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First published online 19 December 2007
doi: 10.1242/dev.013268
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Department of Developmental Biology, Washington University School of Medicine, St Louis, MO 63110 USA.
* Author for correspondence (e-mail: dornitz{at}wustl.edu)
Accepted 26 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: FGF, FGF receptor, Apical ectodermal ridge (AER), Limb bud development, Chondrogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Surgical
removal of the AER results in skeletal truncation that is limited to more
distal segments when the AER is removed at progressively later stages
(Rowe and Fallon, 1982;
Summerbell, 1974). The AER
functions by elaborating signals to the adjacent mesenchyme, and members of
the fibroblast growth factor (FGF) family have been identified as key
components of AER signaling (Martin,
1998). It has been demonstrated that FGF-soaked beads are
sufficient to rescue the skeletal phenotypes that result from AER removal
(Fallon et al., 1994;
Niswander et al., 1993). Of
the four FGFs expressed in the AER, Fgf4 and Fgf8 are
necessary for limb skeletal formation and when both genes are disrupted prior
to limb bud initiation, the entire limb skeleton fails to form
(Boulet et al., 2004;
Sun et al., 2002).
FGFs elicit their biological functions by binding and activating a family
of four high-affinity receptors (FGFRs), a sub-class of receptor tyrosine
kinases (Ornitz and Itoh,
2001; Zhang et al.,
2006). Two members of the FGFR family, Fgfr1c and
Fgfr2c, are expressed in nascent limb bud mesenchyme
(Orr-Urtreger et al., 1991).
In vitro studies demonstrate that both FGFR1c and FGFR2c are capable of
interacting with AER-expressed FGFs
(Ornitz et al., 1996;
Zhang et al., 2006). After
ligand binding, activated FGFRs trigger several downstream intracellular
signaling cascades (Eswarakumar et al.,
2005). During limb development, the MAP kinase signaling pathway
is activated in mesenchymal cells immediately adjacent to the AER, indicating
active AER-FGF signaling in distal limb mesenchyme
(Corson et al., 2003).
Previous studies indicate that AER-FGF signals are important for
maintaining mesenchymal cell survival during limb development
(Boulet et al., 2004;
Sun et al., 2002). However,
although AER-FGF signals are limited to distal mesenchyme, cell death induced
by loss of AER-FGFs appears in proximal mesenchyme, far away from the AER.
This is inconsistent with current knowledge that FGFs act as paracrine factors
due to their interaction with heparan sulfate proteoglycans (HSPGs) in the
extracellular matrix (ECM) (Ornitz,
2000). Moreover, when Fgf4 and Fgf8 are
disrupted after limb bud initiation, transient AER-FGF signaling allows
formation of a severely hypoplastic skeleton
(Sun et al., 2002). Notably,
despite significant size and number reduction of skeletal elements, all three
PD segments are still recognizable. By contrast, AER removal, after limb bud
initiation, results in a truncated skeleton owing to loss of all skeletal
elements in distal segments. Phenotypic differences between loss of the AER
and loss of AER-FGFs suggest that the AER possesses additional functions,
which are either not mediated by FGF4 and FGF8 or are redundantly mediated by
other AER factors such as FGF9, FGF17, or other signaling molecules
including WNTs and BMPs (Barrow et al.,
2003; Wang et al.,
2004; Yamaguchi et al.,
1999). All of these phenotypes, which undoubtedly have mechanistic
implications, are not readily explained by the current model(s) of AER-FGF
functions during limb development.
To further understand the functions of AER-FGF signals during limb development, we used a genetic analysis in the mouse embryo to compare phenotypes resulting from loss of the AER with phenotypes resulting from loss of AER-FGF signals. We also compared phenotypes resulting from partial loss of AER-FGF signaling with phenotypes resulting from complete loss of both Fgf4 and Fgf8 in the AER. We conclude that AER-FGF signals regulate multiple molecular and cellular processes during limb development and that the skeletal agenesis after complete loss of both Fgf4 and Fgf8 is due to combined mesenchymal defects, including increased cell death, decreased proliferation and failure of chondrogenic differentiation. We present a model to explain how AER-controlled proliferation and FGF-regulated mesenchymal differentiation organize the formation of the skeletal primordia along the PD axis during normal limb development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/+
(Trokovic et al., 2003);
Fgfr2flox/flox and
Fgfr2/+
(Yu et al., 2003);
Msx2-Cre (Sun et al.,
2000); Prx1-Cre
(Logan et al., 2002).
