Division of Developmental Biology, Department of Cell and Molecular Biology, and The Center for Bioenvironmental Research, Tulane University, New Orleans, LA 70118, USA
* Author for correspondence (e-mail: kmuneoka{at}tulane.edu)
Accepted 7 July 2003
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SUMMARY |
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Key words: Msx1, BMP4, Regeneration, Digit, Mouse
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Introduction |
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The regeneration of amputated distal digit/finger tips has been reported in
various mammals, including humans and rodents, and represents a mammalian
system where successful epimorphic regeneration can occur (see
Muller et al., 1999). In
humans, regenerative potential is restricted to the distal tip of the finger
in a region associated with the nail organ
(Douglas, 1972
;
Illingworth, 1974
).
Experimental studies using rodent models demonstrate that regenerative
capacity is level specific and, similarly, is associated with the proximal
extent of the nail forming organ (Borgens,
1982
; Zhao and Neufeld,
1995
). A role for the nail organ in the regeneration response is
supported by experiments showing that excluding the nail organ from the
amputation wound results in no regenerative response; however, nail organ
grafts result in ectopic bone formation and do not induce regeneration
(Zhao and Neufeld, 1995
;
Mohammad et al., 1999
). These
experimental results, together with clinical studies in humans, provide
evidence that the nail organ plays some role in the stimulation of adult digit
tip regeneration. Genes specifically expressed in, or associated with, the
nail organ are candidates for regulating this regenerative response. In
previous studies, we demonstrated that the homeobox-containing genes
Msx1 and Msx2 are expressed in association with the nail
organ of neonatal digits, and at the apex of developing digits in the
nail-forming region (Reginelli et al.,
1995
). Furthermore, mapping the regenerative ability of embryonic
and fetal digit tips demonstrated that regenerative capacity correlated with
amputation within the Msx1, but not the Msx2, expression
domain in developing digits. These studies suggest a role for MSX genes in the
digit regeneration response.
In all tetrapod vertebrates, Msx1 and Msx2 are
co-expressed in the apical mesenchyme during limb formation. In animals that
regenerate their appendages, MSX genes are upregulated during the regeneration
response and downregulated in association with re-differentiation
(Crews et al., 1995;
Simon et al., 1995
;
Yokoyama et al., 2001
). In the
regenerating urodele limb, Msx2 is rapidly induced in the healing
epidermis and subjacent tissues following amputation or simple wounding,
whereas Msx1 expression is restricted to blastemal cells
(Koshiba et al., 1998
;
Carlson et al., 1998
). During
fetal digit tip regeneration in the mouse, both Msx1 and
Msx2 are expressed in the regenerating digit mesenchyme, whereas
neither is expressed following proximal amputations that fail to regenerate
(Reginelli et al., 1995
).
Beyond these descriptive studies, the role that MSX genes play in regeneration
of fish fins, amphibian limbs or mammalian digits is largely unexplored.
However, the roles of MSX genes during limb development and in cultured cells
have been extensively studied. Expression of MSX genes in the apical
mesenchyme of the limb bud is dependent on signaling from the apical
ectodermal ridge (AER), and also on interactions with neighboring mesenchymal
cells (Davidson et al., 1991
;
Ros et al., 1992
;
Wang and Sassoon, 1995
). A
number of factors crucial for limb formation have been shown to regulate MSX
gene expression, including members of the FGF
(Watanabe and Ide, 1993
;
Fallon et al., 1994
;
Wang and Sassoon, 1995
;
Vogel et al., 1995
), BMP
(Wang and Sassoon, 1995
;
Ganan et al., 1996
;
Marazzi et al., 1997
;
Hofmann et al., 1996
;
Pizette and Niswander, 1999
;
Pizette et al., 2001
) and
TGFß (Ganan et al., 1996
)
signaling families, and retinoic acid
(Yokouchi et al., 1991
;
Wang and Sassoon, 1995
). One
function of Msx1 in early limb development is to mediate a BMP
signaling pathway that leads to the induction of the AER
(Pizette et al., 2001
).
Nevertheless, mice carrying a targeted deletion of the Msx1 gene form
normal limbs, thus indicating that AER formation can occur in the absence of
Msx1 function (Satokata and Maas,
1994
). Because Msx1 and Msx2 are co-expressed in
the apical mesenchyme it remains possible that these genes function
redundantly during limb formation.
