Download Postaxial polydactyly in forelimbs of CRABP-II mutant

Development 121, 671-679 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
671
Postaxial polydactyly in forelimbs of CRABP-II mutant mice
Diana Fawcett1, Peter Pasceri1, Robert Fraser1, Melissa Colbert2,†, Janet Rossant3,4 and Vincent Giguère1,4,*
1Division of Endocrinology, Research Institute, Hospital for Sick Children, Toronto, Canada M5G 1X8
2Department of Microbiology, Duke University School of Medicine, Durham, NC 27710
3Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, Toronto, Canada
M5G 1X5
4Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada
†Present
address: Division of Molecular and Cardiovascular Biology, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3093, USA
*Present address and address for all correspondence: Molecular Oncology Group, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Canada H3A 1A1
SUMMARY
The cytoplasmic retinoic acid (RA)-binding protein
CRABP-II is expressed widely throughout early morphogenesis in mouse embryo, but its expression becomes more
restricted as organogenesis progresses. CRABP-II
expression remains strong in the developing limb bud suggesting a role for this protein in limb patterning. Here, we
show that the CRABP-II promoter can direct expression of
a lacZ transgene in a specific posterior domain during limb
bud development. In order to investigate in more detail the
role played by CRABP-II in RA signal transduction, we
have also generated mice homozygous for a null mutation
of this gene. CRABPII−/− mice are viable and fertile but
show a developmental defect of the forelimb, specifically an
additional, postaxial digit. This digit is generally, but not
exclusively, limited to a single forepaw of an individual
animal. The penetrance of the phenotype varies according
to the genetic background, occurring most frequently on
the inbred 129Sv background (50%), less frequently on the
C57Bl/6 background (30%) and rarely on the outbred CD1
background (10%). This developmental abnormality
implies a role for CRABP-II in normal patterning of the
limb.
INTRODUCTION
tion are the cytoplasmic RA-binding proteins, of which two
have so far been identified, named CRABP-I (Chytil and Ong,
1984) and CRABP-II (Giguère et al., 1990). These are small,
closely related peptides of approximately 16×103 Mr which
bind RA with high affinity, although the affinity of CRABPII for all-trans RA is 15-fold lower than that of CRABP-I
(Fiorella et al., 1993; Fogh et al., 1993). Both CRABP genes
are expressed by embryonic day 7.5, as detected by in situ
hybridization (Ruberte et al., 1992; Lyn and Giguère, 1994).
Throughout development there is some overlap in the patterns
of expression, but also regions where expression of one gene
is unique. CRABP-I is expressed in regions of the developing
embryo which are particularly sensitive to exogenous RA
(Ruberte et al., 1992) and is thought to play an important role
in regulation of intracellular RA levels. The binding protein
may facilitate metabolism of RA to polar metabolites, or may
simply sequester RA making it unavailable to the nuclear
receptors (Fiorella and Napoli, 1991). Further evidence in
support of this role is provided by the ability of CRABP-I to
reduce RA-induced activation of transcription by RARs in F9
teratocarcinoma cells (Boylan and Gudas, 1991). The role of
CRABP-II is still unclear. By analogy, it is possible that
CRABP-II plays a similar role to CRABP-I as it is also
expressed in RA-sensitive tissues, but it is also expressed in
tissues not known to be sensitive to RA (Ruberte et al., 1992).
Unlike CRABP-I, CRABP-II expression is inducible by RA
(Giguère et al., 1990). The promoter region of the gene
contains two RAREs (Durand et al., 1992), and an additional
Normal development of vertebrate embryos requires retinoic
acid (RA). Regulation of physiological levels is vital, as RA is
a potent teratogen. In both deficiency and excess, RA can cause
a syndrome of developmental defects known as retinoic acid
embryopathy (RAE) (Lammer et al., 1985). These traits
include craniofacial and limb abnormalities, heart defects and
malformations of the central nervous system. Although the role
of RA in both normal and teratogenic development has been
the subject of much investigation, its fundamental role in the
process of morphogenesis is still uncertain. However, the
mechanism of transduction of the RA signal is becoming more
clear.
