1 Laboratory of Genetics, University of Wisconsin-Madison, 425G Henry Mall,
Madison, WI 53706, USA
2 Cancer and Developmental Biology Laboratory, National Cancer Institute,
Frederick Cancer Research and Development Center, Frederick, MD 21702,
USA
3 Genetics of Development and Disease Branch, NIDDK, NIH, 10/9N105, 10 Center
Drive, Bethesda, MD 20892, USA
4 University of Florida College of Medicine, Department of Molecular Genetics
and Microbiology, Gainesville, FL 32610-0266, USA
* Author for correspondence (e-mail: xsun{at}wisc.edu)
Accepted 18 July 2005
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SUMMARY |
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Key words: Cre-mediated recombination, FGF, Fgfr1 signaling, Limb development, Mouse, Patterning, Shh
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Introduction |
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There are four mouse FGF receptors (FGFRs), each characterized by three
extracellular immunoglobulin (Ig)-like domains, a single transmembrane domain
and an intracellular split cytoplasmic tyrosine kinase domain
(Itoh and Ornitz, 2004).
Alternative splicing within the third Ig loop of FGFR1-3 produces two splice
variants: IIIb and IIIc which display different ligand specificities. For
example, mitogenic assays in cultured cells show that FGF4 and FGF8
preferentially activate the IIIc isoform of FGFR
(Ornitz et al., 1996
).
Conversely, genetic data suggest that FGF10, which is expressed in the LBM,
preferentially activates the IIIb isoform
(Min et al., 1998
;
Revest et al., 2001
;
Sekine et al., 1999
). In
addition, the IIIb and IIIc isoforms are often differentially expressed, with
IIIb preferentially in the epithelium and IIIc in the mesenchyme
(Finch et al., 1995
;
Orr-Urtreger et al., 1993
;
Peters et al., 1992
).
Two Fgfrs, Fgfr1 and Fgfr2 are expressed in the early
limb bud (Orr-Urtreger et al.,
1993; Peters et al.,
1992
; Xu et al.,
1998
; Yamaguchi et al.,
1992
). Fgfr2-IIIc, although expressed in early LBM, is
only essential at a later stage in ossification
(Eswarakumar et al., 2002
;
Yu et al., 2003
). A role for
FGFR1 in limb has been implicated through studies of chimera and hypomorphic
mutants, which bypass the gastrulation defect that causes
Fgfr1-/- mutants to die prior to limb initiation
(Ciruna and Rossant, 2001
;
Deng et al., 1994
;
Yamaguchi et al., 1994
). These
milder mutants exhibit deformed limb buds and varying degrees of reduction in
limb skeletal elements (Deng et al.,
1997
; Partanen et al.,
1998
; Xu et al.,
1999
). However, the precise role of FGFR1 in limb formation awaits
definition. Here, we dissect FGFR1 function by conditional inactivation in
mouse using the Cre/loxP approach. Our results show that FGFR1 plays
multiple roles in limb bud establishment, outgrowth and patterning.
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Materials and methods |
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RT-PCR analysis
For normal and Tcre;Fgfr1 mutant limb buds (two pairs each), the
LBM was dissected from the ectoderm and total RNA was prepared using TRIzol
(Invitrogen). First-strand synthesis was carried out using the Superscript
First Strand cDNA Synthesis Kit (Invitrogen). PCR was performed using the
following primer pairs: for Fgfr1,
5'-TCTGGAAGCCCTGGAAGAGAGA-3' and
5'-TCTTAGAGGCAAGATACTCCAT-3'; for Gapdh,
5'-ACCACAGTCCATGCCATCAC-3' and
5'-TCCACCACCCTGTTGCTGTA-3'.
Embryo isolation and phenotype analyses
Embryos were dissected from time-mated mice, counting noon on the day the
vaginal plug was found as embryonic day (E) 0.5. Whole-mount in situ
hybridization was performed as previously described
(Neubuser et al., 1997). The
Fgfr1 in situ probe was prepared from a plasmid containing
Fgfr1 exon 9 cDNA. This cDNA was generated by PCR using primer pair:
5'-TCTGGAAGCCCTGGAAGAGAGA-3' and
5'-TGCGCAGAGGGATGCTCTTG-3'.
Limb buds for histological analysis were fixed in 4% paraformaldehyde after whole-mount in situ hybridization and embedded in JB-4 plastic resin (Polysciences) according to the manufacturer's protocol. Sections were cut at 5 µm and counterstained with 0.1% nuclear fast red. Skeletal preparations were performed with Alcian Blue and Alizarin Red using a standard protocol.