MSX gene function is implicated in the control of cellular differentiation
during embryogenesis (see Bendall and
Abate-Shen, 2000). The expression pattern of MSX genes during limb
development is consistent with a role in the control of cell proliferation
and/or cell differentiation: Msx1 and Msx2 are expressed in
the apical mesenchyme in association with undifferentiated proliferating
cells, whereas proximal tissues where MSX genes are not expressed are
associated with reduced growth and tissue differentiation. Forced expression
implicates Msx1 in the inhibition of myogenesis
(Song et al., 1992
;
Woloshin et al., 1995
), and
there is evidence that this inhibitory activity can be generally extended to
the differentiation of a number of mesenchymal (e.g. adipose, cartilage and
bone) and epithelial (e.g. mammary) cell types
(Hu et al., 2001
). A similar
conclusion can be drawn from the results of loss-of-function studies that
indicate that both Msx1 and Msx2 mutants display defects in
the formation of certain skeletal elements and ectodermally derived organs
(Satokata and Maas, 1994
;
Satokata et al., 2000
). An
intriguing discovery that is potentially relevant to limb regeneration is the
demonstration that regulated Msx1 expression in differentiated C2C12
myotubes induces a de-differentiation response resulting in the establishment
of multi-potent progenitor cells (Odelberg
et al., 2000
). In addition, an extracellular activity derived from
newt blastema extract possesses a similar de-differentiation activity, thus
suggesting that an intercellular signal, presumably acting through MSX1, is
involved in de-differentiation during the early stages of limb regeneration
(McGann et al., 2001
).
The availability of mice carrying targeted deletions of the Msx1 and Msx2 genes, and the established regenerative response of fetal digit tips, allowed us to carry out a functional analysis of the MSX genes in regeneration. In this study, we have established a fetal digit tip regeneration model in cultured explanted autopods of E14.5 digits. Using this model, we discovered that Msx1, but not Msx2, mutant digits displayed a regeneration defect. Gene expression studies demonstrated that the Msx2 expression domain was expanded into the Msx1 domain in the Msx1 mutant digit, thus showing that Msx1 and Msx2 are not functioning in a redundant manner in digit regeneration. By contrast, the Bmp4 expression domain, which coincides with that of Msx1 in wild-type digits, was apically restricted in the Msx1 mutant digit, and Bmp4 transcripts were not detected in the Msx1/Msx2 double-mutant digit tip. Exogenous application of BMP4 was found to rescue the Msx1 mutant-digit regeneration defect in a dose-dependent manner, and exogenous noggin application inhibited the regeneration response in wild-type digits. These studies provide functional evidence linking Msx1 function and BMP signaling to the control of digit tip regeneration in the mammalian fetus.
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Materials and methods |
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Digit amputation
For in vivo studies, stage 11 fetal digit tips were amputated at E14.5
using exo utero surgical techniques as described
(Reginelli et al., 1995;
Ngo-Muller and Muneoka,
2000a
). Briefly, timed-pregnant mice were anesthetized with sodium
pentobarbital (60 µg/g body weight), fentany (1.6 µg/animal) and
droperidol (80 µg/animal). The abdomen was opened with a mid-ventral
incision and fetuses were exposed by incision of anti-placental uterine wall.
Access to the hindlimb was through an incision in the extraembryonic membranes
and the hindlimb was teased out with a blunt probe. The three central hindlimb
digits, digits 2, 3, and 4, were amputated at either a distal level,
approximately 75 µm from the digit tip (see
Fig. 1H), or a proximal level
through the presumptive terminal interphalangeal joint. The uterus with
fetuses attached was positioned within the abdominal cavity and the abdominal
wall of female mouse was closed. Operated fetuses were allowed to develop for
2 to 4 days exo utero (Muneoka et al.,
1986
) after which the hindlimbs were collected for analysis of the
digits.
