage 10) (Fig.
7H). Since these wings were
examined at a later
stage (stage 39), the feather bud defect is
more evident. These
alterations of the skeletal elements and
feather buds are likely
to be a consequence of changes in the
expression patterns of Msx1-responsive
genes, as well as direct effects
of Msx1 on cellular proliferation
and differentiation (
1a).
In contrast to the defects observed with retroviruses expressing
Msx1 or
Msx1(R31A), infection with the
Msx1(R31P)-expressing
retrovirus in the prospective
forelimb did not produce any significant
alteration in the size or
shape of the infected wings (Fig.
7D
to F, H, and I and Table
1). In
fact, the wings infected with
either the mouse or chicken
Msx1(R31P)-expressing retroviruses
were
indistinguishable from the uninfected wings, suggesting that
Msx1(R31P) is inactive in vivo as well as in vitro.
To examine whether
Msx1(R31P) affects the activity
of
Msx1 in vivo, we infected the prospective forelimb with a
1:1 mixture
of
Msx1- and
Msx1(R31P)-expressing retroviruses. In control
experiments,
we verified the efficacy of coinfection by using a 1:1
mixture
of the
Msx1- and AP-expressing retroviruses, which
produced the
Msx1 phenotype, as well as AP activity
throughout the limb (Table
1 and data not shown). Coinfection with the
Msx1- and
Msx1(R31P)-expressing
retroviruses
produced a wing phenotype that is indistinguishable
from that produced
by infection with
Msx1 alone (compare Fig.
7D and G and
Table
1). These findings indicate that
Msx1(R31P) does
not affect the activity of
Msx1 in vivo.
Conclusions.
In summary, we have found that Msx1(R31P) is
partially or completely inactive in vitro and in vivo because of a
perturbation of structure and decreased stability that results from the
introduction of a proline residue within helix II of the homeodomain.
Furthermore, Msx1(R31P) does not appear to influence the activity
of wild-type Msx1, nor does it display any novel activities in the
assays performed. We therefore propose that the phenotype in affected
individuals with selective tooth agenesis is due to haploinsufficiency.
These findings raise several interesting questions regarding the mode
of action of
MSX1 and its particular importance in tooth
morphogenesis.
Msx1 is expressed throughout the tooth
mesenchyme
(
17,
19,
20) as well as other embryonic regions
(reviewed
in reference
7), and complete loss of
Msx1 function in mice
results in a failure of tooth
development (
6,
23). Yet the
missense mutation of
MSX1 in humans selectively affects certain
teeth,
particularly the second premolars and third molars (
30).
Apparently, the reduced dosage of
MSX1 in other embryonic
regions,
and even in other teeth, is tolerated, suggesting that
morphogenesis
of the affected teeth requires a greater dosage of
MSX1. This
idea is supported by the clinical observation
that while individuals
affected with selective tooth agenesis always
fail to develop
second premolars and third molars, flanking teeth are
more variably
affected (
30). Alternatively, tooth
morphogenesis in the affected
family may be particularly susceptible to
a reduced
MSX1 dosage
because of the specific effects of
genetic background. It is noteworthy
that mice heterozygous for
Msx1 exhibit no abnormalities in tooth
development
(
23). Although the absence of this phenotype may
simply
reflect the fact that mice lack premolars, it would be
of interest to
examine tooth morphogenesis in heterozygous mice
in different genetic
backgrounds.
Interestingly, a missense mutation in human
MSX2, which is
the cause of Boston-type craniosynostosis, also results in the
substitution of a single residue within the homeodomain
(
16).
In this case, substitution of a proline for a
histidine renders
the protein more stable than wild-type MSX2, and the
resulting
phenotype is believed to occur from a gain-of-function
activity
(
18,
32). In combination with the present study,
these findings
highlight the significance of structural integrity and
protein
stability as a means of regulating the activities of proteins
such as MSX1. Furthermore, this study demonstrates the importance
of a
detailed analysis of the biochemical and biological consequences
of
missense mutations for understanding the molecular bases of
genetic
disease.
| |
ACKNOWLEDGMENTS |
G.H., H.V., and A.J.B. contributed equally to this work.
We are indebted to Cliff Tabin (Harvard Medical School) for generous
assistance with the chick retroviral expression studies and for many
helpful discussions and Lee Niswander (Memorial Sloan Kettering) for
the gift of the GMsx1 probe. We thank Michael Shen and Aaron
Shakin (CABM) for helpful comments on the manuscript and Qing Li for
assistance with the CD analysis.
This work was supported by NIH grant HD29446-06 to C.A.-S.; HHMI
funding to C.E.S., J.G.S., and H.V.; predoctoral grants from the
American Heart Association to G.H. and H.Z.; and a grant from the
American Association of Orthodontists Foundation to H.V. C.A.-S. is a recipient of an NSF Young Investigator award.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CABM, 679 Hoes
Lane, Piscataway, NJ 08854. Phone: (732) 235-5161. Fax: (732) 235-4850. E-mail: abate{at}mbcl.rutgers.edu.
Present address: Division of Growth and Developmental Sciences and
Division of Basic Sciences, New York University College of Dentistry,
New York, NY 10010.
Present address: Department of Molecular and Cellular Biology,
Division of Genetics, University of California, Berkeley, CA 94720.
§
Present address: Center for Cancer Research, Department of Biology,
MIT, Cambridge, MA 02139.
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Top
Abstract
Introduction