Fgfr2Msx2-Cre mice were generated by mating
Fgfr2+/; Msx2Cre/+
mice with Fgfr2flox/flox mice.
Fgfr1Prx1-Cre, Fgfr2Prx1-Cre and
Fgfr1/2Prx1-Cre mice were generated by mating
Fgfr1+/;
Fgfr2+/; Prx1Cre/+
compound heterozygous mice with Fgfr1flox/flox;
Fgfr2flox/flox double homozygous mice. In all matings,
Fgfr1 and Fgfr2 alleles
were incorporated to increase the efficiency of Cre-mediated recombination.
Mouse embryonic skeletons were prepared as described previously
(Yu et al., 2003).
Staining, immunohistochemistry and in situ hybridization
Embryos or tissues were fixed in 4% paraformaldehyde in PBS and embedded in
paraffin. Sections were stained with Hematoxylin and Eosin (H&E). For
β-galactosidase histochemistry, embryos were fixed in 0.2% glutaraldehyde
in PBS for 60 minutes, washed in PBS and stained in β-gal staining buffer
[5 mM K3Fe (CN)6,5 mM K4FE
(CN)6*3H2O, 1 mM MgCl2, 0.01%
sodium desoxycholate, 0.009% NP40, 0.002% X-Gal] for 8 hours at 4°C. After
post-fixing in 4% paraformaldehyde, embryos were embedded in paraffin and
sections counterstained with nuclear fast red.
Immunohistochemistry was carried out using LAB-SA Detection System (Zymed) according to the manufacturer's instructions. Primary antibodies are anti-cleaved caspase 3 (Cell Signaling) and anti-phosphohistone H3 (pHH3) (Sigma). TUNEL assay was carried out using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. Distal mesenchymal proliferation was analyzed on transverse sections of limb buds and pHH3-labeled mesenchymal cells were counted in a 1.5x104 µm2 area immediately adjacent (within 100 µm) to the AER.
Fgf8 whole-mount in situ hybridization was performed as described
previously (Sun et al.,
2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), conditional inactivation of Fgfr2, using
the limb ectoderm-specific Msx2-Cre transgene
(Sun et al., 2000), could be
used to terminate AER functions during limb development. Msx2-Cre/+;
Fgfr2flox/
(Fgfr2Msx2-Cre) mice appeared normal except for the
absence of hindlimbs and severely truncated forelimbs
(Fig. 1A-F), consistent with
expression of Msx2-Cre before hindlimb bud initiation and after
forelimb bud initiation (Lewandoski et
al., 2000). Fgfr2Msx2-Cre forelimbs formed a
normal stylopod (humerus) and zeugopod (radius and ulna), but completely
lacked an autopod (Fig. 1D,E).
The mutant forelimb skeleton was very similar to the truncated chicken wing
skeleton that results from AER removal at around stage 23
(Summerbell, 1974), indicating
that genetic methods can mimic the effects of surgical means to completely
ablate the AER during limb development. In Fgfr2Msx2-Cre mice, forelimb buds were initiated normally, but showed reduced distal outgrowth after embryonic day 10.5 (E10.5). By E11.5, mutant forelimb buds were only comparable in size to control limb buds at E10.5 and showed distal morphological deformities (Fig. 1R, dotted line). By E12.5, mutant forelimbs appeared truncated, lacking any autopod primordial structures (Fig. 1C,F). By contrast, mutant hindlimbs only developed a small bulge protruding from the body wall and never demonstrated further outgrowth (Fig. 1F, arrow and dotted line).
Examination of histological sections indicated that a stratified AER was never formed in Fgfr2Msx2-Cre hindlimb buds (Fig. 1G-J). Consistent with agenesis of the AER in mutant hindlimbs, Fgf8 was not detected at any stage examined (Fig. 1O,P and data not shown). In mutant forelimbs, transient epithelial thickening was observed in the prospective AER region at E10, which rapidly regressed to a single layer of epithelial cells by E10.5 (Fig. 1K-N). Fgf8 expression was detected in the AER at E10.5, but was prematurely lost by E11.5 (Fig. 1O-R).
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). As the truncation level correlates with the timing of AER
removal in chick experiments, these results indicate that AER functions are
lost at a later stage in Fgfr2Msx2-Cre forelimbs than in
β-cateninMsx2-Cre forelimbs. Since the same
Msx2-Cre transgene was used in both studies, it suggests that AER
ablation in the forelimbs is not determined by the time of gene targeting but
more likely by the time of protein inactivation.
