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First published online May 30, 2007
doi: 10.1242/10.1242/dev.001677
1 Laboratory of Cancer and Developmental Biology National Institutes of Health,
Frederick, MD 21702, USA.
2 SAIC, NCI-Frederick, National Institutes of Health, Frederick, MD 21702,
USA.
3 Laboratory of Reproductive and Developmental Toxicology, National Institute of
Environmental Health Sciences, Research Triangle Park, NC, USA.
* Author for correspondence (e-mail: mlewandoski{at}mail.ncifcrf.gov)
Accepted 10 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Apoptosis, BMP, FGF, Interdigit, Limb development, Programmed cell death
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Niswander,
2002; Tickle,
2006).
Vertebrate limbs begin as buds of the lateral plate mesoderm, jacketed in
surface ectoderm. In the mouse, at about embryonic day (E) 10.5 and 11.0 in
the forelimb and hindlimb bud, respectively, cells have finished moving from
the ventral ectoderm of the early bud (the `preAER') to the bud apex to form a
stratified columnar epithelial structure called the apical ectodermal ridge
(AER) (Kimmel et al., 2000;
Loomis et al., 1998;
Guo et al., 2003). Although
the AER itself is fated to undergo cell death, it is a source of secreted
factors required for limb bud outgrowth. This is demonstrated by the surgical
removal of the AER in chick embryos, which results in proximal-distal
truncations (Dudley et al.,
2002; Saunders,
1948; Summerbell,
1974).
The AER-specific activity required for limb bud outgrowth is encoded by
members of the FGF family (reviewed by
Martin, 1998). FGF protein can
rescue limb development in chick embryos that have a surgically removed AER
(Fallon et al., 1994;
Niswander et al., 1993). Four
Fgf genes (Fgf4, 8, 9 and 17, referred to here collectively
as AER-FGFs) are expressed in the AER
(Martin, 1998;
Sun et al., 2000). The
Fgf8 expression domain is the largest spatially, being strongly
expressed throughout the anterior-posterior extent of the AER, and is also the
longest temporally, occurring earliest, in the preAER
(Lewandoski et al., 2000), and
ending during AER regression (E12.5-13.5)
(Crossley et al., 1996;
Mahmood et al., 1995;
Salas-Vidal et al., 2001). By
contrast, the other three AER-FGF genes display more restricted expression
domains within the posterior two-thirds of the AER, with expression initiating
later and ceasing earlier than Fgf8
(Sun et al., 2000). In the
mouse, single-gene inactivation studies reveal that, of the four AER-FGFs,
only Fgf8 is required for normal limb development, although most limb
skeletal elements still form in limb buds lacking Fgf8
(Colvin et al., 2001;
Lewandoski et al., 2000;
Moon et al., 2000;
Moon and Capecchi, 2000;
Sun et al., 2000;
Xu et al., 2000). However,
when Fgf4 and 8 are simultaneously inactivated throughout
the preAER, mice are born without limbs, indicating that these two Fgf genes
are genetically redundant (Sun et al.,
2002; Boulet et al.,
2004). Thus, Fgf4 and 8 may be considered the
principal AER-FGFs.
Another set of molecules playing a role in limb development are the members
of the bone morphogenetic protein (BMP) family, which, in contrast to FGFs,
have been implicated in a variety of diverse aspects of limb development
including digit identity, anterior-posterior and dorsal-ventral patterning of
the limb and AER regulation (Niswander,
2002; Tickle,
2006). Early in limb bud development, BMP signaling is required
for normal formation of the AER (Ahn et
al., 2001; Pizette et al.,
2001). As the AER develops, regulation of BMP activity by the
antagonist gremlin is required to activate Fgf4, 9 and 17
(Capdevila et al., 1999;
Khokha et al., 2003;
Michos et al., 2004;
Zuniga et al., 1999;
Zuniga et al., 2004).
Later in limb development, BMPs also play a role in programmed cell death
(PCD) of the interdigit region, which is required to separate digits and
prevent soft tissue syndactyly (webbing) (reviewed by
Chen and Zhao, 1998;
Zuzarte-Luis and Hurle, 2002;
Zuzarte-Luis and Hurle, 2005).
Prior to cell death, Bmp2, 4 and 7 are upregulated within
the interdigit mesenchyme (Chen and Zhao,
1998; Hogan, 1996)
and inhibition of BMP signaling in chick embryos, via the retroviral
expression of transgenes encoding dominant-negative BMP receptors, inhibits
interdigit PCD (Yokouchi et al.,
1996; Zou and Niswander,
1996). Furthermore, in the mouse, syndactylous limbs are caused by
inhibiting BMP signaling through the ectopic expression of the BMP antagonist
noggin (Guha et al., 2002;
Wang et al., 2004).
