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First published online March 21, 2008
doi: 10.1242/10.1242/dev.018945
Department of Anatomy and Program in Developmental Biology, University of California at San Francisco, San Francisco, CA 94143-0452, USA.
* Authors for correspondence (e-mail: pengfei.lu{at}ucsf.edu, zena.werb{at}ucsf.edu)
Accepted 11 February 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Apoptosis, Autopod progenitors, Cell proliferation, FGF signaling, Limb patterning, Mouse, Wnt signaling
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Martin,
1998; Niswander,
2003). The importance of the AER was demonstrated by classic
experiments in chicken embryos showing that AER removal at progressively later
stages of limb development causes a progressive loss of distal elements of the
limb (Saunders, 1948;
Summerbell, 1974). Although
different models have been proposed to explain limb skeletal patterning along
the proximal-distal (PD) axis, AER function in this process has remained
largely unclear. For example, the Progress Zone (PZ) model postulates that the
AER provides permissive signals to keep PZ cells labile until they exit the
zone, at which time these cells are autonomously specified by ceasing to
acquire `positional information'
(Summerbell et al., 1973). By
contrast, the Early Specification (ES) model proposes that PD elements are
specified, rather than progressive, at the earliest stages of limb bud
development and that the AER regulates subsequent expansion of progenitor
pools by promoting cell proliferation and survival
(Dudley et al., 2002).
Furthermore, recent models suggest that PD elements are specified via dynamic
interactions between the flank of the lateral plate mesoderm and the AER
(Mercader et al., 2000;
Tabin and Wolpert, 2007).
A transient structure, the AER undergoes a series of morphogenetic changes
consisting of four stages - initiation, maturation, maintenance and regression
- each characterized by distinctive changes of cell shape and gene expression
(Altabef et al., 1997;
Kimmel et al., 2000). Briefly,
AER initiation in the mouse forelimb starts at approximately embryonic day (E)
9, when the limb bud is first discernable and the gene expression in the
ventral ectoderm that marks the future AER becomes apparent. During AER
maturation, pre-AER cells in the ventral ectoderm migrate towards the distal
tip and undergo a compaction process, whereby a distinctive narrow band of
stratified columnar epithelium forms at 
E10
(Loomis et al., 1998). The
mature AER is then maintained for an additional 2-3 days, while mesenchymal
skeletal progenitors continue to proliferate and differentiate until a fully
patterned limb emerges. The AER then regresses via programmed cell death and
eventually flattens to a simple cuboidal epithelium
(Guo et al., 2003).
At the molecular level, AER initiation involves interactions of several
major signaling pathways. The prevailing model holds that
Wnt2b-Wnt8c/β-catenin signaling in the lateral plate mesoderm is required
for Fgf10 expression in the presumptive limb bud mesenchyme (LBM)
(Kawakami et al., 2001), which
in turn regulates Wnt3/β-catenin signaling in the overlying ectoderm to
induce AER formation (Barrow et al.,
2003; Kengaku et al.,
1998). BMP signaling also plays a role in this process, as mice
lacking Bmpr1a in the ectoderm fail to form the AER
(Ahn et al., 2001). The
molecular basis of the remaining stages of AER morphogenesis is less clear.
Studies have shown, however, that engrailed 1 (En1) plays a role
during migration and compaction of AER progenitor cells
(Loomis et al., 1998), whereas
Wnt/β-catenin signaling is required to maintain the AER after AER
initiation and maturation (Barrow et al.,
2003). By contrast, BMP signaling promotes destruction of the AER
during the regression process (Pizette and
Niswander, 1999), despite its early role in initiating the
AER.