Areas of cell death were detected by staining with LysoTracker Red DND-99
(Molecular Probes) using a modified protocol
(Zucker et al., 1999).
Shh-expressing cell lineage analysis
For lineage analysis, the Cre reporter line R26R
(Soriano, 1999) was introduced
into the mutant background to generate mutant
Shhcre/+;Fgfr1co/co;R26R/+ and control
Shhcre/+;Fgfr1co/+;R26R/+ embryos. In embryos
of either genotype, lacZ is expressed in Cre-expressing cells and
their progeny (Shh-expressing lineage). These cells are visualized by
ß-galactosidase (ß-gal) staining using a standard protocol.
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Results |
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Using the same analysis, in Shhcre;Fgfr1, we can detect a domain lacking Fgfr1 expression in the posterior mesenchyme of E10.5 limb buds, shortly after the commencement of Shhcre activity (Fig. 1D,G,H). Spry4 and Mkp3 gene expression is reduced in this domain (Fig. 1K,L and Fig. 5G,H).
Inactivating Fgfr1 in LBM affects the size and shape of all three limb skeletal segments
Tcre;Fgfr1 mutants die at birth probably owing to neural tube and
axial skeletal defects (see below). In E17.5 mutant forelimbs (n=8),
we found that the stylopod is shortened by an average of 15%. The zeugopod,
reduced by 12%, is often fused at the distal end
(Fig. 2A,B, n=4/8
mutant forelimbs). The mutant autopod often consists of three digits, one
tri-phalangeal digit flanked by two bi-phalangeal digits
(Fig. 2E,F, n=5/8
forelimbs).
The Tcre;Fgfr1 mutant hindlimb is more severely affected than the
forelimb (Fig. 2C,D,G,H). One
explanation for the increased severity is based on a possible reduction of LPM
cell number in the prospective hindlimb, but not forelimb region prior to limb
initiation. This reduction is deduced from a combination of phenotypes,
including irregular somite size and an expanded open neural plate at E9.5, and
misshapen ribs and axial vertebrae at E17.5 (data not shown). These defects
are probably due to a previously described requirement of Fgfr1 in
mesoderm production (Ciruna and Rossant,
2001; Ciruna et al.,
1997
; Deng et al.,
1997
; Yamaguchi et al.,
1994
). In Tcre;Fgfr1 hindlimb, a defect in mesoderm
production will lead to reduced prospective LBM cell number, and in turn a
reduced limb skeleton. By contrast, none of the phenotypes indicative of
mesoderm reduction is observed within and rostral to the forelimb region,
suggesting that the phenotypes observed in the forelimb are not compounded by
earlier defects in mesoderm production. Thus, to address the specific role of
FGFR1 in limb development, we have concentrated on the forelimb of the
Tcre;Fgfr1 mutant for subsequent analyses.
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Based on phalanx number, the two bi-phalangeal digits in the
Tcre;Fgfr1 forelimb might be of digit 1 character. To address whether
this conclusion is also supported by molecular characteristics, we assayed for
Hoxd12 and Hoxd13 expression
(Fig. 2M-P). Previous studies
show that digit 1 identity is marked by the absence of Hoxd12 and
presence of Hoxd13 expression
(Fromental-Ramain et al.,
1996; Knezevic et al.,
1997
; Zakany et al.,
1997
). Their expression in Tcre;Fgfr1 mutant limb buds
supports that the most anterior digit is digit 1, while the most posterior
digit is not, despite being biphalangeal. These results show that in
Tcre;Fgfr1 limbs, digit 1 is present while some of the posterior
digits are absent.
Fgfr1 regulates nascent limb bud shape and cell number
We found that the Tcre;Fgfr1 forelimb bud is misshapen at E10.0
shortly after limb initiation. Although shorter along the PD axis, it is wider
along the AP axis and thicker along the DV axis
(Fig. 3A,B,E,F). By E10.5, the
differences in all three axes are further exaggerated
(Fig. 3C,D,G,H). A previous
study of Fgfr1 hypomorphic mutants show that a posterior shift in
Hoxb9 expression in the LPM may be responsible for the expansion of
limb bud AP width (Partanen et al.,
1998). A careful examination of Hoxb9 expression in
Tcre;Fgfr1 LPM shows that there is no posterior shift of expression
in this mutant to explain the increase in width (data not shown).