These data are generally construed to signify that BMPs act as direct
effectors of PCD within the interdigit region
(Chen and Zhao, 1998;
Zou and Niswander, 1996;
Zuzarte-Luis and Hurle, 2002;
Zuzarte-Luis and Hurle, 2005).
However, FGF-soaked beads can prevent interdigital PCD in chick embryos
(Buckland et al., 1998;
Ganan et al., 1996;
Macias et al., 1996;
Ngo-Muller and Muneoka, 2000),
suggesting that interdigital PCD might be induced by the loss of AER-FGF
signaling that occurs during AER regression
(Salas-Vidal et al., 2001;
Yokouchi et al., 1996;
Zou and Niswander, 1996). On
the other hand, application of FGF-soaked beads can also increase interdigital
apoptosis, consistent with a model of cooperation between FGFs and BMPs acting
as cell death triggers (Ganan et al.,
1998; Montero et al.,
2001). Thus, the relationship between BMP and FGF signaling during
interdigital PCD is unclear.
Here, we define a relationship between BMP and FGF signaling during interdigital PCD. First, we inactivate the gene encoding the BMP receptor, Bmpr1a, specifically in the limb bud preAER or AER via Cre-mediated DNA recombination. Our data reveal a dynamic role for BMPR1A signaling: early BMP activity is required for AER formation and later activity is required for cessation of AER-FGF expression, which is correlated with interdigital PCD. Then, by analyzing mice carrying different combinations of double and triple AER-specific gene inactivations of Fgf4, 8 and Bmpr1a, we provide genetic evidence that BMPs regulate interdigit cell death through the regulation of AER-FGF signals.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Mishina et al., 2002;
Mishina et al., 1995;
Sun et al., 2000). Mutants
were generated on a mixed CD1/129SvEv/C57BL/6 background. Msx2-Cre;
Bmpr1aflox/null mice were produced as described in
Fig. 1. Msx2-Cre;
Bmpr1aflox/null mutants that also carry mutated Fgf4
(or Fgf8) alleles were generated by mating
BmpR1aflox/flox; Fgf4flox/flox (or
BmpR1aflox/flox; Fgf8flox/flox)
females with Msx2-Cre; BmpR1anull/wt;
Fgf4null/wt (or with Msx2-Cre;
Fgf8null/wt) males. Likewise, to generate triple mutants,
BmpR1aflox/flox; Fgf4flox/flox,
Fgf8flox/flox females were mated with Msx2-Cre;
BmpR1anull/wt; Fgf4null/wt, Fgf8null/wt)
males. Noon of the day on which the vaginal plug was detected was considered
E0.5. More precise age of E9.5-10.5 embryos was determined by counting the
somites posterior to the forelimb, scoring the first as somite 13.
Whole-mount in situ hybridization, ß-galactosidase (ß-gal) and Lysotracker staining
RNA localization by whole-mount in situ hybridization was performed as
described (Pizard, 2004), with
a minor change: detection of AER transcripts required no proteinase K
treatment. ß-gal and Lysotracker Red (Molecular Probes) staining was
performed as previously described (Chi et
al., 2003; Zucker et al.,
1999).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) will inactivate the Bmpr1aflox
allele. In the course of performing the required genetic crosses, we
discovered that the Msx2-Cre transgene is integrated on chromosome 14, very
closely linked to the Bmpr1a locus. Therefore, in order to generate
mutant mice, we needed to first produce mice carrying both the Msx2-Cre
transgene and the Bmpr1anull allele in cis on chromosome
14. To achieve this, we screened approximately 1300 progeny of Msx2-Cre;
Bmpr1anull/+ mice crossed to wild-type mice before identifying
one offspring with a recombinant chromosome 14 containing both genetic
markers. This mouse and its progeny served as sires in the genetic cross
indicated in Fig. 1A, in which
50% of the offspring are mutant and 50% (Bmpr1aflox/+) are
controls.
We and others (Barrow et al.,
2003; Sun et al.,
2000) have previously shown that Msx2-Cre is active earlier in the
prospective AER cells of the hindlimb region where expression is widespread by
the 24-somite stage (ss), as compared with the forelimb where extensive
expression occurs by 28ss. To determine whether the kinetics of
Bmpr1a inactivation followed this pattern, we performed RNA in situ
hybridization (ISH) on mutant embryos using a riboprobe derived from those
sequences in the Bmpr1aflox allele deleted by Cre.
