Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0618, USA
* Author for correspondence (e-mail: meislerm{at}umich.edu)
Accepted 16 February 2004
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SUMMARY |
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Key words: Dkk1, Lrp6, Wnt, Limb, Polydactyly
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Introduction |
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Dkk1 was identified in a screen for head inducers in
Xenopus (Glinka et al.,
1998). Overexpression of Dkk1 in early Xenopus
blastomeres led to embryos with enlarged anterior head structures, whereas
injection of antibody to Dkk1 induced microcephaly. The role in head
development is conserved in mammals. Mice lacking Dkk1 exhibit
incomplete development of structures anterior to the midbrain, resulting in
perinatal death (Mukhopadhyay et al.,
2001
). The null mice also display polydactyly and syndactyly,
demonstrating the role of Dkk1 in limb development.
Mammalian Dkk1 is a 266-residue protein with two cysteine-rich domains
(Fedi et al., 1999). The
second cysteine-rich domain is required for binding to Lrp6 and Kremen 2
(Li et al., 2002
;
Mao and Niehrs, 2003
). Three
vertebrate paralogs, Dkk2, Dkk3 and Dkk4, were identified by
sequence homology (Glinka et al.,
1998
; Krupnik et al.,
1999
). Dkk2 and Dkk4 bind Lrp5/6 and inhibit Wnt signaling with
lower affinity than Dkk1. Dkk2 can activate Wnt signaling in cells lacking
Krm2 (Mao and Niehrs, 2003
).
Dkk3 does not bind to Lrp5/6 or Kremen, but may play a role in Wnt signaling
(Krupnik et al., 1999
;
Mao et al., 2001
;
Mao et al., 2002
).
Lrp5 and Lrp6 are widely expressed during embryonic
development and in many adult tissues
(Brown et al., 1998;
Hey et al., 1998
;
Pinson et al., 2000
). In in
vitro assays, the affinity of Dkk1 for Lrp6 is greater than for Lrp5
(Bafico et al., 2001
). The
phenotypic consequences of mutations in Wnt proteins and Lrp receptors are
closely related. Lrp6 null mice exhibit defects in neural tube
closure and midbrain/hindbrain like Wnt1 null mice, axial truncation
and loss of hindlimbs like Wnt3a hypomorphs, and urogenital defects,
loss of posterior digits and double ventral forelimbs like Wnt7a null
mice (Pinson et al.,
2000
).
Deficiency of Lrp5 results in low bone mass, failure of postnatal
regression of eye vasculature, and abnormal metabolism of cholesterol and
glucose (Kato et al., 2002;
Fujino et al., 2003
).
Loss-of-function mutations of human LRP5 result in
osteoporosis-pseudoglioma syndrome, a recessive disorder with low bone mass
and disruptions in ocular vasculature
(Gong et al., 2001
), whereas
gain-of-function mutations in LRP5 result in excess bone density
(Little et al., 2002
;
Boyden et al., 2002
;
Van Wesenbeck et al., 2003
).
Dkk inhibitors have been suggested as potential therapeutic agents for
osteoporosis (Patel and Karsenty,
2002
).
The doubleridge mutant mouse was identified in a screen for
recessive, transgene-induced insertional mutants
(Adamska et al., 2003). The
doubleridge transgene randomly inserted into an SJL-derived segment
of chromosome 19 in a microinjected (C57BL/6xSJL)F2
fertilized egg. Homozygous doubleridge mice exhibit defective limb
development. In this report we describe cloning of the doubleridge
insertion site and demonstration that doubleridge is a hypomorphic
allele of Dkk1. We used this viable mutant to investigate the
interaction of Dkk1 with other components of Wnt signaling during
development.
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Materials and methods |
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Genotyping
Dkk1 genotype of doubleridge mice was determined by amplification
of the proximal transgene junction with three primers, F1 (5' GTT TCA
GCC CCA AAG ACT GCA TAG), R1-genomic (5' TTC ATT GAC GCT TTC CTT TCC
AAG) and R-2 transgenic (5' GAA TGT TGA GAG TCA GCA GTA GCC) using the
following PCR conditions: incubation at 94°C for 2 minutes followed by 30
cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2
minutes, followed by a final extension of 7 minutes at 72°C. The Dkk1
targeted null allele was detected with primers for the Neomycin-resistance
cassette: Dkk1-Neo F: 5'CTT GGG TGG AGA GGC TAT TC and Dkk1-Neo R:
5'AGG TGA GAT GAC AGG AGA TC. Genotypes for Lrp5 and Lrp6 null mutations
were determined with the following primers: Lrp5-NeoF, 5'GCA GCG CAT CGC
CTT CTA TC; Lrp5-genomicF, 5'GAG CTC TCA AGC TCA GCC AG; Lrp5-genomicR,
5'CTT CTC TCC AGA CTC CCA AAG C; Lrp6-genomic3F, 5'CAG GCA TGT AGC
CCT TGG AG; Lrp6-genomic4R, 5'ACT ACA AGC CCT GCA CTG CC and Lrp6-insert
R, 5'GTA GAG TTC CCA GGA GGA GCC.
