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First published online 24 November 2005
doi: 10.1242/dev.02172
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1 Department of Anatomy and Program in Developmental Biology, School of
Medicinè University of California at San Francisco San Francisco, CA
94143-2711, USA.
2 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Case
Western Reserve University, School of Medicine, University Hospitals of
Cleveland, Cleveland, OH 44106, USA.
Author for correspondence (e-mail:
gmartin{at}itsa.ucsf.edu)
Accepted 27 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cutaneous syndactyly, FGF signaling, Limb development, Apical ectodermal ridge, SHH/FGF loop, Interdigital programmed cell death, Polydactyly, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Capdevila and Izpisua
Belmonte, 2001). In the mouse, AER formation begins at the stage
when the limb bud can first be discerned. It involves the initiation of gene
expression that later marks the mature AER, movement distally of cells within
the epithelium (particularly on the ventral side of the limb bud) and changes
in cell shape. After the AER matures into a narrow band of pseudostratified
columnar cells, it persists for 3-4 days while the limb bud enlarges and cells
in the underlying mesenchyme become determined to form the progenitors of the
skeletal elements and some of the other mesodermal cell types in the mature
limb. At the end of this period, cells in the AER die and the AER regresses
(Guo et al., 2003).
The importance of the AER has been well-established by experiments in the
chicken embryo showing that AER removal causes limb truncations
(Saunders, 1948;
Summerbell, 1974;
Rowe and Fallon, 1982), and
that this is due primarily to death of the underlying limb bud mesenchyme
(Rowe et al., 1982;
Dudley et al., 2002). The fact
that such limb truncations can be rescued by implanting beads soaked in FGF
proteins (Niswander et al.,
1993; Fallon et al.,
1994) indicates that an important function of the AER is to
produce FGFs. In addition to sustaining cell survival in limb bud mesenchyme
(Sun et al., 2002;
Boulet et al., 2004), FGF
signaling from the AER is thought to be an essential component of a positive
gene regulatory feedback loop that maintains the expression of sonic hedgehog
(Shh) and its downstream targets in the mesenchyme
(Laufer et al., 1994;
Niswander et al., 1994), and
also to regulate the expression of other genes vital for limb development
(Martin, 1998;
Capdevila and Izpisua Belmonte,
2001).
Of the 22 mouse genes that encode FGF ligands
(Itoh and Ornitz, 2004), four
(Fgf4, Fgf8, Fgf9 and Fgf17) are expressed in the AER but
not in other cells in the limb bud. Fgf8 is unique among these
`AER-FGFs' in several respects. Fgf8 expression is first detected in
AER progenitors in the ventral ectoderm, and subsequently along the entire
anteroposterior (AP) length of the AER until it regresses
(Crossley and Martin, 1995;
Mahmood et al., 1995). By
contrast, Fgf4, Fgf9 and Fgf17 expression commences only
after the AER has fully formed, is restricted to the posterior half to
two-thirds of the AER, and ceases at least 1 day before AER regression
(Sun et al., 2000).
Furthermore, gene knockout studies have shown that when each of the AER-FGFs
is individually inactivated, only loss of Fgf8 function causes limb
abnormalities (Lewandoski et al.,
2000; Moon and Capecchi,
2000); no defects have been detected in Fgf4
(Moon et al., 2000;
Sun et al., 2000),
Fgf9 (Colvin et al.,
2001) or Fgf17 (Xu et
al., 2000) null limbs. However, Fgf4 is essential in the
absence of Fgf8, because if both Fgf4 and Fgf8 are
never expressed in the limb bud, the limb does not form
(Sun et al., 2002;
Boulet et al., 2004).
One model to explain these observations is based on the premises that a certain amount of AER-FGF is required at each stage of limb development, and that the contributions made by FGF4 and FGF8 (and perhaps the other AER-FGFs) to the ligand pool are functionally similar. According to this model, there are two reasons why inactivation of Fgf8, but not of other individual AER-FGF genes, causes abnormalities in limb development. First, at some stages (e.g. in the early and late limb bud) Fgf8 is the only AER-FGF gene that is expressed and thus is contributing to the ligand pool. Second, at stages when all four AER-FGF genes are co-expressed in the posterior limb bud, Fgf8 is expressed more abundantly and more broadly than the others, and therefore makes a more substantial contribution to the ligand pool. In either case, when any other AER-FGF gene is individually is inactivated, the total amount of ligand in the pool does not fall below the level needed for normal limb development. However, when both Fgf4 and Fgf8 are completely inactivated, there is insufficient ligand to sustain limb development. An alternative model is that FGF8 has unique functions in limb skeletal patterning that FGF4 (and presumably the other AER-FGFs) cannot perform.