|
In situ hybridization
For in situ hybridization, digoxigenin-labeled riboprobes complementary to
Msx1 (Ngo-Muller and Muneoka,
2000b), Msx2
(Ngo-Muller and Muneoka,
2000b
), Bmp4 (Jones
et al., 1991
), Hoxc13
(Godwin and Capecchi, 1998
)
and Ihh (St-Jacques et al.,
1999
) were used in whole-mount or paraffin-sectioned preparations
as described (Schaller and Muneoka,
2001
; Omi et al.,
2002
). Fetal digit tissues were fixed by immersion in 4%
paraformaldehyde and post-natal digits were fixed by injection of fixative
into the digits followed by immersion in 4% paraformaldehyde. For tissues
processed in parallel, all aspects of the in situ hybridization staining
protocol were carried out in synchrony and imaged identically. For
Msx1/Msx2 double-mutant tissue that is negative for Bmp4
expression, we used the expression of Bmp2 in limb tissues as a
positive control for tissue viability.
Histology and cell proliferation
For differentiating skeletal analysis in the developing digits, tissues
were stained with Alcian Blue/Alizarin Red S according to methods described by
McLeod (McLeod, 1980). For
BrdU incorporation studies, BrdU was added to the culture 1 hour prior to
tissue fixation following a protocol recommended by the manufacturer (Roche).
Paraffin-sectioned tissue samples were incubated with anti-BrdU and
differentiated with anti-mouse Ig-alkaline phosphatase. Incorporated BrdU was
detected using NBT and X-phosphate as substrates for alkaline phosphatase.
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Results |
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In addition to Msx1 and Msx2 expression, Bmp4 is expressed in the stage 11 distal digit mesenchyme in a pattern similar to that of Msx1 (Fig. 1J). At this stage, the onset of endochondral differentiation of the terminal phalanx is indicated by the expression of Ihh at the distal tip of the digit (Fig. 1K), and the initiation of nail organ formation is indicated by the expression of Hoxc13 on the dorsal surface of the digit (Fig. 1L). In neonatal digits (E18.5 and postnatal), the anatomy of the terminal digit region is complete. In the dorsal mesenchyme separating the nail organ and the terminal phalanx, Msx1 is expressed prominently, both at birth (not shown) and postnatally (Fig. 1B), whereas Bmp4 is expressed weakly at birth (data not shown) but is expressed strongly postnatally (Fig. 1D). Msx2 expression is downregulated in the dorsal mesenchyme and its expression in the epidermis is restricted to the nail bed (Fig. 1C). Hoxc13 expression remains associated with the forming nail and is prominent in the neonatal nail bed (Fig. 1F). The formation of the terminal phalanx itself is characterized by the distal expression of Ihh (Fig. 1E).
Digit regeneration in vivo
In vivo amputation of the stage 11 digit tip of E14.5 fetuses results in a
wound healing response that is followed by the regeneration of the digit tip
blastema and the eventual formation of an anatomically complete digit. This
process is completed in a 4 day period so that at birth (E18.5) the digit tip
has a relatively normal appearance, albeit somewhat shorter by comparison to
non-amputated control digits (compare the regenerated central digits with the
non-amputated peripheral digit in Fig.
2A). Gene expression studies of the 4-day-regenerated digit tip
corroborate anatomical observations and indicate that the regenerate is
normal. Msx1 is expressed in the dorsal mesenchyme between the nail
organ and the terminal phalanx (Fig.
2B). Msx2 is downregulated in the dorsal mesenchyme, but
is expressed in the epidermis associated with the nail organ
(Fig. 2C). Bmp4 is
weakly expressed in the dorsal mesenchyme
(Fig. 2D). The differentiation
markers, Ihh and Hoxc13, are expressed in the terminal
phalanx and the forming nailbed, respectively
(Fig. 2E,F). Analysis of the
regenerating digit tip 2 days post-amputation shows that the response involves
the reformation of a digit blastema distal to, and surrounding, a central
cartilaginous element. Within this digit blastema marker genes are expressed
largely in a manner similar to those of the developing digit tip
(Fig. 2H-L). Thus, Msx1,
Msx2 and Bmp4 are all expressed in the regenerating digit
blastema mesenchyme, Msx2 and Hoxc13 are expressed in the
apical epidermis, and Ihh is expressed in the central cartilaginous
element. However, one difference is that the Msx2 expression domain
in the distal mesenchyme is expanded so as to coincide with that of
Msx1 (Fig. 2I). Amputation of the digit tip at a more proximal level does not result in a
regenerative response, and distal digit marker genes (Msx1, Msx2 and
Bmp4) are not expressed at the site of amputation injury
(Reginelli et al., 1995)
(Fig. 2G).