RA affects expression of responsive genes via the action of
two distinct families of nuclear receptors, known as the RAR
and RXR families. Belonging to the steroid/thyroid hormone
superfamily of nuclear receptors, these RA receptors are
ligand-dependent transcriptional regulators and act by binding
to specific DNA sequences (RA response elements (RAREs))
contained in the regulatory regions of responsive genes
(reviewed in Giguère, 1994). At least three genes have been
identified within each receptor family, and each gene gives rise
to multiple isoforms, some of which are inducible by RA. Differential expression of these isoforms (which act as RAR/RXR
heterodimers) could give rise to a wide variety of isoform combinations which may possess specific regulatory functions.
A second class of proteins involved in RA signal transduc-
Key words: vitamin A, retinoic acid, limb, mouse, CRABP-II,
polydactyly
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D. Fawcett and others
level of control is provided by a post-transcriptional
mechanism (MacGregor et al., 1992). CRABP-II is largely coexpressed with another autoregulated member of the RA signal
transduction pathway, namely RARβ2 (de Thé et al., 1990;
Hoffman et al., 1990; Sucov et al., 1990).
Further insight into gene function can be gained by studying
the effects of specific mutations. To this end the technique of
gene targeting by homologous recombination in embryonic
stem cells has proved highly useful. Recently, several different
RAR and RXR isoform-specific mutations have been reported,
as well as total null mutations (Li et al., 1993; Lohnes et al.,
1993; Lufkin et al., 1993; Sucov et al., 1994) Surprisingly,
individual isoforms of each receptor seem to be dispensable for
normal development. At least two isoforms must be eliminated
before any obvious defect is produced, an effect seen when
isoforms from either the same or different genes within the
family are targeted (Lohnes et al., 1993; Lufkin et al., 1993);
Luo and Giguère, unpublished results).
To investigate the role of CRABP-II in RA signal transduction, we have disrupted the CRABP-II gene to give a null
mutation. Mice homozygous for the mutation are viable and
fertile. Defects are limited to the forelimb in which there is an
additional, postaxial digit, which is generally, but not exclusively, unilateral. This limb abnormality can be related to the
expression domains of a CRABP-II promoter-lacZ transgene
within the developing limb. The possibility that loss of
CRABP-II may result in an alteration of RA levels on the
posterior side of the limb bud is discussed.
MATERIALS AND METHODS
Transgenic mice: production and expression analysis
The vector p2.7CIIlacZ was made by inserting a 2.7 kb BglII fragment
containing the CRABP-II promoter and 121 bp of 5′ untranslated
sequence (MacGregor et al., 1992; see also Fig. 2A) upstream of the
E. coli lacZ reporter gene in pSDKlacZpA. Transgenic mice were
generated on an outbred CD-1 background by standard procedure
(Hogan et al., 1986), and transgenic offspring were identified by
Southern blots of tail DNA with a lacZ DNA probe. Transgenic males
mice were mated with CD-1 females, and fetuses were examined in
mid-gestation for β-galactosidase expression. Only those males (Tg18 and Tg15-3) that showed CRABP-II-like expression were retained
for further breeding and analysis. Expression analysis was performed
as previously described (Rossant et al., 1991).
Targeting vector construction
The genomic clones for the mouse CRABP-II gene was isolated from
a mouse strain 129Sv genomic library. The targeting vector
pNTC2.KO was constructed as follows. A 2.7 kb BglII fragment containing the promoter region of the CRABP-II gene and the 5′ untranslated region of exon 1 was cloned into the XhoI site of plasmid pNT
(Tybulewicz et al., 1991) to form pNT2.7. An 8.3 kb BglII fragment
originating within intron 1 and containing the remaining 3′ sequences
of the gene was cloned into the BamHI site of pNT2.7 to form
pNTC2.KO. The 0.5 kb BglII fragment containing exon 1 sequences
including the translational start site and the splice donor site, is thus
replaced by the neo cassette (Fig. 2).
ES cell culture, detection of homologous recombinants
and generation of chimeras
The targeting vector was introduced into the D3 ES cell line
(Doetschman et al., 1985) by electroporation, and the cells were maintained under G418/GANC double selection for 8 days. Genomic DNA
was isolated from surviving colonies, digested with BglII and
screened by Southern blotting for homologous recombinants using the
1.6 kb EcoRI probe derived from the shorter vector arm (probe A in
Fig. 2). Positive clones contained a 4.3 kb mutant band as well as a
2.7 kb wild-type band. These clones were verified by a second
genomic digest (XhoI), probed with an external 3′ probe (probe B,
Fig. 2) which gave a 9 kb wild-type and 11 kb mutant band. Cells
from 3 targeted lines were introduced into C57Bl/6 blastocysts by
microinjection as described (Papaioannou and Johnson, 1993).