Primer extension/chain termination assay
Embryos from timed matings were dissected as follows: E7.0 whole embryo;
E8.5 head and tail; E9.5 head, forelimb and tail; E10.5 head, forelimb,
hindlimb and tail; and E13.5 head, forelimb, hindlimb and tail. Genomic DNA
was prepared from extraembryonic membranes for genotyping. Tissues from
littermates with identical genotypes were pooled and RNA was prepared with the
TRIzol reagent (Invitrogen). RNA (<1 µg) was treated with
Dnase I (Invitrogen) and first-strand cDNA was synthesized with Superscript II
reverse transcriptase (Invitrogen) using random hexamer primers. The 3'
untranslated region (UTR) of Dkk1 was amplified with primers 1 (5'AGG
GGA AAT TGA GGA AAG CAT C) and 2 (5'TTG GAA GGT ATT GTC GGA ATG C) using
PfuTurbo DNA polymerase (Stratagene). The 499 bp RT-PCR product was separated
from the 570 bp product of genomic DNA by gel purification. Thermosequenase
(Stratagene) was used for the extension reactions from primer 3 (5'TGC
CAG AGA CAC TAA ACC GAC AGT C) in the presence of dATP, dCTP, dCTP and
33P-ddGTP. The relative amount of 31 bp product from the C3H
allele and 33 bp product from the SJL allele was determined by densitometry
using the BioRad Molecular Imager FX with Quantity One software, as described
previously (Kearney et al.,
2002
; Buchner et al.,
2003
).
Wholemount in situ hybridization
Embryos were genotyped by PCR of genomic DNA from embryonic membranes. E0.5
was considered to be noon of the day when the vaginal plug was found.
Hybridization was performed with a single digoxygenin-labelled probe
(Bober et al., 1994) using BM
Purple (Roche) as the alkaline phosphatase substrate. The Dkk1 probe was
described by Glinka et al. (Glinka et al.,
1998
).
Skeletal preparations
Samples were prepared and stained as described
(Kimmel and Trammell, 1981),
using alcian blue to stain cartilage and alizarin red to stain bone.
Northern blots
RNA was prepared from adult tissues and analyzed as described
(Kearney et al., 2001). cDNA
probes were amplified by RT-PCR. The 972 bp mannose binding lectin 2
(Mbl2) fragment was amplified with primers F (5'CTT GCC TCC TGA
GTC TTT GCT G), R (5'TTT TCA GAA CAA ACT GCG GAC G); Prkg1 (818 bp) F
(5'CAT TTA CAG GGA CCT CAA GCC G), R (5'GCT TTG CTT CAG GAC CAC
CAT G); AK006729 (359 bp) F (5'TCT GGC AAC ATA AAC GGA AGT G) R
(5'TGG ATT GAG AAG CGT GTA GGA G).
Histology
After fixation in Bouin's solution, E17.5 embryos were embedded in paraffin
and sectioned. Six micron cross-sections of forelimbs were stained with
hematoxylin and eosin.
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Results |
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The only transcript deleted by the doubleridge mutation is a testis EST represented by a singleton clone, AK006729, in the NCBI database. This EST contains two exons but lacks an open reading frame. There is no corresponding human EST, and the lack of sequence conservation in the human genome (<50% sequence identity) suggests that this is not a functional transcript. An LTR element of the RMER15 class in exon 1 accounts for 74/148 bp of exon 1 and a SINE repeat accounts for 56/259 bp in exon 2. RT-PCR from 20 wild-type mouse tissues indicated that transcription is restricted to testis. The fertility of doubleridge males demonstrates that the transcript is not required for reproduction. It is unlikely that deletion of this EST contributes to the doubleridge phenotype.
We compared the human and mouse sequences spanning the 60-kb deletion using
the programs PipMaker and Vista, in order to detect functional noncoding
elements (Schwartz et al.,
2000; Mayor et al.,
2000
). Seven conserved non-coding elements
100 bp in length
with
75% sequence identity were identified (data not shown), but none of
these were conserved in the corresponding regions of the Fugu or
zebrafish genomes (Ensembl sequence assembly of 3/3/03 for Fugu and
7/2/03 for zebrafish).