FGFR2 functions in both limb ectoderm and mesenchyme during limb
development (Itoh and Ornitz,
2004). Although mouse embryos completely lacking Fgfr2
function fail to develop beyond implantation stages
(Arman et al., 1998), those
with partial loss of Fgfr2 function, including ones specifically
lacking the 2b isoform (De Moerlooze et
al., 2000; Revest et al.,
2001), survive to later embryonic stages, but fail to develop
limbs (Arman et al., 1999;
Gorivodsky and Lonai, 2003;
Xu et al., 1998). These
results suggest that AER initiation requires ectodermal FGFR2 function to
respond to mesenchymal FGF10 signaling, as mice lacking mesenchymal
Fgf10 are also limbless (Min et
al., 1998; Sekine et al.,
1999). However, mesenchymal expression of FGFR2
(Coumoul et al., 2005), as well
as of FGFR1 (Li et al., 2005;
Verheyden et al., 2005), is
essential for skeletal progenitor cells to respond to AER-FGFs to ensure
normal skeletal formation and patterning. In this study, we used a conditional
approach based on the Cre/lox system to modify the AER. We found that
AER maintenance requires FGFR2 function and is essential for distal limb
development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the Msx2-cre transgene
(Lewandoski et al., 2000) or a
conditional gain-of-function (GOF) allele of β-catenin,
Catnblox(ex3) (Harada et
al., 1999) were kindly provided by Drs David Ornitz, Gail Martin
and Makoto Mark Taketo, respectively. Mice carrying the BAT-Gal transgene, a
reporter of Wnt/β-catenin signaling activities
(Maretto et al., 2003), were
purchased from the Jackson Laboratory (Stock #005317). All mice were
maintained on a mixed genetic background and genotyped based on previously
published reports.
Phenotypic analysis
Noon of the day when a vaginal plug was detected was considered E0.5.
Embryos were collected in cold PBS, fixed in 4% paraformaldehyde (PFA), and
stored in 100% methanol at -20°C. To stage embryos more precisely, the
somites posterior to the forelimb bud were counted and the total number of
somites was determined by scoring the first one counted as somite 13. Each
somite stage is 2 hours. E10.5 is equivalent to 35-sominte stage and
E11.5 is equivalent to 45-somite stage. Standard protocols were used for
RNA in situ hybridization and skeletal preparations as previously described
(Lu et al., 2006). The area of
Hoxa13 expression was measured using ImageJ. β-galactosidase
(β-Gal) activity was conducted using standard protocol. Bright-field
images were captured with a Leica DMR HC microscope using a 40x/0.75
Plan Apochromat air objective.
Immunofluorescence, cell proliferation and cell death analyses
Embryos were embedded in agarose, and 40 µm sections were cut using a
Leica vibratome. Samples were blocked in PBS containing 5% BSA and 0.5% Tween
20 for 1 hour before they were subjected to incubation with primary
antibodies, anti-β-catenin mAb (BD-Biosciences), anti-CD44
(BD-Biosciences) and anti-phospho-Histone H3 (pH3, Upstate Biotechnology).
Goat-anti-rabbit secondary antibodies (AlexaFluor488, Invitrogen) were used to
detect the primary antibodies.
Cell proliferation analyses were based on either detection of
5-bromodeoxyuridine (BrdU) incorporation or pH3 immunofluorescence, which
marks nuclei in cells undergoing mitosis. For BrdU analysis, mice were
injected with BrdU (5 mg/100 g body-weight) 1 hour prior to euthanasia.
Embryos were harvested and fixed in 4% PFA. Transverse paraffin sections (5
µm) were cut using microtome and five sections, 50 µm apart from the
middle region of each limb bud were used for BrdU detection. Antigen was
retrieved in 10 mM sodium citrate (pH 6.0) in a microwave oven and BrdU was
detected using a kit from Roche (Cat#1296736). Alternatively, 40 µm
vibratome sections were used for pH3 immunofluorescence. All samples were
counterstained with To-Pro-3 (Invitrogen). The total number of nuclei and
BrdU-positive nuclei or pH3-positive nuclei in a 3.1x104
µm2 area (
=200 µm) of the sub-AER limb mesenchyme were
counted in each section. BrdU incorporation and pH3 immunofluorescence were
quantified using ImageJ. Confocal microscopy was performed on a ZeissLSM510
confocal.
