1 Department of Molecular Biology, University of Texas Southwestern Medical
Center at Dallas, TX 75390-9148, USA
3 Department of Pathology, University of Texas Southwestern Medical Center at
Dallas, TX 75390-9148, USA
2 Department of Molecular, Cellular and Craniofacial Biology, Birth Defects
Center, School of Dentistry, University of Louisville, Louisville, KY 40292,
USA
* Present address: Department of Cell Biology and Genetics, Erasmus Medical
Center Rotterdam, Dr Molewaterplein 50, 3015GE Rotterdam, The
Netherlands
Author for correspondence (e-mail:
eolson{at}hamon.swmed.edu)
Accepted 12 December 2002
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SUMMARY |
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Key words: dHand, Craniofacial development, Neural crest, Cleft palate
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INTRODUCTION |
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The peptide ligand endothelin-1 (ET-1; also known as Edn1) plays a key role
in regulating branchial arch development. Targeted mutations of the genes
encoding ET-1, the G protein-coupled endothelin receptor A (ETA, EndrA) and
endothelin converting enzyme-1 (ECE-1), show identical phenotypes
characterized by abnormalities in branchial arch-derived skeletal elements,
arteries and the cardiac outflow tract
(Clouthier et al., 1998;
Kurihara et al., 1995
;
Kurihara et al., 1994
;
Yanagisawa et al., 1998
). ET-1
is secreted by the surface epithelium and the paraxial mesodermal core of the
branchial arches, and acts on surrounding ectomesenchymal cells that express
ETA. Pharmacological interventions with an ETA antagonist in chick embryos
showed that ET-1/ETA-mediated signaling is critical for development of the
lower beak and other distal branchial arch derivatives during the time period
corresponding to colonization of EndrA-positive post-migratory neural
crest cells (Kempf et al.,
1998
). In addition, the gene responsible for the sucker
mutation in zebrafish was shown to encode ET-1, and defects observed in
sucker mutants, such as severe hypoplasia of the lower jaw and
malformations of distal (ventral) branchial arch cartilages can be rescued by
injection of ET-1 orthologs or administration of human recombinant ET-1
(Miller et al., 2000
). These
findings suggest that a common signaling pathway involving ET-1/ETA is
conserved between zebrafish, birds and mammals, and is essential for
development of branchial arch-derived structures.
ET-1 is required for expression of the basic helix-loop-helix (bHLH)
transcription factor genes dHAND/Hand2 and
eHAND/Hand1 in the mesenchyme of the anterior branchial
arches (Thomas et al., 1998;
Clouthier et al., 2000
). We
have shown that a 208 bp enhancer upstream of the dHAND gene is
sufficient to drive expression of dHAND in the mandibular component
of branchial arch 1 and branchial arch 2 (hyoid arch) in mice, and that
activity of this enhancer is completely abolished in
EdnrA/ null embryos, suggesting that it is a
downstream target for ETA signaling
(Charité et al., 2001
).
This enhancer contains a series of conserved ATTA motifs that correspond to
the consensus-binding motif for many homeodomain proteins. Mutation of these
sites abolishes expression of a linked transgene in branchial arches 1 and 2
of transgenic mouse embryos at E10.5, suggesting that binding of homeodomain
transcription factors to these sites is essential for enhancer activity.
Consistent with this notion, the distal-less homeodomain protein Dlx6 binds
these sites and is expressed in a pattern that overlaps that of dHAND
in the branchial arches. Expression of Dlx6 is undetectable in the
distomedial branchial arches of EdnrA/
embryos, suggesting that Dlx6 is a key transcription factor involved in
ETA-dependent regulation of dHAND in the branchial arch.
Because mice homozygous for a dHAND null allele die from cardiac abnormalities prior to branchial arch development, the specific role of dHAND in development of these structures has been unclear. To address this question, we generated mutant mice in which the ETA-dependent neural crest enhancer of dHAND was deleted by homologous recombination. Mice homozygous for this enhancer deletion fail to express dHAND in the ventrolateral region of the first and second branchial arches and show lethal craniofacial abnormalities that include cleft palate and malformations of the mandible and Meckel's cartilage. However, expression of dHAND in the ventral region of the branchial arches is retained in these mutant mice, demonstrating the involvement of additional cis-regulatory elements in the control of branchial arch expression of dHAND. These findings demonstrate an essential role for dHAND in craniofacial development and reveal unanticipated molecular heterogeneity in the transcriptional pathways that subdivide cells within the branchial arch neural crest.
