1 Genetics of Development and Disease Branch, NIDDK, NIH, 10/9N105, 10 Center
Drive, Bethesda, MD 20892, USA
2 Department of Craniofacial Biology and Cellular and Developmental Biology,
University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver,
CO 80262, USA
3 Laboratory of Protein Dynamics and Signaling, Center for Cancer Research,
National Cancer Institute, NCI-Frederick, Frederick, MD 21702, USA
* Author for correspondence (e-mail: chuxiad{at}bdg10.niddk.nih.gov)
Accepted 26 August 2005
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SUMMARY |
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Key words: Apoptosis, BMP4, MKP3, DKK1, ALX4
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Introduction |
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The AER is a specialized region of thickened ectoderm (epithelium) covering
the tip of the limb bud. It is essential for the sustained outgrowth of the
limb along the PD axis, and the patterning of the limb through its interaction
with the underlying mesenchyme. The ZPA is a region of mesenchyme at the
posterior margin of the limb bud, where sonic hedgehog (SHH) provides the
spatial cues for the growth of the limb bud along the AP axis. The PZ is
composed of undifferentiated mesenchymal cells lying beneath the AER.
According to the PZ model, the components of the limb (the autopod, zeugopod
and stylopod) are determined by the length of time that progenitor cells spend
in the PZ (Summerbell et al.,
1973). It has been shown that fibroblast growth factors (FGFs) and
FGF receptors (FGFRs) are expressed in the limb bud and play critical roles in
the initiation, outgrowth and patterning of the limb (reviewed by
Coumoul and Deng, 2003
;
Mariani and Martin, 2003
;
Martin, 1998
;
Tabin, 1995
;
Tickle and Munsterberg, 2001
;
Xu et al., 1999
).
FGF4, 8, 9 and 17 are expressed in the AER, while FGF10 is expressed in
mesenchymal cells underlying the AER
(Crossley and Martin, 1995;
Martin, 1998
;
Savage et al., 1993
;
Tickle and Munsterberg, 2001
).
It has been shown that placing beads soaked in a particular FGF (FGF2, 4, 8 or
10) on the flank of embryos induces the formation of a limb, while limb
truncation, caused by removal of the AER, can be overcome by implanting beads
soaked with FGF2 or FGF4 into the limb mesenchyme
(Fallon et al., 1994
;
Niswander and Martin, 1993
).
In addition, targeted deletion of Fgf4 and Fgf8 in the AER
generates limbless embryos at birth (Boulet
et al., 2004
; Sun et al.,
2002
). Notably, Fgf4 and Fgf8 double mutant limb
buds are abnormally small although they initiate normally. Increased apoptosis
was found in limb mesenchyme, suggesting that AER FGF serves as a survival
factor regulating the number of precursor cells of the nascent limb.
FGF receptors contain, in their full-length form, a hydrophobic leader
sequence, three immunoglobulin-like (IgI, II, and III) domains, an acidic box,
a transmembrane domain, and a divided tyrosine kinase domain. Many isoforms
can be generated through alternative splicing and polyadenylation. For
example, alternative splicing at the 5' region of the Fgfr1
locus generates FGFR1 (containing three Ig loops), FGFR1ß
(containing two Ig loops) and FGFR1
(identical to FGFR1ß except it
lacks the signal sequence for membrane translocation). Similarly, variable
splicing of exons encoding the IgIII domain yield b and c isoforms of FGFR1, 2
and 3 (Hou et al., 1991
;
Werner et al., 1992
). FGFR2b
and FGFR2c isoforms are differentially expressed in ectoderm and mesenchyme of
the limb bud and mediate signals of mesenchyme-based FGFs (i.e. FGF10) and
ectodermal-based FGFs (i.e. FGF8), respectively. Thus, targeted disruption of
Fgfr2 blocks FGF signaling from both mesenchyme and ectoderm, leading
to mutant embryos without limb buds (Arman
et al., 1999
; Li et al.,
2001
; Xu et al.,
1998
). Mutant embryos carrying hypomorphic mutations of FGFR2
exhibit abnormal development of limbs with varying severity
(Revest et al., 2001
).
Unlike Fgfr2, Fgfr1 is primarily expressed in the mesenchyme of
developing limb buds. Several lines of evidence indicate that FGFR1 also has
important roles in limb development. Human patients carrying a missense
mutation (Pro252Arg) that activates FGFR1 display broad toes and split thumbs
(Muenke et al., 1994).
