1 Departments of Pediatrics and Medicine, UCSD Comprehensive Cancer Center,
University of California, San Diego, 9500 Gilman Drive, La Jolla, CA
92093-0627, USA
2 Institute of Molecular Medicine, University of California, San Diego, 9500
Gilman Drive, La Jolla, CA 92093-0641, USA
3 Genetic Disease Research Branch, National Human Genome Research Institute,
National Institutes of Health, Bethesda, MD 20814, USA
4 University of Maryland School of Pharmacy, Department of Pharmaceutical
Sciences, 20 N. Pine Street, Baltimore, MD 21201, USA
5 Departments of Neurobiology, Pathology, and Physical Medicine and
Rehabilitation, University of Alabama at Birmingham, 1530 Third Avenue South,
Birmingham, AL 35294-0021, USA
* Author for correspondence (e-mail: awynshawboris{at}ucsd.edu)
Accepted 19 September 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dvl2, Cardiac neural crest, DORV, PTA, Somitogenesis, Neural tube closure
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The similarity between dsh mutants and the wg phenotype
in Drosophila (Perrimon and
Mahowald, 1987) indicates that an overlap in phenotype might be
expected between Wnt mutants and Dvl knockout mice. All three of the
murine Dishevelled genes are widely expressed in embryonic and adult
tissues suggesting that there may be functional redundancy among the three
genes. These findings make it difficult a priori to predict specific classes
of defects that may occur in mammals after disruption of each of these genes.
Surprisingly, null mutants for Dvl1 exhibit deficits in social
interaction and sensorimotor gating (Lijam
et al., 1997
). By contrast, mouse mutants that contain null
mutations in Wnt genes result in mice with varied brain and
developmental abnormalities (Cadigan and
Nusse, 1997
). For example, mice that are deficient in
Wnt1 display midbrain and hindbrain (cerebellar) abnormalities
(McMahon and Bradley, 1990
;
Thomas and Capecchi, 1990
),
and Wnt1/Wnt3a double mutants have additional deficiencies in the
neural crest (Ikeya et al.,
1997
). The neural crest is a migratory group of cells that emanate
from the dorsal neural tube and give rise to various cell populations,
including melanocytes, the dorsal root ganglia and the cardiac neural crest.
Cardiac neural crest cells originate from the occipital neural tube and aid in
septation of the outflow tracts and in aortic arch development.
The murine Dvl1 and Dvl2 genes are 64% identical and it is unknown whether Dvl2 has a similar role as Dvl1 in development. To address this question and to define further the role of individual Dishevelled genes in mammalian development, mice were generated that were deficient in Dvl2. We found that Dvl2 is essential for normal cardiac morphogenesis, somite segmentation, and neural tube closure. In addition, there is functional redundancy between Dvl1 and Dvl2 in somite development and neural tube closure, as Dvl1/2 double mutants display more severe defects than the Dvl2 single mutants.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoblot analysis
Brain tissue (0.2 mg) from wild-type and Dvl2-/- mice
was homogenized in 1 ml of RIPA buffer containing Pefabloc (Boehringer
Mannheim). Homogenates were subjected to centrifugation for 1 minute at full
speed in a microcentrifuge. An aliquot of the supernatants was saved for A280
measurement (for normalization of gel loading), while the remainder was mixed
with an equal volume of 2x SDS gel loading buffer and boiled for 3
minutes. Aliquots (20 µl) were loaded on a 7.5% polyacrylamide SDS minigel
and subjected to electrophoresis and transfer to nitrocellulose membrane for
western blot analysis. A polyclonal antibody to the C terminus of Dvl1
(Luo et al., 2002) and
monoclonal antibodies to the C terminus of Dvl2 (2-10B5)
(Song et al., 2000
) and Dvl3
(3-4D3) (Strovel and Sussman,
1999
) were used in conjunction with peroxidase-conjugated
anti-mouse IgG (Sigma) for chemiluminescent detection (Amersham Pharmacia
Biotech).
Scanning electron microscopy
Embryo hearts were collected at 18.5 dpc or embryos were collected at
8.5-10.5 dpc and fixed in 3% aldehyde solution (1.5% paraformaldehyde, 1.5%
glutaraldehyde) in 0.1 M phosphate buffer pH 7.5, dehydrated in a graded
ethanol series, then stored in 100% ethanol until scanning. Samples were
critically point dried, mounted and then coated with 300 Angstrom
gold-palladium. Specimens were viewed and photographed with a Cambridge
Instrument Stereoscan 360 scanning electron microscope (Scripps Institute of
Oceanography Analytical Facility).
Whole-mount in situ hybridization
Embryos were prepared for in situ using the protocol detailed by Wilkinson
(Wilkinson, 1992) with
modifications. Embryos were dissected at the appropriate ages, fixed in 4%
paraformaldehyde in PBS then dehydrated in a methanol series. Embryos were
rehydrated in PBT (0.01% Tween-20 in PBS), treated with 6%
H2O2 to remove endogenous peroxidases and then
permeabilized with proteinase K. Hybridization was performed at 70°C
overnight in hybridization solution (50% formamide, 5xSSC pH 4.5, 50
µg/ml yeast tRNA, 1% SDS, 50 µg/ml heparin) using an RNA probe labeled
with digoxigenin-UTP (Boehringer/Mannheim). Afterwards embryos were washed
extensively in TBST (TBS with 0.1% Tween-20, Sigma) and blocked in 1% sheep
serum in TBST. Transcripts were detected with anti-dig Fab' conjugated
with alkaline phosphatase followed by color reaction with X-phosphate/NBT
substrate. Reactions were stopped with PBT and embryos stored in 80%
glycerol/PBT until photographed.
