1 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15
2TT, UK
2 Weatherall Institute of Molecular Medicine, The John Radcliffe Hospital,
Headington, Oxford OX3 9DS, UK
Author for correspondence (e-mail:
j.k.heath{at}bham.ac.uk)
Accepted 14 October 2003
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SUMMARY |
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Key words: Fgf, Signalling, Skeleton
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Introduction |
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Specific hypermorphic mutations in Fgfr1 and Fgfr2 are the cause of seven
clinically distinct craniosynostosis syndromes
(Passos-Bueno et al., 1999),
the phenotypic hallmark of which is the obliteration of sutures separating
calvarial bones (reviewed by Wilkie and
Morriss-Kay, 2001
; Ornitz and
Marie, 2002
). Craniosynostosis is, in particular syndromes,
accompanied by limb defects ranging in severity from a broadening of the
thumbs and great toes in Pfeiffer syndrome, to boney and soft tissue
syndactyly of the digits in Apert syndrome. The extent and severity of
phenotypes correlates with the type and position of the mutations, and with
the Fgfr gene involved. Apert syndrome (which exhibits strong limb and skull
defects) generally involves specific missense substitutions in two adjacent
residues of Fgfr2: Ser252Trp and Pro253Arg. These both lie in the `linker'
region joining Ig-like domains II and III
(Oldridge et al., 1997
;
Ibrahimi et al., 2001
). The
phenotypically milder Pfeiffer syndrome arises from a missense substitution in
Fgfr1 - Pro252Arg - in the topologically equivalent `linker' residue to Fgfr2
Pro253Arg.
Fgfr2 `linker' mutations have been shown to exhibit increased affinities
for specific Fgf ligands (Anderson et al.,
1998; Ibrahimi et al.,
2001
) because of the additional receptor/ligand contact site
introduced by the missense substitution. This finding suggests that
Apert/Pfeiffer-type linker domain substitutions result in receptors that are
activated at lower concentrations of ligand compared with their wild-type
counterparts, and that exhibit quantitatively distinct ligand-dependant
signalling dynamics. As receptor activation is initiated by ligand-mediated
receptor dimerisation (reviewed by
Schlessinger, 2000
), only
mutant receptor homodimers exhibit mutant signalling dynamics. This
consideration predicts that the phenotypic consequences of mutant receptor
signalling will be dictated by the ratio of mutant to wild-type receptors, as
elevating the ratio will favour the formation of mutant homodimer complexes in
the presence of appropriate ligands. Thus, varying the expression of mutant
receptors provides a means to study the consequences of quantitative changes
in Fgfr signalling dynamics.
Here we employ a novel bacterial artificial chromosome (BAC)-based
transgenic system (Lalioti and Heath,
2001) to dissect the molecular consequences of mutant Fgfr action
in vivo. We have introduced into the mouse germ line a BAC encoding the entire
mouse Fgfr1 gene that harbours a single nucleotide substitution
corresponding to the human Pfeiffer syndrome mutation Pro252Arg
(Muenke et al., 1994
). We find
that, in contrast to BACs harbouring silent single nucleotide substitutions,
the presence of the mutant BAC transgene yields skeletal defects that involve
both membranous and endochondral modes of ossification, resembling those seen
in human Pfeiffer patients. Doubling the BAC gene copy number - thereby
incrementally elevating signalling through mutant receptors - yields an
increase in the severity of the ossification defects, and an unexpected de
novo appearance of pre-axial polydactyly of the hindlimbs and homeotic
transformation of the vertebrae. These findings demonstrate the existence of a
Fgfr1 signalling threshold governing the development of digit I and vertebral
patterning. This study also shows - as predicted by Freeman and Gurdon
(Freeman and Gurdon, 2002
) -
that a single signalling pathway can exhibit both morphogen and threshold
signalling properties depending on the level of receptor activation.
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Materials and methods |
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Mutant Fgfr1 fragments were generated by overlap PCR. Essentially,
two complementary primers covering the region to be mutated were used in
conjuction with a pair of external primers to produce two overlapping
fragments using Pfu polymerase (Promega). The two fragments were then joined
together in a second reaction using only the external primers. For the
Pro252Arg mutation (BAC16) the complementary primer pairs were (16L)
5'-GAACGATCCCGGCACCGAC-3', and external primers were (21L) and
(21R) (see above). These products were then cloned into the
BamHI-SalI sites of the pKOV-kan shuttle vector in
preparation for modification of the BAC as described previously
(Lalioti and Heath, 2001).