The AER is essential for proliferation of adjacent limb mesenchyme
Previous studies indicated that AER extirpation results in reduced
proliferation and/or increased cell death in distal mesenchyme
(Dudley et al., 2002;
Niswander and Martin, 1993;
Rowe et al., 1982;
Sun et al., 2002). Mesenchymal
proliferation and cell death were examined in
Fgfr2Msx2-Cre limb buds using anti-phosphohistone H3
(pHH3) and anti-caspase 3 immunohistochemistry (IHC), respectively. Consistent
with the complete absence of AER functions, mutant hindlimb buds showed
decreased mesenchymal proliferation beginning at the limb bud initiation stage
(E10) (Fig. 2A,B;
Table 1). By E10.5, mesenchymal
proliferation was further decreased in mutant limb mesenchyme, and few
pHH3-positive cells were found throughout the whole hindlimb field
(Fig. 2E,F;
Table 1). In contrast to the
reduced proliferation observed at early stages, mesenchymal cell death did not
appear in the hindlimb buds until E10.5, when massive cell death was detected
throughout the entire limb mesenchyme, adjacent to the prospective AER
(Fig. 2C,D,G,H;
Table 1). Mutant forelimbs
showed normal mesenchymal proliferation at E10
(Fig. 2I,J;
Table 1). However, by E10.5,
reduced proliferation was evident in distal mesenchyme
(Fig. 2M,N;
Table 1). At E11.5, mutant
forelimb buds lacked highly proliferative distal mesenchyme
(Fig. 2Q,R). No abnormal
mesenchymal cell death was found in mutant forelimbs at E10
(Fig. 2K,L). At E10.5, mutant
forelimbs showed a slight increase in cell death in distal mesenchyme
(Fig. 2O,P;
Table 1). At E11.5, apoptotic
cells were no longer detected in distal mesenchyme
(Fig. 2S,T). This is consistent
with the reduced or absent cell death observed after AER extirpation at later
stages of chick limb development (Dudley et
al., 2002; Rowe et al.,
1982), further indicating that loss of AER functions occurs at a
later developmental stage in Fgfr2Msx2-Cre forelimbs.
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), which do
not show reduced distal mesenchymal proliferation at early stages, with
Fgfr2Msx2-Cre mice, revealed that either the AER
contributes to mesenchymal proliferation independently of FGF4 and FGF8, or
that mesenchymal proliferation is redundantly regulated by these FGFs and
other AER signals. As pointed out in previous studies
(Sun et al., 2002), it is
puzzling that in Fgf4/8Msx2-Cre limb buds, the cell-death
zone is located in proximal mesenchyme, whereas AER removal leads to cell
death in distal mesenchyme. However, what was not rigorously considered during
such a comparison was whether loss of all AER functions versus loss of
AER-FGFs happens at the same developmental stages. As demonstrated in previous
studies, removal of the AER at different times can lead to different
mesenchymal defects and skeletal phenotypes
(Dudley et al., 2002). Thus, to
understand functional differences between the AER and AER-FGFs, it is
necessary to choose and compare phenotypes that occur at the same stages of
development. Hindlimb phenotypes of Fgfr2Msx2-Cre mice and
Fgf4/8Msx2-Cre mice allow such a comparison.
Fgf8, which is expressed before limb bud initiation, mediates
AER-like functions before formation of a morphologically identifiable AER. In
Fgfr2Msx2-Cre hindlimbs, Fgf8 was never
expressed, similar to the situation for Fgf4/8Msx2-Cre
hindlimbs, but, additionally, the AER was never formed. By contrast, a
morphologically normal AER is present in Fgf4/8Msx2-Cre
hindlimbs at early stages (Sun et al.,
2002). Thus, it is conceivable that before or during limb bud
initiation, all AER functions are lost in Fgfr2Msx2-Cre
hindlimbs, whereas only FGF signals are lost in
Fgf4/8Msx2-Cre hindlimbs. However, despite the loss of all
AER-derived signals, mesenchymal cell death in
Fgfr2Msx2-Cre hindlimbs occurs at the same time as that of
Fgf4/8Msx2-Cre hindlimbs (35 somites, E10.5), strongly
suggesting that the survival function of the AER is solely mediated by FGF
signals. Phenotypic comparison of Fgf4/8Msx2-Cre and
Fgfr2Msx2-Cre hindlimbs also suggests that the proximal
location of the cell-death domain, after loss of AER-FGF signals, is likely to
be due to continued proliferation of distal mesenchyme, which is not affected
by loss of AER-FGF signals at early stages
(Sun et al., 2002). As
apoptosis is delayed, continual proliferation gradually separates dying
mesenchymal cells from the AER. Without distal mesenchymal proliferation, the
same cell-death domain will remain adjacent to the ectoderm/AER, as shown in
Fgfr2Msx2-Cre hindlimbs
(Fig. 2H).