Although we could detect Bmpr1a expression at E10.5 in the control
forelimb (including the AER) and demonstrate Bmpr1a inactivation in
the mutant AER (Fig. 1A,
inset), we could not reproducibly detect Bmpr1a expression at limb
bud stages prior to AER formation. However, using a full-length
Bmpr1a riboprobe, we were able to detect expression in the preAER
(data not shown), although this riboprobe cannot discriminate between mRNA
transcribed from the unrecombined floxed allele and the recombined derivative.
Therefore, we conclude that Bmpr1a is expressed in the limb bud
surface ectoderm/preAER, but below detectable levels when using the floxed
Bmpr1a sequences as an ISH riboprobe, and we confirm a previous
report that these sequences function inadequately as a riboprobe in ISH
analysis (Liu et al.,
2005).
As an alternative measure of whether the kinetics of Msx2-Cre activity
occurred as expected, we stained for the Cre-dependent ß-gal activity of
the R26R reporter allele
(Soriano, 1999) in
Msx2-Cre; Bmpr1aflox/null; Rosa26R26R/+
embryos. As expected, ectoderm in the mutant hindlimb territory was
extensively recombined by 24ss, whereas the mutant forelimb preAER was
extensively recombined by 28ss (Fig.
1B,C).
Because Msx2-Cre is active earlier in the prospective hindlimb ectoderm relative to forelimb Cre activity, we expected a more severe hindlimb mutant phenotype. We found that in all skeletal preparations examined, mutant hindlimbs never formed (n=16) and the pelvic bones were reduced to two rudimentary elements, each resembling a fused ischium and ilium (Fig. 1D-G). All forelimb skeletal elements were present (n=15), with excess material in the digit tips (n=15) (Fig. 1D,E,H,I). Furthermore, 20% (3 of 15) contained a forelimb preaxial extra digit and 33% (5 of 15) displayed a split preaxial digit (data not shown). Surprisingly, these mutant phenotypes are not similar defects that vary in severity, but rather the hindlimb phenotype (no outgrowth) and the forelimb phenotype (extra outgrowth) are opposite in nature, suggesting that BMP signaling plays a dynamic role during limb development.
No hindlimb PreAER forms in Msx2-Cre; Bmpr1aflox/null embryos
The AER is derived from the preAER, a broad domain of thickened ventral
limb bud ectoderm (Kimmel et al.,
2000). To determine whether the lack of hindlimbs in mutants is
due to a failure in preAER/AER formation, we examined histological sections of
mutant and control hindlimb buds from E9.5 (31ss) through to E10.5. The
morphological preAER and AER were both absent in all mutant hindlimb buds
(Fig. 2A-D). We used ISH to
assay for expression of Fgf8, the earliest expressed AER-FGF,
normally present in the hindlimb preAER by 27ss
(Lewandoski et al., 2000). At
this stage, Fgf8 expression was not detected in mutant hindlimb bud
preAER (Fig. 2E,F).
Furthermore, we never detected expression of Fgf8 (or other AER
markers: Fgf4, Msx2, Jag2, Bmp2, 4 and 7) at later stages of
hindlimb development through to E12.5 (data not shown).
|
|
;
Sekine et al., 1999).
Fgf10 expression was normally induced in mutant hindlimb buds at 29ss
(Fig. 2G,H), but maintenance
was lost by 33ss (Fig. 2I,J).
Thus, we conclude that early Bmpr1a inactivation in the hindlimb
prevents both the induction of Fgf8 in the preAER and the formation
of a morphological AER. Owing to the lack of AER-FGFs, mesenchymal
Fgf10 expression is not maintained.
Decreased interdigital cell death leads to webbing in Msx2-Cre; Bmpr1aflox/null forelimbs
The adult forelimbs of Msx2-Cre; Bmpr1aflox/null mice
display soft tissue syndactyly (webbing between the digits)
(Fig. 7A,B). In the mouse,
between E12.5 and E13.5, PCD eliminates cells in the interdigit region,
leading to the separation of individual digits
(Fernandez-Teran et al., 2006;
Zuzarte-Luis and Hurle, 2002;
Zuzarte-Luis and Hurle, 2005).
To determine whether mutant webbing was due to a PCD defect, we examined
E12.5-13.0 control and mutant embryos by staining with the fluorophore
Lysotracker Red, and observed a significant reduction in the degree of
interdigital cell death in mutant forelimbs
(Fig. 3A,B).