Transgenic mice bearing four copies of Pro250Arg genomic DNA exhibited an
extra digit 1 (Hajihosseini et al.,
2004
). Conversely, reduced FGFR1 signaling affected development
and patterning of the distal limb structures
(Partanen et al., 1998
). We
have previously shown that mutant embryos carrying a targeted deletion of the
FGFR1
isoform exhibited distal truncation before they died at E12.5
(Xu et al., 1999
). As the tip
of the limb is the position of the PZ, it is conceivable that FGFR1-mediated
signals play an important role in PZ formation and/or maintenance.
The potential impact of a complete loss of FGFR1 function on limb
development has not been assessed because of the early post-implantation
lethality of the FGFR1-null mutant (Deng
et al., 1994; Yamaguchi et
al., 1994
). Therefore, we have created an Fgfr1
conditional allele by flanking exons eight to 14 with loxP sites. In this
study, we have used two different Cre lines to ablate FGFR1 at two slightly
different time points during the early stages of limb development. This has
revealed a narrow window in which FGFR1 signaling is essential for cell
survival and autopod morphogenesis occurring at later stages.
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Materials and methods |
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Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out as described previously
(Riddle et al., 1993).
Anti-sense RNA probes were synthesized using the DIG RNA Labeling Kit (Roche
Diagnostics, Mannheim, Germany) according to the manufacturer's
recommendations. Probes for the following genes were used: aristaless 4
(Alx4), bone morphogenic protein (Bmp4), dickkopf homolog 1
(Dkk1), engrailed 2 (En2), Fgf4, Fgf8, Fgfr1, homeo
box D11, 12 and 13 (Hoxd11, Hoxd12, Hoxd13), dual specificity
phosphate 6 (MKP3/Dusp6), homeo box, msh-like 1 and 2 (Msx1,
Msx2), sonic hedgehog (Shh), SRY-box containing gene 9
(Sox9), T-box 2 and 3 (Tbx2, Tbx3) and wingless-related MMTV
integration site 7A (Wnt7a).
X-gal staining
Embryos were stained in X-gal overnight at 37°C as described previously
(Mansour et al., 1993).
Embryos were washed in PBS twice after staining, then fixed in 4%
paraformaldehyde for 1 hour, dehydrated through a graded alcohol series,
treated with xylene, and embedded in paraffin. Eight µm sections were
prepared and counterstained with Harris Hematoxylin according to standard
procedures.
Whole-mount skeletal preparation
Animals were sacrificed by asphyxiation in CO2. After removing
the skin, carcasses were eviscerated, fixed in 95% ethanol, stained with
Alizarin Red S and Alcian Blue, cleared by KOH treatment, then stored in
glycerol as described previously (McLeod,
1980).
Detection of apoptotic and proliferating cells
The TUNEL assay was used to detect apoptotic cells in 6 µm sections of
paraffin-embedded tissue using a kit obtained from Chemicon International
(Temecula, CA, USA). Cell death was detected by staining with LysoTracker Red
DND-99 (Molecular Probes, Eugene, OR, USA) using a procedure described by
Zucker et al. (Zucker et al.,
1999). For detecting cell proliferation in embryos, pregnant mice
were injected intraperitoneally with BrdU at a dose of 250 µg/g, and were
sacrificed 2 hours later. Immunohistochemical staining for BrdU was performed
using an antibody from Becton-Dickinson (South San Francisco, CA, USA)
according to the manufacturer's instructions.
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Results |
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The mouse forelimb bud initiates at early embryonic day 9 (16-17 pairs of
somites). Using the Rosa26-ß-gal reporter line (R26R)
(Soriano, 1999), the Cre
activity was first detected in the distal portion of embryos with 20 pairs of
somites (Fig. 1A). As the limb
bud continued to grow, the area of ß-gal-positive cells gradually
extended proximally (Fig. 1B,C)
and by E11.5 covered the entire limb bud except for the AER
(Fig. 1E,F). Of note, the
distribution of ß-gal-positive cells was biased toward the
anterior-distal portion of the forelimb buds at E10.5
(Fig. 1C). Similarly, Cre
activity was also first detected at the tip of the hindlimb bud and the area
of ß-gal-positive cells gradually extended proximally to the entire limb
bud at E11.5 (Fig. 1E,F). The
biased pattern of ß-gal-positive cells observed in the forelimb was less
obvious in the hindlimb buds (Fig.
