PLoS ONEplosplosonePLoS ONE1932-6203Public Library of ScienceSan Francisco, USAPONE-D-10-0191810.1371/journal.pone.0016260Research ArticleBiologyAnatomy and physiologyEndocrine systemEndocrine
physiologyGrowth factorsMusculoskeletal systemDevelopmental biologyMorphogenesisBirth defectsOrganism developmentTeratologyEmbryologyMolecular developmentGeneticsAnimal
geneticsHistologyModel organismsAnimal
modelsMouseGenetics and GenomicsPhysiologyDevelopmental BiologyGenetic Analysis of Hedgehog Signaling in Ventral Body Wall
Development and the Onset of Omphalocele FormationOmphalocele Phenotypes with Hh Signal
ModulationMatsumaruDaisuke1HaraguchiRyuma1¤aMiyagawaShinichi1¤bMotoyamaJun2NakagataNaomi3MeijlinkFrits4YamadaGen1*Global COE "Cell Fate Regulation
Research and Education Unit", Department of Organ Formation, Institute of
Molecular Embryology and Genetics (IMEG), Kumamoto University, Kumamoto, JapanDepartment of Medical Life Systems,
Doshisha University, Kyoto, JapanCenter for Animal Resources and
Development (CARD), Kumamoto University, Kumamoto, JapanHubrecht Institute, KNAW and University
Medical Center, Utrecht, The NetherlandsShamMai
HarEditorThe University of Hong Kong, China* E-mail: gensan@gpo.kumamoto-u.ac.jp
Conceived and designed the experiments: DM RH SM GY.
Performed the experiments: DM RH SM JM NN. Analyzed the data: DM RH FM GY.
Wrote the paper: DM GY.
Current address:
Department of Molecular Pathology, Ehime University Graduate School of Medicine,
Ehime, Japan
Current address:
Okazaki Institute for Integrative Bioscience, National Institutes of Natural
Sciences, Aichi, Japan
The authors have declared that no competing interests
exist.
2011201201161e162601192010121220102011Matsumaru et alThis is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.Background
An omphalocele is one of the major ventral body wall malformations and
is characterized by abnormally herniated viscera from the body trunk. It has
been frequently found to be associated with other structural malformations,
such as genitourinary malformations and digit abnormalities. In spite of its
clinical importance, the etiology of omphalocele formation is still controversial.
Hedgehog (Hh) signaling is one of the essential growth factor signaling pathways
involved in the formation of the limbs and urogenital system. However, the
relationship between Hh signaling and ventral body wall formation remains
unclear.
Methodology/Principal Findings
To gain insight into the roles of Hh signaling in ventral body wall formation
and its malformation, we analyzed phenotypes of mouse mutants of Sonic
hedgehog (Shh), GLI-Kruppel family member
3 (Gli3) and Aristaless-like homeobox 4
(Alx4). Introduction of additional Alx4Lst
mutations into the Gli3Xt/Xt background resulted
in various degrees of severe omphalocele and pubic diastasis. In addition,
loss of a single Shh allele restored the omphalocele and
pubic symphysis of Gli3Xt/+; Alx4Lst/Lst
embryos. We also observed ectopic Hh activity in the ventral body wall region
of Gli3Xt/Xt embryos. Moreover, tamoxifen-inducible
gain-of-function experiments to induce ectopic Hh signaling revealed Hh signal
dose-dependent formation of omphaloceles.
Conclusions/Significance
We suggest that one of the possible causes of omphalocele and pubic diastasis
is ectopically-induced Hh signaling. To our knowledge, this would be the first
demonstration of the involvement of Hh signaling in ventral body wall malformation
and the genetic rescue of omphalocele phenotypes.
This work is supported by Grant-in-Aid
for Scientific Research B, for Scientific Research on Innovative Areas; Molecular
mechanisms for establishment of sex differences (22132006), and the Global
COE program Cell Fate Regulation Research and Education Unit from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan, and a grant
for Child Health and Development (20-3) and Health Sciences Research Grant
from the Ministry of Health, Labor, and Welfare, Japan. This work was also
supported by National Institutes of Health Grant R01ES016597. The funders
had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.Introduction
The embryonic visceral organs transiently protrude out of the body trunk
during mid-gestation, where they are covered with the peritoneal membrane.
Subsequently they return to the peritoneal cavity in both mouse and human
embryos. This transient embryonic hernia of the viscera is termed the physiological
umbilical hernia [1], [2]. According
to previous reports, protrusion of the midgut loop through the umbilical ring
is due to the rapid expansion in the volume of visceral organs, exceeding
the space of the peritoneal cavity [1], [3]. However,
the molecular mechanisms underlying the ventral body wall formation, including
physiological umbilical herniation, are still unclear.
An omphalocele is a major ventral body wall malformation characterized
by a severe umbilical defect with herniation of visceral organs covered with
peritoneum and amnion [2], [4], [5]. The frequency is reported to
be approximately 1 in 4,000 live births [6]–[8]. In spite of
its high incidence, the cause of omphalocele is controversial; it might be
due to the failure of recovery of the physiological umbilical hernia or to
a midline defect at the transition zone between the ectoderm and mesoderm [7], [9]–[12]. Omphaloceles are frequently
associated with other structural malformations such as cardiac, anorectal
and digit malformations in more than 50% of cases [5], [13], [14]. For instance,
patients with omphalocele-exstrophy-imperforate anus-spinal defects complex
(OEIS complex, OMIM: 25840) or bladder exstrophy (OMIM: %600057) exhibit
defects not only in the body wall region but also in urogenital organs and
its adjacent tissues, including the pelvic girdle [15]–[18]. Our understanding
of these malformations is hampered by the complexity of these syndromes. Even
the nomenclature and definitions for syndromic congenital malformations are
still controversial [19]–[21].