Using Fgf8 expression as a marker for AER, we found that in
Tcre;Fgfr1 forelimb buds the AER is wider along DV axis starting at
E10.0 (Fig. 3I-L) and shorter
along AP axis starting at E10.5 (Fig.
3C,D). This suggests that Fgfr1 inactivation in the
mesenchyme can influence AER morphology in a cell non-autonomous manner. A
possible mediator for this function is GREMLIN, a secreted antagonist of BMP
signaling. Gremlin null mutants show a similar AER phenotype
(Khokha et al., 2003;
Michos et al., 2004
), and its
expression is downregulated in Tcre;Fgfr1 limb buds
(Fig. 3M,N).
To address whether the whole limb bud shape change is accompanied by cell number changes, we counted LBM cell number (see Table S1 in the supplementary material). Our result shows that there is an approximate increase of 65% and 31% in the number of cells in the Tcre;Fgfr1 forelimb buds compared with controls at E10.0 and E10.5, respectively. At these stages, using phosphorylated Histone H3 antibody staining, no significant difference in cell proliferation is detected between mutant and normal to account for the increase in cell number (data not shown).
The initial excess cell number in Tcre;Fgfr1 limb buds is quickly
negated by increased cell death observed starting at E10.5
(Fig. 3O,P). By E11.5, a mutant
limb bud on average has 26% fewer cells compared with normal (see Table S1 in
the supplementary material). We found that the expression of Dkk1, a
mediator of limb bud cell death (Grotewold
and Ruther, 2002; Mukhopadhyay
et al., 2001
), is increased in Tcre;Fgfr1 limb buds
(Fig. 3Q,R). This provides a
possible molecular mechanism for the cell death phenotype. Despite the
increased number of dying cells, the death domain in Tcre;Fgfr1 limb
buds is confined to the proximal and anterior LBM, similar to normal
(Fig. 3O,P). We hypothesize
that no cell death is detected in the distal mesenchyme because these cells
are protected by residual FGF signaling, as indicated by Spry4
expression (Fig. 3S,T).
Complete inactivation of Fgfr1 in LBM affects the expression of key patterning genes
In search of additional molecular changes, we found that the expression of
Shh is reduced to a very small domain in Tcre;Fgfr1 forelimb
buds (Fig. 4A,B). Accordingly,
Ptch1 and Gli1 expression, which is responsive to the SHH
signal, is detected in reduced domains (data not shown). In addition,
Alx4 expression, which is restricted by SHH signal to the anterior
mesenchyme (Takahashi et al.,
1998), is detected in a larger domain in the mutant
(Fig. 4C,D). These results
indicate that SHH signaling is reduced in the absence of FGFR1. The AER
expression of all three BMP genes implicated in limb bud patterning, Bmp2,
Bmp4 and Bmp7 is slightly upregulated in intensity in
Tcre;Fgfr1 limb buds, possibly owing to the widened AER
(Fig. 4E-H; data not shown).
Interestingly, the mesenchymal expression of Bmp4 and Bmp7
is slightly reduced, while that of Bmp2 is upregulated in the
Tcre;Fgfr1 limb buds. This suggests that FGFR1 regulates Bmp gene
expression in a complex manner.
A previous study of a hypomorphic Fgfr1 mutant shows that
Hoxd13 expression is downregulated in those limbs
(Partanen et al., 1998).
Consistent with this, we found that in E10.5 Tcre;Fgfr1 limb buds,
expression of both Hoxd13 and the paralogous Hoxa13 is
reduced, with Hoxa13 more severely downregulated
(Fig. 4I-L). Interestingly, by
E12.5 Hoxd13 expression in Tcre;Fgfr1 limb buds appears to
have recovered (compare Fig. 2P
with Fig. 4L). A plausible
explanation for this change is that early phase and late phase Hoxd13
expression may be differentially controlled. This was also exemplified by a
previous observation that in a limb-specific Shh chick mutant termed
oligozeugodactly (ozd), early phase Hoxd13
expression is absent while late phase expression is present
(Ros et al., 2003
). In
summary, our molecular data suggest that a combination of these gene
expression changes may account for the phenotypes in the Tcre;Fgfr1
mutant.