1D). Using whole-mount in situ hybridization we detected
diminished Fgfr1 transcripts in both the forelimbs and hindlimbs of
Fgfr1Co/CoAp-2Cre mice
(Fig. 1G, and not shown). These
observations indicate that Ap-2Cre may be suitable for studying gene
function at stages immediately past limb bud initiation.
Abnormal anterior digit formation in the Fgfr1Co/Co;Ap-2Cre mice
Limb buds of Fgfr1Co/Co;Ap-2Cre mice initiated
normally, consistent with our observation that the Cre activity was not
detected at the earliest stages of limb initiation. However, abnormal
development was detected in both forelimb and hindlimb buds of E11.5
Fgfr1Co/Co;Ap-2Cre embryos, which was characterized by an
anterior-distal truncation (Fig.
2A, and not shown). This phenotype was also found in forelimb buds
of E10.5 mutant embryos with less severity (not shown). At E12.5, when
mesenchyme cells condense to form digits in control mice, the pattern of digit
formation was abnormal in the mutant limbs
(Fig. 2B). This finding was
highlighted by an altered pattern of Sox9 expression, a gene
expressed in digit primordia, which demonstrated that the anterior portion of
both the forelimbs and hindlimbs of the Fgfr1Co/Co;AP-2Cre
mice had a reduced number of digits and abnormal digit formation
(Fig. 2B, and not shown).
Abnormal development of anterior digits was confirmed by in situ hybridization
with Hoxd12, which labels digits two to five, but not the first digit
in E12.5-14.5 wild-type limbs (Fig.
2C,D, and not shown). We found that all (n>20) the
mutant forelimbs (Fig. 2C) and
hindlimbs (Fig. 2D) contained
only three (Fig. 2C,D Mt-1) or
four (Fig. 2C,D, Mt-2) digits,
and they were all Hoxd12 positive, indicating that the first digit
was missing.
Limb development was analyzed further by Alizarin Red and Alcian Blue staining. Analysis of new born (Fig. 2E,F,G) and postnatal (P) 10 day (Fig. 2H,I) mice revealed that all mutant limbs only had three or four digits with partial fusion at multiple positions (arrows in Fig. 2F,G,H). The first digit of mutant forelimbs was either missing (not shown) or, more frequently, fused with the second digit so that only one abnormal digit was formed (Fig. 2E,F). In contrast, fusion of the first two digits was not observed in mutant hindlimbs. Instead, the limbs only had four digits (Fig. 2G), or three digits (Fig. 2I). No obvious abnormalities in stylopod and zeugopod cartilage in mutant limbs were observed.
To study the underlying mechanisms leading to abnormal digit formation and
patterning, we analyzed a number of genes that are expressed along the PD
and/or the AP axes. Whole-mount in situ hybridization with Fgf8
revealed that mutant limb buds have an enlargement of the AER, primarily of
the anterior half (Fig. 3A,B).
The expression of Fgf8 in the posterior portion of the mutant AER was
either unchanged or only slightly stronger
(Fig. 3A,C). FGF signaling in
the AER plays an essential role in maintaining expression of SHH in the ZPA
(Laufer et al., 1994;
Niswander et al., 1994
).
Consistent with the observation that there was no significant alteration of
Fgf8 and Fgf4 (not shown) in the posterior half of the AER,
expression of Shh was not altered
(Fig. 3C), indicating that this
function of AER FGF signaling is not disrupted. Moreover, examination of a
number of other genes, including Msx1 and Msx2
(Fig. 3D,E), Hoxd11-13
(Fig. 3F, and not shown), and
Bmp4 (Fig. 3G), did
not reveal obvious changes in their expression in the posterior portions of
the E11.5 (Fig. 3D-G) and E12.5
(Fig. 3H, and not shown) limbs.
Thus, deletion of Fgfr1 using Ap-2Cre does not have an
obvious effect on the posterior-distal portion of the limb at its early stages
of development, which may account for the relatively normal morphology of
posterior portions of the limbs at later stages of development
(Fig. 2E-I).
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In summary, our analysis of Ap2-Cre deletion of Fgfr1 reveals
specific loss in the distal mesenchyme resulting in abnormal development of
the anterior digits. There is only a mild impact on the formation of posterior
digits, and no or rare effects on proximal skeletal elements. These
observations suggest that FGF/FGFR1 signaling plays important roles in
anterior digit formation and is less critical for the formation of the
posterior digits. However, recent data suggests that the precursors for some
skeletal elements of the PD axis might be predetermined at very early stages
of limb bud development (Dudley et al.,
2002; Sun et al.,
2002
). Thus, although deletion of Fgfr1 eventually
spreads to the entire limb prior to any detectable morphological signs of
mesenchyme differentiation, AP-2Cre-mediated deletion of
Fgfr1 may simply be too late to affect the development of posterior
digits and the proximal portion of the limb.