Histology
Tissues were dissected and placed in 20 volumes of 10% buffered formalin,
dehydrated, embedded in paraffin wax, sectioned at 8 µM and stained by
routine methods at the UCSD Histology Core. Photographs were taken using a
Leica DMR light microscope mounted with a Spot 2 camera.
Bone and cartilage stain
Differential staining of mouse fetuses was carried out according to the
procedure of McLeod (McLeod,
1980). Embryonic 17 and 18 dpc fetuses were skinned and
eviscerated, fixed in a 95% ethanol solution for 5 days, then treated in
acetone for 4 days to remove fat. Fetuses were stained with 0.3% Alcian Blue,
0.1% Alizarin Red S ethanol/acetic acid solution for 3 days followed by
clearing in a graded series of glycerol/1% KOH (20-80% glycerol, 1 week each
step). Skeletons were stored in 100% glycerol and photographed using a Leica
dissection microscope and Spot 2 camera.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Perinatal lethality of 50% of Dvl2-/- mice
We examined mutant mice in inbred 129/SvEv or mixed 129/SvEvxNIH
Black Swiss backgrounds and found that Dvl2-/- mice can
survive to adulthood and are fertile, but they were born in reduced numbers
from heterozygous crosses. Of 352 offspring at weaning, there were 130
Dvl2+/+, 180 Dvl2+/- and only 42
Dvl2-/- mice, a reduction of more than 50% of expected
numbers. However, at 18.5 dpc, Dvl2+/+,
Dvl2+/- and Dvl2-/- embryos were
recovered in the expected mendelian ratios, suggesting that death occurred in
the perinatal period. This was confirmed by directly observing litters at
birth, where some newborn Dvl2-/- pups failed to thrive.
Such pups appeared to have difficulty breathing, were often cyanotic, did not
feed and displayed reduced mobility. These pups died within 24 hours. The
perinatal lethality seemed to be unaffected by genetic influences caused by
mouse strain modifiers as the survival percentages for
Dvl2-/- mice were identical for both the mixed (16
observed out of 31 expected, n=125) and inbred strains (six observed
out of 13 expected, n=51). Surprisingly, surviving
Dvl2-/- mutants were predominantly female in either strain
background (Table 1),
suggesting an interaction between genotype and sex.
|
Dvl2-/- mice that survived beyond 24 hours grew
normally into adulthood (2+ years) but 25% (n=17) of the
surviving mutant mice had kinked tails. A percentage of surviving
Dvl2 mutants also exhibited scoliosis and in rare instances displayed
vestigial tail (n=3). This phenotype is very similar to the
haploinsufficiency phenotypes seen in Wnt3A and T
(brachyury) mutants (Yamaguchi et al.,
1999
). Embryo analysis in early gestation at 9.5 dpc revealed that
2-3% of Dvl2-/- embryos had incomplete thoracic neural
tube defects (spina bifida) and exencephaly (see below). Serial sections of
adult Dvl2-/- tissues showed no other morphological
abnormalities (data not shown). Unlike the Dvl1-/- mice,
Dvl2-/- homozygous mice displayed no abnormalities in
social interaction or sensorimotor gating (data not shown).
Dvl2-/- lethality is due to cardiac
anomalies
Dvl2-/- newborns had no abnormalities of the palate,
trachea or lungs (data not shown). We next examined the hearts of 13, 18 and
20 dpc. Dvl2-/- animals obtained from
Dvl2+/- crosses. Sixty-eight mice (16
Dvl2+/+, 36 Dvl2+/- and 16
Dvl2-/- mice) were examined for cardiovascular defects.
Eight Dvl2-/- (50%) of the mutant mice displayed
cardiovascular abnormalities (Table
2). An additional embryo displayed a cardiac abnormality but was
not included in Table 2 as
genotype information for remaining littermates could not be determined because
of a technical error. Eight Dvl2-/- embryos had
structurally normal hearts, similar to the wild type
(Fig. 2A,B). Double outlet
right ventricle (DORV) was the most common defect and the placement of the
aorta varied. Six mutant embryos had DORV in conjunction with ventricular
septal defects (Fig. 2C,D,
Fig. 3B). In one case of DORV
the aorta emerged parallel to the pulmonary valve where the orientation of
both great vessels were inverted and arose from the right ventricle
(Fig. 2C,D). In fact, this was
the most frequent manner in which DORV presented. One mutant had transposition
of the great arteries (TGA). In addition, two of the mutant embryos had
persistent or common truncus arteriosis (TA), a condition in which the outflow
tract fails to divide into an aorta and pulmonary artery
(Fig. 2E,F). These defects were
clearly seen in serial sections of abnormal Dvl2-/- hearts
(Fig. 3). TGA was evident in
one heart (Fig. 3C) where the
aorta emerged from the right ventricle. The severity of these defects would be
sufficient to account for the perinatal lethality of
Dvl2-/- mice. None of the Dvl2+/- or
Dvl2+/+ mice examined displayed any conotruncal
abnormalities or other heart defects.
|
|
|
In separate matings, Dvl1-/-;Dvl2-/- embryos were examined for cardiac defects, to see if there was evidence for redundancy between Dvl1 and Dvl2 in conotruncal development. Of eight double homozygous embryos examined between 14.5 and 18.5 dpc, three displayed conotruncal defects (Table 2), including DORV, TGA and PTA. These results are consistent with a primary role for Dvl2 in cardiac outflow tract development, and strongly suggest that Dvl1 and Dvl2 have non-redundant roles in heart morphogenesis.