To distinguish between these models and to determine the consequences of increasing Fgf4 expression in the limb bud, we employed a conditional gain-of-function (GOF) approach. We found that activating an Fgf4GOF transgene in wild-type limb buds, in a domain that largely resembles that of Fgf8 in the normal limb bud, caused several abnormalities, including formation of a supernumerary posterior digit (postaxial polydactyly) and inhibition of programmed cell death in the interdigital mesenchyme, resulting in retention of tissue between the digits (cutaneous syndactyly); together these phenotypes are termed polysyndactyly. Activation of the Fgf4GOF transgene in an Fgf8-null limb bud still caused polysyndactyly, but it also rescued all the skeletal defects that result from loss of Fgf8 function. Together, these data support the hypothesis that AER-FGF signaling plays a role in regulating digit number and cell death in the interdigital mesenchyme, and that FGF4 and FGF8 perform similar functions in patterning the limb skeleton.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (kindly
provided by Dr Andras Nagy) with a fragment of a mouse cDNA containing the
entire Fgf4 open reading frame. The Fgf4 and loxP
segments of the resulting construct (Fig.
1A) were sequenced to confirm that no mutations had arisen during
vector construction.
The construct was electroporated into a subclone of E14Tg2a ES cells
(Hooper et al., 1987) isolated
by A. Smith (kindly provided to us by W. Skarnes). A total of 384
G418-resistant clones were isolated, and an aliquot of each clone was assayed
for ß-galactosidase (ß-gal) activity
(Lobe et al., 1999). Of these,
the 36 clones with the strongest and most uniform staining were selected for
further study. An aliquot of each was frozen, and another aliquot was expanded
and cultured under conditions appropriate for embryoid body formation
(Martin and Evans, 1975). To
obtain differentiation to a wide variety of cell types, the embryoid bodies
from each clone were allowed to attach to a tissue culture surface. After
8-10 days of culture, when differentiated cells including rhythmically
contracting myocardial cells were observed, the cells were fixed and stained
for ß-gal activity. Nine of the clones were chosen for further analysis
because their differentiated derivatives almost all displayed strong
ß-gal activity; in the remaining clones, ß-gal activity was either
weak or not detected in their differentiated derivatives. Chromosome analysis
was performed on six out of these nine clones, and more than 80% of the cells
examined for each clone were found to contain 40 chromosomes. When assayed by
Southern blotting using a variety of restriction enzymes, the transgene in all
of these six clones appeared to have inserted into a single site.
Two of these ES cell clones were injected into C57BL/6 blastocysts by the Stanford University transgenic facility, and germ-line transmission of the Fgf4GOF transgene was obtained from male chimeras produced from one of these cell lines. The mouse line thus established transmitted the transgene at Mendelian frequency. When crossed inter se, viable homozygotes were obtained, but they bred poorly. The Fgf4GOF transgene was maintained on a mixed genetic background.
Genotyping and phenotypic analysis
The genotypes of embryos and adult mice were determined by PCR assays using
DNA extracted from embryonic yolk sacs or tails as a template. The presence of
the Fgf4GOF allele was detected using primers that amplify
the lacZ sequence in the transgene
(5'-GTCTCGTTGCTGCATAAACC-3' and
5'-TCGTCTGCTCATCCATGACC-3'). Genotyping for Fgf8 alleles
and for the presence of the Msx2-cre transgene was performed as
previously described (Sun et al.,
2002).
Embryos were collected in cold PBS, fixed in 4% paraformaldehyde and stored
in 70% methanol at -20°C. Noon of the day when a vaginal plug was detected
was considered approximately embryonic day (E) 0.5. 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. Standard protocols were employed for RNA in situ hybridizations and
skeletal preparations. Assays for cell death were performed in whole mount, by
staining with LysoTracker (Molecular Probes L-7528)
(Grieshammer et al.,
2005).