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It has been shown that SHH negatively regulates expression of the
aristaless-like transcription factor 4 (Alx4)
(Takahashi et al., 1998).
Along with markedly reduced Shh expression, we detected a significant
expansion of Alx4 expression in the anterior of mutant limb buds
(Fig. 5D). This expansion also
correlates well with the hypoplasia of the anterior portion of the mutant
limbs, which is consistent with a finding that targeted disruption of
Alx4 resulted in preaxial polydactyly of the anterior digits
(Qu et al., 1997
). To
determine the effect of FGFR1 deficiency on the development of posterior
portion of the limb, we examined expression of Tbx2 and
Tbx3. Both are expressed in the interdigit region between the fourth
and fifth digits and in the posterior margin of the limb, and play roles in
patterning the most posterior two digits in the mouse
(Davenport et al., 2003
) and
chicken (Suzuki et al., 2004
).
Our data revealed that expression of both Tbx2 and Tbx3 was
maintained in the posterior region of the mutant limbs
(Fig. 5E,F), suggesting that
this part was less affected. All the mutant limbs examined at E11.5-E12.5 also
exhibited increased length along the dorsal-ventral (DV) axis
(Fig. 5B,G). However, our in
situ hybridization using probes for Wnt7a and En2, which
play a key role in the establishment of the DV axis, did not reveal any
obvious changes of their expression patterns and intensities (not shown),
suggesting that this phenotype may be secondary to the reduced height of the
PD axis.
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Phenotypes of forelimbs of Fgfr1Co/Co;Hoxb6-Cre mice
Our analysis of mutant limbs thus far indicates that severe phenotypes are
associated with FGFR1 deletion at the earliest stages of limb development,
while deletion at slightly later stages immediately after initial budding has
a much milder effect. Because Hoxb6-Cre is first expressed in the
posterior portion of the forelimb prior to its budding and the expression
domain gradually extends to the anterior at later stages, we predict that
abnormalities may only occur in the most posterior part of the forelimb, while
the anterior portions, which express Cre at slightly late stages, should only
have minor or even no visible defects. Indeed, analysis of forelimbs of
Fgfr1Co/Co;Hoxb6-Cre mice revealed a reduced height for
the PD axis (Fig. 7A-D) and an
increased length along the DV axis (Fig.
7C) only in the posterior region. These phenotypes started at
E10.75 and were obvious by E11.5 with some variations. Whole-mount in situ
hybridization using Hoxd12 revealed a normal appearance of the first
two or three digits while the development of the posterior digits was abnormal
(Fig. 7E, and not shown). These
data indicate that deletion of FGFR1 does not affect anterior digit formation
despite Cre activity having spread through the entire distal portions of the
limb prior to digit formation (Fig.
4G). Of note, the areas showing abnormalities in mutant embryos
examined from E9.5-E12.5 (n>30) were always smaller than the
ß-gal positive areas in reporter mice. This indicates the abnormalities
only occur in the region where Hoxb6-Cre is first expressed, i.e.
prior to or during the initial budding, and not in the more anterior regions,
to where Cre expression spreads by slightly later stages. This is consistent
with what was observed in Fgfr1Co/Co;AP-2Cre mice.
Next, we stained the forelimbs of Fgfr1Co/Co;Hoxb6-Cre
mice using Alcian Blue and Alizarin Red staining. This revealed that most
mutant forelimbs contained only four digits
(Fig. 7G), while one mutant
limb had only three digits (not shown). Whole-mount in situ hybridization of
E12.5 limbs using Tbx2 and Tbx3 revealed relatively normal
expression in the interdigit region between the fourth and the fifth digits
and in the posterior margin of the mutant forelimbs
(Fig. 7D and not shown). This
suggests that the mutant limb contains the primordium for the fifth digit at
this stage of development. Therefore we postulate that, similar to the
hindlimb of Fgfr1Co/Co;Hoxb6-Cre mice, the last digit in
the forelimb is digit five and it was able to grow. This is perhaps because of
the expression of SHH and/or HOXD12 and 13
(Fig. 7B,E,F, and not shown),
all of which are known to play an important role in digit formation,
patterning and specification (Chen et al.,
2004; Chiang et al.,
1996
; Davis and Capecchi,
1996
; Zakany and Duboule,
1996
; Zakany et al.,
2004
).