Perturbation of cardiac neural crest expression in
Dvl2-/- mice
The defects observed in the morphology of the great vessels in
Dvl2-/- mice (DORV, TGA and PTA) are congenital heart
defects that occur in embryogenesis due to abnormalities in outflow tract
septation (Chien, 2000;
Srivastava and Olson, 2000
).
Proper formation of the cardiac great vessels involves an early looping event
occurring between 8.5-9.0 dpc followed by rotation of the developing
conotruncal arteries. Between 11.5 and 12 dpc the developing conotruncus first
rotates, followed by the development and fusion of the conotruncal cushions.
As the endocardial cushions grow and migrate they develop chirality along the
outflow tract. In the final step, the cushions fuse to form the
aorticopulmonary septum. Cardiac neural crest cells are believed to migrate
into the truncal cushions as they form and contribute to septation. Neural
crest ablation studies have identified a subpopulation of neural crest cells
at the level of the fourth and sixth aortic arch that affects proper septation
termed the cardiac neural crest (Kirby et
al., 1983
; Creazzo et al.,
1998
; Kirby and Waldo,
1995
).
Therefore, markers for early cardiac neural crest were employed, such as
Pitx2 and plexin A2. Pitx2 is co-expressed with
Pax3, connexin 43 and the endothelin receptor A in the cardiac neural
crest, and Pitx2 mutant mice display similar outflow tract defects as
Dvl2 (Kioussi et al.,
2002). We used a Pitx2 probe for whole mount analysis at
10.5 dpc In wild-type embryos, Pitx2 is normally expressed in a number of
tissues, including the fourth branchial arch and migrating cardiac neural
crest (Fig. 4A,C,E).
Dvl2-/- embryos (Fig.
4B,D,F) and Dvl1/2 double mutants (data not shown) had
nearly undetectable levels of Pitx2 in the branchial arches and in
neural crest cells migrating to the cardiac outflow tract
(Fig. 4B), as well as in the
outflow tract (Fig. 4D,F).
These results suggest that the outflow tract defects seen in
Dvl2-/- mutant embryos are associated with cardiac neural
crest defects. Subsequently, the expression of the cardiac neural crest marker
Plexin A2 (Brown et al.,
2001
) was examined. Plexin A2 expression was greatly reduced in
hearts from Dvl2-/-
(Fig. 4H) mutant embryos
compared with wild-type hearts (Fig.
4G), further supporting the notion that the conotruncal defects
displayed by Dvl2-/- mutants result from neural crest
abnormalities.
|
Skeletal defects in Dvl2-/- and Dvl1/2
double mutant mice
As part of our phenotypic analysis, we examined bone and cartilage
development using Alizarin Red and Alcian Blue staining in 18
Dvl2-/- newborns. Nearly all (90%) of the Dvl2
mutant mice displayed mild abnormalities of the vertebral bodies and ribs
(Fig. 5,
Table 3). Most defects were
localized dorsally in the vertebral ribs and vertebral bodies
(Fig. 5A-E). Many of the
abnormal thoracic vertebrae were disorganized
(Fig. 5A) and fusion of the
ribs near the tubercle was evident (Fig.
5B). We determined whether the normal rib number was altered in
any of the mutant mice. In all but one case we found that in the presence of
either forked or fused ribs the total rib number on both the left and right
sides was 13. These findings suggest that the normal number of ribs was
specified, but segmentation did not occur properly. Dvl1/2 double
homozygotes had more severe skeletal malformations of the type seen in
Dvl2-/- mice (Fig.
5F). For example, a 14.5 dpc. double mutant embryo demonstrated
numerous collapsed vertebrae and extensive fusion of the ribs along the
vertebral column. These defects were not observed in
Dvl1-/- mice (Lijam et
al., 1997), suggesting that functional redundancy restricted this
phenotype in either Dvl1-/- or Dvl2-/-
single mutants.
|
|
A sternal defect was detected in one newborn mouse, and affected the sixth sternebra and the xiphoid process (Fig. 5H). A completely split or perforated xiphoid process was evident. Seven ribs are attached to the sternum but no malformations were detected in any of the seven sternal ribs. Overall, these data demonstrate that Dvl2 is essential for proper formation of ribs, vertebral bodies and sternum.
Somite analysis using paraxial mesodermal markers
Somites originate as cells that pinch off from the presomitic mesoderm. As
the somite condenses, it develops into a disorganized ball of cells internally
surrounded by a layer of columnar epithelial cells. The basolateral wall of
the epithelial cell undergoes an epithelial-to-mesenchymal transition that
results in the formation of sclerotome ventrally and dermomyotome dorsally.
The sclerotome and dermomyotome collectively give rise to the cartilage, bone,
muscle and connective tissue (Keynes and
Stern, 1988; Huang et al.,
2000
). In addition, anterior and posterior domains exist within
the sclerotome as demonstrated with mesodermal probes that identify these
distinct cell populations. To investigate somite differentiation in the
Dvl2 single and Dvl1/2 double mutants, we performed
whole-mount in situ hybridization using a variety of somite markers.