Immunohistochemistry
10.5 dpc embryos were fixed in 4% paraformaldehyde, infiltrated with
30% sucrose and prepared for immunocytochemistry as described
(LaMantia et al., 1993). The affinity purified anti-CRABP-II polyclonal antibody was diluted to 1-2 µg/ml and the monoclonal antiNCAM (Chemicon) antibody was used at 1 µg/ml for immunohistochemical staining. Antibody binding was detected using
DTAF-conjugated species-specific secondary antibodies (Chemicon
or Accurate chemicals) and photographed using a Zeiss fluorescence
microscope (Axioscope).
RNA isolation, skeletal staining and RA treatment of
pregnant females
Total RNA was prepared from embryos using TRIZOL reagent
(BRL). Newborn pups and adult limbs were prepared for skeletal
analysis by alcian blue/alizarin red staining as described (Lufkin et
al., 1992). RA treatment was performed as described (Lyn and
Giguère, 1994).
RESULTS
A CRABP-II promoter lacZ/transgene is expressed in
the posterior domain of the developing limb bud
Previous northern blot and in situ hybridization studies of the
pattern of expression of CRABP-II have demonstrated that
CRABP-II mRNA is abundantly expressed in the mesenchymal cells of the developing limb bud (Giguère et al., 1990;
Ruberte et al., 1992; Lyn and Giguère, 1994). In order to study
the transcriptional regulation of the CRABP-II gene during
limb bud development, we have generated transgenic mice
expressing a 2.7 kb CRABP-II promoter-lacZ fusion gene. Two
transgenic males produced litters in which the expected 50%
showed essentially identical and very specific patterns of
expression. Fetuses derived from Tg15-3 show the strongest
staining, with blue color being apparent within 15 minutes of
immersing in substrate (see below), and the analysis of
expression patterns was performed on litters sired by mouse
Tg15-3. During early development, the CRABP-II promoterlacZ fusion gene exhibits a pattern of β-galactosidase activity
reminiscent of the sites that have been demonstrated to express
CRABP-II mRNA (Fraser, Lyn and Giguère, unpublished
data). At a later stage, intense β-galactosidase activity is
restricted to the developing limb buds (Fig. 1A) while weak
lacZ expression could still be detected in the frontonasal and
pharyngeal arch mesenchyme (data not shown). Interestingly,
strong lacZ expression is detected in the posterior mesenchyme
of the developing limb buds as staining was apparent in the
posterior domain between 15 and 30 minutes after initiation of
staining (Fig. 1A-C). Longer incubation with X-gal (Fig. 1D,
1.5 hours) revealed a graded expression of the lacZ transgene
both in the anteroposterior and dorsoventral axes. Overnight
staining showed an homogenous distribution of lacZ
expression throughout the mesenchyme (data not shown), with
Polydactyly in CRABP-II mutant mice
the exception of anterior, ventral mesenchyme of the limb. This
latter observation is consistent with previously reported pattern
of CRABP-II expression in the limb bud (Ruberte et al., 1992).
These data reveal that transcriptional activity of the CRABP-II
promoter is higher in the posterior mesenchyme of the developing limbs, and further suggest that, if CRABP-II plays a role
in limb development, disruption of the CRABP-II gene may
lead to patterning defects preferentially affecting the posterior
region.
Generation of homozygous CRABP-II null mutants
The vector pNTC2.KO (Fig. 2A) was electroporated into ES
cells and five independent lines containing a mutated CRABPII allele were detected by Southern blotting (Materials and
Methods). A male chimera derived from one of these lines
transmitted the ES genotype when mated with either C57Bl/6,
CD1 or 129Sv females. As expected, 50% of the ES-derived
progeny were heterozygous (CRABP-II+/−) for the mutant
allele, and these animals appeared phenotypically indistinguishable from their wild-type (CRABP-II+/+) littermates. Heterozygotes were crossed to generate homozygous mutant mice.