|
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Msx2 compensation in Msx1 mutant digits
The variability of the Msx1 mutant digit regeneration phenotype
suggests the existence of a redundant activity that might replace Msx1
function in the regenerative response. An obvious candidate in the forming
digit is Msx2 because: (1) its expression is associated with digit
regeneration, (2) Msx1 and Msx2 display similar biochemical characteristics
(Bendall and Abate-Shen, 2000),
and (3) the Msx2 expression domain overlaps with Msx1 in the
distal digit mesenchyme. To explore this possibility, we carried out an
analysis comparing Msx2 expression in wild-type and Msx1
mutant digits. In initial studies, we processed wild-type and Msx1
mutant digits side by side for whole-mount in situ hybridization for
Msx2 expression, and discovered that Msx2 expression is
visibly enhanced in the Msx1 mutant digit as compared with wild-type
(Fig. 4A,B), thus suggesting
that Msx2 expression was compensating for the absence of
Msx1. When the spatial pattern of Msx2 expression was
analyzed in the Msx1 mutant digit, we discovered that the mesenchymal
Msx2 expression domain was expanded to encompass the wild-type
Msx1 expression domain (Fig.
4C). These observations suggest that Msx1 functions to
restrict the Msx2 expression domain during digit formation, although
this inhibition must involve additional activities, as both genes are
co-expressed in the distal mesenchyme. These data are consistent with a
redundancy in Msx1 and Msx2 function during digit
development; however, because the Msx1 mutant digit displays a
regeneration defect, the evidence suggests that Msx1 and
Msx2 are functioning in a partially redundant way in
regeneration.
|
To test this hypothesis, we carried out rescue experiments with exogenous BMP4. Amputated hindlimb digits obtained from Msx1+/- crosses were cultured in medium containing recombinant human BMP4 at two different concentrations, 200 ng/ml and 1000 ng/ml. Treatment at the lower BMP4 concentration resulted in a partial rescue of the regeneration response (73%, Table 1), whereas treatment at the higher concentration enhanced the regeneration response to a level comparable to that seen in wild-type controls (86%, Table 1). In parallel studies, application of BMP2 (200 ng/ml) had no effect on Msx1 mutant digit regeneration (27.8%, Table 1), indicating that the rescue effect is specific to BMP4. The BMP4 rescue of the Msx1 regeneration defect is associated with a striking upregulation of Msx2 expression in the apical epidermis (Fig. 5A), and of Bmp4 expression in the induced digit blastemal mesenchyme (Fig. 5B). Hoxc13 is also expressed in the dorsal epidermis in the BMP4-induced regenerate (Fig. 5C), and Ihh is expressed in the forming terminal skeletal element (data not shown). In summary, these results show that BMP4 functions in a dose-dependent manner downstream of Msx1, and identifies BMP4 as an essential regulator of the regeneration response.
|
Cell proliferation correlates with the regeneration response
In tetrapod vertebrates, epimorphic regeneration of adult and developing
limb tissues is associated with a localized growth response at the site of
injury. To analyze cell proliferation during the fetal digit tip regeneration
response, we carried out BrdU incorporation studies of amputated wild-type and
Msx1 mutant digits after treatments to modulate the regeneration
response. After 2 days in culture, ectodermal wound closure is complete and a
morphological response is evident. In wild-type digits, we found localized
incorporation of BrdU associated with outgrowth of the regenerating digit
blastema (Fig. 6A). By
contrast, we observed little BrdU incorporation at the injury site in
Msx1 mutant digits that fail to mount a regeneration response
(Fig. 6B). BMP4-treated
Msx1 mutant digits displayed BrdU-labeled cells associated with the
rescued regeneration response (Fig.
6C), providing evidence that BMP4 induces a proliferative response
in regenerating digit blastema cells. Consistent with this conclusion, BrdU
incorporation was reduced in noggin-treated wild-type digits in which
regeneration is inhibited (Fig.
6D), which indicates that BMP signaling is required for
proliferation. These studies provide evidence that fetal digit tip
regeneration is an epimorphic response associated with an apical growth zone,
and suggests a role for Msx1 and BMP4 in the control of cell
proliferation following amputation injury.