No differences were found between wild-type and Dvl2-/- mutants using several markers, including the caudal somite markers Uncx4.1, delta 1 and lunatic fringe, as well as the rostral marker delta 3 (data not shown). To determine whether the axial defects that affect rib fusion could be due to abnormalities that precede somite division into caudal and rostral halves, we used probes that identify the presomitic mesoderm. There were no alterations in the coordinated expression of Notch1 or Notch2 in wild-type and Dvl2-/- mutants (Fig. 6C-F). However, myogenin, a myotomal marker, was occasionally attached between two somites of Dvl2 mutants as a bridge (Fig. 6A,B), suggesting that abnormal, incomplete segmentation could be the cause of the somite defects seen in these mutants.
|
As Dvl1/2 double mutants display rib and vertebral defects of the same type seen in Dvl2-/- mice, but of more severe extent and at higher frequency, we examined the expression pattern of somite markers in the double mutants, hoping to increase the chance that relevant abnormalities in marker expression would be observed. Similar to the results for myogenin in Dvl2-/- mice, Uncx4.1 was detected in a fused band between adjacent somites, indicating incomplete segmentation of these two somites (Fig. 6I,J) in the Dvl1/Dvl2 double mutant embryo. The caudal probe delta 1 was expressed throughout the appropriate regions of the somite, but the spacing was irregular in the double mutant (Fig. 6K,L). In both of these cases, the abnormal somites appear to be split, consistent with the normal ultimate rib and vertebral numbers of the Dvl2 and Dvl1/2 mutants. Lunatic fringe transcripts were present in the forebrain, placodes, neural tube and dermomyotome of the wild-type embryo (Fig. 6G). Overall, this pattern was repeated for the Dvl1/2 mutant but expression in the dermomyotome was markedly depressed (inset Fig. 6H). No differences were found in Dvl1/2 double mutants using delta 3. Thus, minor abnormalities were evident in somite precursors of Dvl2 mutants, which were more severe in the Dvl1/2 double mutant. These defects appear to be due to mild abnormalities in somite segmentation.
Neural tube defects in Dvl2-/- and
Dvl1/2 double mutant mice
Approximately 2-3% of Dvl2 homozygotes have incomplete thoracic
neural tube defects (Fig. 7B),
often associated with exencephaly, compared with normal neural tube closure in
wild-type embryos (Fig. 7A).
Although the penetrance was low, we reasoned that there may be functional
redundancy among the Dvl genes because of their overlapping patterns of
expression and structural similarity. To test for functional redundancy,
Dvl1+/-;Dvl2+/- double heterozygous
crosses and Dvl1-/-;Dvl2+/- crosses
were performed. Litters were genotyped at weaning. No
Dvl1-/-;Dvl2-/- mice were identified
(20 expected, none observed, n=230). Embryos were dissected at
various times from these crosses. Surprisingly, we found that
Dvl1-/-;Dvl2-/- embryos
(Fig. 7C) displayed completely
open spinal neural tubes and exencephaly (cranio-rachischisis). The neural
tube closure defects resulted in the development of the brain outside of the
cranium, and the spinal cord was completely open to the base of the tail. The
face was fused, as was the tail, suggesting that some regions of the neural
tube closed normally. At 9.5-10.5 dpc, the neural tubes of wild-type embryos
were completely closed in the midbrain, hindbrain and thoracic region
(Fig. 7D), while the neural
tubes of Dvl1/Dvl2 double mutants were completely open from the
midbrain to the tail (Fig. 7E).
Of 380 embryos dissected between 8.5 and 10.5 dpc from both
Dvl1+/-;Dvl2+/- double heterozygous
crosses and Dvl1-/-;Dvl2+/- crosses,
40 Dvl1-/-;Dvl2-/- double homozygotes
were observed (45 expected). Of these, 31 had open unfused neural tubes, one
had an open neural tube with fused epidermal ectoderm, six had partially open
neural tubes and two appeared normal.
|
There were clear defects in the developing spinal cord and brain resulting from closure defects. In spite of such defects, there appeared to be recognizable regional differentiation of forebrain, midbrain and hindbrain regions, even though there was marked disorganization secondary to exencephaly (Fig. 7F,G). Similarly, the dorsal and ventral regions of the mutant open spinal cord were recognizable (Fig. 7H,I).
Thus, Dvl2 is essential for neural tube closure in mice, and there are overlapping functions between Dvl1 and Dvl2 in neurulation.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice with targeted inactivation of the Dvl1 gene have been
previously described (Lijam et al.,
1997). These mice were found to exhibit alterations in
sensorimotor gating and social interaction. Surprisingly Dvl2 does
not seem to play a similar role in the regulation of social behavior or
sensorimotor gating, as no abnormalities in these processes were observed.
These observations suggest a unique function for Dvl1 in social
behavior and sensorimotor gating.
The heart phenotype manifested by the loss of Dvl2 was specific to
the outflow tract. Proper alignment and development of the aorticopulmonary
septum involves migration and fusion of the endocardial cushions, as well as a
correct pivoting event along the axis that forms the AV canals. Genes that
affect early cardiac looping such as Nkx2.5, Mef2c, as well as the
bHLH proteins Hand1 and Hand2 have been identified
(Olson and Srivastava, 1996;
Thomas et al., 1998
;
Srivastava et al., 1995
;
Lyons et al., 1995
;
Lin et al., 1997
;
Bruneau et al., 2000
). Improper
cardiac looping can lead to outflow tract defects and tetratolgy of Fallot
based on studies in the mouse (Srivastava,
2000
; Creazzo et al.,
1998
; Olson and Srivastava,
1996
).
In the case of Dvl2 mutants, a defect in cardiac neural crest
appears to be responsible for the observed outflow tract defects. Cardiac
neural crest cells are essential for normal development of the outflow tract.