Quantification of Fgf4 RNA
Limb buds were dissected from 5
Msx2-cre;Fgf4GOF and two control
(Msx2-cre hemizygous) embryos at E10.5 and preserved in RNAlater
(Ambion, 7020). Each sample contained either the two forelimb buds or the two
hindlimb buds from an individual embryo. Total RNA was isolated using an
RNeasy Kit (Qiagen 74104). First-strand cDNA synthesis was performed by the
Biomolecular Resource Center at UCSF using a Bio-Rad kit (170-8891).
Quantitative PCR was performed using an ABI Prism 7900 HT Sequence Detection
System. The primers amplified a 57 bp sequence spanning sequences at the
3' end of exon 2 to sequences at the 5' end of exon 3 of the
Fgf4 gene: forward primer 5'-GAAAGGCACACCGAAGAGCTT-3' and
reverse primer 5'-GCCAGCCGGTTCTTCGT-3'. The sequence of the
Fgf4 TaqMan probe was 5'-CCTGCTGCTCATAGC-3'.
Gapdh sequences were also amplified and the amount of product was
used to normalize each sample.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), drives
expression of a floxed (flanked by loxP sites) ß-Geo
reporter gene in most or all cells of the embryo
(Fig. 1B, and data not shown).
After Cre-mediated recombination, which excises the intervening floxed
ß-Geo sequences, the promoter drives expression of an
Fgf4 cDNA (Fig. 1A) in
the cells in which recombination occurred and their descendants, presumably
for as long as they survive. Fgf4GOF mice were crossed to
animals carrying Msx2-cre, a transgene that expresses cre in
early limb bud ectoderm, as well as in other tissues of the embryo. We
previously used Msx2-cre to inactivate Fgf4
(Sun et al., 2000),
Fgf8 (Lewandoski et al.,
2000), and both Fgf4 and Fgf8
(Sun et al., 2002) during limb
development. At birth, Msx2-cre;Fgf4GOF offspring were
present at Mendelian frequency and were viable, but generally grew more slowly
than did their wild-type littermates. All Msx2-cre;Fgf4GOF
animals displayed limb abnormalities, as described below, as well as
hyperplasia of the eyelids, malformations of the external genitalia and other
defects (not shown). These animals carrying the activated transgene (A-Tg) are
described here in comparison with their `normal' littermates, which were
wild-type or carried either Msx2-cre or Fgf4GOF,
but not both transgenes.
During normal limb development, Fgf4 is expressed only after the
AER has fully formed, and therefore Fgf4 RNA is not detected until
the 29- to 30-somite stage (ss) (E10.0) in forelimb (FL) and 33-34 ss
(E10.25) in hindlimb (HL) buds (Sun
et al., 2000) (Fig.
1D,F,H,J). At these stages and later, Fgf4 expression is
normally restricted to the posterior two-thirds of the AER
(Niswander and Martin, 1992)
(Fig. 1H,L,M), and ceases by
E11.5 in FL and E12.5 in HL buds
(Lewandoski et al., 2000)
(Fig. 1N-Q). By contrast, in
Msx2-cre;Fgf4GOF embryos, as a result of
Msx2-cre activity in the early limb bud
(Sun et al., 2000),
Fgf4 RNA was already detected at 28 ss in FL and 30 ss in HL buds, in
a domain that was abnormally extended anteriorly
(Fig. 1E,G,I,K, and data not
shown). At E10.5, Fgf4 expression was detected throughout the
entire AP length of the AER (Fig.
1L,M). Moreover, Fgf4 expression persisted for at least 1
day longer than normal, until the AER itself regressed
(Fig. 1N-S). Together, these
data show that in Msx2-cre;Fgf4GOF limb buds,
Fgf4 expression is initiated earlier, extends throughout a longer
domain and persists for longer than in normal limb buds. In these respects,
the expression of Fgf4 in
Msx2-cre;Fgf4GOF limb buds was more similar to
that of Fgf8 than Fgf4 in normal limb buds.