FGFR1 and FGFR2 mediate AER-FGF signaling during early limb development
To further understand the mechanism of AER-FGF functions, we disrupted FGF
signal transduction in limb mesenchyme by targeting mesenchymal FGFRs. To
overcome potential functional redundancy between Fgfr1c and
Fgfr2c, we inactivated both Fgfr1 and Fgfr2 in limb
mesenchyme with a Prx1-Cre transgene (Prx1 is also known as
Prrx1 - Mouse Genome Informatics)
(Logan et al., 2002).
Prx1-Cre is expressed in both forelimb and hindlimb mesenchyme, but
is activated in the prospective forelimb region prior to limb bud initiation
and targets forelimb mesenchyme exclusively at initiation stages of limb bud
development (Fig. 3A). Double
conditional knockout mice with the genotype Prx1-Cre/+;
Fgfr1flox/;
Fgfr2flox/
(Fgfr1/2Prx1-Cre) were born alive along with compound
single conditional knockout littermates, Fgfr1Prx1-Cre
(Prx1-Cre/+; Fgfr1flox/;
Fgfr2flox/+) and Fgfr2Prx1-Cre
(Prx1-Cre/+; Fgfr1flox/+;
Fgfr2flox/).
Fgfr1Prx1-Cre mice showed mild limb-skeletal defects
(Fig. 3B,C and see Fig. S1 in
the supplementary material) and Fgfr2Prx1-Cre skeletons
were indistinguishable from wild-type skeletons at post-natal day 0 (P0) (data
not shown), consistent with previous findings
(Eswarakumar et al., 2002;
Li et al., 2005;
Verheyden et al., 2005;
Yu et al., 2003). In contrast
to the single-gene knockouts, Fgfr1/2Prx1-Cre mice showed
severe skeletal hypoplasia in both forelimbs and hindlimbs
(Fig. 3B,D and see Fig. S1 in
the supplementary material). All three segments of the forelimb skeleton were
severely affected and bony fusion was found at the elbow joint and between
skeletal elements in the autopod. These results suggest that the functions of
Fgfr1 and Fgfr2 are partially redundant during limb skeletal
development and that FGFR1 has unique functions in distal limb skeletal
formation.
When examined at initiation stages (E9.5),
Fgfr1/2Prx1-Cre forelimb buds appeared normal. However, by
E10, Fgfr1/2Prx1-Cre forelimb buds were significantly
smaller (72%, P<0.03) than control limb buds along the PD axis
(Fig. 3E,H,I,L;
Table 2), a phenotype not found
in either Fgfr1Prx1-Cre or
Fgfr2Prx1-Cre littermates
(Fig. 3E-G,I-K;
Table 2). Smaller limb bud size
in Fgfr1/2Prx1-Cre embryos could result from decreased
mesenchymal cell proliferation or increased cell death. However, proliferation
in the distal mesenchyme of Fgfr1/2Prx1-Cre forelimb buds
was not affected (Fig. 3M-P),
similar to the observations in Fgf4/8Msx2-Cre limb buds
(Sun et al., 2002). Increased
mesenchymal cell death was not detected in mutant forelimb mesenchyme at E9.5
(25 somites; data not shown); however, at E10 (30-32 somites), increased
levels of cell death in Fgfr1/2Prx1-Cre forelimb
mesenchyme were clearly present, as shown by caspase 3 IHC
(Fig. 3Q,R) and TUNEL labeling
(see Fig. S2 in the supplementary material). Apoptotic cells were found in
both proximal and central regions of the limb bud, but were excluded from the
most-distal limb mesenchyme. At E10.5, no abnormal cell death was observed in
Fgfr1/2Prx1-Cre forelimbs
(Fig. 3S,T). As reported
previously, after loss of AER-FGFs, forelimbs show similar mesenchymal
defects, including increased proximal cell death and reduced limb bud size,
that occur at similar stages of development (E10)
(Boulet et al., 2004;
Sun et al., 2002),
demonstrating that AER-FGF signals are mediated by both FGFR1 and FGFR2 during
early mesenchymal development.