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To address expression regulation in the context of Shhcre;Fgfr1 limb buds, we first established Shhcre/+;Fgfr1co/+ limb buds as a proper control. This is important because Shhcre is generated by insertion of Cre into the Shh-coding region. Thus, only one wild-type copy of Shh remains in Shhcre;Fgfr1 (Shhcre/+;Fgfr1co/co) mutants. We found that the Shhcre/+;Fgfr1co/+ control limb buds at E10.75 show robust Shh expression, despite having only one wild-type Shh allele and one wild-type Fgfr1 allele (Fig. 5C). By contrast, in E10.75 Shhcre;Fgfr1 mutant limb buds, Shh expression is reduced to a punctate pattern (Fig. 5D), demonstrating that Fgfr1 cell-autonomously regulates Shh expression at the RNA level.
We noted that Shh expression is more reduced distally than
proximally (asterisk in Fig.
5D). There is evidence that in a normal limb bud, endogenous
Shh is expressed at a higher level distally than proximally (Dr Cliff
Tabin, personal communication). Based on this, we favor the explanation that
in Shhcre;Fgfr1 limb buds, the PD difference in Shh
inactivation is a result of differential Shhcre activity, and hence
differential Fgfr1 inactivation. Consistent with this, the expression
patterns of Fgfr1 and Mkp3 at E10.75
(Fig. 5G,H; data not shown)
indicate that FGF signal reception is efficiently reduced in the distal
two-thirds, while it remains in the proximal one-third of the
Shhcre active domain (compare Fig.
5H with
5B). At later stages,
Shh expression is progressively more reduced in Shhcre;Fgfr1
limb buds, and eventually absent at E11.25, a time when it is still expressed
in control limb buds (data not shown). To investigate factors that may mediate
Fgfr1 regulation of Shh, we examined Hand2
(previously known as dHAND) expression, as it is necessary and
sufficient to induce Shh expression
(Charite et al., 2000).
Hand2 expression does not change in Shhcre;Fgfr1 limb buds
(data not shown), suggesting that HAND2 may act upstream of FGFR1, or that
FGFR1 and HAND2 regulate Shh expression in parallel pathways.
As an example of non-cell-autonomous regulation, reduction of
Ptch1 expression is not restricted to the
Fgfr1-/- domain, consistent with the idea that FGFR1
regulates Ptch1 expression through secreted SHH
(Fig. 5E,F). Previous studies
show that Fgf10 expression in the mesenchyme is dependent on AER-FGF
signaling (Boulet et al., 2004;
Ohuchi et al., 1997
). However,
Fgf10 expression is unaltered in Shhcre;Fgfr1 limb buds
(data not shown), suggesting that FGFR1 is not essential for Fgf10
expression. Of the three Bmp genes investigated above, Bmp2
expression remains normal, while Bmp4 and Bmp7 expression is
reduced only within Fgfr1-/- cells, suggesting
cell-autonomous regulation (Fig.
5I,J; data not shown).
The utility of Shhcre;Fgfr1 mutant limb buds in gene expression studies is best demonstrated by data on Hoxa13 and Hoxd13. In E11.5 Shhcre;Fgfr1 limb buds, we consistently detected reduced Hoxd13 expression in a wedge of cells in the anterior region of the Fgfr1-/- domain (n=5/5) (Fig. 5K,L). This suggests that FGFR1 cell-autonomously regulates the expression of Hoxd13, and that in the posterior Fgfr1-/- domain, its function may be redundant with other regulators of Hox expression. Interestingly, the expression of Hoxa13 is unaltered in Shhcre;Fgfr1 limb buds (data not shown), in contrast to our finding in Tcre;Fgfr1 limb buds. These data together demonstrate that the analysis of Shhcre;Fgfr1 limb buds has led to novel findings in FGF regulation of gene expression.
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The absence of a digit could be due to reduced growth or a defect in AP patterning. We uncovered evidence to support the latter hypothesis. In E11.5 Shhcre;Fgfr1 hindlimb buds, while limb bud size remains normal, only four condensations are detected by Sox9 expression (Fig. 7A,B). Compared with wild type, the two middle condensations in the mutant are each wider and farther apart than any of the wild-type digit pairs. The differences are more pronounced in E11.5 forelimb buds (Fig. 7C,D). These observations suggest that when starting with a mesenchymal field of equivalent size, the presence or absence of FGFR1 function can lead to a difference in the number of digits placed.
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Inactivating Fgfr1 affects Shh lineage
The deduction that digit 3 is absent in Shhcre;Fgfr1 mutant led us
to investigate the mechanism behind this defect. Two recent studies proposed
that the Shh-expressing cell lineage and the SHH-responsive cell
lineage are important for the identities of digits 2-5
(Ahn and Joyner, 2004;
Harfe et al., 2004
). In
particular, digit 3 forms at the anterior boundary of the
Shh-expressing lineage, and is proposed to require the participation
of Shh-expressing cells, as well as the influence of secreted SHH
signaling (Harfe et al.,
2004
). We found that a change in the Shh-expressing
lineage may account for the digit defect in the Shhcre;Fgfr1
mutant.