Assays for cell death via TdT-mediated dUTP nick-end-labeling (TUNEL)
analysis on vibratome sections were performed according to manufacturer's
protocol (Roche Cat#1684817), while those via LysoTracker
(MolecularProbesL-7528) staining were conducted as previously reported
(Lu et al., 2006). A total of
over 180 embryos were examined between the 28- and 45-somite stages by TUNEL
or Lysotracker staining (at least five mutant embryos were examined for each
stage).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sun et al., 2000).
Importantly, in the hindlimb Msx2-cre acts before limb bud induction,
while in the forelimb cre is not expressed until 20 hours after
AER initiation (Sun et al.,
2002). We crossed male mice homozygous for the Msx2-cre
transgene and heterozygous for the Fgfr2-null allele,
Fgfr2
(Yu et
al., 2003), with female mice homozygous for the Fgfr2
conditional allele, Fgfr2fl
(Yu et al., 2003). All
Msx2-cre;Fgfr2fl/ progeny
(Fgfr2AER-KO) were viable and displayed anomalies,
including hair overgrowth (not shown), when compared with their
morphologically normal `control' littermates
(Msx2-cre;Fgfr2fl/+). We found that Fgfr2 removal via Msx2-cre before AER initiation eliminated the hindlimb of Fgfr2AER-KO embryos at E18.5 (Fig. 1A,B; n=18). By contrast, Fgfr2 removal at the 26- to 28-somite (s) stage after AER initiation in the forelimb eliminated only distal bones (Fig. 1C-F). In these mutants, although the humerus, radius and ulna were present and of normal sizes, the autopod (hand-plate) was absent except for a few carpal bones (Fig. 1C-F, n=18). Thus, FGFR2 is required for later stages of mouse forelimb development.
The loss of distal elements in Fgfr2AER-KO forelimbs
resembles the limb truncations of chicken embryos in AER removal studies
(Saunders, 1948;
Summerbell, 1974). To
determine whether the AER is maintained in mutant forelimb buds, we examined
the histological and molecular consequences of FGFR2 removal during AER
morphogenesis. AER histology was assessed by the immunofluorescence of CD44,
an AER marker (Sherman et al.,
1998), on vibratome sections of early limb buds at stages after
cre activation. In control embryos, we noted a distinctive AER
composed of stratified epithelium at the 29 s stage, which became
progressively more compact along the dorsal-ventral (DV) axis at later stages
(33-45s) (Fig. 1G,I,K,M). In
mutant limb buds, the AER was also distinctive at the 29 s stage, although it
was slightly thinner than normal (Fig.
1H). During later stages, the mutant AER became progressively
thinner and did not compact along the DV axis
(Fig. 1J,L). By the 45 s stage,
the AER in mutant limb buds was lost, as shown by the lack of stratified
epithelium in limb ectoderm and the absence of CD44 expression
(Fig. 1N). The loss of the AER
was also shown by the lack of expression of additional AER markers, including
Bmp4, Dlx2, Sp8 and En1, in mutant limb buds at E11.5 (not
shown). Thus, FGFR2 is required for AER maintenance during mouse limb
development.
We then analyzed how expression of Fgf8, the primary
AER-Fgf mediating AER function
(Lewandoski et al., 2000;
Moon and Capecchi, 2000), is
affected by premature AER loss. Using RNA in situ hybridization on whole-mount
embryos, we found that Fgf8 was expressed at a relatively normal
level in mutant limb buds at the 28-somite stage
(Fig. 1O-P'). However, by
the 33-somite stage, Fgf8 expression was noticeably less in mutant
limb buds than in control (Fig.
1Q-R'). Moreover, Fgf8 expression became
discontinuous, and gaps lacking Fgf8 expression were often observed
in mutant limb buds at later stages, including the 35 s (not shown) and 39 s
stages (Fig. 1S-T'). By
the 45 s stage, Fgf8 expression was essentially absent, except
occasionally in a tiny patch of the residual AER
(Fig. 1V). No Fgf8
expression was observed afterwards (not shown). Thus, FGFR2 removal causes
premature AER regression and progressive AER-FGF reduction.