Several genetically-modified animals have been reported to display abnormalities
in the body wall region. Such reports include cases of mutants of Msh-like
homeobox 1 and 2 (Msx1/2), Transcription
factor AP-2 alpha (Tcfap2α), Paired-like
homeodomain transcription factor 2 (Pitx2), Insulin-like
growth factor 2 (Igf2), Igf2 receptor
(Igf2r), Transforming growth factor beta 2
and 3 (Tgfβ2/3), Bone morphogenetic
protein 4 (Bmp4) and Bmp receptor type Ia
(BmprIa) [3], [22]-[33]. Of note, most of these animals
had accompanying limb deformities.
The Hedgehog (Hh) signaling pathway is an essential growth factor signaling
pathway involved in many developmental contexts, including digit formation.
One of the Hh ligands, Sonic hedgehog (Shh), is secreted from the posterior
mesenchymal region of limb buds, the zone of polarizing activity. It is suggested
that the anterior-posterior Shh gradient, together with a temporal gradient
of exposure to Shh signaling, may specify digit number and identity [34]–[39]. Previous
studies suggested that digit abnormalities such as polydactyly frequently
accompany ectopic Hh signal induction in anterior limb buds [40], [41]. Both inactivation of Patched
1 (Ptc1, a Hh signal repressor gene) and constitutive
activation of Smoothened (Smo, a Hh signal
transducer gene) or GLI-Kruppel family member 2 (Gli2,
a Hh signal transcription factor gene) resulted in polydactylous phenotypes [42]–[45]. As for the
mutants with body wall phenotypes, BmprIa, Msx1/2
and Tcfap2α mutants exhibited ectopic expression of Shh
gene or its signaling genes (Gli1 or Ptc1)
in their limb buds or other tissues [46]–[48]. Of note,
the mutants of GLI-Kruppel family member 3 (Gli3)
or Aristaless-like homeobox 4 (Alx4) also
displayed body wall abnormalities, polydactylies and ectopic Hh signal activity
in limb buds [40], [41], [49]–[58]. However,
the correlation between omphalocele formation and Hh signaling has not yet
been examined.
In this study, we investigated the participation of Hh signaling in ventral
body wall formation and the pathogenic mechanisms leading to its malformation
by utilizing a series of genetically-modified mouse systems. The phenotypic
coordination of ventral body wall, digit and pelvic girdle formations is also
discussed. We analyzed the lower body wall phenotypes of combinatorial mutants
of Shh, Gli3 and Alx4 genes.
We also analyzed conditional gain-of-function mutants of Hh signaling and
revealed the Hh signal dose-dependent pathogenesis of omphalocele and pubic
diastasis phenotypes. These results suggest that Hh signaling regulates omphalocele
formation and shed light on the pathogenic mechanisms underlying a broad spectrum
of lower body malformations.
Materials and MethodsMouse strains and embryos
The mutant mice used herein were Shh[59], Gli3Xt
(XtJ) [50], Alx4Lst
(LstJ) [54], [55], Gli1-CreERT2[60], Shh-CreERT2[61], Rosa26R[62], CAGGS-CreER™[63], Rosa26-SmoM2[64] and del5-LacZ
reporter[65], [66]. The genotypes
of each strain were determined as reported previously. To obtain Gli3Xt;
Alx4Lst; Shh compound mutant embryos, single, double or
triple heterozygous male and female mice were crossed. Noon of the day when
the vaginal plug appeared was designated as embryonic day 0.5 (E0.5). Embryos
for each experiment were collected from more than three independent pregnant
females. All experimental procedures and protocols for animal studies were
approved by the Committee on Animal Research of Kumamoto University (B22-198,
B22-200, B22-201 and B22-202).
Preparation of tamoxifen
The tamoxifen (TM)-inducible Cre recombinase system removes the floxed
sequence of the target genome [67]–[69]. TM (Sigma,
St. Louis, MO, USA) was dissolved in sesame oil (Kanto chemical, Tokyo, Japan)
at a final concentration of 10 mg/ml [65], [70], [71].
Hh-responded cell contribution analysis
To analyze the cell contribution that responded to Hh signaling, we utilized
the Gli1-CreERT2; Rosa26R system [60]. The Gli1-CreERT2
mice were crossed with Rosa26R Cre-indicator (R26R)
mice to obtain Gli1-CreERT2/+; R26R/R26R
males, which were subsequently crossed with ICR females [60], [72].
Time-mated ICR females were administered TM (2 mg per 40 g maternal body weight
(bw)) orally with a gavage needle. Mouse embryos were processed for whole-mount
X-gal staining.
Hh signal gain-of-function experiments
For gain-of-function experiments of Hh signaling, the Rosa26-SmoM2
(R26-SmoM2) homozygous female mice were crossed with the
Cre-driver mice, such as CAGGS-CreER™ transgenic male
mice [63], [64], [73]. The pregnant R26-SmoM2
females were treated with TM (1 mg, 2 mg or 4 mg per 40 g bw) orally with
a gavage needle. Embryos were collected, and their morphology was investigated
between mid-gestation and perinatal stages. No overt teratological effects
were observed in wild-type embryos after TM administration under these conditions [65], [70], [71].
Histological analyses
Mouse embryos were fixed overnight in 4% paraformaldehyde (PFA)
(Sigma) with PBS, dehydrated through methanol, embedded in paraffin, and 8 µm
serial sections were prepared. Hematoxylin and Eosin (HE) staining and X-gal
staining were processed by standard procedures [65], [74], [75]. For skeletal
staining, dehydrated embryos were skinned, eviscerated and refixed in 95%
ethanol for several days. Cartilage staining was performed for two days by
incubation in 0.03% Alcian blue 8GX (Sigma), dissolved in 80%
ethanol/20% acetic acid. After washing the embryos in 95% ethanol
for five days, they were stained with 0.0025% Alizarin red S (Sigma)
in 1% KOH for two days. Subsequently, they were treated with 1%
KOH for 6 hours. Finally, the embryos were cleared with 20%, 40%
and 60% glycerol and stored in 60% glycerol.
Statistical analysis
For the statistical analyses of the length of an extra digit, the length
was measured with a slide gauge. Data were analyzed using a Student's t-test
(two tailed). A probability of less than 0.001 was considered to indicate
statistical significance. Values are given as the means±SD.