Table 1 shows the number of each genotype obtained (Fig. 2B
shows a sample litter). No under-representation of homozygote
(CRABP-II−/−) offspring was found, indicating that the
CRABP-II−/− mutation is not lethal at an embryonic stage and
that the pups are viable until at least 3 weeks. No adult CRABPII−/− animals have died unexpectedly and the oldest mice have
lived at least 10 months. Homozygote males and females were
crossed with heterozygous mice to assess fertility. In both
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Table 1. Viability of CRABP-II−/− mice
Genotype (3 weeks)
+/+
+/−
−/−
Number of mice
Expected (if 1:2:1)
72
62
121
124
55
62
Numbers indicate offspring from heterozygous intercross. DNA was made
from tail snips for determination of genotype.
types of cross, several litters of average size were obtained
showing that homozygotes are fully fertile.
CRABP-II−/− mutant mice are true nulls
To ensure that the targeting event produced a truly null
phenotype, CRABP-II−/− embryos were examined for both
residual protein and mRNA. CRABP-II protein was evaluated
by immunocytochemical staining with a polyclonal antibody
specific for CRABP-II (M. Colbert, unpublished). Transverse
sections through the spinal cord were taken at the level of the
forelimbs from 10.5 dpc embryos produced by heterozygote
crossess. At this gestational stage the expression of the
CRABP-II gene is high within this region (Ruberte et al., 1992,
1993). As shown in Fig. 3A, the cells within the developing
motor regions in the basal plate of the spinal cord as well as
the cells in the dorsal root ganglia are the most intensely
stained by the antibody against CRABP-II in the wild-type
embryos. The staining pattern of similar sections from heterozygotes is identical although it appears quantitatively less
than the wild-type embryos (compare Fig. 3C with A). No
Fig. 1. Transgenic CRABP-II promoter-lacZ 10.5 day embryos stained with X-gal. (A) Embryo showing intensive staining in the posterior
domain of the limb buds after a 30 minute incubation with X-gal. Close-up of forelimb (B) and hindlimb (C). (D) The same embryo showing
more widespread staining after 90 minute incubation with X-gal. Staining extends more posteriorally and proximally in the limb bud and can
now be observed in the frontonasal and pharyngeal arch mesenchyme.
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CRABP-II protein, however, could be detected by the antibody
in the CRABP-II−/− embryos (Fig. 3E). Serial sections from
each genotype were also stained with antibodies against
NCAM as control. In these sections, the patterns were indistinghuishable (Fig. 3B,D,F). As additional verification, total
RNA was isolated from 12.5 dpc embryos of each genotype
(littermates from heterozygote intercross) and compared by
band intensity in a northern blot. No transcripts could be
detected in CRABP-II−/− embryos (Fig. 4). Again, the heterozygote shows a weaker signal that
the wild type.
penetrance of this phenotype varies according to the genetic
background (Table 2). It occurs most frequently in the 129Sv
background (approximately 50%), less frequently in the
C57Bl/6 background (approximately 30%) and rarely occurs
in the CD1 background (approximately 10%). No other defect
in the limb was found, for example in the length of the digits
or the long bones. Other regions of the skeleton were examined
but no defects were detected. No limb defects were observed
in heterozygous or wild-type mice on any genetic background.
Expression of CRABP-I, RARβ2
and a CRABP-II promoter/lacZ
transgene are unaffected in
CRABP-II−/− mice
We investigated the possibility that in
CRABP-II−/− mice the loss of CRABPII may be compensated for by an
increase in CRABP-I. Total RNA
isolated from 12.5 d.p.c. embryos was
again compared by band intensity on a
northern blot. The level of CRABP-I
RNA was the same in CRABP-II−/−
embryos as in wild-type or heterozygous littermates (Fig. 4). We also used
the same RNA to investigate expression
of RARβ2, a RA-responsive gene,
which may show an elevated level of
expression in the absence of CRABP-II
if this results in more free RA (or conversely a decrease in expression if
CRABP-II has the opposite function).
Again we could detect no major change
in the level of expression of the RARβ2
gene (Fig. 4). To study whether
CRABP-II participates in an autoregulatory mechanism, we transmitted onto
the CRABP-II−/− mutant background the
CRABP-II promoter/lacZ transgene that
expresses lacZ in the posterior mesenchyme of the limb bud (see above).
Expression of lacZ was not affected by
the loss of CRABP-II (data not shown).
CRABP-II−/− mice have an
additional postaxial digit
A superficial examination of CRABPII−/− adult mice revealed the presence of
an additional postaxial digit (Fig.