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Discussion |
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Regeneration in vitro
The capacity of embryonic/fetal tissues to undergo enhanced regenerative
repair identify developing tissues as relatively simple models for
investigating regenerative responses (see
Muller et al., 1999). The
regenerating mammalian digit tip is no exception to this rule. In mice, mature
digit tip regeneration is level-specific, associated with the nail organ, and
proceeds very slowly, requiring weeks to months to complete. Fetal digit tip
regeneration is also level-specific and associated with the nail anlagen, but
is completed within a 4 day period. Because the regeneration response occurs
so rapidly in fetal digits, we have been able culture this regenerating
structure in vitro, where the regeneration environment can be manipulated. The
fact that a regeneration response can be elicited in explanted limb tissue
indicates that components that are likely to play crucial roles in mature
digit regeneration, such as functional innervation, vascularization and the
availability of circulating hormones, are not required for fetal regeneration.
Thus, the simplicity of the regeneration response provides a means to
experimentally dissect underlying regulatory mechanisms that might otherwise
be inaccessible in the mature digit.
Regeneration studies on the mature mammalian digit tip have focused almost
exclusively on the role of the nail organ and the distal growth of the
terminal phalanx (see Zhao and Neufeld,
1995). This association is derived from both clinical studies in
humans and experimental studies on rodents
(Douglas, 1972
;
Illingworth, 1974
;
Borgens, 1982
). By studying
both developing and mature digits, we found that regenerative potential is
associated with the expression domain of Msx1
(Reginelli et al., 1995
), but
that Msx1 expression in mature digits is restricted to loose
connective tissue fibroblasts subjacent to the nail, and is not found in the
nail organ itself. The demonstration that Msx1 mutant digits display
a regeneration defect indicates that Msx1-expressing cells play a
crucial role in the response. These findings suggest that digit tip
regeneration may not be dependent on the nail organ, but rather on the
connective tissue cells underlying the nail organ. Because this population of
cells is closely associated with the nail organ it would be difficult to
distinguish between a nail organ effect and the influence of the underlying
connective tissues. The importance of this cell population in mature digit
regeneration is also supported by histological observations noting a streaming
of fibroblastic cells toward the regenerating digit tip
(Revardel and Chebouki, 1987
;
Muller et al., 1999
), and by
the observation that a regeneration response itself need not include the nail
plate (Reginelli et al.,
1995
).
MSX genes in digit formation and regeneration
The expression patterns of the MSX genes at the digit apex represent a
simple nested relationship that partitions the digit apex into a distal
domain, in which both MSX genes are expressed, and a sub-distal domain, in
which only Msx1 is expressed. Digit formation occurs normally in
Msx1 and Msx2 mutant mice, indicating that individual MSX
gene function is not essential during outgrowth
(Satokata and Maas 1994;
Satokata et al., 2000
). The
co-expression of Msx1 and Msx2 in apical mesenchymal cells
raises the possibility that these two genes function redundantly during digit
development. The expansion of the Msx2 expression domain in
Msx1 mutant digit tips suggests a compensatory response by
Msx2 in the absence of Msx1, and is consistent with a
redundancy hypothesis. This response also suggests that Msx1 plays an
inhibitory role in Msx2 expression within the sub-distal cells where
only Msx1 is expressed in wild-type digits. However, if MSX1 inhibits
Msx2 expression, its activity must be repressed in the distal zone
where both genes are co-expressed. Dlx5 is also expressed in the
distal digit mesenchyme (Robledo et al.,
2002
) (M.H., unpublished), and has been shown to compete with
and/or repress the activity of MSX genes (see
Bendall and Abate-Shen, 2000
),
thus it represents a candidate for modulating MSX activity in the digit
tip.
Our studies indicate that MSX1 functions in a regeneration-specific manner.
In the absence of MSX1, digit formation is normal yet digit regeneration is
defective, thus MSX1 function is necessary for regeneration but not for
development. In addition, Msx2 compensation in the Msx1
mutant suggests an incomplete or partial redundancy of function that is
restricted to digit regeneration. Studies on cultured cells indicate that one
activity of MSX1 involves the control of cell differentiation (see
Hu et al., 2001). Based on
amphibian limb regeneration studies, a significant regeneration-specific event
is the de-differentiation of cells at the wound site to form the blastema.