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MATERIALS AND METHODS |
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Linearized targeting vector DNA was electroporated into SM-1 embryonic stem
(ES) cells, which were subsequently selected under G418 and FIAU as described
previously (Yanagisawa et al.,
2000). Genomic DNA was prepared from ES cell clones and digested
with SacI for hybridization with a 5' probe, and with
NdeI and XbaI for a 3' probe. Targeted clones were
expanded and injected into blastocysts from C57BL/6 mice and resultant
chimeras were bred to C57BL/6 mice to obtain germ line transmission.
Heterozygous mutant mice for the dHAND branchial arch enhancer
(+neoBAenh) were intercrossed to obtain
+neoBAenh/ homozygous mutants. To obtain
heterozygous mutants for the branchial arch enhancer without a
neor cassette
(
neoBAenh), +neoBAenh+/
heterozygous mutants were bred to transgenic mice expressing Cre recombinase
under the cytomegalovirus immediate early enhancer-chicken ß-actin hybrid
promoter (CAG) (Sakai and Miyazaki,
1997
). The resulting
neoBAenh+/
heterozygous mutants were bred to the +neoBAenh+/
heterozygous mutants to obtain
neoBAenh/ homozygous mutants.
Genotyping and PCR
Genotyping was performed by Southern blot analysis using genomic DNA
isolated from tail biopsies or yolk sac preparations. We performed PCR
amplifications of the neo gene
(5'-TTCCACCATGATATTCGGCAAGCAGG-3' for an upstream primer and
5'-TATTCGGCTATGACTGGGCACAACAG-3' for a downstream primer), and the
BAenh sequence (5'-TCTGATCTCCTTTCAAACT-3' for an upstream
primer and 5'-ATTTCCAGCAAGCATCCTGC-3' for a downstream primer) to
identify +neoBAenh+/,
+neoBAenh/ or the
neoBAenh/ mutants. For detection of
the Cre transgene, PCR primers (5'-AGGTTCGTTCACTCATGGA-3' for an
upstream primer and 5'-TCGACCAGTTTAGTTACCC-3' for a downstream
primer) were used. For a control, PCR primers
(5'-TGGATAATACAATGATGTGGAAAATGGGA-3' for an upstream primer and
5'-AGCTCCTAGCTATGGGTTCTC-3' for a downstream primer) were used.
Southern blot analysis was performed to distinguish wild-type mice from the
neoBAenh+/ mice.
Histology and skeletal analysis
For routine histological analysis, embryos were fixed in 10% neutral
buffered formalin, embedded in paraffin and sectioned at 5 µm. Paraffin
sections were stained with Hematoxylin and Eosin. For skeletal analysis,
postnatal day 1(P1) embryos were collected, prepared and stained with Alizarin
Red and Alcian Blue to examine bone and cartilage formation, respectively
(Yanagisawa et al., 1998).
Cartilaginous fetal skeletons (E14.5) were prepared and stained with Alcian
Blue as previously described (Jegalian and
De Robertis, 1992
).
In situ hybridizations
E10.5 embryos were harvested and fixed in 4% paraformaldehyde overnight at
4°C. Riboprobes for dHAND, eHAND and Dlx6 were prepared
as described previously (Charité et
al., 2001; Thomas et al.,
1998
) with 35S-UTP (Amersham) using the Maxiscript In
Vitro Translation Kit (Ambion). In situ hybridizations were performed as
described previously (Shelton et al.,
2000
).