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Discussion |
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Mechanism of FGFR1 function in limb development
The combined data from Tcre;Fgfr1 and Shhcre;Fgfr1
mutants lead us to propose that signaling through FGFR1 impacts limb skeletal
formation in three phases (Fig.
8). In the early phase, FGFR1 is required for elongating the
nascent limb bud along the PD axis and restricting it along the other two
axes. In the middle phase, FGFR1 is required for mesenchymal cell survival. In
the late phase, FGFR1 is required for autopod patterning by influencing digit
placement and identity. The skeletal defect in Tcre;Fgfr1 mutant is
probably due to a combined cellular deficiency in all three phases of limb
development, while loss of one digit in Shhcre;Fgfr1 limbs is due to
lack of FGFR1 function in the late phase.
At the molecular level, the reduced expression of several key patterning
molecules, including Shh, Hoxa13 and Hoxd13, in the two
mutants may explain their zeugopod and autopod defects. Certain
Hoxa13;d13 homozygous/heterozygous combination mutants exhibit
reduction of digit number and size, similar to that of Tcre;Fgfr1
mutant (Fromental-Ramain et al.,
1996). Although not fully characterized, it is worth noting that
the Tcre;Fgfr1 hindlimb skeletal phenotype closely resembles that of
the Shh-/- mutant hindlimb
(Fig. 2D)
(Chiang et al., 2001
;
Kraus et al., 2001
). As
Shh expression is drastically reduced in the Tcre;Fgfr1
hindlimb buds (data not show), the phenotypic similarity is suggestive of a
causal relationship. Furthermore, as discussed in more detail below, the lack
of digit 3 in Shhcre;Fgfr1 mutant is probably due to the reduction of
Shh expression in these limb buds. We note that Tcre;Fgfr1
mutant exhibits stylopod reduction that is not observed in either
Shh-/- or 5'-Hox mutants. This
suggests that there are additional factors mediating FGFR1 function.
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Fgfr1 expressed in LBM is essential for cell survival
Similar excess cell death phenotypes are observed in Tcre;Fgfr1
mutant and in AER-Fgf mutants
(Boulet et al., 2004;
Lewandoski et al., 2000
;
Sun et al., 2002
), suggesting
that AER-FGF signaling mediated by FGFR1 is essential for mesenchymal cell
survival. In a wild-type limb bud, limited cell death is observed in the
proximal mesenchyme (Fig. 3O),
in agreement with previous findings (Dawd
and Hinchliffe, 1971
; Milaire
and Rooze, 1983
). We hypothesize that these cells die because they
are out of the range of AER-FGF signaling. This is supported by several lines
of evidence. First, beads soaked in FGF protein can rescue cell death
following AER removal, suggesting that FGF can maintain LBM cell survival
(Fallon et al., 1994
). Second,
secreted FGFs exhibit a limited range, possibly as a result of endocytosis and
subsequent degradation in lysosomes
(Scholpp and Brand, 2004
).
Third, using phosphorylated-ERK as an indicator of FGF signal activation, it
has been shown that only cells in the distal mesenchyme of the limb bud are
under the influence of FGF (Corson et al.,
2003
). This is confirmed by the expression patterns of Spry genes
and Mkp3, which are transcriptionally regulated by FGF signaling
(Eblaghie et al., 2003
;
Kawakami et al., 2003
;
Minowada et al., 1999
).
Consistent with our hypothesis, the expression pattern of Spry4
complements the observed cell death domain in normal limb buds (compare Fig.
3S with
3O). This limited range of FGF
pathway activation is likely due to restricted diffusion of the ligands, as
there is no evidence for a high-distal/low-proximal gradient in the expression
of Fgfr1 and Fgfr2 in LBM cells
(Orr-Urtreger et al., 1991
;
Peters et al., 1992
;
Yamaguchi et al., 1992
).
Based on our hypothesis, we would argue that the increase in proximal cell
death observed in Tcre;Fgfr1 and AER-Fgf mutant limb buds is
due to reduced FGF reception and reduced FGF signaling range, respectively,
leading to fewer proximal cells being protected from cell death compared with
wild type. This is supported by the reduced domain of Spry2,4 and
Mkp3 expression in these mutants
(Fig. 3T; X.S., unpublished).