Webbing in Msx2-Cre; Bmpr1aflox/null forelimbs isnot correlated with changes in mesenchymal BMP signaling
BMP signals to the interdigit region have been implicated as direct
effectors or triggers of PCD (Ganan et
al., 1996; Macias et al.,
1997; Merino et al.,
1999a; Yokouchi et al.,
1996; Zou and Niswander,
1996). Therefore, we examined the expression of genes related to
mesenchymal BMP signaling to determine whether mutant forelimb webbing might
be explained by Bmpr1a inactivation in the AER indirectly causing a
diminution of interdigital BMP signaling. Bmp2, 4 and 7 are
normally upregulated at E12.5-13.5 in the interdigit region, suggesting that
they are candidate ligands for activating BMP signaling in this tissue
(Hogan, 1996;
Salas-Vidal et al., 2001). All
three Bmp genes were induced normally in the interdigit region in both control
and mutant E12.5 forelimbs (Fig.
3C-H), with enhanced Bmp4 expression in mutant subapical
mesenchyme (Fig. 3F). Gremlin
antagonizes mesenchymal BMP activity
(Zuniga et al., 1999) and its
enhanced expression in the interdigit region of ducks correlates with the
decrease in interdigital PCD occurring in these birds, as compared with
chickens (Merino et al.,
1999b). However, we found no significant difference between
mutants and controls in interdigital gremlin expression
(Fig. 3I,J).
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).
Furthermore, misexpression of Msx2 induces cell death in the chick
limb bud (Ferrari et al.,
1998; Merino et al.,
1999b) and Msx1-/-; Msx2-/- embryos
retain their interdigit mesenchyme
(Lallemand et al., 2005). We
detected no decrease in the expression of Msx1 or 2 in
mutant forelimbs at E12.5 (which would have been indicative of a reduction of
BMP signaling), but did note an expanded expression domain that is likely to
occur as a consequence of the retention of interdigit tissue
(Fig. 3K-N).
AER-specific Msx2 expression
(Coelho et al., 1991;
Ferrari et al., 1998) was
however absent in Msx2-Cre; Bmpr1aflox/null forelimbs from
E10.5-12.5 (Fig. 4A,B and data
not shown), demonstrating that Msx2 is downstream of BMPR1A signaling
in the AER. We also detected a reduction of AER-specific expression of
Bambi (Fig. 4C-F),
which is also a BMP target and encodes a natural dominant-negative BMP
receptor (Onichtchouk et al.,
1999). Together, these data suggest that only AER-specific BMP
targets are affected in Msx2-Cre; Bmpr1aflox/null
forelimbs, and that mesenchymal BMP-signaling is relatively normal.
Fgf4 and Fgf8 are upregulated in the forelimb AER of Msx2-Cre; Bmpr1aflox/null mutants
To analyze AER function in mutant forelimbs, we examined the expression of
the four AER-FGFs (Fgf4, 8, 9 and 17). We found no
significant changes in Fgf9 expression, but did note a slight
increase in Fgf17 expression (data not shown). At 22ss, we detected
no change in Fgf8 expression between normal and mutant forelimbs
(Fig. 5A,B). However, by E10.5
(approximately 24 hours after forelimb Msx2-Cre activation), Fgf8
expression in the mutant forelimb was less constricted dorsal-ventrally, but
normally extended anterior-posteriorly
(Fig. 5C,D). At E11.5, this
expanded Fgf8 pattern was more pronounced in the anterior AER of
mutant forelimbs (Fig. 5E,F)
relative to the posterior AER. By E12.5, normal AER-specific Fgf8
expression was ceasing over the interdigit region, but remained over the
developing digits (Fig. 5G),
consistent with published reports
(Crossley and Martin, 1995;
Salas-Vidal et al., 2001).
However, in Msx2-Cre; Bmpr1a mutants, this cessation of Fgf8
expression failed to occur, although we did note some infrequent small
discontinuities in Fgf8 expression at the anterior and posterior end
of the AER that did not correlate with the digit/interdigit pattern
(Fig. 5H). By E13.5, normal
Fgf8 expression was present only over the digits
(Fig. 5I,K), whereas punctate
Fgf8 expression occurred throughout the mutant AER
(Fig. 5J,L), with regions of
expression still present in the interdigit region associated with convex
outgrowths of the distal autopod (see Fig.