We detected one significant difference between Fgf4 expression in
Msx2-cre;Fgf4GOF limb buds and the normal
expression patterns of both Fgf4 and Fgf8. In the normal
embryo, Fgf4 RNA is never detected in ventral limb bud ectoderm, and
Fgf8 RNA is detected in that domain only transiently, presumably in
AER progenitors before they move distally and become incorporated into the
nascent AER (Guo et al.,
2003). By contrast, because Msx2-cre is active in a few
cells that remain in the ventral ectoderm
(Barrow et al., 2003),
Fgf4 RNA was detected at a low level in the ventral ectoderm of
Msx2-cre;Fgf4GOF limb buds from early stages
until at least E10.5 and presumably longer
(Fig. 1I; data not shown).
To quantify the increase in Fgf4 expression in
Msx2-cre;Fgf4GOF limb buds at E10.5, we performed
a quantitative RT-PCR assay (see Materials and methods). The level of
Fgf4 RNA was found to be 3.5-fold higher in FL and twofold higher in
HL buds from Msx2-cre;Fgf4GOF embryos than from
control (Msx2-cre hemizygous) embryos
(Fig. 1C). From our in situ
hybridization data, it is clear that at this stage, some of the Fgf4
expressed by the activated Msx2-cre;Fgf4GOF transgene is
localized in two ectopic domains, i.e. regions in which Fgf4 is never
expressed in the normal limb bud: one in the anterior AER, where Fgf8
is normally expressed, and the other in the ventral ectoderm, where AER-FGF
gene expression is not normally detected. Because Msx2-cre is known
to function throughout the AER (Sun et
al., 2000), the remainder of the Fgf4 produced by the
activated Fgf4GOF transgene is presumably localized within
the normal Fgf4 expression domain in the posterior AER. However,
expression of Fgf4 in this domain occurs both earlier and for longer
than normal in Msx2-cre;Fgf4GOF limb buds.
Effects of activating Fgf4GOF on the AER-FGF/Sonic Hedgehog positive feedback loop
Before assessing the effects of the excess/ectopic Fgf4 expression
on limb morphogenesis, we examined Msx2-cre;Fgf4GOF limb
buds to determine whether the AER itself or the expression of other AER-FGF
genes was perturbed. Fgf8 expression appeared normal in
Msx2-cre;Fgf4GOF limb buds at E10.5 and E11.5
(Fig. 2A-B', and data not
shown), indicating that the AER, both with respect to its length along the AP
axis and its width along the dorsoventral (D-V) axis, was normal. The
expression of the other AER-FGF genes, Fgf9 and Fgf17, also
appeared similar in Msx2-cre;Fgf4GOF and normal limb buds
(data not shown). Significantly, we found that at E13.5, Fgf8
expression was detected in both normal and
Msx2-cre;Fgf4GOF limb buds in a thin domain at the distal
tip of each of the developing digits, but not in the regions overlying the
interdigital mesenchyme (Fig.
2C,D, and data not shown). These observations indicate that the
AER regresses at the same stage in
Msx2-cre;Fgf4GOF and normal limb buds. One
consistent difference we observed was that by E11.5 the limb buds of the
Msx2-cre;Fgf4GOF embryos were slightly larger than those
of their normal littermates (Fig.
1N-Q).
As signaling by AER-FGFs is thought to influence outgrowth and patterning
of the limb bud mesenchyme, at least in part via its contribution to a
positive feedback loop involving Sonic hedgehog (Shh) and its
downstream target Gremlin (Grem1)
(Laufer et al., 1994;
Niswander et al., 1994;
Zuniga et al., 1999;
Khokha et al., 2003;
Michos et al., 2004;
Scherz et al., 2004), we next
investigated whether Shh or Grem1 expression was affected in
Msx2-cre;Fgf4GOF limb buds. We found that both
Shh and Grem1 expression patterns appeared normal at
E10.5 (Fig. 2E-H).