Despite reduction, these indicators of FGF signaling are still expressed in
the distal mesenchymal cells of Tcre;Fgfr1 limb buds
(Fig. 3T; data not shown). This
may explain why cell death in Tcre;Fgfr1 limb buds is not detected in
the distal mesenchyme, unlike the situation following AER removal
(Dudley et al., 2002;
Rowe et al., 1982
). We
speculate that excess cell death in the Tcre;Fgfr1 limb buds
contributes to the later reduction of limb skeleton, in particular the
proximal elements.
FGFR1 influences digit number and identity
In both Tcre;Fgfr1 and Shhcre;Fgfr1 mutants, digit(s) are
missing. However, we propose that the mechanism leading to the loss of digits
is different in the two mutants. In the Tcre;Fgfr1 mutant, a
reduction in progenitor cell number is probably the main cause, as evidenced
by reduced limb bud size prior to digit condensation
(Fig. 4). By contrast, in the
Shhcre;Fgfr1 mutant, limb bud size remains normal when only four
instead of five digit condensations are observed
(Fig. 7A,B). This suggests that
FGFR1 influences the selection of cells from the mesenchyme that constitute
digit condensations.
We propose that this influence is achieved through FGFR1 regulation of
Shh expression. In the Shhcre;Fgfr1 mutant, the observed
downregulation of Shh RNA level would presumably result in reduced
SHH production. It has been shown that a reduction in SHH
production/distribution leads to loss of digits
(Lewis et al., 2001).
We propose that at a later stage, FGFR1 also influences the determination
of digit identity by regulating Shh expression. Recent studies of
Shh-expressing and SHH-responsive cell lineages suggest that these
are key parameters in the hypothesized rules of digit determination
(Ahn and Joyner, 2004;
Harfe et al., 2004
). As
discussed at the end of the Results section, based on these rules, the
observed reduction of Shh-expressing lineage in Shhcre;Fgfr1
limb buds may be responsible for the failure to form a normal digit 3 in this
mutant.
Fgfr1 is required for the expression of key genes in limb bud patterning
The Shhcre;Fgfr1 mutant is the first mutant in the FGF signaling
pathway that offers a rigorous setting to test FGF regulation of gene
expression during limb development. We report unequivocal evidence that FGF
signaling regulates Shh at the RNA level, providing genetic support
for the Fgf/Shh feedback loop
(Laufer et al., 1994;
Niswander et al., 1994
). In
addition, this mutant yields new data on whether FGFR1 regulates gene
expression in a cell-autonomous manner. For example, as it was shown that SHH
regulates the expression of Bmp genes
(Laufer et al., 1994
;
Yang et al., 1997
), it is
reasonable to hypothesize that FGFR1 regulates Bmp4 expression non
cell-autonomously through the regulation of Shh by FGFR1. However, in
Shhcre;Fgfr1 limb buds, the sharp boundary between
Bmp4-expressing and non-expressing cells corresponds well with the
anterior boundary of Fgfr1 inactivation (arrows in
Fig. 5H,J), suggesting that
FGFR1 cell-autonomously regulates Bmp4 expression, instead of acting
through secreted SHH.
The effectiveness of using Shhcre;Fgfr1 limb buds to assay gene expression is also demonstrated by our expression analysis of the paralogous group Hoxa13 and Hoxd13. Both genes are downregulated in Tcre;Fgfr1 limb buds (Fig. 4I-L), while only Hoxd13 is downregulated in Shhcre;Fgfr1 limb buds (Fig. 5L; data not shown). One possible explanation for this difference is that Fgfr1 may regulate Hoxd13 cell-autonomously and Hoxa13 non-cell-autonomously. Thus, the effect on Hoxa13 gene expression is evident only when Fgfr1 is inactivated in a larger domain in Tcre;Fgfr1 limb buds. Alternatively, Hoxa13 downregulation in Tcre;Fgfr1 limb buds could be largely due to reduction of the distal mesenchymal cell population that normally expresses Hoxa13. The combined gene expression data demonstrate that novel insights can be gained by revisiting FGF regulation of gene expression in Shhcre;Fgfr1 limb buds. As discussed above, Shh and 5'-Hox genes can mediate only a subset of FGFR1 function in limb skeleton formation. Our goal in the near future is to use Shhcre;Fgfr1 limb buds as a unique setting to identify other candidate mediators of FGF function in limb development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/19/4235/DC1
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ACKNOWLEDGMENTS |
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