Generation and screening of transgenic animals
Modified BACs were prepared for pronuclear injection as described
(Chrast et al., 1999;
Lalioti and Heath, 2001
), and
injected as linearized DNA (0.3-3 ng/µl) into fertilized oocytes. Genomic
tail DNA from offspring was subjected to the following PCR conditions: 1 cycle
of 5 minutes at 94°C; 10 cycles of 20 seconds at 94°C, 20 seconds at
60°C (with 1°C decrease per cycle), 30 seconds at 72°C; 20 cycles
of 20 seconds at 94°C, 20 seconds at 50°C, 30 seconds at 72°C; 1
cycle of 5 minutes at 72°C. Primer pairs used were exon 7 (31L):
5'-TGACATGCCTGTCCTCTCTGTG-3' and (34R)
5'-CCCTACTAGGAGATAGTATGTG-3'. The PCR products were then digested
for 2 hours with MspI (for BAC16) or PstI (for BAC15), and
resolved on 3% high resolution agarose gels (Elchrom Scientific).
To test expression of the transgene, 100 ng of total liver RNA isolated using Trizol® Reagent (GibcoBRL) was subjected to One Step RT-PCR (Qiagen) using the primers (8L) 5'-ACCTACCAGCTTGACGTCGTG-3' and (6R) 5'-CATTTCCTTGTCGGTGGTATTAAC-3', as per manufacturer recommendations.
Skeletal staining
Skeletons from embryos, new born or adult mice were stained with Alcian
Blue and/or Alizarin Red as described by McLeod
(McLeod, 1980). A full listing
of the animals examined with phenotypic annotation is provided in
supplementary Tables S1 and S2.
Analysis of BAC-transgene copy number and sites of integration by fluorescence in situ hybridisation (FISH)
FISH analysis was carried out as described by Buckle and Rack
(Buckle and Rack, 1993).
Essentially, chromosomes were isolated from splenic cells, previously cultured
for 48 hours in the presence of lipopolysaccharide
(Triman et al., 1975
), and
hybridised with a mixture containing a bitoin 16-UTP-labelled murine
Fgfr1 BAC probe and either Cy3-labelled mouse chromosome 8 paint or
FITC-labelled mouse chromosome 4 paint (Cambio, Cambridge, UK). The
hybridisation mixture contained 100 ng of biotinylated probe, 3 µg mouse
COT-1 DNA (Invitrogen) and 20 µg salmon sperm DNA (Sigma), together with
the appropriate amounts of paint, according to the manufacturer's
instructions. The biotinylated Fgfr1 probe was detected with either
avidin Cy3.5 (Amersham Pharmacia) or avidin FITC (Vector Laboratories).
Chromosomes were counterstained with 4',6-diamidino-2-phenylindole
(DAPI) at 1.5 µg/ml and analysed using an Olympus BX-60 microscope equipped
with a Pinkel filter wheel. Images were captured and analysed using a SenSys
cooled CCD camera (Photometrics, Tuscon), and MacProbe version 4.3 software
(Applied Imaging, UK).
Quantitative PCR analysis of BAC copy number/transgene expression
Genomic DNA was amplified with primers (31L) and (34R) (see above; the
reverse oligonucleotide was fluorescently labelled) using the following
cycling conditions: 1 cycle of 10 minutes at 94°C; then 25 cycles of 30
seconds at 94°C, 30 seconds at 58°C and 30 seconds at 72°C;
followed by 10 minutes at 72°C. The PCR products were digested for 1 hour
with MspI and the fragments separated on an ABI 377 automated
sequencer and analysed by GeneScan software (Applied Biosystems). cDNA (2
µl from a 40 µl first strand synthesis reverse transcription reaction
using 2-5 µg total RNA) was amplified with primers (8L) and (6R) (see
above; the reverse oligonucleotide was fluorescently labelled), using the same
cycling conditions as for genomic DNA except the annealing temperature was
55°C. PCR products were digested for 2 hours with MspI and
analysed as above.