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), in which
complete loss of FGF4 and FGF8 activity results in skeletal agenesis,
Fgfr1/2Prx1-Cre forelimbs were hypoplastic but formed a
normally segmented skeleton. Notably, the Fgfr1/2Prx1-Cre
skeletal phenotype was similar to that of Fgf4/8Msx2-Cre
forelimbs, in which AER-FGF signals are transiently present at early stages.
This suggested that in Fgfr1/2Prx1-Cre forelimbs, early
mesenchymal cells still receive FGF signals, albeit at a significantly reduced
level. Consistent with this, it has been reported that complete mesenchymal
deletion mediated by Prx1-Cre may occur by as late as E12.5
(Hill et al., 2006;
Kmita et al., 2005;
Lewis et al., 2001),
suggesting incomplete mesenchymal inactivation of FGFRs by Prx1-Cre
during early limb development. However, the hypomorphic nature of the
Fgfr1/2Prx1-Cre mutation allows transient FGF signaling,
which can then be contrasted with both pre-limb bud initiation and post-limb
bud initiation deletions of Fgf4 and Fgf8. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sun et al., 2002). However,
when compared with Fgf4/8RAR-Cre forelimbs, which
completely lack a limb skeleton, both Fgf4/8Msx2-Cre and
Fgfr1/2Prx1-Cre forelimbs show similar patterns of
mesenchymal cell death but they do form a skeleton
(Fig. 4B,D,E). It is worth
noting that although proximally located cell death domains overlap with the
prospective stylopod region, the humerus is the least affected skeletal
element in Fgf4/8Msx2-Cre forelimbs. These observations
suggest that increased mesenchymal cell death is not sufficient to cause
skeletal agenesis.
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).
However, skeletal defects following X-irradiation are very different from
those following loss of both Fgf4 and Fgf8. After
X-irradiation, proximal skeletal elements are either not formed or reduced in
size, whereas skeletal phenotypes in the autopod are much less severe. It is
worth noting that in the X-irradiation model, similar levels of cell death
occur in proximal and distal mesenchyme. By contrast, after loss of both
Fgf4 and Fgf8, even though mesenchymal cell death is limited
to proximal mesenchyme, distal skeletal elements are either not formed or are
severely hypoplastic. As AER functions are not affected by X-irradiation
(Wolpert et al., 1979), such
phenotypic differences strongly suggest that loss of AER-FGFs must cause
additional mesenchymal defects that result in severe distal skeletal
phenotypes.
Continual proliferation of distal mesenchyme is essential for distal skeletal formation
Previous studies concluded that skeletal defects, after loss of both
Fgf4 and Fgf8, are not due to reduced mesenchymal
proliferation, as distal mesenchymal proliferation was not affected by loss of
AER-FGF signals when examined before E10.5
(Sun et al., 2002). Although
AER-FGF signals are dispensable for mesenchymal proliferation, they are
essential to maintain mesenchymal gene expression. One such gene is
Fgf10, which is abundantly expressed in distal mesenchyme throughout
early limb development (Ohuchi et al.,
1997). After loss of both Fgf4 and Fgf8, Fgf10
expression is greatly reduced in distal mesenchyme and is barely detectable at
later stages (Boulet et al.,
2004; Sun et al.,
2002). During limb development, Fgf10 functions to
maintain FGFR2b signaling in the AER and the integrity of the AER
(Min et al., 1998;
Ohuchi et al., 2000;
Sekine et al., 1999). This is
supported by the observation that after loss of both Fgf4 and
Fgf8, there is a higher than normal level of cell death in the AER
between E10.5 and E11.5, when Fgf10 expression is no longer
maintained (Boulet et al.,
2004; Sun et al.,
2002).
As illustrated by Fgfr2Msx2-Cre forelimb defects (Fig. 4F), the integrity of the AER is important for maintaining distal mesenchymal proliferation. Therefore, we suggest that after loss of both Fgf4 and Fgf8, distal mesenchymal proliferation is eventually reduced following downregulation of Fgf10 and premature degeneration of the AER. Thus, in the absence of both Fgf4 and Fgf8, increased mesenchymal cell death results in an initial reduction in limb bud size. The extreme reduction in limb size that occurs by E11.5 is likely to be due to the combined effects of reduced distal mesenchymal proliferation and sustained cell death that result in the complete cessation of limb bud outgrowth.