5K,L). Normal AER Fgf8 expression completely ceased by
E14.5, although mutant expression could still be detected at E15.5 (data not
shown).
|
;
Sun et al., 2000). In mutants,
Fgf4 activation occurred more extensively in the anterior-posterior
axis and in several distinct patches (Fig.
5N), and by E10.5 extended more anteriorly with some anterior
discontinuities in expression (Fig.
5P). By E11.5, Fg4 expression had ceased in normal
forelimbs, whereas in mutants expression remained robust in the posterior
region of the AER with weaker expression in the anterior AER
(Fig. 5Q,R). Punctate
AER-specific expression of Fgf4 remained through E12.5 and E13.5
(Fig. 5S-X). To determine whether the expression of other AER markers was temporally extended, we examined Bmp2, 4 and 7 expression in the mutant AER at E12.5-13.5. Bmp4 expression was not upregulated in the AER, although expression was increased in the subapical region (Fig. 3F and data not shown). At these stages, mutant AER-specific expression of Bmp2 and 7 was either absent or upregulated in a few posterior cells (Bmp2, 3 of 13 limbs; Bmp7, 5 of 7 limbs) (data not shown). Thus, considering the extent of their upregulation, Fgf4 and 8 were uniquely affected, owing to the lack of BMPR1A-mediated signaling to the AER.
FGF4 and FGF8 act as survival factors for the interdigit region
The upregulation of the principal AER-FGFs (Fgf4 and 8)
in mutant forelimbs raises the possibility that webbing might be due to
prolonged expression of these FGFs acting as survival factors for the
underlying interdigital mesenchyme. To test this, we examined webbing in
mutant animals in which we genetically lowered the AER-FGF levels in the
forelimb AER by generating Msx2-Cre; Bmpr1aflox/null
animals that also carry null or floxed alleles of Fgf4 and/or
8. However, we could not assess the complete contribution of both
Fgf4 and 8 to the mutant webbing phenotype (i.e. in animals
of the genotype Msx2-Cre; Bmpr1aflox/null;
Fg4flox/null; Fgf8flox/null) because Msx2-Cre
inactivation of both Fgf genes causes a severe disruption of limb development,
preventing analysis of interdigital PCD
(Sun et al., 2002).
Nevertheless, we could evaluate the effect of less extreme genotypes (i.e.
with at least one copy of Fgf4 or Fgf8 intact), which reduce
AER FGF4/8 activity to a level above zero, on the webbing phenotype of
Msx2-Cre; Bmpr1aflox/null progeny.
|
).
Thus, this hypodactyly phenotype was rescued in Msx2-Cre;
Bmpr1aflox/null; Fgf8flox/null animals. We
speculated that the levels of Fgf4 expression in Msx2-Cre;
Bmpr1aflox/null;Fgf8flox/null animals might be
above a threshold level required to rescue digit formation and maintain lower
interdigit PCD. We also reasoned that Fgf4 expression levels in
Msx2-Cre;Fgf8flox/null animals, although elevated
(Lewandoski et al., 2000),
might be below this threshold as hypodactyly but no webbing occurs in these
mutants. Indeed, semi-quantitative ISH revealed that Fgf4 expression
in E11.5 and E12.5 forelimbs was higher in Msx2-Cre;
Bmpr1aflox/null;Fgf8flox/null than in forelimbs in
which only Fgf8 had been deleted by Msx2-Cre
(Fig. 6D-I).
Next we examined the phenotype of mutant animals in which only one copy of
Fgf8 was deleted (i.e. of the genotype
Bmpr1aflox/null;Fgf8flox/+, or
Bmpr1aflox/null; Fgf8null/+; n=26
limbs). We reasoned that webbing might be reduced in these mice because we do
not expect a compound increase in Fgf4 expression as Fgf8
heterozygotes do not affect AER Fgf4 expression
(Lewandoski et al., 2000).
Webbing was partially reduced to a variable extent in this genotype
(Fig. 7D). Lastly, we found
that reducing FGF4 levels by generating Msx2-Cre;
Bmpr1aflox/null;Fgf4null/+;Fgf8flox/null
animals resulted in forelimbs in which interdigit PCD was rescued as these
limbs displayed little or no webbing (n=14 limbs)
(Fig. 7E,F). Furthermore, the
hypodactyly phenotype due to loss of Fgf8
(Lewandoski et al., 2000) was
usually restored (10 of 14 forelimbs had four digits and two of 14 had three
digits, and in each of six skeletal preparations generated, at least one digit
lacked a phalanx, see Fig. 7F
inset), demonstrating that elevated AER Fgf4 expression rescues digit
formation and causes webbing in the absence of Fgf8 and
Bmpr1a. Taken together, these data provide genetic evidence that
normal interdigit PCD is regulated by the BMP-mediated downregulation of
AER-FGFs, which act as cell survival factors of the interdigit mesenchyme.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Pizette et al., 2001;
Soshnikova et al., 2003;
Zou and Niswander, 1996).