Cellular de-differentiation has been best documented for multinucleated
amphibian myotubes that are induced to form individual cells in vitro and,
after grafting, in vivo (Lo et al.,
1993
). Studies using differentiated C2C12 myotubes and regulated
Msx1 expression provide evidence that MSX1 induces de-differentiation
in vitro, and that subsequent suppression of Msx1 expression can lead
to transdifferentiation to multiple cell types, including cartilage, bone,
adipose and muscle (Odelberg et al.,
2000
). Although these de-differentiation studies are specific to
muscle tissue, forced Msx1 expression studies demonstrate that
multiple mesenchymal and epithelial cell types are inhibited from
differentiation in culture, and that differentiation of mammary epithelial
tissue is impaired in vivo (Hu et al.,
2001
). Thus, the available evidence suggests that MSX1 functions
in multiple cell types to control differentiation and, in the context of
regeneration, de-differentiation. As regenerative potential is restricted to
domains of Msx1 expression in the mature and fetal digit tip, we
hypothesize that MSX1 functions to maintain a population of undifferentiated
mesenchymal cells that can participate in a regeneration response. One
significant difference between amphibian limb regeneration and regeneration of
the mammalian digit tip is that Msx1 expression is induced in
response to amputation in amphibian limbs, whereas, in mammals, regenerative
potential is linked to regions where Msx1 expression is maintained in
the mature digit.
BMP signaling and regeneration
BMP signaling is crucial for fetal digit regeneration. A number of lines of
evidence support this conclusion. First, BMP4 rescues the Msx1 mutant
regeneration defect in a dose-dependent manner. Second, Bmp4
expression is downregulated in the Msx1 mutant digit, which is
consistent with the idea that residual regenerative capability is associated
with this reduced level of BMP availability. Third, noggin-treated
Msx1 mutant digits display a more severe phenotype associated with
the absence of Bmp4 expression. Fourth, noggin treatment of wild-type
digits results in a more than 5-fold reduction in regenerative capability.
Noggin is not expressed within the forming digit tip, but is
expressed in chondrofying skeletal elements at proximal digit levels that lack
regenerative potential (Brunet et al.,
1998; Capdevila and Johnson,
1998
; Merino et al.,
1998
). Other BMP signaling antagonists, such as follistatin,
gremlin (Cktsf1b1 - Mouse Genome Informatics) and
chordin, are also expressed in developing limbs
(D'Souza and Patel, 1999
;
Merino et al., 1999
;
Zhang et al., 2002
). The
expression of multiple BMP signaling antagonists associated with digit
formation suggests that the inability of proximal digit amputations to
regenerate may be a consequence of regulated BMP activity associated with
skeletal differentiation.
The sole function of MSX1 in digit regeneration lies in the regulation of
Bmp4 expression. BMP4 rescue of Msx1 mutant digits indicates
that MSX1 function is not required downstream of BMP signaling. Successful
regeneration of a low percentage of Msx1 mutant digits correlates
with a reduced level of Bmp4 expression and provides definitive
evidence that successful digit regeneration can occur in the absence of MSX1
function. During digit formation, we have identified Bmp4 as
functionally downstream of both Msx1 and Msx2. The shift of
the Bmp4 expression domain to coincide with the wild-type
Msx2 expression domain is consistent with the hypothesis that
Bmp4 is regulated by both Msx1 and Msx2 in the
distal digit domain, but by only Msx1 in the sub-distal domain.
However, the compensatory response of Msx2 in the Msx1
mutant complicates this interpretation and indicates the presence of an
Msx2-dependent Bmp4 regulatory component that is
co-expressed in the distal digit compartment. It is interesting that in vitro
studies of the transcriptional regulator Runx2 (previously known as
Cbfa1) indicate that it is regulated by MSX2
(Shirakabe et al., 2001) and
that it regulates Bmp4 (Helvering
et al., 2000
), and that we find it co-expressed with Bmp4
in the developing digit tip (M.H., unpublished). Thus, Runx2
represents a candidate for mediating the regulation of Bmp4
expression by MSX genes. Unravelling the details underlying the regulation of
BMP4 signaling in the digit tip should provide key insights into the control
of fetal digit regeneration.
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ACKNOWLEDGMENTS |
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