In situ hybridization for gene expression analysis
In situ hybridization was performed on PFA-fixed and dehydrated
embryos. Samples were rehydrated, and incubated in 6% hydrogen peroxide
solution for 1 hour. After washing in PBS containing 0.1% Tween 20,
samples were incubated in 1 µg/ml ProteinaseK for 18 minutes, and refixed
with fixing solution (4% PFA/0.2% glutaraldehyde) for 10 minutes.
After washing with PBS containing 0.1% Tween 20, overnight incubation
was performed in a buffer (50% formamide, 5x saline sodium citrate,
50 µg/ml yeast tRNA, 1% sodium dodecyl sulfate, 50 µg/ml
heparin) at 65°C. Subsequent overnight hybridization was performed in
a buffer with 0.5 µg/ml riboprobes at 65°C. Samples were washed
in 50% formamide, 5x saline sodium citrate, 1% sodium dodecyl
sulfate and 50% formamide, 2x saline sodium citrate for each 1 hour
at 65°C, then 140 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 25 mM Tris-HCl
(pH 7.5) for 5 minutes at room temperature before incubating with blocking
solution (25% heated FBS in 140 mM NaCl, 2.7 mM KCl, 25 mM Tris-HCl
(pH 7.5), 0.1% Tween 20) for 1 hour. Samples were treated with anti-digoxigenin
antibody (Roche, Mannheim, Germany) in a blocking solution overnight at 4°C.
After washing, samples were equilibrated in 100 mM NaCl, 50 mM MgCl2,
0.1% Tween 20, and 100 mM Tris-HCl (pH 9.5) including 2 mM levamisole
(Sigma) and incubated in BM purple AP Substrate solution (Roche). Myogenin
(kindly provided from Dr. Shosei Yoshida) and Gli1[65] probes
were used. The preparation of the digoxigenin-labeled probes was performed
according to the manufacturer's instructions (Roche).
Cell death analysis
Embryos were collected in PBS, rinsed in PBS and stained with 500 ng/ml
Acridine Orange base (Fluka, St. Gallen, Switzerland) for 30 minutes. These
procedures were performed at 37°C. Samples were then rinsed briefly in
PBS, followed by fluorescence microscopy.
ResultsVentral body wall formation and the developmental coordination between
the ventral body wall and the pelvic girdle
We analyzed the development of the embryonic body wall in a series of wild-type
murine embryos. The protrusion of embryonic viscera covered with a peritoneal
membrane (physiological umbilical hernia) was apparent by E12.5 (Fig. 1A,B) [1], [2]. It was subsequently
recovered from E16.5 onwards when the ventral body wall closed (Fig. 1C). As a result, only the umbilical cord
could then be observed outside of the ventral body wall (Fig. 1C,D). We also analyzed pelvic girdle
morphogenesis because patients with several congenital diseases, such as exstrophy
of the cloaca, display malformations not only in the body wall region but
also in the urogenital organs and the pelvic girdle [15]–[18]. The bilateral primordia
(cartilaginous elements) of the pelvic girdle started to be perceptible from
E11.5 (Fig. 1E) [76] and they
were positioned in parallel along with the body trunk at E12.5 (Fig. 1F). Subsequently, the edges of the pubic
bones started to close, but were not yet connected at the stage of the physiological
umbilical hernia (at E14.5) (Fig.
1G). Consistent with the recovery of the physiological umbilical hernia,
the pubic symphysis was formed at about E16.5 or later (Fig. 1H,I).
10.1371/journal.pone.0016260.g001
The development of the ventral body wall and the pelvic girdle.
Wild-type embryos exhibited a physiological umbilical hernia in the ventral
body wall at E12.5 and E14.5 (A, B). Black arrows indicate the
physiological umbilical hernia. The physiological umbilical hernias were recovered
in wild-type late staged embryos at E16.5 and E18.5 (C, D). The
anlagen of the pelvic girdle start to be observed at around E11.5 (E;
black arrowheads). The jointing of the hip bones (pubic symphysis) was not
formed yet in wild-type embryos at E12.5 and E14.5 (F, G). The
embryonic pelvic girdle develops to the midline at E16.5 and the pelvic ring
is formed at E18.5 (H, I). Red arrowheads indicate the midline
edges of the pelvic girdle primordia (future symphysial surfaces of the pubis).
f: femur, gt: genital tubercle, hl: hind limb, il: iliac bone, is: ischial
bone, pu: pubic bone, t: tail, uc: umbilical cord.
Genetic interaction between Gli3 and Alx4
genes and their involvement in the Hedgehog signaling pathway
According to previous studies, several human patients and genetically-modified
mouse models with body wall phenotypes often have accompanying digit abnormalities [3], [20], [26], [28], [47], [51], [53]. Judging by the causative genes
of digit abnormalities, we hypothesized that Hedgehog (Hh) signaling may also
be involved in the onset of body wall malformation. To examine this hypothesis,
we analyzed combinatorial mutants for Hh and putative Hh signaling related
genes: Shh, Gli3 and Alx4.
Hence, we analyzed the phenotypes of the hind limb, which is a well-analyzed
system for examining genetic relationships among developmental genes. Wild-type
and Shh+/− mice displayed normal digit
morphology (Fig. 2A).
Both Gli3Xt/+ and Alx4Lst/+
single heterozygotes showed preaxial polydactyly (Fig.
2B,D) [40], [49], [54]. The size of the extra digit
in Gli3Xt/+; Shh+/−
mice was smaller than that of Gli3Xt/+ mice
(Fig. 2C). On the other
hand, this digit phenotype was completely restored in Alx4Lst/+;
Shh+/− mice (Fig.
2E). Moreover, Gli3Xt/+; Alx4Lst/+
mice displayed severe polydactyly (two extra digits) (Fig. 2F). This phenotype was also partially
restored by the addition of the Shh mutation (Fig. 2G). To quantify the effects of the gene
mutations, we analyzed the significance of the length of the extra digit (Fig. 2H). The introduction of
an additional Shh mutation significantly reduced the length
of the extra digit (by a comparison between Gli3Xt/+
versus Gli3Xt/+; Shh+/−:
1.44±0.30, n = 30 versus 0.90±0.24, n = 26; P<0.001).