However, at E11.5 we detected a significant abnormal expansion of the
Shh expression domain both ventrally and especially proximally, with
the effect being much more pronounced in HL than in FL buds
(Fig. 2I,J). Grem1
expression was similarly affected, but to a lesser extent
(Fig. 2K,L). Analysis of
sections through the limb buds demonstrated that these ectopic Shh
and Grem1 expression domains were localized exclusively in the
mesenchyme (data not shown). Such ectopic mesenchyme gene expression in the
Msx2-cre;Fgf4GOF limb buds might be due to either excess
FGF4 produced in the AER, or more likely to ectopic Fgf4 expression
in the ventral ectoderm (Fig.
1I; data not shown), because the expansion of the Shh and
Grem1 expression domains was detected in ventral but not dorsal limb
bud mesenchyme, and was not observed in the distal-most region near the
AER.
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; Akiyama et al.,
2002), showed that as early as E12.5, a supernumerary skeletal
condensation was present posterior to the condensation representing digit V
(Fig. 3G-J). At this stage,
there were no obvious supernumerary condensations on the anterior ventral side
of Msx2-cre;Fgf4GOF limb buds. These data indicate that
formation of the supernumerary postaxial digit occurred during limb bud stages
(prior to the regression of the AER).
Activation of Fgf4GOF results in persistence of interdigital tissue
Another abnormality observed in Msx2-cre;Fgf4GOF
autopods was the presence of soft tissue between the digits
(Fig. 4A-D), a phenotype
presumably caused by a lack of the interdigital programmed cell death (IPCD)
that normally occurs in the mouse limb at E12.5-13.5. To confirm this, we
assayed Msx2-cre;Fgf4GOF embryos at E13.5 by staining
with LysoTracker, which labels acidic compartments (lysosomes) within
apoptotic cells themselves as well as in healthy cells that are engulfing
apoptotic debris (Zucker et al.,
1999; Schaefer et al.,
2004). In hindlimbs of normal embryos, intense LysoTracker
staining was observed in the interdigital mesenchyme
(Fig. 4E,E'); by
contrast, we saw little or no LysoTracker staining in the hindlimbs of their
Msx2-cre;Fgf4GOF littermates
(Fig. 4F,F'). These data
suggest that abnormally prolonged expression of Fgf4 in the AER (see
Fig. 1N-Q), or possibly a low
level of ectopic expression in the ventral ectoderm, prevents cell death in
the interdigital mesenchyme of Msx2-cre;Fgf4GOF
embryos.
There is an extensive literature indicating that IPCD in the chicken embryo
is mediated by BMP signaling and showing that IPCD can be prevented by ectopic
expression of BMP antagonists
(Zuzarte-Luis and Hurle,
2005). To determine whether the observed lack of IPCD in
Msx2-cre;Fgf4GOF limb buds was correlated with changes in
the level of expression of genes involved in BMP signaling, we assayed for
Bmp2 and Bmp7, two members of the BMP family known to be
expressed in the interdigital mesenchyme
(Hogan, 1996), for
Msx2, which is thought to be a downstream target of BMP signaling in
the limb (Pizette et al.,
2001), and for Grem1, which encodes an antagonist of BMP
signaling in the limb (Hsu et al.,
1998; Khokha et al.,
2003; Michos et al.,
2004). In situ hybridization assays showed no reproducible
difference in the expression of these genes in
Msx2-cre;Fgf4GOF when compared with normal limb buds at
E12.5 (Fig. 4G-J),
approximately 1 day earlier than the stage at which IPCD was detected in
normal but not in Msx2-cre;Fgf4GOF hindlimb buds. These
data suggest that excess/ectopic Fgf4 expression prevents IPCD via a
mechanism that does not involve changes in the expression of genes involved in
BMP signaling.
Activation of Fgf4GOF rescues the skeletal defects caused by loss of Fgf8 function
Finally, we sought to determine whether the Fgf4 expressed by the
activated Fgf4GOF transgene can functionally replace
Fgf8 expression in the limb bud. We crossed male mice carrying both
Msx2-cre and an Fgf8 null allele,
Fgf8
, to females carrying an Fgf8
conditional allele, Fgf8flox, and
Fgf4GOF, to generate
Msx2-cre;Fgf8/flox;Fgf4GOF
mice. In these animals, Msx2-cre-mediated recombination inactivates
Fgf8 by converting Fgf8flox to a null allele and
concomitantly activates Fgf4 expression from the
Fgf4GOF transgene.