Second, we demonstrate that a secreted signal, downstream of Bmpr1a,
emanating from the early limb bud ectoderm to the underlying lateral plate
mesenchyme, is required for normal pelvic bone development. Lastly, we define
a relationship between BMP and FGF signaling and the roles that these pathways
play during interdigital PCD. We provide genetic evidence that BMP signaling
to the AER is required for the patterned downregulation of Fgf gene
expression, which in turn encodes cell survival activity for the interdigit
tissue.
BMP signals are required for AER induction and cessation of AER-FGF expression
Msx2-Cre is active before formation of the hindlimb preAER but after
forelimb preAER induction (Sun et al.,
2000). In other studies, in which Msx2-Cre has been used to study
preAER/AER gene function, this pattern resulted in a phenotype that affected
the hindlimb more than the forelimb (Barrow
et al., 2003; Lewandoski et
al., 2000; Pan et al.,
2005; Sun et al.,
2000; Sun et al.,
2002). However, in the case of Msx2-Cre-mediated Bmpr1a
inactivation, opposite phenotypes are observed: forelimb AER function is
longer-lived owing to prolonged AER-FGF expression, whereas the hindlimb
preAER/AER never forms. We have performed confocal imaging of forelimb
interdigit mutant and normal ectoderm at E12.5-E13.5 and could not demonstrate
a longer-lived morphological AER (data not shown). Moreover, we examined cell
proliferation (by immunostaining for phosphorylated histone H3) and cell death
(by Lysotracker Red staining) in the normal and mutant forelimb AER, and found
no qualitative differences between the two (data not shown). Therefore, we do
not suggest that BMPR1A signaling is required for the normal regression of the
AER that occurs at about E12.5, but rather that BMPR1A signals specifically
regulate the cessation of AER-FGF expression.
|
;
Pizette and Niswander, 1999).
Thus, we interpret our data as supporting a hypothesis in which, within each
limb bud, BMP signals to the early ectoderm are required to induce the preAER,
and later signals to the AER are required to cease AER-FGF expression. This is
consistent with the data of Ahn et al. (Ahn
et al., 2001), in which a variable degree of hindlimb AER
disruption occurred due to the AER-specific inactivation of Bmpr1a.
The Cre line used by Ahn et al. is active in the hindlimb ectoderm at about
E9.75, which is later than Msx2-Cre activation (at 24ss, 
E9.5). Hence we
propose that E9.75 is in the temporal window when BMP signaling is beginning
to cease functioning in hindlimb AER induction.
The molecular basis for this switch in BMP function might be an intrinsic
property of preAER/AER cells, i.e. over time the competence of these cells
changes such that at early stages they respond to BMPR1A signals by forming an
AER, and at later stages by shutting down AER-FGF expression. Alternatively,
the integration of BMP with other signals might translate to different
cellular behaviors. In support of this, mesenchymal FGF10 signals and
ectodermal WNT3 signals are both required for AER formation
(Barrow et al., 2003;
Min et al., 1998;
Sekine et al., 1999).
A preAER signal is required for normal hip girdle formation
Surgical removal of chick ectoderm prior to limb bud formation prevents
normal pelvic bone development, suggesting that a preAER factor signals to the
lateral flank mesoderm for pelvic girdle development
(Malashichev et al., 2005).
Also, certain mouse mutations that prevent hindlimb AER induction also affect
pelvic bone development, including defects in p63
(Trp63-Mouse Genome Informatics), Wnt3 or
ß-catenin (Barrow et al.,
2003; Mills et al.,
1999; Yang et al.,
1999). Our observation that the pelvic bones in Msx2-Cre;
Bmpr1aflox/null mice are reduced to two elements resembling
rudimentary ischia/ilia, indicates that the preAER factor is downstream from
BMPR1A and secreted from the Msx2-Cre expression domain.
Pelvic bones are also diminished in Fgf10-/- animals
(Min et al., 1998;
Sekine et al., 1999). Given
that mesenchymal Fgf10 expression is not maintained in Msx2-Cre;
Bmpr1aflox/null hindlimb buds (owing to a lack of AER-FGF
signals), one might consider that loss of Fgf10 expression causes the
Msx2-Cre; Bmpr1aflox/null pelvic girdle phenotype.