Non-complementation of doubleridge and Dkk1
The limb abnormality in doubleridge mice is very similar to that
of Dkk1 null mice (Mukhopadhyay
et al., 2001). To determine whether doubleridge is an
allele of Dkk1, we performed a complementation test with the null
allele. Homozygous doubleridge females were crossed with a
heterozygous Dkk1+/ male and forelimbs were
examined at E18.5. Two phenotypic classes of offspring were obtained in equal
numbers: 9/18 with normal limbs (Fig.
2A) and 9/18 with postaxial polysyndactyly of the forelimb
(Fig. 2B). Genotyping
demonstrated that all of the offspring with normal forelimbs inherited the
Dkk1 wild-type allele, whereas all of the offspring with
polysyndactyly inherited the Dkk1 null allele from the heterozygous
parent. The forelimb abnormalities in the affected offspring are similar to
those in doubleridge and Dkk1 null homozygotes
(Fig. 2C,D). The failure of the
doubleridge chromosome to complement the limb abnormality, together
with its chromosomal location, demonstrate that the doubleridge
mutation generated a new allele of Dkk1, designated
Dkk1d. The Dkk1d/ compound
heterozygotes are fully viable on the mixed genetic background of this
cross.
|
Quantitation of Dkk1 expression from the doubleridge allele
The doubleridge mutation arose on an SJL chromosome
(Adamska et al., 2003). To
develop an assay for quantitation of Dkk1 transcripts, we first
identified a single nucleotide polymorphism in the 3' UTR that differed
between the founder strain SJL and a control strain, C3H. The relative
abundance of the two allelic transcripts in (SJL X C3H) F1 heterozygous mice
was determined with a primer extension/chain termination assay based on the
polymorphic nucleotide (Fig.
3A). RNA from heterozygous embryos was amplified by RT-PCR using
primers 1 and 2, followed by a primer extension step using primer 3 in the
presence of
33P-ddGTP. The radiolabelled product obtained
from the C3H allele is 31 bp in length, whereas the product of the SJL allele
is 33 bp in length. The two products were separated by gel electrophoresis and
visualized on X-ray film or with a phosphorimager.
|
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Other genes are not affected by the doubleridge transgene insertion
To determine whether the doubleridge insertion affects the
expression of other genes in the Dkk1 region, we examined the
expression of the two closest genes, Mbl2 and Prkg1
(cGMP-dependent protein kinase 1)
(Fig. 1B). Northern blot
analysis of RNA from adult tissues demonstrated no difference between mutant
and wild-type expression of Mbl2 in adult liver, and no difference in
expression of Prkg1 in brain and heart (data not shown). The size and
abundance of the transcripts was consistent with previous reports
(Pfeifer et al., 1998;
Hansen et al., 2000
).
Prkg1 expression in E13.5 limb bud was examined by semiquantitative
RT-PCR and did not differ between doubleridge and wild-type embryos.
The adjacent genes thus appear to be unaffected by the transgene
insertion.
Effect of reduced Dkk1 expression on head development
In the allelism tests described above, Dkk1d/
compound heterozygotes on a mixed genetic background were viable and fertile,
with complete head development like the Dkk1d/d homozygote
(Fig. 4A). On a predominantly
C57BL/6J genetic background, however, a variety of severe cranial defects were
observed in compound heterozygotes (Fig.
4B), including hydrocephaly with micrognathia
(Fig. 4C). The most severely
affected Dkk1d/ compound heterozygotes exhibit
neonatal lethality with anophthalmia and hypoplastic anterior head structures
(Fig. 4E). Nonetheless, these
defects are less severe than those of Dkk1 null mice, which exhibit
anterior truncation of the head (Fig.
4F,G). The level of Dkk1 expression in compound
heterozygotes appears to be close to the threshold required for development of
anterior head structures.
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Improved viability of Lrp6 null embryos with reduced expression of Dkk1
Homozygous Lrp6/ embryos are recovered in
reduced numbers late in gestation (Stump
et al., 2003) (B.T.M., unpublished). To determine whether
reduction of the Wnt antagonist Dkk1 would compensate for the loss of
the Lrp6 receptor, we generated Lrp6 null embryos that were
Dkk1d/+ or Dkk1+/. A total of
82 embryos were collected at E16.5, five litters from the cross
Lrp6+/ X
Lrp6+/Dkk1d/+ and five litters
from the cross Lrp6+/ X
Lrp6+/Dkk1+/. Nine of
the Lrp6/ embryos were heterozygous for one
of the Dkk1 mutations, consistent with the prediction of 10/82
(P>0.8). Only three of the Lrp6/
embryos were wild-type for Dkk1, significantly fewer than the
predicted 10/82 (P<0.05). Thus, reduction of Dkk1
increased the viability of Lrp6 null embryos at E16.5.