Expression of key mesenchymal patterning genes is altered when the AER fails to be maintained
To determine how the mesenchymal gene expression is affected, we examined
Mkp3 expression, a downstream target of FGF signaling
(Kawakami et al., 2003). We
found that Mkp3 expression was not obviously different in
Fgfr2AER-KO and control forelimb buds at the 27 s (not
shown) or 30 s stage (Fig.
2A,B). However, the Mkp3 expression domain was reduced by
the 32 s stage in mutant limb buds (Fig.
2C,D) and became progressively smaller at later stages
(Fig. 2E-H). By the 46 s stage,
while the proximal domain of Mkp3 expression was not affected, the
Mkp3-positive domain in the sub-AER mesenchyme was absent
(Fig. 2G,H) because the distal
limb bud failed to form in mutant limbs (see below). The Mkp3
expression domain was also lacking in the distal mesenchyme of mutant limbs at
E12.5, when extensive Mkp3 expression was evident in the autopod of
control limb buds (not shown). Thus, FGF signaling activities continuously
decrease in mutant limb buds.
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In addition, we found that expression of Alx4, Meis2 and
Hoxa11, markers for the anterior mesenchyme
(Fig. 2O), the proximal
mesenchyme (Fig. 2Q), and the
slightly more distal mesenchyme (Fig.
2S), respectively, was not changed in mutant limb buds at E11.5
(Fig. 2P,R,T). Likewise, the
proximal domain of Hoxd11 expression, which marks the proximal
mesenchyme, was not affected in mutant limb buds
(Fig. 2U,V). By contrast, the
distal domain of Hoxd11 expression, which marks the future autopod
(Tarchini and Duboule, 2006)
(see below), was completely absent in mutant limb buds at E11.5
(Fig. 2U,V). As Hoxd11
expression in these two domains results from two separate transcriptional
events, first in the proximal then in the distal mesenchyme
(Tarchini and Duboule, 2006),
the lack of Hoxd11 expression in the distal domain could be due to
either delayed transcription in the distal mesenchyme or an absence of the
future autopod. No Hoxd11 expression in the distal domain was
observed at E12.5 (not shown), thus arguing against the possibility of a
transcriptional delay of Hoxd11. These data suggest that the
molecular events causing loss of the distal autopod in mutant embryos have
occurred by E11.5.
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) and Fgfr2
(Arman et al., 1999;
Min et al., 1998;
Revest et al., 2001) are
expressed, was greater in mutant forelimb buds than in control limb buds at
the 30 s stage (Fig. 3A,B;
n=8). A similar pattern of cell death was observed in mutant limb
buds up to the 36 s stage (Fig.
3C,D; n=6), when AER morphology was no longer distinct.
Thus, FGFR2 acts as a survival factor to maintain the AER and, in its absence,
the AER regresses prematurely owing to increased cell death.
Next, we examined cell proliferation in mutant limb buds by assaying the
percentage of cells in an area [3.1x104 µm2,
approximately the size of the hypothetical PZ
(Dudley et al., 2002;
Summerbell et al., 1973)] of
the sub-AER mesenchyme that incorporated BrdU after a 1-hour pulse. We found
no significant differences between the percentages of BrdU-positive cells in
the distal mesenchyme of mutant and control limb buds either at the 34 s or 44
s stage (Fig. 3E-H',M).
We also examined the mitotic index as indicated by pH3 immunofluorescence in
the sub-AER mesenchyme but again found no significant differences between
mutant and control limb buds at the 36 s, 39 s or E11.5 stages
(Fig. 3I-L,N). Thus, based on
two independent methods, our results indicate that global reduced cell
proliferation is unlikely to account for loss of the distal autopod in mutant
limbs.
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49 s stage (Fig.