On the other hand, the additional Alx4Lst mutation
induced the opposite effect (Gli3Xt/+; Shh+/−
versus Gli3Xt/+; Alx4Lst/+; Shh+/−:
0.90±0.24, n = 26 versus 2.03±0.74, n = 10; P<0.001).
From these results, we suggest that both Gli3 and Alx4
genes may negatively regulate Hh signaling.
10.1371/journal.pone.0016260.g002
The digit phenotypes of Shh, Alx4
and Gli3 heterozygotes.
Shh heterozygous mutants (Shh+/−)
showed a normal number of digits (A). Both Gli3
and Alx4 heterozygotes (Gli3Xt/+
or Alx4Lst/+) displayed polydactyly phenotypes
in the hind limbs (B, D). Polydactylies were partially restored
(C) or fully restored (E) by the addition of a Shh
heterozygous mutation in Gli3Xt/+ or Alx4Lst/+
heterozygotes. The Gli3Xt/+; Alx4Lst/+
double heterozygotes displayed polydactyly with more than two extra digits
(F). Polydactylies in the Gli3Xt/+; Alx4Lst/+
double heterozygotes were partially restored by the additional introduction
of a Shh heterozygous mutation (Gli3Xt/+;
Alx4Lst/+; Shh+/−) (G).
Red arrows indicate extra digits. The length of the extra digit was measured
for each genetic combination (H). An asterisk indicates statistical
significance based on the comparison of each mutant by Student's t-test.
The results are presented as the means±SD. *P<0.001.
Compound allelic series of Alx4 and Gli3
mutants display omphalocele and pelvic girdle abnormalities
We generated graded levels of mutations for Hh signaling by introducing
the Alx4Lst allele into a Gli3Xt/Xt
background, and analyzed the resultant compound mutant embryos at E18.5 (Fig. 3A–D,A'–D').
The physiological umbilical hernia was recovered, and pubic symphysis was
formed in wild-type embryos at E18.5 (Fig.
1D,I and Fig. 3A,A').
Decreasing wild-type Alx4 alleles accelerated the degree
of omphalocele in the Gli3Xt/Xt embryos (Fig. 3B–D). In Gli3Xt/Xt;
Alx4Lst/+ embryos and Gli3Xt/Xt;
Alx4Lst/Lst embryos, the upper (dorsal) side of the genital
tubercle was hypoplastic, in addition to the presence of an omphalocele (Fig. 3C,D). The development
of the pelvic girdle also showed severe malformations in these mutants. The Gli3Xt/Xt
embryos showed pubic diastasis (Fig.
3B'). The Gli3Xt/Xt; Alx4Lst/+
embryos displayed pubic diastasis and partial loss of pubic bones (Fig. 3C'). The Gli3Xt/Xt;
Alx4Lst/Lst embryos showed more severe truncation and
separation of the pubic bones than the Gli3Xt/Xt; Alx4Lst/+
embryos (Fig. 3D').
Thus, all of these mutants with omphalocele phenotypes displayed pubic diastasis.
10.1371/journal.pone.0016260.g003
The omphalocele, pubic diastasis, loss of pubic bones and polydactyly
in Gli3Xt; Alx4Lst; Shh combinatorial
mutants.
The lateral view of the embryonic ventral body wall (A–D, E,
H) and frontal view of the pelvic girdle (A'–D',
F, I). Gli3Xt/Xt embryos (B), Gli3Xt/Xt;
Alx4Lst/+ embryos (C) and Gli3Xt/Xt;
Alx4Lst/Lst embryos (D) showed a graded extent
of omphaloceles by the introduction of additional Alx4Lst
alleles into the Gli3Xt/Xt background (B–D;
white arrows). The dorsal parts of the genital tubercle were hypoplastic in Gli3Xt/Xt;
Alx4Lst/+ and Gli3Xt/Xt; Alx4Lst/Lst
embryos (C, D; red arrowheads). The pubic symphysis of wild-type
embryo was already formed at E18.5 (A'; asterisk). Pubic
diastasis also became evident by the introduction of Alx4Lst
mutation (B'–D'). Gli3Xt/Xt;
Alx4Lst/+ and Gli3Xt/Xt; Alx4Lst/Lst
embryos showed partial loss of pubic bone components (C', D';
yellow arrowheads). Black arrows indicate the unclosed pelvis. Gli3Xt/+;
Alx4Lst/Lst embryos showed omphalocele (E;
red arrow), severe polydactyly (G), pubic diastasis and loss
of pubic bones (F). Black arrows show the unclosed pelvis. Gli3Xt/+;
Alx4Lst/Lst; Shh+/− embryos did
not show omphalocele phenotypes (H). The pubic symphysis was
formed but pubic bones were lost (I; asterisk). Polydactyly was
still observed in Gli3Xt/+; Alx4Lst/Lst;
Shh+/− embryos (J). Black arrowheads
indicate extra digits. f: femur, fl: fore limb, gt: genital tubercle, il:
iliac bone, is: ischial bone, om: omphalocele, ps: pubic symphysis, pu: pubic
bone, t: tail, u: umbilical cord.
Phenotypic recovery of omphalocele and pubic diastasis, but not polydactyly
and pubic bone hypoplasia, results from reducing the Shh
allele
We further analyzed the effects of mutations in Hh signaling related genes.
The Gli3Xt/+; Alx4Lst/Lst embryos
also exhibited multiple deformities, including an omphalocele, polydactyly
and the loss of pubic bones and their diastasis (Fig.
3E–G). Introducing a Shh mutation could restore
some of these phenotypes in Gli3Xt/+; Alx4Lst/Lst
embryos (Fig. 3H–J).
The omphalocele observed in Gli3Xt/+; Alx4Lst/Lst
embryos (Fig. 3E) was
restored completely in Gli3Xt/+; Alx4Lst/Lst;
Shh+/− embryos (Fig.