Reduction of Dkk1 levels improves axial development in the Lrp6 null embryo
Homozygous Lrp6 null embryos exhibit pleiotropic defects including
axial truncation distal to the lumbar/sacral vertebrae similar to that seen in
hypomorphic Wnt3a mutants (Pinson
et al., 2000). Diminished signaling through Wnt3a in the
presomitic mesoderm is the probable cause of the axial truncations.
Dkk1 is also expressed in the presomitic mesoderm during
somitogenesis. Reduction of the Wnt inhibitor Dkk1 is predicted to increase
Wnt3a signaling and correct axial development in the Lrp6 null
embryos. To test this hypothesis, we generated an allelic series of
Lrp6 null embryos with decreasing levels of Dkk1
expression.
Lrp6/ homozygotes display axial truncation, fusion of thoracic ribs, and severely defective hind limbs (Fig. 7A). As predicted, axial development was improved in Dkk1d/+Lrp6/ and Dkk1d/dLrp6/ double mutants (Fig. 7B,C). The rescue of hindlimb development is striking, and is more extensive in the Dkk1d/d homozygotes, which have lower expression of Dkk1 than the Dkk1d/+ heterozygotes. Formation of caudal vertebrae is also more complete in the Dkk1d/d mice (Fig. 7C). Nonetheless, the Dkk1d/dLrp6/ double mutants do not survive beyond P1 and are significantly smaller than their Dkk1d/d littermates (Fig. 7D). The lumbar vertebrae of Dkk1d/dLrp6/ embryos exhibit spondylosis (anterior-posterior fusion) (Fig. 7E, bracket), and the distinct organization of individual vertebrae is lost in the sacral region. The major improvement in the axial structure of Dkk1d/dLrp6/ mice compared with the Lrp6 null provides additional evidence of in vivo interaction between Dkk1 and Lrp6.
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Discussion |
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Two mechanisms may be considered to explain the negative effect of the
doubleridge insertion on Dkk1 expression. The replacement of
60 kb of endogenous DNA with 50 kb of transgene may have affected chromatin
structure. In some cases, insertion of multiple transgene copies has been
shown to induce DNA methylation and silencing of nearby genes
(Garrick et al., 1998;
McBurney et al., 2002
).
However, the normal expression of the adjacent genes in doubleridge
mice suggests that the effect of this insertion is restricted to the
Dkk1 gene.
A second reasonable hypothesis is that the 60-kb deletion removed a
transcriptional enhancer of Dkk1 located 150 to 200 kb downstream of
the 3' UTR. Several examples of transcriptional regulatory elements
located more than 100 kb from the gene promoter are now known
(Bedell et al., 1995;
Calhoun and Levine, 2003
). In
another recently described transgene-induced limb mutant, the
Sasquatch mouse, the insertion site is located 1 Mb from the promoter
and disrupted a cis-acting regulatory element
(Lettice et al., 2002
;
Lettice et al., 2003
). The
sequence of the Sasquatch enhancer of Sonic hedgehog is
well-conserved in fish, and mutations in the enhancer were identified in human
patients with limb defects. We attempted to identify an evolutionarily
conserved regulatory element in the 60-kb doubleridge deletion by
sequence comparison with the corresponding region of human chromosome 10q21.
We found seven segments of 100 bp with sequence identity greater than 75%, but
none were conserved in fish genomic sequences. When more sequence is available
from chicken and other vertebrate genomes, we may be able to find a
doubleridge enhancer by this method. There is no match for the
Sasquatch enhancer in the doubleridge deletion.
Head development in Dkk1 mutants
Head induction in the vertebrate embryo is dependent on inhibition of Wnt
and Bmp signaling (Niehrs,
2001). The Six3 transcription factor represses Wnt1
expression, and loss of Six3 results in anterior head defects because
of excess Wnt signalling (Lagutin et al.,
2003
). The null mutation of the Wnt antagonist Dkk1 also
results in failure to develop anterior structures of the head
(Mukhopadhyay et al., 2001
).