Whole-mount in situ hybridisation (WMISH)
Whole embryos were isolated at appropriate stages of development from
time-mated mice, counting noon of the day on which the vaginal plug was found
as zero. Embryos were fixed overnight in 4% paraformaldehyde, dehydrated the
following day through ascending concentrations of methanol/PBS containing 0.1%
Tween-20, and stored until use in absolute methanol at -20°C. The genotype
of each embryo (i.e. whether carrying low or high copies of the transgene) was
verified by PCR (described above), using genomic DNA isolated from yolk
sacs.
For WMISH, embryos were rehydrated in descending concentrations of methanol
in PBS/Tween-20, treated with proteinase K, and hybridised at the appropriate
temperatures (55-69°C) with sense (control) or anti-sense riboprobes
generated from linearized plasmids containing the gene of interest (see Table
S3 at
http://dev.biologists.org/supplemental/).
The protocol followed for WMISH was as previously described
(Hajihosseini et al., 2001),
with the exception that the colour development (NBT/BCIP) reaction was
performed in the presence of polyvinyl alcohol (Sigma).
Detection of apoptotic cell by neutral red staining
Embryos were stained live as previously described
(Hajihosseini and Heath,
2002). Briefly, soon after isolation, embryos were rinsed in
freshly prepared 3% BSA/PBS and stained in the dark for 25 minutes (for E12.5)
at room temperature in the same solution containing 0.05% Neutral Red. Excess
dye was discarded and embryos were rinsed several times in 3% BSA/PBS, then in
PBS and fixed for 15 minutess in 4% paraformaldehyde solution. Stained embryos
were then photographed immediately.
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Results |
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These observations show that Fgfr1 signalling resulting from expression of
the Pro252Arg mutant receptor selectively affects the development of the
skeleton, targets distinct bones and sutures, and affects both membranous and
endochondral modes of ossification. The range of defects described here are
similar to those reported by other studies of hyperactive Fgfr function in
mice (Zhou et al., 2000;
Hajihosseini et al., 2001
;
Wang et al., 2001
), and to the
cranial and skeletal defects observed in Pfeiffer patients
(Muenke et al., 1994
;
Roscioli et al., 2000
). In
this respect, it should be noted that some phenotypic features of the 2C-BAC16
model, such as sternal fusions, have not previously been described in Pfeiffer
patients. However, it is possible that these defects do occur in Pfeiffer
syndrome but have not been previously looked for. It should also be noted that
the 2C-BAC16 mice, like the model of Zhou et al.
(Zhou et al., 2000
), did not
show defects in the development of the halluces described in Pfeiffer
syndrome.
The phenotype of 4C BAC16 mice: limb and axial skeleton defects
A valuable feature of the BAC model employed in these experiments is the
ability to manipulate mutant gene copy number and the consequent expression of
mutant transcripts. In particular, it is predicted that a doubling of gene
copy number by intercrossing 2C-BAC16 mice would further accentuate signalling
via mutant receptors, as it favours the formation of mutant receptor
homo-dimers, which are required for mutant gene function in the presence of
ligand. 2C-BAC16 mice were intercrossed to produce 4C-BAC16 mice
(Fig. 1D), which were recovered
in the expected Mendelian ratios.
4C-BAC16 mice presented more severe cranial and sternal phenotypes. The curvature of the maxillary process was accentuated (Fig. 2G,J), resulting in mis-aligned jaws and overgrown or in-growing teeth, particularly the incisors. Fusion of zygomatic arch bones was also accentuated, such that in some 4C-mutants both the anterior and posterior joints showed precocious ossification (not shown). This indicates that increasing signalling via mutant Fgfr1 accentuates the severity of the endochondral and membranous bone defects in a quantitative manner.
A key finding, however, was that 4C-BAC16 mice presented a novel set of phenotypes, involving homeotic transformations of the axial skeleton and pre-axial polydactyly of the hind limbs.
Defects in axial patterning
The mouse vertebral column is composed of distinct sets of bones that show
unit and segmental identity resulting from the expression of a particular set
of Homeobox (Hox) genes (reviewed by
Gaunt, 2000). A subset of
4C-BAC16s (n=7/15) presented fusion and homeotic transformations of
the axial skeleton in a posterior direction. Cervical vertebrae 6 (C6)
acquired a C7 identity, and C7 acquired a Thoracic vertebrae 1 (T1) identity
(Fig. 3B-D). In one mutant,
these transformations were accompanied by fusion of the first and second ribs
with an accompanying sternal defect (Fig.