However, we speculate that this is not the case, because Fgf10
expression is also not maintained in Msx2-Cre; Fgf4flox/null;
Fgf8flox/null mice and yet there is no similar pelvic bone
phenotype in these mutants (Sun et al.,
2002). [A very subtle pelvic bone phenotype occurs, but with all
pelvic bones present in Msx2-Cre; Fgf4flox/null;
Fgf8flox/null mutants (B. Rosenman and C. O. Lovejoy, personal
communication)]. Indeed, our observations suggest the novel interpretation
that the Fgf10-/- pelvic girdle phenotype might be
non-cell-autonomous in that it could be a secondary effect resulting from the
lack of a preAER signal.
Consideration of the relatively normal pelvic bones in Msx2-Cre;
Fg4flox/null; Fgf8flox/null mutants also indicates
that neither of the principal AER-FGFs, FGF4 and FGF8, is the secreted factor
downstream of BMPR1A required for pelvic development. WNT signaling might be
involved as BMPR1A has been shown to control WNT-mediated limb bud outgrowth
(Soshnikova et al., 2003) and
Msx2-Cre inactivation of Wnt3 similarly affects pelvic bone
development (Barrow et al.,
2003).
BMP signals control interdigital PCD by regulating AER-FGF expression
In Msx2-Cre; Bmpr1aflox/null mutants, a lack of normal
interdigital PCD causes forelimb cutaneous syndactyly to occur between all
forelimb digits. During normal development, elevated BMP levels within the
interdigit region have been proposed to be a direct effector of cell death
within this region (Zuzarte-Luis and
Hurle, 2002; Zuzarte-Luis and
Hurle, 2005). However, no change indicative of a reduction of BMP
signaling occurred in the mesenchymal gene expression levels of BMP ligands
(Bmp2, 4 and 7), the antagonist gremlin, or the downstream
targets Msx1 and 2, indicating that this proposed BMP role
does not cause mutant webbing. Instead, a prolonged period of AER
Fgf4 and Fgf8 expression occurred in mutants. By examining
Msx2-Cre; Bmpr1aflox/null mutants in which we reduced
AER-FGF activity by also inactivating Fgf4 and/or 8, we
observed a partial rescue of normal interdigit PCD with one copy of
Fgf8 inactivated, and a more complete rescue with both copies of
Fgf8 and one copy of Fgf4 inactivated. Thus, we provide
genetic evidence that one mechanism by which BMPs can regulate interdigit PCD
is through the regulation of AER-FGFs acting as cell survival factors.
|
).
However, given that during normal development, AER Fgf4 expression
ceases by E11.5 (Lewandoski et al.,
2000) and that Fgf8 expression ceases at E12.5 first over
the interdigit regions preceding interdigital cell death
(Salas-Vidal et al., 2001), we
postulate that FGF8, not FGF4, is the principal AER-FGF that maintains
interdigit cell survival, until it is downregulated through BMPR1A-mediated
signaling (Fig. 8).
The insight that FGF8 controls interdigital cell death is reminiscent of
studies in which Fgf8 inactivation causes an abnormal increase in
cell death in the first branchial arch
(Trumpp et al., 1999), the
brain (Chi et al., 2003;
Storm et al., 2003), the early
limb bud (Moon and Capecchi,
2000; Sun et al.,
2002) and the kidney
(Grieshammer et al., 2005;
Perantoni et al., 2005).
However, the current study is the first report demonstrating that the embryo
regulates Fgf8 expression to control normal cell death during
development.
Our genetic experiments support speculations, based on gene expression
patterns (Salas-Vidal et al.,
2001) and the effects of FGF-bearing bead insertion into chick
limb buds (Montero et al.,
2001; Macias et al.,
1996), that AER FGF8 activity might act as a cell survival
activity for the interdigit region. Genetic proof for this idea was not
obtainable by a simple Msx2-Cre-mediated inactivation of Fgf8 itself
(in which one would expect premature interdigit cell death), because the
overall development of the autopod is retarded
(Lewandoski et al., 2000).