4F, Fig. 5O). Thus,
Hoxd13 expression at E10.5 is also likely to mark progenitors of both
the proximal limb and the autopod.
Hoxa13 was expressed only in the distal autopod as Hoxd13
at E12.5 (Fig. 5K). Unlike
Hoxd13, however, Hoxa13 was not expressed in the proximal
limb bud at the 35 s stage (Fig.
4G). Rather, at this stage, it was expressed in a thin strip of
the posterior-distal mesenchyme, correlating with the area that is going to
give rise to the future autopod based on fate-mapping studies of the chicken
limb buds at the equivalent stages (Sato
et al., 2007; Vargesson et
al., 1997). Looking more closely over time, we found that
Hoxa13 was first expressed in the posterior-distal mesenchyme in
forelimb buds at the 31-32 s stages (Fig.
5A). As the limb bud grew out at later stages, the
Hoxa13-positive domain expanded concomitantly and extended anteriorly
until a well-patterned limb emerged at E12.5
(Fig. 4G-I;
Fig. 5C,E,G,I,K,U,U').
Importantly, we have never observed a loss of expression domain for
Hoxa13, as we did for Hoxd13, during the course of early
limb bud development, suggesting that Hoxa13 expression is unlikely
to mark progenitors of the proximal mesenchyme. Thus, consistent with studies
showing that Hoxa13 is initially expressed in a subpopulation of
autopod progenitors (Nelson et al.,
1996; Sato et al.,
2007), our data suggest that Hoxa13 expression in the
early limb bud marks autopod progenitors of the distal limb.
Generation of autopod progenitors is delayed in mutant limb buds
Next, we examined development of autopod progenitors by analyzing
Hoxa13 expression in mutant limb buds. We found that the onset of
Hoxa13 expression was delayed by two somite stages beginning at the
33-34 s stages instead of at the 31-32 s stages in
Fgfr2AER-KO limb buds
(Fig. 5C,D;
Table 1). At later stages, the
Hoxa13 expression domain expanded at a relatively stable pace, as in
the control. Thus, at the stages examined after its initiation, the area of
the Hoxa13-expression domain in the mutant was 45% of that in
the control until about the 45 s stage, when it reached a plateau and barely
expanded afterwards (Fig.
5F,H,J,L,U,U'). Hoxd13 expression, marking slightly
more distal autopod, confirmed that autopod expansion was indeed stalled by
the 47 s and E12.5 stages (Fig.
5M-P).
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Wnt/β-catenin and FGFR2 signaling interact to maintain the AER
To examine whether Wnt/β-catenin signaling is affected by
Fgfr2 removal, we used the reporter allele of the BAT-Gal transgene
to directly examine Wnt/β-catenin signaling
(Maretto et al., 2003). We
found that Wnt/β-catenin signaling was greatly reduced in the AER of
Fgfr2AER-KO forelimb buds at the 33 s stage
(Fig. 6A, n=6;
Fig. 6B, n=4),
although ectopic activities was also observed in the distal-dorsal mesenchyme
(Fig. 6B, arrow). In addition,
expression of Lef1, which plays a role in limb development
(Galceran et al., 1999), was
reduced in mutant limb buds than in control at E10.5 (35 s stage)
(Fig. 6C,D). Tcf1 and
Wnt3 expression were not significantly changed (not shown). Thus,
FGFR2 is required for normal Wnt/β-catenin signaling during AER
maintenance.
Next, we asked whether gain of Wnt/β-catenin signaling in the AER may
prevent premature AER loss and restore autopod bones in the mutant limb. We
crossed male mice homozygous for the Msx2-cre transgene and
heterozygous for the Fgfr2 allele with female mice
homozygous for the Fgfr2fl allele and heterozygous for the
Catnblox(ex3) allele, a conditional GOF allele of
β-catenin (Harada et al.,
1999), to generate
Msx2-cre;Fgfr2fl/;Catnblox(ex3)
embryos (referred to as
Fgfr2AER-KO;β-catGOF
hereafter). In these embryos, Msx2-cre-mediated recombination
inactivates Fgfr2 by converting Fgfr2fl to a null
allele and concomitantly activates β-catGOF
expression from the Catnblox(ex3) conditional allele. We
found that β-catenin overexpression
(Fig. 6E-H) caused ectopic
Fgf8 expression in the ventral ectoderm at E11.75
(Fig. 6I,K), when it was
already absent in the mutant limb bud (Fig.