Msx2-cre;Fgf8/flox littermates
from this cross displayed an Fgf8 loss-of-function phenotype similar
to, but somewhat less severe than that previously described
(Lewandoski et al., 2000): the
FL stylopod was usually mildly hypoplastic and missing the deltoid tuberosity
(Fig. 5A,E), whereas the HL
stylopod was often severely reduced (Fig.
5B,F); the zeugopod elements were mildly hypoplastic
(Fig. 5A,B,E,F); and the
autopod was frequently missing a digit: digit II or III in the forelimb
(Fig. 5C,G, and data not shown)
and digit I in the hindlimb (Fig.
5D,H). In addition, FL digits I and V and HL digits II and V were
frequently missing a phalanx (Fig.
5C,D,G,H; data not shown).
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/flox;Fgf4GOF skeletons.
Thus, the deltoid tuberosity was always present
(Fig. 5I), the femur appeared
normal (Fig. 5J), and digits
I-V were all present in both the FL and HL autopod and contained the normal
number of phalanges (Fig.
5K,L). However, all
Msx2-cre;Fgf8/flox;Fgf4GOF limbs
examined (n=7) still displayed the abnormalities observed when the
Fgf4GOF transgene was activated by Msx2-cre in
embryos that were wild type for Fgf8
(Fig. 3), including enlargement
of the calcaneus and a supernumerary posterior digit
(Fig. 5I-L), as well as
cutaneous syndactyly (data not shown). It is not clear precisely which aspects
of the Msx2-cre-activated Fgf4GOF expression
pattern are responsible for each of these specific abnormalities (see
Discussion). What is most important about these data is that they clearly
demonstrate that Fgf4 is capable of performing the functions of
Fgf8 that are essential for normal limb skeletal development. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AER-FGF function in skeletal patterning
Based on the results of AER-FGF gene inactivation studies, it has been
proposed that a key role of AER-FGF signaling is to establish the number of
chondrocyte progenitors available to form limb skeletal elements
(Sun et al., 2002), as a
result of an anti-apoptotic effect and/or stimulation of cell proliferation.
Our data, showing that the excess/ectopic Fgf4 expression in
Msx2-cre;Fgf4GOF limb buds results in the formation of
supernumerary skeletal elements, are consistent with this hypothesis. However,
because the normal pattern of Fgf4 expression is altered in several
ways in Msx2-cre;Fgf4GOF limb buds (including increased
and prolonged expression in the posterior AER, and ectopic expression in the
anterior AER and ventral ectoderm), we can only speculate on the source of the
FGF4 that causes the observed defects in the autopod skeleton.
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;
MacDonald et al., 2004) or in
which the BMP antagonist Noggin is ectopically expressed in the AER
(Wang et al., 2004) display an
increase in AER-FGF expression and have a supernumerary digit on the posterior
side. However, if the supernumerary posterior digit in
Msx2-cre;Fgf4GOF limbs is indeed due to excess FGF
expression in the AER, then why does an extra digit still form in
Msx2-cre;Fgf8/flox;Fgf4GOF
limbs, which lack Fgf8 expression? A possible explanation comes from
the observation that in the absence of Fgf8 function in the AER,
endogenous Fgf4 expression is significantly upregulated
(Lewandoski et al., 2000;
Moon and Capecchi, 2000),
although obviously not to the level needed for formation of the normal
complement of skeletal elements. Thus, we suggest that the AER-FGF produced by
the expression of the activated Fgf4GOF transgene in
combination with the normal levels of endogenous Fgf4 and
Fgf8 expression in Msx2-cre;Fgf4GOF limbs or in
combination with upregulated endogenous Fgf4 expression in
Msx2-cre;Fgf8/flox;Fgf4GOF limb buds, is
sufficient to produce six digits.
We were surprised to find that Msx2-cre;Fgf4GOF limbs
did not contain one or more supernumerary digits on the anterior side of the
limb, because there are numerous mouse mutants in which Fgf4
expression in the anterior AER is correlated with such preaxial polydactyly
(Masuya et al., 1995;
Qu et al., 1997;
Hayes et al., 1998;
Yang et al., 1998;
Yada et al., 2002;
Zhang et al., 2003). However,
in those mutants the anterior expansion of the Fgf4 expression domain
appears to be a secondary response to ectopic Hedgehog pathway activity in the
anterior mesenchyme (Talamillo et al.,
2005; Liu et al.,
2005). As yet, there are no data addressing the issue of whether
such ectopic Fgf4 expression in the anterior AER is necessary for the
formation of the supernumerary preaxial digit(s).