3H). On the other hand, polydactyly was partially rescued, but was
still observed in these mice. While Gli3Xt/+; Alx4Lst/Lst
embryos displayed polydactyly (Fig.
3G), the number of extra digits was reduced in the Gli3Xt/+;
Alx4Lst/Lst; Shh+/− embryos (Fig. 3J). With regard to pelvic
girdle development, parts of the pubic bones were still not observed but the
midline symphysis of the pelvic girdle was formed in Gli3Xt/+;
Alx4Lst/Lst; Shh+/− embryos (Fig. 3I; asterisk). Taken together,
these results suggest the possible involvement of Hh signaling in omphalocele
and pubic diastasis phenotypes.
Ectopic Hh-signal activity is observed in Gli3Xt/Xt
mutants
In order to analyze the contribution of Hh-responded cells, we utilized
the Gli1-CreERT2; R26R system. In Gli1-CreERT2
mice, a TM-inducible form of Cre recombinase (CreERT2)
was knocked into the Gli1 locus (Gli1-CreERT2),
which is one of the direct target genes of Hh signaling [60], [68], [69], [77]. The Gli1-CreERT2
hemizygotes correspond to Gli1+/− mutants,
and displayed normal morphology in the ventral body wall (data not shown).
By crossing Gli1-CreERT2/+; R26R/R26R males
and ICR females, we could obtain Gli1-CreERT2/+; R26R/+
embryos. We treated pregnant ICR females once with 2 mg/40 g bw of TM at 8.5,
9.5, 10.5, 11.5 or 12.5 days post coitum, and embryos were collected at E14.5
(Fig. 4A–E) or at
E13.5 (Fig. 4F). The recombination
period in this system was estimated to occur within 6–12 hours and to
continue for up to 36 hours after TM administration [60], [63].
Our protocols were expected to detect Hh-responded cells during an embryonic
period approximately from E8.75 to E14.0. Under these TM treatment conditions,
we could not detect a significant LacZ-positive population in the ventral
body wall region (Fig. 4A–F).
10.1371/journal.pone.0016260.g004
The analysis of Hh-responded cells in the ventral body wall region.
A schematic diagram of Hh-responsive cell contribution assays. The R26R
allele contains the LacZ gene and a floxed stop cassette under the Rosa26
promoter. The Gli1-CreERT2 allele contains an
insertion of TM-inducible Cre recombinase into the Gli1 gene
locus. By crossing the Gli1-CreERT2; Rosa26R and
ICR mice, gene recombination in Hh-responded cells could be achieved specifically
under the control of TM. The embryos were stained with X-gal and dissected
horizontally at the umbilical cord level. The stages of TM administration
and estimated recombination periods in A–E are depicted.
Under these TM treatment conditions, few LacZ-positive cells were observed
in the ventral body wall (A–E; red arrowheads). The lateral
view of the embryo also showed few Hh-responded cells (F). Red
arrow indicates the LacZ-positive population in visceral organs. The del5-LacZ
transgenic mice, the Hh signal indicator strain, displayed relatively high
ectopic Hh signal activity in the Gli3Xt/Xt background
compared with the control at E12.5 (G, H; black
arrows).
We also employed a reporter mouse strain (del5-LacZ) to
locate active Hh signaling in vivo. The del5-LacZ
model employs Gli-responsive binding sites identified in the upstream sequence
of the Foxa2 gene [65], [66]. In the ventral
body wall region, we could not observe Hh signal activities in the del5-LacZ
strain at E12.5 (Fig. 4G).
This result was consistent with Hh-responded cell contribution analysis. In
contrast, we observed ectopic Hh activity by del5-LacZ staining
with the Gli3Xt/Xt mutation at E12.5 (Fig. 4H). These results imply that Hh signaling
may not play essential roles in normal development of the embryonic ventral
body wall, but may be implicated in omphalocele pathogenesis.
Augmented Hedgehog signaling results in omphalocele phenotypes
To assess the effects of ectopically-induced Hh signaling, we analyzed
gain-of-function mutants of Hh signaling (hereafter designated as Hh-GOF)
by utilizing the TM-inducible gene recombination system. Ectopic induction
of Hh signaling was achieved by utilizing R26-SmoM2 and CAGGS-CreER™
mice. The CAGGS-CreER™ mice display Cre activity throughout
the body upon TM treatment [63].
The R26-SmoM2 allele possesses the constitutively activated
form of Smoothened (SmoM2) and a floxed
stop cassette under the ubiquitous Rosa26 promoter [64], [73]. By crossing R26-SmoM2
mice and the TM-inducible form of Cre-driver mice, activation of Hh signaling
was achieved. We analyzed mutant embryos that were treated once with various
doses of TM (1 mg, 2 mg or 4 mg/40 g bw) at various time points on E9.5, E10.5,
E11.5, E12.5 or E13.5, respectively. No noticeable toxic effects were observed
for any of these TM treatment protocols [65], [70], [71]. Upon administration
of TM at E9.5, E10.5 or E11.5, mutant embryos displayed omphalocele and polydactyly
phenotypes (Fig. 5B,B',D,D';
data not shown). The phenotypic differences induced by the different doses
of TM were present following administration at E10.5 (Fig. 5C,C',D,D'). Omphaloceles were
prominently observed in embryos from dams treated with the higher dose of
TM (2 mg/40 g bw) but not with the lower dose (1 mg/40 g bw) (Fig. 5C,D). In contrast to the mutants treated
with TM at E10.5, Hh-GOF mutants did not display an omphalocele even with
the higher dose of TM treatment (4 mg/40 g bw) at E12.5 (Fig. 5E). On the other hand, the mutants exhibited
an omphalocele induced by the lower dose of TM treatment (1 mg/40 g bw) at
E9.5 (Fig. 5B). With regard
to the phenotypes for digits and the pelvic girdle, the mutants with omphaloceles
also showed severe polydactyly (Fig.
5B',D') compared with the non-omphalocele mutants in their
hind limbs (Fig. 5C',E').