5C,D). This digit is small, containing a
single phalangeal bone, and is not
therefore a true duplication of digit 5.
The defect is apparently restricted to the
forelimbs as no alteration of the
hindlimbs has been observed. In general
only one forelimb per individual is
affected, although we have found
several animals in which both forelimbs
carry the defect (on both C57Bl/6 and
129Sv backgrounds). Interestingly, the
Fig. 2. Schematic representation of the CRABP-II targeting vector and recombination at the
CRABP-II locus. (A) Map of the CRABP-II locus from the 129Sv mouse strain. Top, the wildtype locus. Coding regions of exons are shown as shaded boxes, non-coding regions as open
boxes. Fragments used to probe Southern blots are indicated. Centre, the targeting vector
pNTC2.KO. Bottom, the structure of the disrupted CRABP-II locus, showing replacement of
genomic sequences by the neo cassette. The genomic fragments detected by probe A (BglII
digest) and probe B (XhoI digest) are illustrated. (B) Southern blot of a typical litter from a
heterozygote intercross. Genomic DNA was digested with BglII and probe A was used to
distinguish wild-type and mutant alleles.
Polydactyly in CRABP-II mutant mice
Table 2. Frequency of occurrence of polydactyly in
CRABP-II−/− adult mice, according to the genetic
background of the mutation
Forelimbs affected
Genetic background*
One limb
Both limbs
Neither limb
Total
129Sv
C57Bl/6
CD1
3
4
1
1
1
0
4
11
8
8
16
9
*Embryos were obtained by mating a male chimera to 129Sv, C57Bl/6 or
CD1 females.
Loss of CRABP-II causes no gross organ
abnormalities
CRABP-II is expressed widely throughout embryonic development (Ruberte et al., 1992; Lyn and Giguère, 1994). We
performed a histological examination of several adult tissues
including brain, liver, kidney and gut, all sites of CRABP-II
expression in the embryo, and also adult skin, the predominant
site of expression in the adult (Giguère et al., 1990). In no case
could we detect any change in morphology when comparing
CRABP-II+/+ and CRABP-II−/− individuals (data not shown).
CRABP-II−/− mice show no change in sensitivity to
exogenously administered RA during early
embryogenesis
If CRABP-II is involved in catabolism and/or sequestration of
RA, CRABP-II−/− embryos may be more sensitive to exogenously administered RA as their ability to catabolize and/or
sequester RA is reduced. Conversely, if CRABP-II has the
opposite effect CRABP-II−/− embryos may be less sensitive to
exogenously administered RA. To test this, heterozygous
matings were set up. At day 7.5 p.c. pregnant females were
treated with RA at a range of concentrations from 5 mg/kg to
50 mg/kg, and the embryos removed at day 10.5 p.c. By this
method a gross response to RA is readily observable by examination of size and morphology of the embryos, although a
more detailed analysis will be required to monitor subtle morphological differences (R. Conlon, personal communication).
At high doses (50 mg/kg and 20 mg/kg), embryos were greatly
reduced in size, the morphology was grossly abnormal and
there were several resorbtions. All embryos analyzed (17 and
26 embryos from mothers treated with RA at concentrations of
50 mg/kg and 20 mg/kg, respectively), irrespective of
genotype, responded to the same degree. At the lowest dose,
the affect was much milder. The embryos (12 embryos from
mothers treated with RA at a concentration of 5 mg/kg) were
of fairly normal size and morphology with only slight defects
in the head region, and all embryos were affected to the same
extent, irrespective of genotype.
DISCUSSION
CRABP-II is presumed to play a role in transduction of the RA
signal, but the nature of that role is unclear. We have generated
a null mutation of the CRABP-II gene in order to gain an
insight into function of this protein. The results of this study
indicate that CRABP-II is involved in the patterning of the
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forelimb, as loss of this protein causes postaxial polydactyly.
It is surprising, given the widespread expression of the
CRABP-II gene throughout development, that the limb defect
is the only obvious consequence of this mutation. This may
reflect compensation for the loss by other RA-binding proteins,
such as CRABP-I and possibly others which have yet to be
identified (Giguère et al., 1990). Expression of CRABP-I
appears to be unchanged in CRABP-II−/− mice, but as the
affinity of CRABP-I for RA is relatively much higher than that
of CRABP-II there may be no requirement for more CRABPI if the protein is not normally saturated. If other binding
proteins can compensate for loss of CRABP-II, then it might
be expected that where CRABP-II is uniquely expressed there
would be structural abnormalities. However, we found no such
defects at the level of gross morphology. It is possible there
are changes affecting the cytoarchitecture, which could have
more subtle consequences. For example, such a defect within
the telencephalon could lead to alterations in sensory functions,
or behavior.