In contrast, Dkk1d/d homozygotes express one-third of
normal Dkk1 levels during head induction, and this provides
sufficient reduction in Wnt signalling to permit normal head development in
most individuals. Dkk1d/ compound heterozygotes
exhibit variable development of rostral head structures, suggesting that the
threshold requirement for Dkk1 during head induction is in the range
of 15 to 20%.
Dkk1 and vertebral development
Segmentation in the vertebrate embryo is achieved through the oscillating
expression of Notch signaling genes and the intracellular Wnt inhibitor
Axin2 (Pourquie et al., 2003). Expression of Wnt3a in the
presomitic mesoderm is required for caudal somitogenesis, as demonstrated by
the missing somites in the vestigial tail mouse mutant, a hypomorph
of Wnt3a (Greco et al.,
1996; Ikeya and Takada,
2001
). Overexpression of Wnt3a in the posterior
presomitic mesoderm reduced the size of the somites in chick embryos
(Aulehla et al., 2003
). The
expression of Dkk1 in the presomitic mesoderm overlaps the
Wnt3a expression domain. We observed small, irregularly shaped
vertebrae in mice deficient for Dkk1, which progressed to more
anterior positions with lower levels of Dkk1 in the allele series. Reduced
Dkk1 expression thus results in a phenotype similar to overexpression
of Wnt3a in the chick, indicating that Dkk1 modulates Wnt
signaling during development of vertebral structures.
Interaction between Dkk1 and Lrp6
Functional interaction of Dkk1 and Lrp6 has been observed
in Xenopus embryos and in cultured mammalian cells, and direct
biochemical interaction between the two proteins was demonstrated
(Mao et al., 2001;
Bafico et al., 2001
;
Semenov et al., 2001
). We
provide genetic evidence for interaction of Dkk1 and Lrp6
during mammalian development, based on analysis of double mutants. The ratio
of ligand (Dkk1) to receptor (Lrp6) appears to determine the extent of
abnormalities in developing forelimb (Table
3). Reduction of Dkk1 results in extra digits, whereas
reduction of Lrp6 reduces the number of digits. In
Dkk1d/d Lrp6/ double mutants,
normal digit number is restored (Table
3). This in vivo `titration' provides genetic evidence that the
genes are active in the same pathway and is consistent with a model of direct
interaction. Other aspects of the Lrp6 null phenotype such as the
presence of ectopic ventral tendons are not rescued, perhaps because
Dkk1 is not normally expressed in the dorsal compartment.
When Lrp6 is absent, Lrp5 is thought to mediate canonical Wnt signaling.
Reduction of Lrp5 was less effective in rescuing digit abnormalities,
suggesting that Lrp6 is more important in the developing limb. In
Xenopus embryos, injection of Lrp6 but not Lrp5
induces a secondary axis (Tamai et al.,
2000). Humans and mice with null mutations in Lrp5 do not
exhibit morphological abnormalities, and phenotypes are limited to postnatal
defects of low bone density and impaired regression of the hyaloid vessels in
the eye (Gong et al., 2001
;
Kato et al., 2002
). Dkk
inhibitors have been considered as possible treatments for these conditions.
The Dkk1d/d Lrp5/ mice provide a
viable animal model for evaluating the effect of reduced Dkk1 on
these phenotypes.
The axial truncations in Lrp6/ embryos
are located caudal to the lumbar region and resemble those of hypomorphic
Wnt3a mutants (Pinson et al.,
2000). Caudal development is more complete in
Dkk1d/d Lrp6/ double mutants. No
additional improvement was observed with further reduction of Dkk1
expression in Dkk1d/- and
Dkk1/ double mutants (B.T.M., unpublished).
The Dkk1d/d Lrp6/ double mutants
were able to suckle but exhibited perinatal lethality. The accumulation of
urine in the kidneys and ureter of affected animals suggests that the
lethality may be the result of neurogenic bladder or obstruction of the
urethra. In mice with low levels of Wnt3a, reduced urinary and
excretory function were associated with defects in the S2-S4 autonomic ganglia
(Greco et al., 1996
).
Dkk1 null mice exhibit anterior truncation of the head that is incompatible with postnatal survival. This defect can be ameliorated in Dkk1/ Lrp6+/ double mutants (Fig. 8). Thus, simultaneous reduction of an antagonistic ligand and a receptor can restore Wnt signaling to a balance compatible with life. The dramatic correction of the severe developmental defects of Lrp6 null and Dkk1 null mice observed in double mutants provides support for a direct role of Dkk1 in reducing Wnt signaling through the Lrp6 receptor.
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ACKNOWLEDGMENTS |
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