The pubic symphysis was formed in control embryos at E17.5 (Fig. 5F). The Hh-GOF mutants showed pubic diastasis
when 2 mg/40 g bw of TM was administered at E10.5 (Fig.
5G). These results may indicate that the pathogenesis of omphalocele
is induced by augmented Hh signaling in a time- and dose-dependent manner.
10.1371/journal.pone.0016260.g005
The conditional activation of Hh signaling by the protocols inducing
omphalocele and pubic diastasis phenotypes.
The R26-SmoM2 allele contains the constitutively activated
form of Smoothened and a floxed stop cassette under the control
of the Rosa26 promoter. By crossing R26-SmoM2
mice and the TM-inducible form of Cre-driver mice, administration of TM to
the pregnant mice induced embryonic stage-specific gene recombination, allowing
continuous activation of Hh signaling. The lateral view of the body trunk
and the left hind limb of a wild-type embryo treated with a high dose of TM
at E10.5 (A, A'). Mutant embryos treated with
a low dose of TM at E9.5 (B, B'), a low dose
of TM at E10.5 (C, C'), a high dose of TM at
E10.5 (D, D') and a high dose of TM at E12.5
(E, E'). Embryos were collected at E17.5 (A–G
and A'–E'). Mutant embryos treated with the low
dose of TM at E9.5 and the high dose of TM at E10.5 showed omphalocele phenotypes
(B,D; red arrows). Under such conditions, mutants
displayed polydactyly phenotypes (B'–E'). Control
embryos at E17.5 developed a pubic symphysis (F; asterisk). Mutant
embryos treated with a high dose of TM at E10.5 showed a pubic diastasis phenotype
(G). Black arrows indicate the unclosed pelvis. f: femur, gt:
genital tubercle, hl: hind limb, il: iliac bone, is: ischial bone, om: omphalocele,
pu: pubic bone, t: tail, u: umbilical cord.
Abnormal body wall muscle formation and excessive cell death would
be associated with omphalocele formation in the Hh-GOF mutants
We further analyzed the Hh-GOF mutants in mid-gestation. To confirm the
induction of ectopic Hh signaling, we performed gene expression analyses as
one of the readouts of Hh signaling: Gli1 mRNA in Hh-GOF
mutant embryos. The expression of Gli1 was observed ectopically
throughout the body, including the lateral body wall in Hh-GOF mutants (Fig. 6H). We hypothesized that
two potential causative factors might underlie the etiology of omphalocele
formation. One could be an abnormality in the endodermal organs, such as an
excess bulging out of visceral organs when the physiological umbilical hernia
is observed. Another possibility could be defects in the mesodermal or ectodermal
organs, such as a failure of the ventral body wall muscle formation. We expected
that either or both of these factors could cause omphalocele formation. To
assess these possibilities, we analyzed CAGGS-CreER™; R26-SmoM2
embryos using different TM administration protocols (TM treatment with 2 mg/40
g bw at E10.5 and harvested at E14.5, or TM treatment with 1 mg/40 g bw at
E9.5 and harvested at E12.5 and E13.5). These TM administration protocols
were sufficient to induce an omphalocele in later embryonic stages, and all
of these mutants exhibited similar phenotypes (Fig.
5B,D). Interestingly, the volume of the herniating viscera in the peritoneal
sac appeared smaller in Hh-GOF mutants than in control embryos (Fig. 6A,B; red arrow). On the other hand, excessive
amount of cell death was detected by acridine orange staining in Hh-GOF mutants
during the ventral body wall formation (Fig.
6D). With regard to the muscle differentiation, gene expression analyses
of a muscle marker, Myogenin, suggested that the populations
of muscle precursors in both the lateral body wall and limbs were decreased
and distributed abnormally in Hh-GOF mutants (Fig.
6I–L). The lateral body wall of the mutants seemed to be disorganized
(Fig. 6F; yellow arrowheads).
Moreover, both epaxial and hypaxial muscle precursors seemed to be affected
in Hh-GOF mutants (Fig. 6M;
red arrowheads). These results might suggest that the pathogenesis of omphalocele
in Hh-GOF mutants could be due to the failure of body wall formation and an
abnormally enlarged umbilical ring associated with excessive cell death.
10.1371/journal.pone.0016260.g006
Formation of embryonic abdominal muscles and herniation of the visceral
organs are affected by Hh signal activation.
The CAGGS-CreER™; R26-SmoM2 (Hh-GOF) mutant embryos
showed a moderate degree of herniation into the sac (peritoneal membrane)
of physiological umbilical hernia compared with control embryos (A, B;
red arrow). Hh-GOF mutants also exhibited more prominent cell death compared
with controls as determined by acridine orange staining (C, D;
white arrowheads). In addition, the lateral embryonic trunk was malformed
in mutants (E, F; yellow arrowheads). Expression
analysis of Gli1 confirmed the ectopic induction of Hh signaling
in the ventral body wall (G, H). Myogenin
expression was weaker (J; black arrowheads) and ectopically located
(L; black arrow) in Hh-GOF mutants (I–M).
Red arrowheads indicate affected muscle precursors after Hh activation.
Discussion
Recent advances in developmental biology and human embryology provide a
profound understanding of the organogenesis and the pathogenesis of congenital
diseases [78].
Embryonic organogenesis is potentially influenced by genetic programs, maternal-embryonic
interactions and embryonic physiological conditions [79], [80].
The development of the ventral body wall displays dynamic processes, such
as that observed during the formation and recovery of the physiological umbilical
hernia. These processes proceed with the proper formation of adjacent structures,
including body wall muscles and the pelvic girdle. Although some previous
studies of genetically-modified animals have reported the processes of ventral
body wall formation, our understanding of the ventral body wall formation
and its related dysmorphogenesis remains incomplete. We herein reported that
Hedgehog signaling is one of the causative factors for omphalocele formation,
as demonstrated by utilizing a series of combinatorial mutants for Hh signaling
genes and conditional gain-of-function mutants of the Hh signaling pathway.