3C). In another, C1 was found to be fused to C2 (not shown).
Interestingly though, these defects occurred predominantly in a unilateral
fashion, on the left side. In the lower lumbar region of the same animals, L6
often acquired a Sacral (S)1 identity, but this transformation was always
bilateral (Fig. 3F). These
transformations are similar to those seen in mice carrying a Y766F
gain-of-function mutation in Fgfr1
(Partanen et al., 1998
), and
in those lacking caudal-related genes (Cdx)
(Van den Akker et al.,
2002
).
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These results show a direct relationship between mutant Fgfr1 copy number (or ratio to wild-type alleles) and the severity of phenotypes observed. In particular, cranial and sternal development exhibited `morphogen' characteristics, where the severity of the defect is accentuated by increasing the expression of the mutant gene. However, the development of the axial skeleton and digit I exhibited `threshold' characteristics, in which the mutant phenotypes only appear when mutant genes are present in excess over their endogenous counterparts.
Candidate gene analysis of polydactyly in 4C-BAC16 hindlimbs
Much is known about genes that control growth and patterning of the
vertebrate limbs in the three primary axes (dorsoventral, anteroposterior and
proximodistal) (Martin, 1998;
Capdevila and Izpisua Belmonte,
2001
). Moreover, targeted mutations in mice or spontaneous
mutations in man have implicated a number of genes and pathways in pre-axial
polydactyly (see Table S3 at
http://dev.biologists.org/supplemental/)
(Biesecker, 2002
). This
provided a list of candidate genes that could mediate the threshold-dependent
polydactyly phenotype of 4C-BAC 16 mice. We performed a comprehensive analysis
of this candidate gene set, comparing expression patterns in wild-type,
2C-BAC16 and 4C-BAC16 limbs by whole-mount in situ hybridisation. For each
gene, the analysis focused on embryonic stages previously shown to correspond
with either optimal expression and/or a crucial function.
From the candidate gene set listed in Table S3, the majority, including Shh (Fig. 5A), exhibited a normal expression pattern in both 2C-BAC16 and 4C-BAC16. However, we did detect alterations in the expression patterns of participants in three pathways: the Hox gene pathway, in the form of the d-cluster Homeobox 13 (Hoxd13); the calcium-dependent Wnt pathway, in the form of ligand Wnt5a; and the canonical ß-catenin Wnt pathway, in the form of Dickkopf (Dkk1).
|
At E11.5, Dkk1 is normally expressed throughout the apical
ectodermal ridge (AER) and in the hind limbs, strongly in a region that
roughly corresponds to parts of the anterior necrotic zone (ANZ)
(Fig. 5F,G) (Mukhopadhyay et al., 2001).
In eight out of ten 4C-BAC16 hind limbs, the level of Dkk1 expression
was reduced in the anterior two-thirds of the AER, and lost in the ANZ
(Fig. 5F,H). Cell death in the
developing limbs contributes to the final shape of the autopod, and expression
patterns and functional data suggests that Dkk1 has a role in inducing cell
death (Grotewold and Ruther,
2002
). Thus to examine any potential differences in cell death, we
labelled E12.5 embryos with Neutral Red dye, a faithful marker of apoptotic
cells in developing limbs (Macias et al.,
1996
; Hajihosseini and Heath,
2002
). In normal and 2C hind limbs, extensive cell death was noted
in the mesenchyme anterolateral to the digit I condensate. By contrast, and
consistent with the differences in Dkk1 expression, we found
significantly reduced cell death in the corresponding region of 4C abnormal
limbs (Fig. 5I).
Wnt5a has been highlighted as a member of the Wnt family that
plays a crucial role in distal limb outgrowth, as well as in anteroposterior
patterning of the early limb bud (Dealy et
al., 1993; Parr et al.,
1993
; Kawakami et al.,
1999
; Yamaguchi et al.,
1999
). We thus compared the patterns of Wnt5a expression
between wild-type, 2C- and 4C-mutant limbs, only to discover a higher
intensity of labelling throughout the distal fore and hind limbs of 4C
embryos. More relevant to the described limb phenotypes, Wnt5a
expression was upregulated in the hind limb digit I region of 4C mutants, when
compared to 2C mutants or wild-type mice
(Fig. 5J).