These cells originate from the caudal hindbrain and migrate into the caudal
pharyngeal arches (third, fourth and sixth). A subset of these cells continues
to migrate into the cardiac outflow tract where it will organize the
aorticopulmonary septum. If cardiac neural crest cells are removed prior to
migration, several predictable outflow tract phenotypes are observed after
development of the heart and great arteries is complete, including DORV, TGA
and PTA (Kirby et al., 1983;
Creazzo et al., 1998
)
(reviewed by Kirby and Waldo,
1995
; Chien,
2000
). Pitx2 is co-expressed with Pax3 in the
cardiac neural crest, and Pitx2 mutant mice display similar outflow
tract defects as Dvl2 (Kioussi et
al., 2002
). Plexin A2 has recently been identified as a marker for
cardiac neural crest that is expressed at later times in development
(Brown et al., 2001
).
Therefore, we examined the expression of Pitx2 and plexin A2 in
Dvl2 and Dvl1/2 mutant mice. Expression of these markers
along the migrating cardiac neural crest were impaired in Dvl mutant mice,
implicating the neural crest in the outflow tract defects displayed by
Dvl2-/- mice.
We recently found that Dvl2 and Pitx2 were part of a
common pathway regulating proliferation in specific tissues
(Kioussi et al., 2002). We
found that Dvl2 and Pitx2 genetically interact to produce
cardiac outflow tract abnormalities. We further demonstrated that Pitx factors
can exert essential roles in cardiac neural crest cell development based on
the ß-catenin-dependent transcriptional induction of Pitx2.
Pitx2, in turn, acts upstream of genes required for the cell proliferation
program, including cyclin D1 and cyclin D2. Components of this
Wnt/Dvl/ß-catenin
Pitx2 pathway are required, in a dose-dependent
fashion, for physiological proliferation of specific cells within the cardiac
outflow tract, in particular the cardiac neural crest, and pituitary gland.
These findings strongly support our interpretation that the primary outflow
tract defect in Dvl2 mutant mice is in the cardiac neural crest. In
addition, they suggest that Dvl2 and Pitx2, among other proteins, could be
novel components of a multigenic origin of cardiac outflow tract defects that
occur in the human population. As cardiac outflow tract abnormalities account
for
30% of all cardiovascular malformations in humans
(Chien, 2000
;
Srivastava and Olson, 2000
),
this pathway could be important for human conotruncal defects as well.
These findings support a role for the Wnt pathway in cardiac morphogenesis
through the control of cardiac neural crest development. There is some earlier
evidence that the Wnt pathway plays an important role in cardiac
morphogenesis, beginning with studies using antisense attenuation of
Wnt1 and Wnt3a expression in whole embryo cultures
(Augustine et al., 1993).
Recent reports demonstrate that Wnt inhibition induces cardiogenesis in
Xenopus (Schneider and Mercola,
2001
) and in chick (Marvin et
al., 2001
), but the role of Wnt signaling in mammalian systems was
unclear. So far, no Wnt pathway mutants display cardiac defects. However,
neural crest abnormalities do occur in the Wnt1/Wnt3a double mutant.
In addition, using a floxed ß-catenin allele and the Wnt1-Cre transgenic
mouse that expresses Cre in the neural crest, we were able to delete
ß-catenin completely in the neural crest. These mice displayed similar
conotruncal defects as the Dvl2-/- mice
(Kioussi et al., 2002
),
consistent with a role for Dvl2 in the cardiac neural crest.
Downstream of Dishevelled, the Notch and Wnt
pathways have both been implicated in somite formation and segmentation.
Members of the Notch pathway are intimately involved in controlling
somitogenesis (reviewed by Muskaitch et al., 1994;
Artavanis-Tsakonas et al.,
1999), including Notch1
(Conlon et al., 1995
),
Notch2, delta 3 (Kusumi et al.,
1998
) and lunatic fringe
(Evrad et al., 1998
; Zhang et
al., 1998). Notch is downstream of Dsh in
Drosophila (Axelrod et al.,
1996
; Couso and Martinez
Arias, 1994
). Notch1 and Notch2 expression were
normal in the somites of Dvl2 mutants, indicating that development of
the somites from presomitic mesoderm was unperturbed. However, somite
segmentation was abnormal as defined by expression of the sclerotomal marker
Uncx4.1. These expression alterations were not identical to the
severe defects displayed by Notch pathway mutants. Instead, rather subtle but
distinct alterations in somite boundaries were evident as detected by the
delta 1 and lunatic fringe. Wnt3a is also involved in paraxial
mesoderm differentiation. Wnt3a mutants exhibit posterior truncation
and lack a significant number of posterior somites
(Takada et al., 1994
). The
somite defects in the Dvl2 mutant mice reflect mild defects in
segmentation, rather than conversion of posterior to anterior fates, but a
role for Dvl genes in posterior development cannot be completely ruled
out.
Dvl2 is essential for normal neural tube closure, because a small
number of embryos display thoracic spina bifica and exencephaly. Neural tube
defects (NTDs) are a common class of birth defects in humans with an incidence
that varies from 0.5-8 per 1000 live births
(Elwood et al., 1992). Several
factors are associated with NTDs, including genetic factors, teratogens and
low levels of dietary folate. There is wide variation in the type and severity
of NTDs in humans. Genetic epidemiology studies have suggested that NTDs have
a multifactorial etiology with genetic predisposition because of many genes
and a threshold beyond which environmental factors can trigger NTDs during
crucial fetal periods. Several mouse mutants display NTDs. More than 50 loci
have been identified, and many of the mutant alleles have been cloned
(Juriloff and Harris, 1999
).
We have now identified Dvl2 as an additional locus important for
neural tube closure. In addition, there is functional redundancy between
Dvl1 and Dvl2 in neural tube closure, as the double mutants
display a completely open neural tube between the midbrain and tail, termed
craniorachischisis.