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We next investigated possible causes for the increased apoptosis using a
candidate approach and found alterations in the expression of three genes that
are involved in apoptosis. BMP signals play an important role in inducing
apoptosis (Guha et al., 2002),
and we have shown above that disruption of Fgfr1 by AP-2Cre
results in increased expression of Bmp4 in the AER
(Fig. 3I). We detected a
similar pattern of increased expression of Bmp4 in the AER of
Fgfr1Co/Co;Hoxb6-Cre limbs and decreased expression in the
underlying mesenchyme (Fig.
9E). We also found significant down regulation of MAPK phosphatase
3 (MKP3) in mutant hindlimbs (Fig.
9F). Interestingly, it was recently shown that FGF8 positively
regulates MKP3, and that suppression of MKP3 by small
interfering RNA (siRNA) induced apoptosis in the mesenchyme
(Kawakami et al., 2003
). We
also examined expression of dickkopf 1 (Dkk1), which is a negative
modulator of the Wnt pathway and has been implicated in programmed cell death
during limb development (Mukhopadhyay et
al., 2001
). Dkk1 is normally expressed in the anterior
proximal margin and the posterior proximal margin of the limb (arrowheads,
Fig. 9G). We found that loss of
FGFR1 significantly increased expression of Dkk1 in these areas
(Fig. 9H).
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Discussion |
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AP-2Cre deletion produced a more consistent mutant forelimb
phenotype than Hoxb6-Cre and enabled us to compare the consequences
of removal of FGFR1 activity at later stages of hindlimb outgrowth. Consistent
with the later induction of Cre recombinase, the phenotype of
Fgfr1Co/Co;AP-2Cre hindlimbs was milder than that of
Fgfr1Co/Co;Hoxb6-Cre limbs, but nevertheless reveals an
ongoing requirement for FGF signaling in digit patterning. Moreover, FGFR1
mutant limbs generated by either Cre transgene had several common features,
including malformation of the AER and truncation of distal mesenchyme at
E10.5-E12.5, followed by abnormal digit development and patterning at later
stages. Because neither AP-2Cre nor Hoxb6-Cre is expressed
in the AER, the malformation of the AER must be secondary to abnormalities in
the underlying mesenchyme. For example, the increased Fgf8 expression
found in the AER of mutants could be a feedback response to a signaling block
in the underlying mesenchyme resulting from loss of receptor. Indeed, we have
specifically knocked out Fgfr1 in the AER using Msx2-Cre
(Sun et al., 2000), and did
not find any obvious abnormalities (data not shown).
We have found hypoplasia of the mesenchyme in FGFR1-deficient limbs and
shown that it is associated with an increase in cell death. Increased
apoptosis was also observed in limb buds carrying either an
Fgf4/Fgf8 double mutation
(Boulet et al., 2004;
Sun et al., 2002
) or a
hypomorphic mutation of Fgfr2
(Revest et al., 2001
).
Together, these data provide compelling support for the hypothesis that FGF
signaling, mediated by FGFR1 and FGFR2, acts as a survival program that
regulates the number of precursor cells within the nascent limb mesenchyme
(Boulet et al., 2004
;
Sun et al., 2002
). Our data
indicate that apoptosis first occurs in the mutant AER and gradually spreads
into mesenchyme of the limb. In an effort to identify potential downstream
mediators for FGF signaling in cell survival, we have identified four
candidate genes, Bmp4, Dkk1, MKP3 and Alx4, which show
significant alterations in their expression pattern and intensity in
FGFR1-deficient limbs. Specifically, we detected increased expression of
Bmp4 in the AER, reduced expression of MKP3 in the distal
mesenchyme underneath the AER, an expanded expression domain of Alx4
in the anterior half of the limb, and increased expression of Dkk1 in
both the anterior proximal and the posterior proximal margins of the limb.
Although it remains possible that the altered expression of all or some of
these genes is simply a secondary consequence of FGFR1 mutation, previous
studies have implicated them in apoptosis and/or digit patterning
(Dunn et al., 1997
;
Guha et al., 2002
;
Katagiri et al., 1998
;
Kawakami et al., 2003
;
Mukhopadhyay et al., 2001
;
Qu et al., 1997
). It was shown
that blocking BMP signaling by over expressing a BMP antagonist, noggin, in
limbs of transgenic mice significantly reduced apoptosis, leading to extensive
limb soft tissue syndactyly and postaxial polydactyly
(Guha et al., 2002
).