1N,V; Fig. 6J). In
Fgfr2AER-KO;β-catGOF
limb buds, Fgf8 expression was retained in the mutant AER and was
ectopically expressed in the ventral ectoderm
(Fig. 6L). Moreover, we found
that β-catenin GOF prevented ectopic cell death in the ventral ectoderm
(Fig. 6N, n=3) and
restored normal expression of Hoxa13 in mutant limb buds
(Fig. 6O,P;
Table 1). Furthermore, we found
that the distal limb elements, based on Sox9 expression marking
skeletal progenitors (Fig.
6Q,R) and skeletal staining
(Fig. 6T, n=5), were
present in
Fgfr2AER-KO;β-catGOF
embryos. Together, these data showed that β-catenin GOF prevents AER loss
and restores the autopod in Fgfr2AER-KO limb buds.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Vargesson et al.,
1997). In addition, despite close examination, we never observed a
loss of Hoxa13 expression domain during early limb bud development.
These results suggest that, in contrast to Hoxd13, the initial
Hoxa13 expression domain is unlikely to mark progenitors of the
proximal limb. Indeed, studies have shown that this initial Hoxa13
expression domain marks only a subpopulation of autopod progenitors. For
example, using DiI labeling in chick embryos, Nelson and colleagues
demonstrated that Hoxa13-positive cells in early limb buds ultimately
give rise to only the posterior two-thirds of the autopod
(Nelson et al., 1996).
Likewise, recent studies suggested that this initial Hoxa13
expression domain contributes to the distal two-thirds of the future
autopod (Sato et al., 2007).
The most anterior and proximal autopod progenitors, which express
Hoxa13 at E12.5, must acquire Hoxa13 expression de novo,
thus highlighting a heterogeneous origin of autopod progenitors.
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). Thus, the final pattern of Hoxd13
expression in the autopod is a combined result of downregulation of its
expression in the proximal domain and upregulation of its expression in the
distal mesenchyme (Vargesson et al.,
1997).
Based on data showing that AER removal in chicken limb buds at the stage
20/21 causes loss of Hoxa13 expression, it was suggested that the AER
directly regulates Hoxa13 expression
(Hashimoto et al., 1999;
Vargesson et al., 2001). In
light of recent studies (Dudley et al.,
2002), an alternative explanation is that loss of Hoxa13
expression may be caused by death of autopod progenitors because of AER
removal. Indeed, our data show that Hoxa13 expression is initiated,
albeit with a delay, and its domain is able to expand in the presence of
dwindling AER-FGF signaling activities. In addition, Hoxa13
expression is maintained for at least 24 hours after the AER is lost and
sub-AER mesenchymal FGF signaling is absent by the 45 s stage. Together, these
results suggest that the AER indirectly regulates Hoxa13 expression
by directly controlling development of autopod progenitors (see below).
Generation of autopod progenitors requires normal AER function
Our data support a model in which the AER influences autopod development by
regulating `generation' (see below) of autopod progenitors
(Fig. 7). Previous studies
suggest that skeletal condensation occurs on a fixed schedule in a proximal to
distal wave (Dudley et al.,
2002; Lewandoski et al.,
2000; Sun et al.,
2002; Wolpert et al.,
1979) (reviewed by Mariani and
Martin, 2003). Thus, whether a normal autopod forms depends on the
number of progenitors at the time of autopod condensation. In
Fgfr2AER-KO limbs, the onset of generation of autopod
progenitor is delayed by two somite stages because of premature AER regression
and reduction of AER-FGF signaling activities in the LBM
(Fig. 7B). This delay of
progenitor generation causes an insufficient number of autopod progenitors to
be available at autopod condensation to form normal autopod bones. Remarkably,
autopod progenitors in mutant limbs are initially able to expand at a rate
comparable with that in the control when AER-FGF signaling activities
progressively diminish in the mesenchyme. These data thus suggest that the
primary function of the AER is to promote generation of autopod progenitors
during vertebrate distal limb development.