These results indicate that threshold-dependent hindlimb polydactyly in 4C-BAC16 mice arises through defects in tissue growth and patterning resulting from elevation of mutant receptor signalling. The majority of candidate preaxial polydactyly pathways, such as the Shh or BMP pathway and related genes, appeared unaffected in 4C-BAC16 mutant mice, although we cannot eliminate an increase in the activity of these pathways that does not result in changes in gene expression.
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Discussion |
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Mechanistic properties of signalling thresholds
Threshold signalling may be contrasted with morphogen signalling, in that
in the former there is a `binary' response to receptor activation whereas in
the latter there is a quantitative relationship between receptor occupancy and
response, until saturation of receptors by ligand occurs
(Freeman and Gurdon, 2002). In
both cases, receptor-mediated activation is controlled by the availability of
ligand and the affinity of the ligand for the receptor. In pathways that
exhibit threshold properties, a covalent modification (e.g. phosphorylation or
ubiquitinylation) is usually linked to a process in which the modification is
reversed or inhibited (reviewed by
Ferrell, 1999
;
Salazar and Hofer, 2003
;
Germain, 2001
;
Germain and Stefnova, 1999
).
Thus, the difference between the two types of signalling characteristics is
explained by the presence of limiting inhibitory pathways in threshold
signalling; in morphogen type systems these inhibitory pathways are absent or
compromised (Ortega et al.,
2002
), and there is therefore a direct relationship between
receptor occupancy and signal output. The results reported here therefore show
that the development of the axial skeleton and digit I of the hind limb
involve Fgfr1-mediated signalling pathways that have different regulatory
properties from those involved in the sternum and cranial sutures. This could
include different pathway connectivities and/or the presence or absence of
inhibitory regulators.
Morphogen signalling in cranial and sternal defects
Mice harbouring 2C-BAC16 alleles exhibit both endochondral and membranous
bone defects, including premature fusion of cranial sutures, the zygomatic
bones and the sternum. The precocious ossifications and suture fusions
reported here are consistent with the proposed role of signalling via Fgfr1 in
the induction of osteogenic precursor cell terminal differentiation
(Colvin et al., 1996;
Iseki et al., 1997
;
Iseki et al., 1999
;
Rice et al., 2000
;
Zhou et al., 2000
;
Eswarakumar et al., 2002
;
Huang et al., 2003
). These
sites of action are similar to those observed in the equivalent mutation in
man (Muenke et al., 1994
;
Roscioli et al., 2000
), and in
a knock-in mouse model reported by Zhou et al.
(Zhou et al., 2000
). We were,
however, also able to show that the severity of these defects increased when
the mutant/wild-type expression ratio is doubled, indicating that osteogenic
differentiation is accelerated as signalling via Fgfr1 increases.
The effects of the mutant Fgfr1 gene in these tissues are
restricted to specific bones and sutures. In the skull vault, we observed
synostosis of metopic (frontal) but not coronal or sagittal sutures. In the
skull base, only the joints separating the maxilla from the pre-maxilla were
fused. This differential sensitivity of specific sutures to the presence of
mutant receptors could represent sites of selective distribution or
concentration of FGF ligands, such that Fgfr1 signalling is limited by ligand
availability in susceptible sutures but is in ligand excess in non-susceptible
sutures. Although multiple Fgf ligands are expressed in the developing
calvarial sutures (Hajihosseni and Heath, 2002), little is known about the
distribution or activity state of the corresponding proteins. However, sites
of ligand-limited signalling would be susceptible to the experimental addition
of extra ligand. Indeed, Iseki et al. and Greenwald et al. have demonstrated
that coronal and metoptic sutures undergo premature fusion when exposed to
additional Fgf ligand (Iseki et al.,
1997; Iseki et al.,
1999
; Greenwald et al.,
2001
). Collectively, these findings show that sculpture of the
cranium through differential rates of suture fusion in normal development is
quantitatively controlled by the availability of Fgf ligand.