It is interesting that the one region where a defect is seen is
one in which both CRABPs are expressed, namely the
forelimb. In the developing limb bud there is a complex pattern
of CRABP expression. Although both genes are expressed in
the developing limb buds, their spatial restrictions are quite
different (Ruberte et al., 1992), leading perhaps to localized
regions of higher or lower RA concentration. At the anterior
side of the limb bud, CRABP-I forms a proximodistal gradient,
the highest levels being found at the distal side (Dollé et al.,
1989b). Expression is abundant and more uniform on the
posterior side. In situ hybridization studies have shown that
CRABP-II is apparently expressed more uniformly in these
dimensions, although a dorsoventral gradient was also
observed, the highest levels being found on the dorsal side. In
this study, we have demonstrated that a CRABP-II promoter
directs strong expression of a lacZ transgene in the posterior
mesenchyme of the limb bud. At the posterior side therefore,
where the extra digit is found, there is a high level of both
binding proteins. This is also the location of the ZPA, where
the level of RA is high (Thaller and Eichele, 1987). If the
function of both CRABP-I and II is to bind RA and prevent
access to the receptors, then in CRABP-II−/− embryos there
may be an excess of RA on the posterior side of the limb bud
where there is a lack of CRABP-II. This would assume that the
level of RA is high enough that CRABP-I is saturated.
Until fairly recently, RA itself was considered as a likely
candidate for a limb morphogen, possibly by diffusing across
the limb bud from the ZPA to form a gradient which could give
positional information to the mesenchymal cells (Eichele,
1989). More recent evidence suggests that the product of the
Sonic hedgehog (Shh) gene may act as the limb morphogen, as
Shh is expressed in the putative ZPA and ectopic expression
of Shh leads to limb duplication (Echelard et al., 1993; Krauss
et al., 1993; Riddle et al., 1993). RA has been shown to induce
expression of Shh concomitantly with production of the ZPA,
while sonic hedgehog itself exhibits ZPA-like activity (Riddle
et al., 1993). Thus RA probably acts at an earlier stage in
pattern formation, perhaps by determining the formation of the
ZPA. It is interesting to note that expression of the CRABP-II
promoter-lacZ transgene is preferentially localized to a
posterior domain in the limb bud (Fig. 1).
Both RA and sonic hedgehog act to repattern the limb by
Fig. 3. Immunohistochemical staining of sections through the trunk region of 10.5 d.p.c.
embryos from a heterozygote intercross. (A,C,E) Stained with a polyclonal anti-CRABP-
II antibody, the genotypes are as indicated. (B,D,F) Consecutive sections from the same
embryos as A, C and E respectively, stained with a control anti-NCAM antibody.
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D. Fawcett and others
Polydactyly in CRABP-II mutant mice
Fig. 4. Northern blot analysis of total RNA isolated from 12.5 d.p.c.
embryos. CRABP-II mRNA was detected using a mouse CRABP-II
cDNA probe containing exon 2, 3 and 4 sequences but lacking exon
1 sequence 5′ of the neo insertion site. CRABP-I was detected using
the entire mouse CRABP-I cDNA. Both CRABP signals were
detected after overnight exposure to X-ray film at −70°C. RARβ2
mRNA was detected using a probe derived from RARβ2-specific
exon 1. The signal was weak but detectable after 7 day exposure. A
mouse β-actin cDNA probe was used as a loading control.
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affecting expression of the Hoxd genes. These genes are
activated in a sequential manner to give a pattern of nested
expression across the limb bud; the first gene to be activated
showing the most anterior expression boundary and each successive gene showing a more posterior restriction until five
distinct regions are specified (Dollé et al., 1989a; IzpisúaBelmonte et al., 1991; Nohno et al., 1991). Ectopic administration of RA or ectopic expression of ssh causes Hoxd genes
to be expressed to more anterior positions than normal
(Izpisúa-Belmonte et al., 1991; Riddle et al., 1993). If there is
an excess of RA in the limb bud of the CRABP-II−/− mice then,
the limb defect could be due to interference with normal Shh
function, possibly resulting in mis-expression of Hoxd genes.