Whole-mount in situ hybridization
Embryos were harvested at E10.5 and fixed in 4% paraformaldehyde overnight
at 4°C. Whole-mount in situ hybridizations were performed as previously
described (Clouthier et al.,
2000) using digoxigenin-labeled riboprobes for dHAND, eHAND,
MHox, Msx1 and Msx2 (Thomas
et al., 1998
), Gsc, Dlx2 and Dlx3
(Clouthier et al., 2000
),
Dlx5 [a gift from J. L. R. Rubenstein
(Liu et al., 1997
)] and
Dlx6 (a gift from G. Levi) and Alx3
(ten Berge et al., 1998
). At
least 3 embryos per genotype were examined per probe. Following whole-mount in
situ hybridization, embryos were photographed using an Olympus SZX12
photomicroscope with an attached DP11 digital camera.
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RESULTS |
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The targeting vector was electroporated into ES cells and 480 colonies were
screened by Southern blot analysis. Four independent ES clones containing a
+neoBAenh mutant allele (data not shown) were injected into
blastocysts obtained from C57BL/6 mice, and 6 chimeras were obtained. Three
chimeras from two independent ES cell clones transmitted the mutant allele
through the germline. The +neoBAenh heterozygous mice were then bred
to transgenic mice expressing Cre-recombinase under control of the CAG
promoter to establish heterozygous mutant mice carrying the
neoBAenh mutant allele in the germline. Despite reported activity of
the CAG promoter in female oocytes (Sakai
and Miyazaki, 1997
), Cre recombinase-mediated loxP deletion was
not observed without integration of the transgene in the genome, suggesting
that recombination did not occur in the germ cells of
+neoBAenh+/ mice (data not shown).
The +neoBAenh+/ mice carrying the Cre transgene
were bred to +neoBAenh+/heterozygous mutant mice,
and genotyping of progeny was performed by Southern blot analysis. As shown in
Fig. 1B, hybridization of
SacI-digested tail DNA with a 5' probe resulted in a 13.5 kb
band for the wild-type and neoBAenh alleles, whereas the
+neoBAenh allele gave a 6.5 kb band because of an additional
SacI site in the neor cassette. Hybridization of
NdeI- and XbaI-digested tail DNA with a 3' probe
yielded bands of 16 kb for the wild-type allele, 12 kb for the
+neoBAenh allele and 9.5 kb for the
neoBAenh allele.
Next, we performed PCR analyses to confirm that the branchial arch enhancer
sequence was deleted in +neoBAenh/ and
neoBAenh/ mice. As shown in
Fig. 1C, a 500 bp neo
band was absent in the presence of Cre-recombinase and a 300 bp band
corresponding to the branchial arch enhancer was absent both in the
+neoBAenh/ and the
neoBAenh/ mutants.
Deletion of the branchial arch enhancer is sufficient to cause
craniofacial abnormalities in BAenh/
embryos
Genotyping of postnatal day 28 mice revealed no viable
BAenh/ mice among more than 100 offspring
examined. The BAenh/ mutants with or without
a neor cassette showed an identical phenotype of
hypoplastic jaw (Fig. 2A), and
all died within 24 hours of birth from failure to suckle. The secondary palate
of the mutants failed to fuse along the midline of the oral shelf
(Fig. 2B), and the stomach
contained no milk. In the homozygous mutants there were no other gross
abnormalities related to branchial arch-derived craniofacial structures, the
cardiac outflow tract or the great vessels (data not shown).
|
Histological examinations of P1 BAenh/ mutants showed that the mandibular bones were hypoplastic and displaced laterally compared to those of wild-type mice (data not shown). The palatine processes were elevated and fused to form the secondary palate in wild-type mice (Fig. 3A). In contrast, the secondary palate was defective in BAenh/ mice (Fig. 3B, arrows). Consequently, the mutants had a cleft palate. The muscle fibers of the tongue were also less organized and seemed to be oriented randomly in the BAenh/ mutant compared to wild-type littermates (tg in Fig. 3A,B). The distal symphysis of Meckel's cartilage was present in both the wild-type and the BAenh/ mutant mice (asterisk in Fig. 3C,D), although it was smaller in the BAenh/ mutant (Fig. 3D). Meckel's cartilage was continuously fused to the malleus at the proximal end in both wild-type and BAenh/ mutant mice (Fig. 3E,F). Inner ear structures and middle ear ossicles were all present in the BAenh/ mutants.