Together, the data suggest a model in which excess AER-FGF signaling per se
promotes the formation of a supernumerary digit on the posterior side,
possibly by increasing SHH signaling or by expanding the number of descendants
of Shh-expressing cells, which are known to contribute to posterior
digits (Ahn and Joyner, 2004;
Harfe et al., 2004). By
contrast, changes in the expression of other genes, such as those that cause
ectopic Hedgehog pathway activity in anterior mesenchyme, are required for
supernumerary digit formation on the anterior side. According to this model,
the postaxial and preaxial polydactyly reported in mouse hindlimbs in which
Bmp4 has been conditionally inactivated in the mesenchyme
(Selever et al., 2004) can be
explained by two separate effects: the supernumerary posterior digit forms in
response to an observed increase in AER-FGF gene expression resulting from
expansion of the AER along the DV axis, and the additional supernumerary
anterior digit forms as a consequence of other changes in gene expression in
the anterior mesenchyme. In this context it is interesting that in embryos
null for both Msx1 and Msx2, which are thought to be
required for BMP signaling, preaxial polydactyly seems to correlate with a
very small ectopic anterior domain of Shh expression
(Lallemand et al., 2005).
However, it is important to note that anterior mesenchyme is capable of
forming supernumerary digits in response to FGF signaling, as for example in
transgenic mice expressing a hypermorphic allele of Fgfr1
(Hajihosseini et al., 2004),
and in chicken limb buds implanted with cells producing FGF2
(Riley et al., 1993).
Curiously, in the latter experiments, posterior mesenchyme showed no response
to FGF2-producing grafts. Further studies will be needed to elucidate the
mechanism(s) that regulate digit number and determine the location(s) at which
supernumerary digits form.
AER-FGF function in regulating interdigital programmed cell death
In addition to abnormalities in skeletal pattern,
Msx2-cre;Fgf4GOF limbs have cutaneous syndactyly between
all the digits. As in many mouse mutants, such syndactyly correlates with a
lack of interdigital programmed cell death (IPCD)
(Zuzarte-Luis and Hurle,
2005). A potential link between AER-FGF signaling and IPCD is
suggested by the hypothesis that AER-FGFs provide proliferation/survival
factors for interdigital mesenchyme cells
(Macias et al., 1996), and
that under normal circumstances IPCD is triggered by the loss of these factors
when the AER regresses. Thus, a delay in AER regression should delay or block
IPCD and result in persistence of interdigital tissue. Support for this model
comes from gain-of-function experiments in both chicken limb buds and
transgenic mice, showing that ectopic expression of the BMP antagonist Noggin
results in a delay in AER regression and cutaneous syndactyly
(Pizette and Niswander, 1999;
Wang et al., 2004). Based on
those studies, it was proposed that during normal limb development, one role
of BMP signaling is to promote AER regression. In support of this hypothesis,
mice that lack both Msx1 and Msx2 function
(Lallemand et al., 2005), or
in which Bmpr1a has been inactivated in the AER (M. Lewandoski,
personal communication), display abnormal AER regression and persistence of
interdigital tissue.
However, AER regression does not appear to be delayed in
Msx2-cre;Fgf4GOF limb buds, and other effects besides
delay of AER regression are observed in mouse limb buds lacking
Msx1/Msx2 function, lacking Bmpr1a function in the AER, or
with ectopic Noggin expression in the AER. In particular, the AER is expanded
along its DV axis (Wang et al.,
2004; Lallemand et al.,
2005) (M. Lewandoski, personal communication), raising the
possibility that excess/ectopic AER-FGF expression from the expanded AER,
rather than a delay in AER regression per se, accounts for the observed
persistence of interdigital tissue in those limb buds. If so, then the
cutaneous syndactyly we observed in Msx2-cre;Fgf4GOF limbs
might likewise be explained by excess/ectopic Fgf4 expression in the
AER, or perhaps in the ventral ectoderm. Such FGF signaling might directly
oppose the pro-apoptotic effects of BMP signaling
(Yokouchi et al., 1996;
Zou and Niswander, 1996;
Macias et al., 1997), as
suggested by studies showing that implanting BMP4-soaked beads into
interdigital mesenchyme causes cell death and this is prevented by
co-implanting FGF2-soaked beads (Ganan et
al., 1996). A more complex relationship between FGF and BMP
signaling in the control of IPCD has also been suggested
(Montero et al., 2001).