Threshold responses in the axial skeleton
4C-BAC16, but not 2C-BAC16, mice exhibit defects in axial patterning
whereby distinct vertebrae acquire a more posterior identity. Partanen et al.
(Partanen et al., 1998)
observed very similar axial transformations, but not polydactyly, by creating
a hypermorphic point mutation Y766F in the putative PLC
/Shb
(Cross et al., 2002
) docking
site in Fgfr1. It is significant that this allele does not give rise to
polydactyly, indicating that the underlying threshold-dependent pathways in
limb and axial skeleton development are distinct.
The vertebral column is derived from the somites, which are generated
sequentially and in a rostrocaudal manner from the paraxial mesoderm.
Segmentation of the paraxial mesoderm is believed to be regulated by an
oscillator mechanism involving the Notch signalling pathway (reviewed by
Holley and Takeda, 2002).
Fgf8 is expressed in a graded manner caudal to the emerging somites,
and ligand overexpression studies have led to the proposal that this
corresponds to a wave of Fgf signalling that functions to position segmental
boundaries by maintaining the pre-somitic mesodermal cells in an uncommitted
state (reviewed by Dubrulle and Pourquie,
2002
; Dubrulle et al.,
2001
; Holley and Takeda,
2002
). In this model, the establishment of segment identity occurs
when Fgf signalling falls below a predetermined threshold. Our findings
support this hypothesis, as it would be predicted that, in the presence of a
fixed threshold, the `wavefront' of Fgfr1 signalling would extend in a rostral
direction in 4C-mutant mice compared with normal counterparts, in turn leading
to somites acquiring a more posterior identity
(Dubrulle et al., 2001
).
Digit I exhibits threshold responses to Fgfr1 signalling
Current models of limb development hold that the individual elements
(autopod, zugopod, stylopod) are specified early. One role of Fgfr signalling
is then to expand each of the specified fields to their relevant final size
and shape, by regulating cell proliferation/survival and the expression of
genes that pattern each element (Dudley et
al., 2002; Sun et al.,
2002
). Expression patterns and genetic dissection studies suggest
that in the early limb bud, the effects of AER-derived Fgfs on the adjacent
mesenchyme are transduced by Fgfr1 (Peters
et al., 1992
; Partanen et al.,
1998
; Eswarakumar et al.,
2002
). The preaxial ploydactyly in hindlimbs, which occurs when
hypermorphic BAC transgene copy number is raised to 4C, shows that the
development of digit I, but not digits II-V, is dependent on Fgfr1 signalling
levels whereby extra digit elements are formed when Fgfr1 signalling is
elevated above a threshold, defined here by the ratio of mutant to wild-type
receptors. By contrast, mice harbouring a hypomorphic Fgfr1 allele
specifically lack digit I in the hind limb
(Partanen et al., 1998
). In
addition, conditional inactivation of Fgf8 in hind limbs specifically
affects digit I development (Lewandoski et
al., 2000
). We conclude that digit I exhibits a particular
dependency on Fgfr1 signalling - despite uniform expression of Fgfr1
throughout the early limb bud mesenchyme - and that a role of Fgfr1 signalling
is to regulate the size of the pool of cells destined to give rise to digit I.
If Fgfr1 signalling is compromised, the digit I precursor pool is reduced
leading to the absence of digit I. If, as a result of altered Fgfr1 signalling
dynamics, the precursor pool is amplified above a threshold level, the outcome
is preaxial polydactyly.
How can the dependency of digit I development on Fgfr1 signalling levels be
explained? First, there could be an asymmetric distribution of Fgf ligands
within the AER, such that higher levels in the anterior region corresponding
to the presumptive digit I trigger a higher level of mutant receptor
activation in the adjacent anterior mesenchyme. However, expression pattern
studies to date have shown the converse, with ligands such as Fgf4 being
restricted to the posterior AER (Bueno et
al., 1996). We have previously shown that Fgf20 is
strongly expressed at the anterior and posterior margins of the developing
autopod (Hajihosseini and Heath,
2002
), but BAC16 mice do not develop digit V polydactyly.