Continual proliferation of distal mesenchyme is important for distal
skeletal element formation. When the AER is excised from chick limb buds at
late stages, distal skeletal truncation (loss of autopod elements) correlates
with reduced distal mesenchymal proliferation, as mesenchymal cell death no
longer occurs after AER removal at late stages of development
(Dudley et al., 2002).
Fgfr2Msx2-Cre forelimb phenotypes further indicate that
autopod skeleton formation requires proliferation of distal mesenchymal cells
to form a primordium prior to overt morphological changes. The resurgence of
autopod skeletal formation after X-irradiation is also likely to be due to
restoration of distal mesenchymal proliferation by the intact AER after
X-irradiation. Thus, we suggest that distal skeletal defects after loss of
both Fgf4 and Fgf8 are more likely to be due to reduced
mesenchymal proliferation than to increased mesenchymal cell death.
A role for FGF signaling during limb mesenchymal differentiation
As reported previously, disruption of Fgf8 prior to limb bud
initiation results in a limb skeleton that completely lacks the stylopod
(Fig. 4C). Formation of the
zeugopod and autopod are less affected, presumably owing to increased
Fgf4 expression that maintains some AER-FGF functions
(Lewandoski et al., 2000;
Moon and Capecchi, 2000).
During limb bud development, Fgf8 expression precedes that of other
AER-FGFs, resulting in a window of time in which only FGF8 is present (from
E9.0 to 
E10 in mouse forelimbs). After that, AER-FGF signals comprise
FGF8 and other FGFs (FGF4, 9 and 17) (Sun
et al., 2000; Sun et al.,
2002). In the absence of Fgf8, even though Fgf4
is precociously expressed (Lewandoski et
al., 2000), there is still a period of time (from E9.0 to
E9.75 in mouse forelimbs) when mesenchymal cells fail to receive FGF
signals from the AER. This gap in FGF signaling results in stylopod agenesis.
Moreover, as shown by Fgf4/8Msx2-Cre forelimb phenotypes
(Fig. 4D), when mesenchymal
cells receive FGF signals during this period of time, stylopod formation
proceeds relatively normally and is not affected by extensive proximal cell
death or dramatic limb bud size reduction. Thus, stylopod agenesis after loss
of Fgf8 cannot simply be due to reduced mesenchymal cell number, as
suggested in previous studies (Lewandoski
et al., 2000; Sun et al.,
2002).
Previous studies also indicate that loss of AER-FGF signals do not prevent
mesenchymal cells from expressing Sox9, the earliest marker for
chondrogenic differentiation (Akiyama et
al., 2002; Wright et al.,
1995), but do prevent formation of mesenchymal condensations
(Boulet et al., 2004;
Sun et al., 2002). Normally,
Sox9 functions to commit undifferentiated mesenchymal cells to
osteochondroprogenitors, a cell type that will give rise to both chondrocytes
and osteoblasts and to promote mesenchymal condensation formation
(Akiyama et al., 2002). During
forelimb development, Sox9 expression begins at E10 in a
subpopulation of mesenchymal cells (Akiyama
et al., 2005). It is worth noting that at E9.5, prior to
Sox9 expression, AER-FGF-induced MAP kinase activity is already
present in the entire forelimb mesenchyme
(Corson et al., 2003). Thus, we
hypothesize that AER-FGF signals are required for Sox9-mediated
mesenchymal condensation formation. In support of this hypothesis, previous
studies have shown that FGF signaling through the MAP kinase signaling pathway
can function synergistically with SOX9 to upregulate Sox9 expression
and that the level of SOX9 protein is critical for the formation of a
mesenchymal condensation (Bi et al.,
2001; Murakami et al.,
2000). Thus, stylopod agenesis after loss of Fgf8 is not
due to reduced mesenchymal cell numbers, but rather to the failure of
Sox9-expressing cells to undergo further chondrogenic
differentiation.