How this results in an additional, postaxial digit is not clear,
but could be a result of an excess of mesenchymal cells on the
posterior side of the limb bud. This is seen in the Hoxd-13
mutant mice (Dollé et al., 1993) where a disruption of the Hoxd
pattern resulted in limb defects, including an additional
postaxial digit. In these mice, a large field of uncondensed mesenchymal cells was observed on the posterior side of the limb
bud. The mechanism suggested by Dollé et al. (1993) for digit
formation depends on a ratio between condensed and uncondensed mesenchymal cells; too many uncondensed cells in one
area could spontaneously condense to form an extra digit. We
Fig. 5. Forelimbs and skeletal
preparation of littermates from
heterozygous mating.
(A,C) Photograph of wild-type
(A) and mutant (C) forelimb of
newborn littermates (129Sv
background) taken at the same
magnification (12×).
(C) Mutant forelimb shows an
extra sixth digit.
(B,D) Photograph taken at the
same magnification showing
alcian blue/alizarin red staining
for cartilage and bone,
respectively. The forelimbs are
taken from newborn wild-type
(B) and mutant (D) littermates.
An additional postaxial sixth
digit (VI) is present with one
phalange.
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D. Fawcett and others
do not know if a similar effect is seen in the CRABP-II−/− mice,
but if so it is likely to be less obvious than in the Hoxd-13−/−
mice where the limb defects are more severe. In CRABP-II−/−
mice, the extra digit tends to be more rudimentary than in
Hoxd-13−/− mice and never occurs in the hindlimb.
Recently the disruption of the mouse En-1 gene has been
described (Wurst et al., 1994). One of the defects seen is a
postaxial, mostly unilateral, extra digit. This similarity with the
CRABP-II phenotype is produced despite the fact that En-1
expression in the limbs is initially restricted to the ventral
ectoderm and later to the AER, where CRABP-II and the Hoxd
genes are not expressed. This illustrates a possible interaction
between the ectoderm and the underlying mesenchyme which
is important not just for proximodistal axis specification but
also for the anteroposterior axis. It is known that to be maintained the AER requires posterior mesenchyme and that conversely the AER produces mitogenic factors (possibly FGF-4)
that are needed for maintenance of the ZPA as well as for limb
outgrowth (Laufer, 1993; Vogel and Tickle, 1993). This interaction between mesenchyme and ectoderm may be mediated
by ssh (Riddle et al., 1993). These complex interactions
between mesoderm and ectoderm provide the underlying
reasons for observing similar phenotypes in mice carrying
mutations in two very different genes.
It is interesting that the extra digit is never found on the
hindlimbs, in which the same mechanism of digit specification
is proposed to exist. This could be a reflection of the relative
timing of forelimb and hindlimb development, as hindlimb
development is delayed by approximately one day. It does
illustrate, however, that forelimb and hindlimb development,
despite similarities in gene expression within the limb buds,
are not equivalent.
It has already been noted that more than one RAR or RXR
isoform must be mutated before a phenotype is observed. It is
possible that targeting of two different components of the RA
signal transduction pathway, for example a receptor isoform
and a binding protein such as CRABP-II, will produce a more
severe phenotype than either single mutation. It is clear that
the mechanism of RA signal transduction is more complex than
was initially visualized, with much functional compensation
being revealed amongst the receptors and now the binding
proteins.
We would like to thank Valerie Prideaux for assistance with mouse
breeding, Xiu-Ming Yang for help with phenotypic analyses and Mark
Hanks for critical reading of this manuscript. We would also like to
thank members of the Giguère laboratory for helpful discussions
during the course of this project. This research was supported by
grants from the National Cancer Institute of Canada (V. G.), the
Medical Research Council (MRC) of Canada (V. G. and J. R.) and
Bristol-Myers Squibb Ltd (J. R.). M. C. was supported by NIH postdoctoral fellowship HD07637 and would like to acknowledge the
hepful advice of members of Elwood Linney’s laboratory at Duke
University where the anti-CRABP-II antibody was generated. V. G.
is an MRC Scholar and J. R. is a Terry Fox Career Research Scientist
of the NCIC and a Howard Hughes International Research Scholar.
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(Accepted 23 November 1994)