The second, and in our view more likely, explanation is that the development of digit I versus digits II-V is normally governed by two distinct levels of Fgfr1 signalling. This supposes that an increase in size of the digit I precursor pool depends upon Fgfr1 signalling and is terminated when levels of Fgfr1 signalling fall below a specified threshold. This threshold mechanism does not operate in the development of digits II-V. In 4C-BAC16 mice, Fgfr1 signalling is elevated and fails to fall below the threshold, resulting in the formation of extra digit elements.
Processes that depend on Fgfr1 signalling in the digit I field could
include cell proliferation and/or cell death in the surrounding mensenchyme.
Polydactyly in many experimental models is accompanied by a reduction in
programmed cell death (Chen and Zhao,
1998; Salas-Vidal et al.,
2001
). We showed that at E12.5, prior to digit outgrowth,
significant cell death does indeed normally occur in the mesenchyme around
digit I, and that this is quenched in developing limbs of 4C mutants. We also
showed that, in the presence of the hypermorphic 4C allele (but not in the 2C
mice), Dkk1 was downregulated close to a region destined to form
digit I. Dkk1 expression is associated with regions of programmed
cell death in the limb (Grotewold et al.,
1999
), ectopic expression of Dkk1 in the limb promotes apoptosis
(Grotewold and Ruther, 2002
)
and Dkk1 null mice exhibit polydactyly
(Mukhopadhyay et al., 2001
).
Collectively, this evidence indicates that at least part of the mechanism by
which Fgfr1 regulates the digit I precursor field is mediated by a
threshold-responsive signal that negatively regulates programmed cell death
via target genes such as Dkk1.
The threshold-sensitive relationship between Fgfr signalling and
Dkk1 expression/cell death may also hold true for the normal
development of digits II-V, as later in limb development Dkk1 becomes
downregulated in the growing digits and becomes restricted to inter-digital
mesenchyme (Grotewold and Ruther,
2002). However, as 4C-BAC-16 mice do not develop digit II-V
syndactyly, it is likely that this threshold-sensitive control is governed by
another Fgf receptor. Fgfr2 would be a good candidate, as gain-of-functions
mutations in this gene result in post-axial syndactyly in Apert Syndrome.
4C-BAC16 limbs also exhibit an ectopic anterior shift in the domain of
Hoxd13 expression, which is normally restricted to regions that
generate the triphalangeal digits II-V. This shift, in addition to the reduced
cell death described above, may contribute to the overgrowth in digit I, as
mice with loss of Hoxd13 function have significantly shorter digits
(Dolle et al., 1993;
Bruneau et al., 2001
). However,
because Hox genes play crucial roles in pattern formation, the most likely
consequence of the Hoxd13 shift is the endowment of a triphalangeal
identity to the expanded digit I pool.
As the normal expression pattern of genes such as Shh, dHand
(Hand2 - Mouse Genome Informatics), Gli3 and several Bmps is
unperturbed in 4C-BAC16 limbs, the ectopic Hoxd13 may represent a
novel connectivity between Hoxd13 gene expression and elevated Fgfr
signalling. This may be mediated by members of the caudal-related homeobox
(Cdx) gene family, as Bel-Vialar et al.
(Bel-Vialar et al., 2002) have
shown that Hox genes exhibit differential sensitivity to Fgf signalling via a
Cdx-dependent pathway. In addition, a subset of Cdx1/Cdx2 compound
mutant mice develop digit I polydactyly and show posterior homeotic
transformation of several vertebrae, remarkably similar to those described in
this report (Fig. 4)
(van den Akker et al., 2002
).
However, we have been unable to detect expression of these genes in either
normal or mutant limbs by WMISH.
Developmental significance of signalling dynamics
Our studies of the hypermorphic Pro252Arg allele of Fgfr1 have highlighted
the importance of signalling dynamics in skeletogenesis. In particular, we
have shown that, for Fgfr1, some system responses to ligands are
quantitatively related to ligand availability and others exhibit a binary
switch in response as signalling passes a threshold value. We have argued that
these different categories of response reflect differing designs of downstream
signalling pathways. This enables major changes in the developmental process,
either evolutionary or pathological, to be effected by changing the `values'
of the signalling pathways rather than the connectivity.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: School of Biological Sciences, University of East Anglia,
Norwich NR4 7TJ, UK
These authors contributed equally to this work
Present address: Yale School of Medicine; BCMM 147, 295 Congress Avenue CT
06511 New Haven, USA
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