The analyses of Shh, Gli3 and Alx4 compound
mutant embryos revealed that the introduction of additional Alx4Lst
mutations into the Gli3Xt/Xt background resulted
in the corresponding omphalocele and pubic diastasis. Moreover, the reduction
of a single Shh allele restored omphalocele and pubic symphysis
formation in the Gli3Xt/+; Alx4Lst/Lst
embryos. The CAGGS-CreER™; R26-SmoM2 (Hh-GOF) conditional
mutant analyses revealed the Hh signal-dependent omphalocele formation and
pubic diastasis. This would therefore be the first demonstration of the involvement
of Hh signaling in ventral body wall malformation and the genetic rescue of
omphalocele formation.
The possible factors causing omphalocele formation
In spite of recent advances in embryology and pathology, the etiology of
omphalocele formation is still controversial. It has been suggested that a
failure of the gut to return to the abdominal cavity after physiological herniation
at appropriate developmental stages results in an omphalocele [6], [10], [81]. According
to this hypothesis, the lateral body wall closure has not been considered
to be related to omphalocele formation, because the loops of the bowel are
in the cord and are covered by amniotic membranes [9].
Another possible cause of omphalocele could be a midline defect at the amnio-ectodermal
transition, the transition zone between the ectoderm and mesoderm, which would
result in an enlarged umbilical ring [2], [7], [11], [12].
During normal development, a mature body wall covers the ventral surface surrounding
the ring and the cord. With omphalocele, the mature body wall shows incomplete
closure and it is localized to the periphery of the enlarged umbilical ring [82].
Our current studies suggested that few Hh-responded cells could contribute
to the normal ventral body wall formation (Fig.
4A–F). In contrast to such results, we observed the ectopic Hh
signaling in Gli3Xt/Xt mutants by utilizing the del5-LacZ
Hh reporter mouse strain (Fig.
4H). In the Alx4Lst/Lst mutants, Shh
expression was augmented in the cloacal epithelium, and ectopic Hh signal
activity was observed in the ventral body wall region by del5-LacZ
staining (data not shown). Moreover, gain-of-function mutants of Hh signaling
displayed defects in body wall formation (Fig.
6F,J). These results suggested that omphalocele might be caused by
ectopically-induced Hh signaling.
In this manuscript, we utilized the system for tamoxifen inducible ubiquitous
activation of Hh signaling by CAGGS-CreER™; R26-SmoM2.
However, the identification of such Hh-responded tissues remained unclear
even when utilizing this conditional activation system. Hence, we further
analyzed mutants that display specific activation of Hh signaling in the endodermal
organs. To achieve endodermal activation of Hh signaling, Gli1-CreERT2;
R26-SmoM2 mice and Shh-CreERT2; R26-SmoM2
mice were employed. The Gli1-CreERT2 mice and Shh-CreERT2
mice possess a tamoxifen-inducible form of Cre recombinase in the Gli1
and Shh gene loci, respectively [60], [61]. Shh
is specifically expressed in the endodermal epithelia of many visceral organs,
and Gli1 is expressed mainly in the mesenchyme of visceral
organs (Fig.
S1A–C) [83].
Regardless of the identity of Cre-driver lines, none of the mutants displayed
omphalocele phenotypes (Fig.
S2; data not shown). These results may suggest that Hh signal activation
in endodermal organs may not be sufficient to induce such phenotypes under
the current experimental conditions. In addition, the Gli1
gene is also considered to be one of the direct target genes of Hh signaling [60], [84], [85].
Hence, the allelic combination of Gli1-CreERT2; R26-SmoM2
could result in Hh signal activation in Hh-responded tissues in such mutant
embryos. Based on comparison of the phenotypes of Gli1-CreERT2;
R26-SmoM2 and CAGGS-CreER™; R26-SmoM2 embryos,
we would also suggest that the pathogenesis of the omphalocele was due to
ectopically-induced Hh signaling (Fig.
5D and Fig.
S2A').
With regard to the involvement of various mesodermal and ectodermal tissues
in the pathogenesis of omphalocele formation, our previous study showed that
there were abdominal wall defects with disorganized muscle layers and connective
tissues in Msx1/2 double mutant mice [28]. Likewise, abnormal Alk3-mediated
BMP signaling in mesodermal tissues caused prenatal omphalocele-like defects [26]. Moreover, the
phenotypes of Pitx2 knockout mice and PITX2
mutation in humans indicate a correlation with the omphalocele formation [33], [86]. The Pitx2
gene, which is expressed in abdominal muscles such as the rectus abdominis
and oblique abdominis, has pivotal roles in muscle anlagen formation and maintenance [23], [87]–[89]. Altogether,
these reports suggest essential roles of mesodermal or ectodermal tissues
in omphalocele formation.
Another question arises regarding the onset of omphalocele formation. We
showed the presence of a critical time-window for inducing omphalocele phenotypes
by analyzing temporally-regulated conditional mutants of CAGGS-CreER™;
R26-SmoM2 mice (Fig.
5B,D,E). Hh signal induction before the stage of physiological umbilical
herniation (E9.5 and E10.5) resulted in omphalocele formation (Fig. 5B,D). In addition, activation of Hh signaling
by TM injection at E12.5 did not lead to omphalocele formation (Fig. 5E). These results may imply that the
onset of omphalocele formation thus begins before physiological umbilical
herniation.
The formation of pubic symphysis may be related to body wall dysmorphogenesis
The pathogenic sequences of human patients with dysgenesis of the bladder
and external genitalia have been reported. Such phenotypes often display exstrophy
of the bladder and hypoplasia of the upper (dorsal) side of the penis [90], [91]. Another characteristic symptom
of bladder exstrophy is pubic diastasis (pelvic girdle separation) [15], [92].
These complex congenital defects prompted the idea of coordinated urogenital
organ development [18], [65]. Despite
the significant correlations of clinical conditions, the influence of pelvic
girdle formation on such developmental coordination has previously been little
analyzed.
Our current study revealed the concordant recovery of the physiological
umbilical hernia and the closure of pelvic girdle at late embryonic stages
(from E14.5 to E16.5) (Fig. 1B,C,G,H).