Loop tail (Lp) is one other mouse mutant that displays
cranio-rachischisis (Greene et al.,
1998). The gene for Lp has been cloned
(Kibor et al., 2001
;
Murdoch et al., 2001
) and
codes for a transmembrane protein loopin with a PDZ-binding domain.
Ltap is related to the Drosophila gene Van Gogh,
which is downstream of frizzled and Dishevelled in the
planar cell polarity (PCP) pathway (Taylor
et al., 1998
; Wolff and Rubin,
1998
). These finding suggest the intriguing possibility that
mammalian Dvls and Lp are in a common PCP-like pathway
mediating neural tube closure. In Xenopus, Dishevelled is an integral
part of the PCP pathway that regulates gastrulation via convergent extension
(Wallingford et al., 2000
),
and has also been implicated in neural tube closure in Xenopus
(Wallingford and Harland,
2001
; Wallingford et al., 2002). The Dvl1/Dvl2 phenotype
may be the result in defects of convergent extension mechanisms via the PCP
pathway, suggesting that Dvl1, Dvl2 and Lp are part of a
common PCP pathway regulating neurulation in the mouse.
In summary, we have provided evidence that Dvl2 is important for
cardiac outflow tract development via the cardiac neural crest, somite
segmentation and neural tube closure. These findings, together with previously
published (Lijam et al., 1997)
and unpublished results, demonstrate that Dvl1 and Dvl2 are
partially redundant, but also have unique functions. Somite segmentation and
neural tube closure appear to be mediated by overlapping redundant functions
that are dependent on the dose of these two genes. By contrast, social
interaction and sensorimotor gating defects are unique to Dvl1
mutants, while cardiac defects appear to be unique to Dvl2 mutants.
The Dvl1 and Dvl2 mutant mice will be invaluable tools with
which to continue to sort out the important pathways regulated by
Dishevelled genes to regulate complex behavior, cardiac outflow tract
development and somite segmentation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Artavanis-Tsakonas, S., Rand, M. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
-776.
Augustine, K., Liu, E. T. and Sadler, T. W. (1993). Antisense attenuation of Wnt1 and Wnt 3A expression in whole embryo culture reveals roles for these genes in craniofacial, spinal cord and cardiac morphogenesis. Dev. Genet. 14,500 -520.[Medline]
Axelrod, J., Matsuno, K., Artavanis-Tsakonas, S. and Perrimon, N. (1996). Interaction between wingless and notch signaling pathways mediated by dishevelled. Science 271,1826 -1832.[Abstract]
Axelrod, J., Miller, J., Shulman, J., Moon, R. and Perrimon,
N. (1998). Differential recruitment of Dishevelled provides
signaling specificity in the planar cell polarity and wingless signaling
pathways. Genes Dev. 12,2610
-2622.
Bejsovec, A. and Arias, A. (1991). Roles of wingless in patterning the larval epidermis of Drosophila. Development 113,471 -485.[Abstract]
Boutros, M., Parcio, N., Strutt, D. and Mlodzik, M. (1998). Dishevelled activates JNK and discriminates between JNK pathways in planar ploarity and wingless signaling. Cell 94,109 -118.[Medline]
Brown, C. B., Feiner, L., Lu, M.-M., Li, J., Ma, X., Webber, A.
L., Jia, L., Raper, J. A. and Epstein, J. A. (2001). PlexinA2
and semaphorin signaling during cardiac neural crest development.
Development 128,3071
-3080.
Bruneau, B. G., Bao, Z. Z., Tanaka, M., Schott, J. J., Izumo, S., Cepko, C. L., Seidman, J. G. and Seidman, C. E. (2000). Cardiac exxpression of the ventricle-specific homeobox gene Irx4 is modulated by Nkx2.5 and dHAND. Dev. Biol. 217,266 -277.[CrossRef][Medline]
Cadigan, K. M. and Nusse, R. (1997). Wnt
signaling: a common theme in animal development. Genes
Dev. 11,3286
-3305.
Chien, K. R. (2000). Genomic circuits and the integrative biology of cardiac diseases. Nature 407,227 -232.[CrossRef][Medline]
Conlon, R., Reaume, A. and Rossant, J. (1995).
Notch1 is required for the coordinate segmentation of somites.
Development 121,1533
-1545.
Couso, J. and Martinez Arias, A. (1994). Notch is required for wingless signaling in the epidermis of Drosophila. Cell 79,259 -272.[Medline]
Creazzo, T., Godt, R., Leatherbury, L., Conway, S. and Kirby, M. (1998). Role of cardiac neural crest cells in cardiovascular development. Annu. Rev. Physiol. 60,267 -286.[CrossRef][Medline]
Deng, C., Wynshaw-Boris, A., Kuo, A., Zhou, F. and Leder, P. (1996). Fibroblast growth factor receptor-3 is a negative regulator of bone growth and development. Cell 84,911 -921.[Medline]
Elwood, J. M., Little, J. and Elwood, L. H. (1992). Epidemiology and control of neural tube defects. New York: Oxford University Press.
Evrad, Y., Lun, Y., Aulehia, A., Gan, L. and Johnson, R. (1998). Lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394,377 -381.[CrossRef][Medline]
Greene, N. D., Gerrello, D., van Stratten, H. W. and Copp, A. J. (1998). Abnormalities of floor plate, nototchord and somite differentiation in the loop-tail (Lp) mouse: a model of severe neural tube defects. Mech. Dev. 73, 59 (1998).[CrossRef][Medline]
Huang, R., Zhi, Q., Schmidt, C., Wilting, J., Brand-Serberi, B.
and Christ, B. (2000). Sclerotomal origin of the ribs.