There are several major distinctions between our model and the existing
models, in particular the PZ model
(Summerbell et al., 1973), the
ES model (Dudley et al., 2002)
and other recent models (Mercader et al.,
2000; Tabin and Wolpert,
2007), regarding limb skeletal patterning along the PD axis.
Unlike these models, which are concerned with and differ at when limb elements
are specified, our model deals with events after the distal skeletal elements
are specified. Indeed, Hoxa13 is unlikely to specify the autopod
directly, even though it is required for autopod development
(Fromental-Ramain et al.,
1996). At present, specification markers of the PD elements are
still elusive. It is thus unclear precisely when and where the autopod is
specified, how many progenitor cells are present in the initial pool, to what
extent cell proliferation is involved and whether these earliest autopod
progenitors share similar proliferative properties to cells in other parts of
the mesenchyme. For the sake of simplicity and clarity in discussing our data,
we have used the generic term `generation' to distinguish these early events,
which include autopod specification, of autopod progenitor development
proceeding Hoxa13 expression with the later `expansion' event, which
our data suggest is initially less sensitive to reduced FGF signaling
activities.
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). By contrast, our data show that autopod progenitors in
Fgfr2AER-KO forelimb buds express markers, including
Hoxd13 and Hoxa13, of events that occur after autopod
specification. Our model suggests that the loss of the distal limb is due to
an insufficiency of skeletal progenitors at the onset of autopod condensation,
rather than to failure of specification according to the PZ model. Moreover,
by regulating generation of AER progenitors, the AER may play more than a
permissive role as the PZ model suggests. Finally, whereas the ES model
proposes that the AER regulates skeletal progenitor pools by promoting cell
proliferation and survival (Dudley et al.,
2002), our model shows that, at least for the autopod, this is
accomplished by controlling the number of progenitors that are initially
generated.
It is noteworthy that our model provides new mechanistic insights on
several puzzling skeletal phenotypes reported previously. For example,
although mice lacking AER-Fgf4/8 fail to form the hindlimb autopod,
neither cell proliferation nor cell death is affected in the distal mesenchyme
(Sun et al., 2002). In light
of our results, it is possible that generation of autopod progenitors may be
delayed, or prevented in these mutants. Furthermore, it remains possible that
the AER, in addition to promoting survival of stylopod and zeugopod
progenitors (Barrow et al.,
2003; Dudley et al.,
2002; Sun et al.,
2002), may also regulate their generation. Consistent with this
notion, mouse limb buds that lack Fgf4/8, which fail to form normal
stylopod and zeugopod, are smaller than normal at the earliest stages of limb
development without changes of cell death or cell proliferation in the
mesenchyme (Sun et al.,
2002).
Absence of abnormal cell death and cell proliferation in the distal mesenchyme
The lack of cell death in the distal mesenchyme of
Fgfr2AER-KO forelimbs is consistent with results from
previous studies. As mentioned, cell death is increased only in the proximal
but not in the distal mesenchyme, where autopod progenitors reside, of embryos
lacking AER-Fgf4/8 (Sun et al.,
2002). In addition, whereas AER removal before stage 24 in the
chick [which eliminates proximal limb elements up to the wrist
(Niswander et al., 1993)]
causes cell death in the distal mesenchyme, AER removal after stage 25 (which
results in loss of the distal autopod) does not
(Dudley et al., 2002). It is
interesting to note that, although abnormal cell death was observed in the
limb bud mesenchyme of certain mutant mice
(Revest et al., 2001;
Sun et al., 2002), it was not
observed in the proximal mesenchyme of Fgfr2AER-KO
forelimbs, as reported here and by a recent study
(Yu and Ornitz, 2008). These
differences in the cell death patterns, in addition to the final skeletal
phenotypes, are due to the fact that AER-FGF signaling is reduced earlier
and/or more greatly in limb buds where AER fails to initiate
(Revest et al., 2001) or in
limb buds where AER-Fgf4/8 are directly ablated
(Sun et al., 2002) than in
Fgfr2AER-KO forelimb buds, where AER-Fgf is,
although progressively less, continuously expressed until the 45 s stage.