AER-FGF functions during PD pattern formation
During limb skeletal development, prior to initiation of a mesenchymal
condensation, some mesenchymal cells undergo initial stages of chondrogenic
differentiation, characterized by expression of Sox9, but without
apparent changes in cell morphology (chondrogenic primordia). Initial
Sox9 expression is localized to central regions of the limb bud,
surrounded by undifferentiated mesenchymal cells. Later, the Sox9
expression domain expands following limb outgrowth, but is still excluded from
distal mesenchyme beneath the AER (Akiyama
et al., 2005). Owing to the lack of definitive markers for each PD
segment at early stages, it is difficult to determine whether the initial
Sox9 expression domain contains progenitors for all three PD
segments, or only for the most-proximal segment.
Recent lineage-tracing studies with Shh-Cre and the ROSA26
(R26R) β-galactosidase reporter suggest that the initial
Sox9 expression domain might not contain progenitors for all three PD
segments. Along the PD axis, progeny of Shh-Cre-expressing cells are
found in the autopod and part of the zeugopod, but never in the stylopod
(Harfe et al., 2004). The
origin of Shh-Cre-expressing cells is limited to the zone of
polarizing activity (ZPA) in the posterior edge of limb mesenchyme, and does
not appear to overlap with the central Sox9 expression domain. This
suggests that skeletal lineages in the zeugopod and autopod are not derived
from the initial Sox9 expression domain, but rather are formed at
later stages after their progenitors move out of the ZPA. Thus, Sox9
expression is sequentially regulated along the PD axis, which is consistent
with the conclusion that chondrogenic differentiation along the PD axis
follows a proximal-to-distal sequence
(Summerbell et al., 1973).
We suggest that the initial Sox9 domain demarcates a chondrogenic primordium for the stylopod, in which Sox9-expressing cells only contribute to very proximal skeletal elements. With continual transition of distal undifferentiated cells into Sox9-expressing cells, chondrogenic primordia for the zeugopod and autopod become sequentially established (Fig. 4A). During this sequential differentiation process, AER-controlled proliferation provides a source of undifferentiated mesenchymal cells that continually receive AER-FGF signals and progressively differentiate into more-distal skeletal elements. Such a mechanism ensures continual commitment of mesenchymal cells to the osteochondroprogenitors that are essential for mesenchymal condensation formation along the PD axis. This could explain the different skeletal phenotypes in Fgf8RAR-Cre and Fgf4/8Msx2-Cre as well as Fgfr2Msx2-Cre forelimbs, in which AER-FGFs or all AER functions are inactivated at different times during development (Fig. 4C,D,F).
In addition to temporal differences in Sox9 expression, different
PD segments might also regulate Sox9 expression through distinct
molecular mechanisms. This is supported by recent studies of hindlimb
phenotypes (lack of the stylopod and zeugopod but normal autopod) in
Gli3 and Plzf (also known as Zbtb16 - Mouse Genome
Informatics) double-knockout mice (Barna et
al., 2005). In the absence of both Gli3 and Plzf,
Sox9 is not expressed in early limb mesenchyme, which, as we suggest,
results in failure of proximal chondrogenic primordia formation and subsequent
failure of proximal skeletal element formation. However, owing to normal
mesenchymal proliferation and normal Sox9 expression in distal
mesenchymal cells at later stages, autopod chondrogenic primordia and distal
skeletal development proceeds normally in mutant hindlimb buds.
In a recent review, after examination of segment-specific marker gene
expression, Tabin and Wolpert present ideas that distal proliferation and
proximal differentiation are tightly coupled at early stages and that
segmental primordia are sequentially established along the PD axis
(Tabin and Wolpert, 2007). It
is worth noting that establishment of chondrogenic primordia, marked by
Sox9 expression, occurs in a similar timeframe to this PD
segmentation process that is defined by segment-specific gene expression.
Since, in the absence of Sox9, segment-specific genes themselves are
insufficient to induce the formation of a mesenchymal condensation
(Akiyama et al., 2002), these
results suggest that PD skeletal pattern formation requires coordination of
chondrogenic- and segment-specific differentiation.
In summary, AER-FGF signals directly or indirectly regulate mesenchymal survival, proliferation and differentiation during limb development. The complete skeletal agenesis after loss of both Fgf4 and Fgf8 or all AER functions before limb bud initiation (Fig. 4E,G) is likely to be due to the loss of all of these functions. Finally, in contrast to previous conclusions that AER-FGF signals regulate PD skeletal pattern formation by controlling mesenchymal cell number, our analysis indicates that the AER and AER-FGFs regulate PD skeletal pattern formation through coordinating distal mesenchymal proliferation and chondrogenic differentiation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/483/DC1
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ACKNOWLEDGMENTS |
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