Consistently, polydactyly or ectopic digit formation was observed in the
Bmp4+/ mice that also carried a heterozygous
mutation for Bmp7, Gli3 or Alx4
(Dunn et al., 1997
;
Katagiri et al., 1998
). We
have also found that AER-specific disruption of Smad4, which is a
common mediator for the TGFß superfamily, including all BMPs
(Heldin et al., 1997
;
Massague, 1998
), reduced
apoptosis in interdigit mesenchyme (C.L. and C.-X.D., unpublished data).
Despite these observations, however, there is no direct evidence that over
expression of Bmp4 in the AER can induce apoptosis. This issue could
be directly tested by expressing this gene in the AER using an AER-specific
promoter, such as the Msx2 promoter, which has been used to target
Cre to the AER (Sun et al.,
2000
). It is interesting that in an independent study using two
different transgenic Cre strains, Verheyden et al. also found that the absence
of FGFR1 results in extensive apoptosis in limb mesenchyme
(Verheyden et al., 2005
),
providing additional evidence that FGF/FGFR1 signals play an essential role in
maintaining cell survival during limb development.
We believe it is a significant finding that the absence of FGFR1 results in
down regulation of MKP3. MKP3 is a specific and potent regulator of
the ERK class of MAP kinases. In the mouse embryo, it was recently
demonstrated that regions of FGFR signaling overlap with the strongest domains
of ERK activation (Corson et al.,
2003), and that FGF8 positively regulates MKP3
(Kawakami et al., 2003
).
Moreover, suppression of MKP3 by small interfering RNA (siRNA)
induces apoptosis in the mesenchyme
(Kawakami et al., 2003
),
providing strong evidence that reduced expression of MKP3 in
FGFR1-deficient limbs could be the cause of the increased apoptosis and
reduced number of digits. Future studies should be directed to determining how
FGF/FGFR1 signaling regulates MKP3 expression, and the potential
relationships among MKP3, Alk4 and Dkk1 in terms of
apoptosis and digit development and patterning.
Another significant finding of this study concerns how the timing of FGFR1 removal alters the ultimate patterning of the limb bud. In contrast to the major hindlimb alterations obtained with Hoxb6-Cre, disruption of FGFR1 at a stage immediately after the initial budding of the limb using AP-2Cre results in a less severe phenotype that only affects the formation of the first one or two digits. These observations suggest that the action of FGFR1 signaling in specifying the majority of autopod development and patterning occurs at very early stages of limb development. One possibility is that the precursor cells that will develop into the autopod skeletal elements have already been specified by the very early stages of limb development, and once the fate of these cells is determined, FGFR1 signaling becomes less critical to their development. Therefore, the very early disruption of FGFR1 by Hoxb6-Cre generates a more profound limb phenotype than the slightly later disruption by AP-2Cre even though both transgenes act prior to the overt differentiation of the mesenchyme. Another theoretical explanation for the severe hindlimb abnormalities in Fgfr1Co/Co;Hoxb6-Cre mice is that the removal of FGFR1 in the lateral plate mesoderm causes less mesoderm to be available for recruitment to the limb bud. This hypothesis remains to be tested, although we did find that hindlimbs of E10 and younger Fgfr1Co/Co;Hoxb6-Cre mice were normal in size and contained equivalent number of cells to that of control limbs.
Of note, a new model regarding limb development and patterning along the
proximal-distal axis has recently been proposed
(Dudley et al., 2002;
Sun et al., 2002
). According
to this model, the components of the limb skeleton (autopod, zeugopod and
stylopod) are specified much earlier than assumed by the PZ model
(Summerbell et al., 1973
), and
may be independent of drop-out time. The model further proposes that limb
outgrowth is associated with expansion and sequential differentiation of these
elements, and that a cell's fate may be determined during the early stages of
limb development (Dudley et al.,
2002
; Sun et al.,
2002
). While this model is still controversial
(Saunders, 2002
;
Wolpert, 2002
), our
observations indicate that FGF/FGFR1 signaling may play an indispensable role
in the very early stages of limb development, which affects autopod formation
and digit patterning at later stages of limb development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/21/4755/DC1
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