Together, these data suggest that AER function is required for the survival of
stylopod and zeugopod progenitors, but not of autopod progenitors.
Interestingly, our analyses detected no significant differences of cell
proliferation in the distal mesenchyme between mutant and control limb buds.
These data thus join several previous studies, which also show that cell
proliferation is not affected in mutant limb buds that fail to form skeletal
elements, including the autopod (Barrow et
al., 2003; Revest et al.,
2001; Sun et al.,
2002; Verheyden et al.,
2005). Furthermore, it was observed over three decades ago
(Janners and Searls, 1971)
that cell proliferation is unchanged after AER removal in chicken limb buds.
Taken together, these results support our conclusion that reduced cell
proliferation in the distal mesenchyme is unlikely to account for loss of the
distal autopod. Finally, although the lack of abnormal cell death and cell
proliferation in the distal mesenchyme led us to discovering AER function in
generation of distal limb progenitors, we do not exclude the possibility that,
earlier, the AER may also regulate cell proliferation of the distal
progenitors.
FGFR2 and Wnt/β-catenin signaling interact to maintain the AER
Previous studies have shown that the FGF10-FGF8 loop, presumably via
ectodermal FGFR2, operates to initiate the AER during vertebrate limb
development (Crossley et al.,
1996; Ohuchi et al.,
1997). It was further suggested that FGF10 may be the `AER
maintenance factor' in the posterior limb mesenchyme to maintain AER
morphology (Ohuchi et al.,
1997). In this study, by conditionally removing FGFR2 function, we
demonstrate that loss of Fgfr2 function causes failure of AER
maintenance and, as a consequence, loss of the forelimb autopod. Our results
therefore support the model that FGFR2 signaling maintains the AER after AER
initiation.
Msx2-cre-mediated loss of Fgfr2 function in the AER and
ventral ectoderm increased cell death in these tissues, where both
Fgfr2 and the cre transgene are expressed. These results
suggest that FGFR2 acts as a survival factor to maintain the AER.
Interestingly, cell death is also increased in limb ectoderm of mice lacking
Fgfr2b function, in which the AER fails to initiate
(Revest et al., 2001). Thus,
these results suggest that FGFR2 promotes cell survival in both AER initiation
and maintenance during limb development.
Several lines of evidence indicate that FGFR2 interacts with
Wnt/β-catenin signaling during AER maintenance. Wnt/β-catenin
signaling activities, as measured by β-Gal staining using the BAT-Gal
transgene, are greatly reduced in the AER of Fgfr2AER-KO
limb buds. Expression of Lef1, which together with Tcf1 is
required for AER initiation, is also downregulated in mutant limb buds.
Furthermore, loss of β-catenin (Barrow
et al., 2003) and FGFR2 function via Msx2-cre cause
similar patterns of cell death increase in the forelimb ectoderm, suggesting
that they interact to promote cell survival during AER maintenance. Finally, a
gain of Wnt/β-catenin signaling rescues AER loss and this in turn
restores the autopod bones missing in Fgfr2AER-KO limbs.
FGF signaling interacts with Wnt/β-catenin signaling during early limb
bud development (Barrow et al.,
2003; Galceran et al.,
1999). Together with our data, these studies suggest that
interactions of FGF and Wnt/β-catenin signaling pathways are required at
multiple stages of vertebrate limb development.
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ACKNOWLEDGMENTS |
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