Alternatively, excess FGF signaling might inhibit BMP activity via effects on
genes involved in BMP signaling. However, we saw no effect on the expression
of Bmp2, Bmp4, Msx2 or Grem1 in
Msx2-cre;Fgf4GOF limb buds just prior to the stage when
IPCD normally occurs. Further studies will be needed to unravel the
potentially complex interactions between FGF and BMP signaling in the
regulation of IPCD.
FGF4 can replace FGF8 during vertebrate limb development
Although FGF4 and FGF8 belong to different subgroups of the FGF family
(Itoh and Ornitz, 2004) and
share only 32% identity in the most conserved core domain
(Coulier et al., 1997), they
have been found to have similar activities in various developmental settings.
For example, in the chicken embryo, beads soaked in recombinant FGF4 or FGF8
can induce the formation of an ectopic limb
(Martin, 1998) and induce
cells in the diencephalon to change fate and develop into a midbrain
(Crossley et al., 1996).
However, during gastrulation in the chicken embryo, primitive streak cells are
attracted to a source of FGF4 and repelled by a source of FGF8
(Yang et al., 2002),
indicating that these two FGF family members do not necessarily perform the
same function in all developmental contexts. Genetic analyses in the mouse
have left open the issue of whether FGF4 and FGF8 have similar or different
functions in limb development.
To resolve this issue, we employed a novel approach to assess whether one
gene can functionally replace another in the mouse, rather than the
conventional knock-in strategy (Hanks et
al., 1995), in which the sequence of one gene is inserted into the
genomic locus of a second, thereby inactivating the second gene and expressing
the first in its place. Instead, we replaced the expression of one gene
(Fgf8) with that of another (Fgf4) by using a single
cre transgene to concomitantly inactivate and activate conditional
Fgf8 loss- and Fgf4 gain-of-function alleles, respectively,
in the same cells. The knock-in strategy has the advantage that the gene that
has been inserted should faithfully recapitulate the expression of the gene it
is meant to replace, although this is sometimes difficult to achieve if
regulatory elements that control the expression of the gene being replaced are
perturbed. Using our strategy, the expression of the gain-of-function target
gene may ultimately be broader than the gene expression it replaces, because
it continues in all descendants of the cells in which it was initially
activated. That was indeed the case in our study, as Fgf4 expression,
which is driven by the promoter in the transgene, persisted in the limb bud
ventral ectoderm, whereas Fgf8 expression, although initially
detected in that domain, normally becomes restricted to the AER. As discussed
above, such discrepancies between the expression patterns of the two genes can
make interpretation of the data difficult. However, our strategy has some
distinct advantages. First, an individual conditional gain-of-function
transgene can be readily used in combination with various conditional
loss-of-function alleles to study the functional relationships between
different members of a multigene family. Second, the conditional
gain-of-function transgene can be activated without inactivating any other
gene, allowing analysis of the consequences of excess or ectopic expression of
the gain-of-function target gene, in our case Fgf4 in the limb bud.
In future, Fgf4GOF could be activated in a variety of
developmental settings using appropriate cre transgenes to gain
further insights into the function of FGF signaling.
Here we made use of the Fgf4GOF transgene to show that all of the limb skeletal defects caused by loss of Fgf8 function can be rescued by increasing Fgf4 expression in the limb bud in a pattern that largely resembles that of Fgf8 in normal limb buds. This demonstrates the utility of our strategy, and more importantly, provides conclusive evidence that FGF4 can functionally replace FGF8 in limb skeletal patterning. Furthermore, our data emphasize the importance of mechanisms that control FGF gene expression for obtaining normal digit number and appropriate separation of the digits.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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