According to textbook anatomy, the rectus abdominis arises from the front
of the pubic symphysis and from the pubic crest [93], [94]. Myocytes
of the abdominal musculature are of somitic origin and the connective tissue
elements within the abdominal wall, including tendons, derive from the somatopleure [95]. Moreover,
the entire pelvic elements also originate from the somatopleure [76], [96].
We suggested that both body wall muscle primordia and pelvic girdle primordia
arise from the lateral side of the embryonic trunk and develop toward the
midline of the body (Fig. 1E–I
and Fig. S3A–C).
These observations imply that an unclosed pelvic girdle may be related to
the phenotype with unclosed body wall muscles. In fact, Gli3Xt/Xt
and Alx4Lst/Lst embryos displayed the absence
of the midline muscular structures (Fig. S4C,D).
Hh signaling can affect the formation of midline structures in many developmental
contexts of mammalian embryos, such as craniofacial formation. For instance,
decreased Hh signal activity causes holoprosencephaly and hypotelorism, in
contrast to augmented Hh signaling, which causes hypertelorism and frontonasal
dysplasia [97].
Our studies may also be supported by a broad spectrum of observations covering
several processes of organogenesis. In the current study, the mutants with
omphalocele phenotypes tended to show pubic diastasis. We revealed a possible
association between the degree of omphalocele phenotype and pubic diastasis
phenotype (Fig. 3B–D,B'–D').
In addition, mutants genetically rescued from omphalocele (Gli3Xt/+;
Alx4Lst/Lst; Shh+/−) did form the
pubic symphysis and normal midline muscle structures (Fig. 3I; data not shown). These results may
indicate a role for Hh signaling in the formation of such midline structures,
and a developmental correlation between the ventral body wall and the pelvic
girdle.
The extent of dysmorphogenesis in the body wall, pelvic girdle and
digit formation by ectopically-induced Hedgehog signaling
While abnormal Hh signaling has been implicated as one of the major causes
of polydactyly in mice, its involvement in the omphalocele formation has so
far not been examined. In the current study, we showed that reduction of a
single Shh allele could restore the omphalocele but could
not restore the polydactyly of Gli3Xt/+; Alx4Lst/Lst
embryos (Fig. 3H,J). In
addition, the Hh-GOF mutants (CAGGS-CreER™; R26-SmoM2)
without the omphalocele phenotype still exhibited polydactyly (Fig. 5C',E'). These results may suggest
that the pathogenesis of the polydactyly phenotype seemed to be more sensitive
to ectopically-induced Hh signaling than that of the omphalocele phenotype.
In support of this notion, the frequency of polydactyly is higher than omphalocele,
and has been reported to occur in approximately 1 in 600 human births [98]. On the other
hand, the frequency of omphalocele is relatively low, namely approximately
1 in 4,000 human births [6].
In the current study, we suggested that ectopically-induced Hh signaling
might be one of the causes of a combination of polydactyly, omphalocele and
pubic diastasis phenotypes. These results may offer a clue that can help elucidate
the mechanisms underlying the formation of omphalocele and its associated
syndromic malformations.
Supporting Information
Cre recombinase activities of Shh-CreERT2
and Gli1-CreERT2in the developing gut.
The Shh-CreERT2 activity was not observed in the
ventral body wall at E14.5 upon tamoxifen treatment (4 mg/40 g maternal body
weight) at E9.5 (A). Red arrow indicates the expression in the
developing gut. The expression of Cre recombinase was detected in the endodermal
epithelia of the midgut and posterior part of limb buds (B).
The activity of Gli1-CreERT2 was also observed
in the embryonic gut, including a part of the mesentery (C).
The Gli1-CreERT2; R26R embryo was treated with
4 mg/40 g bw of tamoxifen at E10.5 and harvested at E13.5.
(TIF)
Augmentation of Hh signaling by utilizing Gli1-CreERT2
and Shh-CreERT2driver mouse lines. Both Gli1-CreERT2;
R26-SmoM2 and Shh-CreERT2; R26-SmoM2
embryos did not display omphalocele phenotypes following administration of
4 mg/40 g bw of tamoxifen at E10.5 (A, A', B, B').
gt: genital tubercle, hl: hind limb, t: tail, uc: umbilical cord.
(TIF)
The expression of Myogenin in wild-type embryos
at E10.5, E11.5 and E12.5. The ratio between primordia of hypaxial
musculature (a, a' and a″) and epaxial musculature (b, b'
and b″) was gradually increased during these stages, as hypaxial musculature
(body wall muscle precursors) developed toward the midline (A-C).
(TIF)
Absence of midline structures in Alx4Lst/Lst and Gli3Xt/Xt
embryos. Sagittal sections of a control embryo at E18.5 displayed prominent
pubic symphysis (A; asterisk) and abdominal muscle structures
(B). Muscles were stained with Anti-Skeletal Myosin antibody
(FAST) (Sigma). Neither Alx4Lst/Lst (C)
nor Gli3Xt/Xt (D) mutant embryos
developed pubic symphysis or abdominal muscles, as shown by sagittal sections.
b: bladder, gt: genital tubercle, om: omphalocele, r: rectum, u: urethra.
(TIF)
We thank members of our laboratory, Mika Kamimura, Aki Murashima, Mylah
Villacorte, Yukiko Ogino, Masayo Harada and Kentaro Suzuki for comments and
discussion. We would like to specially thank Drs. Alexandra L. Joyner, Shosei
Yoshida, Andrew P. McMahon, Chi-chung Hui, Hiroshi Sasaki, Sanne Kuijper,
Annemiek Beverdam, Toshihiko Shiroishi, Chin Chiang and Philippe Soriano for
their invaluable support. We would also like to thank Drs. Pierre Chambon,
Shigemi Hayashi, Sho Ohta, Shigeru Makino, Ken-ichi Yamamura, Kenji Shimamura,
Shihuan Kuang, Alexander I. Agoulnik, Rolf Zeller, Richard R. Behringer and
Anne M. Moon for encouragement and suggestions. We would also like to express
our appreciation to Sawako Fujikawa, Keiko Horie and Yuka Endo for their assistance.
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