However, this model is supported by the observation that cutaneous syndactyly
in the classical human craniosynostosis syndrome, Pfeiffer's, is caused by
mutations causing constitutive signaling of the FGFR2c isoform, which is
active in limb bud mesenchyme and is thought to act as an FGF8 receptor. Also,
we propose that excess AER-FGF activity might be the mechanism by which
syndactyly occurs in a number of mouse mutants in which an expanded AER is
evident, which include mouse lines carrying mutations affecting the NOTCH and
WNT pathways (Jiang et al.,
1998; Mukhopadhyay et al.,
2001; Pan et al.,
2005; Sidow et al.,
1997). This proposal can be tested directly by specifically
inactivating Fgf4 and 8 in the AER of these mutants as we
have done here.
A supernumerary pre-axial digit occurs in 25% of Msx2-Cre;
Bmpr1aflox/null forelimbs, as well as in other mouse mutants
with enhanced AER-FGF signaling (Adamska et
al., 2003; Lu et al.,
2006; MacDonald et al.,
2004; Wang et al.,
2004). However, the prolonged AER-FGF expression in mutant
forelimbs was not associated with the formation of extra phalanges. This
contrasts with what has been observed in chick embryos, in which prolongation
of AER Fgf8 expression can cause an additional penultimate phalange
within a digit, supporting a model in which the duration of AER FGF8 signaling
controls elongation of digit primordia and the normal decrease in AER FGF8
signaling generates digit tip formation
(Sanz-Ezquerro and Tickle,
2003). The discrepancy between these studies may be explained by
inherent differences in AER FGF8 function between mouse and chick limb buds.
Alternatively, extra phalange development might also require enhanced SHH as
well as FGF8 signaling, considering that AER Fgf8 expression was
prolonged in the chick studies by the insertion of SHH-soaked beads in the
underlying mesenchyme (Sanz-Ezquerro and
Tickle, 2003). However, consistent with these chick studies we did
observe thickened digit tips in all Msx2-Cre;
Bmpr1aflox/null forelimbs.
In our model (Fig. 8), we
propose that FGF8 acts as a cell survival factor for the interdigit region;
its normal downregulation via BMPR1A-mediated signals allows interdigit PCD to
occur. In our mutant model, Bmpr1a inactivation results in prolonged
FGF4 and FGF8 signaling, leading to abnormal cell survival and webbed limbs.
What is the source of these interdigital BMP signals during normal
development? We have examined Bmp gene expression in the AER prior to and
during the initiation of interdigital PCD (E12.5-13.5), and found that that
Bmp2 and 7 are not expressed in the AER, whereas
Bmp4 is specifically expressed in the AER/subapical region overlying
the digits. However, because Bmp2, 4 and 7 are upregulated
in the interdigit mesenchyme prior to PCD
(Hogan, 1996;
Salas-Vidal et al., 2001), we
speculate in our model that this tissue is the BMP source. It is tempting to
further speculate that elevated BMP activity during development of the digits,
as chondrogenesis proceeds (Yoon and
Lyons, 2004), might cause the cessation of AER Fgf8
expression overlying the digits. Also, earlier studies that described the
inhibition of interdigit PCD in chick embryos through the blocking of BMP
signaling with retroviral expression of transgenes encoding dominant-negative
BMP receptors (Yokouchi et al.,
1996; Zou and Niswander,
1996), may be reinterpreted in the context of our model as these
retroviral vectors might have inhibited BMP signaling to the AER, elevating
Fgf gene expression. Alternatively, our demonstration that BMPs regulate
interdigital cell death through the regulation of AER-FGF expression does not
exclude a direct role for BMPs as effectors of cell death. Evaluating such a
role requires the inactivation of Bmp receptor genes specifically within the
interdigit region without affecting AER morphology or gene expression.
Lastly, these data have significant implications for the evolution of limb
morphology. Considering the well-established role that the AER plays in the
outgrowth of skeletal limb elements, it has been proposed that evolutionary
changes in AER-FGF activity might reduce or extend the different cartilaginous
and bony limb structures that occur in different species
(Cohn and Tickle, 1999;
Freitas et al., 2006;
Richardson and Oelschlager,
2002; Thewissen et al.,
2006). The data presented here suggest that evolutionary
alterations in AER-FGF activity might also contribute to the shaping of
soft-tissue structure in vertebrates, resulting in normal free digits or
webbed limbs. For example, a recent study provides evidence that FGF8-mediated
signaling is pivotal in maintaining the interdigit webs in the bat wing
(Weatherbee et al., 2006).
Future studies into the targets of AER-FGF activity and the interaction of FGF
signaling with other pathways are needed to understand how AER-FGF signaling
differentially affects the patterning of bony structures and soft tissue.
|
ACKNOWLEDGMENTS |
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