Development 127,527
-532.
Ikeya, M., Lee, S., Johnson, J., McMahon, A. and Takada, S. (1997). Wnt signaling required for expansion of neural crest and CNS progenitors. Nature 389,966 -970.[CrossRef][Medline]
Juriloff, D. M. and Harris, M. J. (1999). Mini-review: toward understanding mechanisms of genetic neural tube defects in mice. Teratology 60,292 -305.[CrossRef][Medline]
Keynes, R. and Stern, C. (1988). Mechanisms of vertebrate segmentation. Development 103,413 -429.[Medline]
Kibor, Z., Vogan, K. J., Groulx, N., Justice, M., Underhill, D. A. and Gros, P. (2001). Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tive mutant Loop-tail. Nat. Genet. 28,251 -255.[CrossRef][Medline]
Kioussi, C., Briata, P., Baek, S. H., Rose, D., Hamblet, N. S., Herman, T., Lin, C., Gleiberman, A., Wang, J., Brault, V., Ruiz-Lozano, P., Nguyen, H. D., Kemler, R., Glass, C. K., Wynshaw-Boris, A. and Rosenfeld, M. G. (2002). A Wnt/Dvl->Pitx pathway reveals molecular mechanisms for mediating cell type-specific proliferation during development. Cell (in press).
Kirby, M. L., Gale, T. F. and Stewart, D. E. (1983). Neural crest cells contribute to normal aorticopulmonary septation. Science, 220,1059 -1061.[Medline]
Kirby, M. L. and Waldo, K. L. (1995). Neural
crest and cardiovascular patterning. Circ. Res.
77,211
-215.
Kishida, S., Yamamoto, H., Hino, S., Ikeda., M. and Kikuchi,
A. (1999). DIX domains of Dvl and axin are necessary for
protein inbteractions and their ability to regulate beta-catenin.
Mol. Cell Biol. 19,4414
-4422.
Klingensmith, J., Nusse, R. and Perrimon, N. (1994). The Drosophila segment polarity gene dishevelled encodes a novel protein required for response to the wingless signal. Genes Dev. 8, 118-130.[Abstract]
Klingensmith, J., Yang, Y., Axelrod, J. D., Beier, D. R., Perrimon, N. and Sussman, D. J. (1996). Conservation of dishevelled structure and function between flies and mice: isolation and characterization of Dvl2. Mech. Dev. 58, 15-26.[CrossRef][Medline]
Kusumi, K., Sun, E., Kerrerock, A., Bronson, R., Chi, D., Bulotsky, M., Spencer, J., Birren, B., Frankel, W. and Lander, E. (1998). The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries. Nat. Genet. 19,274 -278.[CrossRef][Medline]
Lijam, N. and Sussman, D. (1996). Organization and promoter analysis of the mosue Disheveled-1 gene. Genome Res. 5,116 -124.[Abstract]
Lijam, N., Paylor, R., McDonald, M. P., Crawley, J. N., Deng, C. X., Herrup, K., Stevens, K. E., Maccaferri, G., McBain, C. J., Sussman, D. J. and Wynshaw-Boris, A. (1997). Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1. Cell 90,895 -905.[Medline]
Lin, Q., Schwartz, J., Bucana, C. and Olsen, E. N.
(1997). Control of mouse cardiac morphogenesis and myogenesis by
transcriptioin factor MEF2C. Science
276,1404
-1407.
Luo, Z. G., Qiang Wang, Q., Zhou, J. Z., Wang, J., Liu, M. Y., He, X., Wynshaw-Boris, A., Xiong, W. C., Lu, B. and Mei, L. (2002). Dishevelled mediates AChR clustering by interacting with MuSK and PAK1. Neuron (in press).
Lyons, I., Parsons, L. M. and Hartley, L. (1995). Myogenic and morphogenetic defects in the heart tubes of muring embryos lacking the nomeobox gene Nkx2.5. Genes Dev. 9,1654 -1666.[Abstract]
Marvin, M. J., DiRocco, G., Gardiner, A., Bush, S. and Lassar,
A. B. (2001). Inhibition of Wnt activity induces heart
formation from posterior mesoderm. Genes Dev.
15,316
-327.
McLeod, M. J. (1980) Differential staining of carilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22,299 -301.[Medline]
McMahon, A. P. and Bradley, A. (1990). The wnt-1 (Int-1) proto-oncogense is required for development of a large region of the mouse brain. Cell 62,1073 -1085.[Medline]
Moon, R., Brown, J. and Torres, M. (1997). Wnts modulate cell fate and behavior during vertebrate development. Trends Genet. 13,157 -162.[CrossRef][Medline]
Moriguchi, T., Kawasaki, K., Kamakura, S., Masuyma, N.,
Yamanaka, H., Matsumoto, K., Kikuchi, A. and Nishida, E.
(1999). Distinct domains of mouse dishevelled are responsible for
the c-jun N-terminal kinase/stress activated protein kinase activation and the
axis formation in vertebrates. J. Biol. Chem.
274,30957
-30962.
Murdoch, J. N., Doudney, K., Paternotte, C., Copp, A. J. and
Stanier, P. (2001). Severe neural tube defects in the
loop-tail mouse result from mutation of Lpp1, a novel gene
involved in floor plate specification. Hum. Mol.
Genet. 10,2593
-2601.
Muskavitch, M. A. (1994). Delta-Notch signaling and Drosophila cell fate choice. Dev. Biol. 166,415 -430.[CrossRef][Medline]
Olson, E. and Srivastava, D. (1996). Molecular pathways controlling heart development. Science 272,671 -676.[Abstract]
Perrimon, N. and Mahowald, A. P. (1987). Multiple functions of segment polarity genes in Drosophila. Dev. Biol. 119,507 -600.
Ponting, C., Phillips, C., Davies, K. and Blake, D. (1997). PDZ domains: targeting signaling molecules to sub-membraneous sites. BioEssays 19,469 -479.[Medline]
Schneider, V. and Mercola, M. (2001). Wnt
antagonism initiates cardiogenesis in Xenopus laevis. Genes
Dev. 15,304
-315.
Semenov, M. V. and Snyder, M. (1997). Human dishevelled genes constitute a DHR-containing multigene family. Genomics 42,302 -310.[CrossRef][Medline]
Smalley, M., Sara, E., Paterson, H., Naylor, S., Cook, D.,
Jayatilake, H., Fryer, L., Hutchison, L., Fry, M. and Dale, T.
(1999). Interaction of axin and dvl-2 proteins regulates
Dvl2-stimulated TCF-dependent transcription. EMBO J.
18,2823
-2835.
Srivastava, D. (2000). Congenital heart
defects, trapping the culprits. Circ. Res.
86, 917.
Srivastava, D. and Olson, E. N. (2000). A genetic blueprint for cardiac development. Nature 407,221 -226.[CrossRef][Medline]
Srivastava, D., Cserjesi, P. and Olsen, E. N. (1995). A new subclass of bHLH proteins required for cardiac morphogenesis. Science 270,1995 -1999.[Abstract]
Song, D. H., Sussman, D. J. and Seldin, D. C.
(2000). Endogenous protein kinase CK2 participates in Wnt
signaling in mammary epithelial cells. J. Biol. Chem.
275,23790
-23797.
Strovel, E. T. and Sussman, D. J. (1999). Transient overexpression of murine dishevelled genes results in apoptotic cell death. Exp. Cell Res. 253,637 -648.[CrossRef][Medline]
Sussman, D. J., Klingensmith, J., Salinas, P., Adams, P. S., Nusse, R. and Perrimon, N. (1994). Isolation and characterization of a mouse homolog of the Drosophila segment polarity gene dishevelled. Dev. Biol. 166, 73-86.[CrossRef][Medline]
Takada, S., Stark, K. L., Shea, M. J., Vassialeva, G., McMahon, J. A. and McMahon, A. P. (1994). Wnt 3A regulates somite and tailbud formation in the mouse embryos. Genes Dev. 7, 197-203.[Abstract]
Taylor, J., Abramova, N., Charlton, J. and Adler, P. N.
(1998). Van Gogh: a new Drosophila tissue
polarity gene. Genetics
150,199
-210.
Thomas, T., Yamagishi, H., Overbeek, P. A., Olsen, E. N. and Srivastava, D. (1998). The bHLH factors, dHAND and eHAND, specify pulmonary and sustemic cardiac ventricles independent of left-right sidedness. Dev. Biol. 196,228 -236.[CrossRef][Medline]
Thomas, K. and Capecchi, M. (1990). Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 346,847 -850.[CrossRef][Medline]
Theisen, H., Purcell, J., Bennet, M., Kansagara, D., Syed, A.
and Marsh, L. J. (1994). Dishevelled is required
during wingless signaling to establish both cell polarity and cell identity.
Development 120,347
-360.
Tsang, M., Lijam, N., Yang, Y., Beier, D. R., Wynshaw-Boris, A. and Sussman, D. J. (1996). Isolation and characterizaation of mouse Dishevlled-3. Dev. Dyn. 207,253 -262.[CrossRef][Medline]
Tybulewicz, V., Crawford, C. E., Jackson, P. K., Bronson, R. T. and Mulligan, R. C. (1991). Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65,1153 -1163.[Medline]
Wallingford, J. B. and Harland, R. M. (2001).
Xenopus Dishevelled signaling regulates both neural and mesodermal convergent
extension: parallel forces elongating the body axis.
Development 128,2581
-2592.
Wallingford, J. B. and Harland, R. M. (2002).
Neural tube closure requires Dishevelled-dependent convergent extension of the
midline. Development
129,5815
-5825.
Wallingford, J. B., Rowning, B. A., Vogeli, K. M., Rothbacher, U., Fraser, S. E. and Harland, R. M. (2000). Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405,81 -85.[CrossRef][Medline]
Wilkinson, D. G. (1992). Whole mount in situ hybridization of vertebrate embryos. In In Situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), pp.75 -83. Oxford: IRL Press.
Wolff, T. and Rubin, G. M. (1998).
Strabismus, a novel gene that regulates tissue polarity and cell fate
decisions in Drosophila. Development
125,1149
-1159.
Yamaguchi, T., Takada, S., Yoshikawa, Y., Wu, N. and McMahon,
A. (1999). T(Brachyury) is a direct target of Wnt3A during
paraxial mesoderm specification. Genes Dev.
13,3185
-3190.
Yang, Y., Lijam, N., Sussman, D. J. and Tsang, M. (1996). Genomic organization of mouse Dishevelled genes. Gene 180,121 .[CrossRef][Medline]
Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., Lee, J. J., Tilghman, S. M., Gumbiner, B. M. and Costantini, F. (1997). The mouse Fused locus encodes axin, an inhibitor of the Wnt signaling pathways that regulates embryonic axis formation. Cell 90,181 -192.[Medline]
Zhang, N. and Gridley, T. (1998). Defects in somite formation in lunatic fringe deficient mice. Nature 394,374 -376.[CrossRef][Medline]