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To further examine craniofacial structures in BAenh/ mutants, we compared bone and cartilage staining of homozygous mutants with wild-type littermates at P1. We observed that the mandible of homozygous mutants was much smaller in size and shortened (ma in Fig. 4B,D), compared with the mandible in wild-type mice (ma in Fig. 4A,C). A ventral view of the skull showed deformity of the mandible in the homozygous mutant (Fig. 4D compare with C). The well-developed Meckel's cartilage was observed extending from the malleus to the middle of the mandible in the wild-type mouse (arrows in Fig. 4C). However in the BAenh/ mutant mouse, Meckel's cartilage was disrupted at the proximal end closer to the junction to the malleus (arrow in Fig. 4D, mc in 4P). Close examination showed that the angle between the right and left mandibular bones was wider in the BAenh/ mutant than in the wild-type mouse (Fig. 4K,L). In addition, the angular process was severely reduced, and an ectopic process was observed extending from the ventral surfaces of the mandible (Fig. 4M,N). Defects in the mandible were already apparent in BAenh/ mutant embryos at E14.5 (Fig. 5B,D). Cartilage staining at E14.5 revealed the well-developed Meckel's cartilage in wild-type embryos (Fig. 5A). In contrast, Meckel's cartilage was obviously truncated in BAenh/ mutant embryos (Fig. 5B). Interestingly, there was a cartilage primordium in the distal part of the mandible both in the wild-type and the BAenh/ mutants (Fig. 5C, arrow in D). Meckel's cartilage normally forms from proximal and distal primordia, and failure of expansion from either primordium could result in a truncated Meckel's cartilage.
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In P1 embryos, the tympanic rings were shortened and deformed in the BAenh/ mutant mouse (compare arrows in Fig. 4E and F). In the wild-type mouse, bilateral palatine processes extended horizontally and fused to form the secondary palate (dotted line in Fig. 4E, arrows in G). In contrast, the palatine processes of the BAenh/ mouse appeared not to be elevated and thus the secondary palate was not formed (dotted line in Fig. 4F, arrows in H). This causes the underlying presphenoid bone to be visible in ventral view (ps in Fig. 4H). The pterygoid bones were also deformed so that the relative angle to the basisphenoid bone was abnormal in the BAenh/ mutant mouse (Fig. 4F). Although the remnants of the palatine processes were detectable in the BAenh/ mutant mouse by close examination (arrows in Fig. 4J), they did not fuse along the midline unlike those of the wild-type mouse (arrows in Fig. 4I). The middle ear ossicles seemed to be less affected, however, the projection of the manubrium of the malleus was abnormal (red arrow in Fig. 4P). Skeletal structures affected in the mutant are summarized in Table 1.
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dHAND expression in the ventral (distal) region of branchial
arches 1 and 2 is unaffected in BAenh null embryos
To examine the effect of deletion of the branchial arch enhancer on
dHAND expression, we compared dHAND expression in the
+neo BAenh/ and the
neoBAenh/ mutants and wild-type embryos by
in situ hybridization. As shown in Fig.
6, dHAND expression in wild-type embryos was observed in
the ventral portions of the first and second branchial arches at E10.5
(Fig. 6A). In contrast,
dHAND expression was abolished in all except the most ventral regions
of branchial arch 1 in the +neo BAenh/
(Fig. 6B,E) and the
neoBAenh/ mutants
(Fig. 6C). As expected,
dHAND expression in the heart and limb was unaffected by deletion of
the branchial arch enhancer (Fig.
6E).
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We also examined whether eHAND expression was attenuated in the
absence of the dHAND branchial arch enhancer. At E10.5,
eHAND is expressed in the most ventral portions of branchial arches 1
and 2 of wild-type embryos (Fig.
6F). The eHAND expression pattern appeared identical in
the branchial arches of BAenh/ embryos
(Fig. 6G and
Fig. 7B), suggesting that a
loss of dHAND expression did not induce compensatory up-regulation of
eHAND expression. Notably, eHAND was not expressed in the
region of the branchial arches where the dHAND neural crest enhancer
is active. Since there is evidence for functional redundancy of dHAND
and eHAND in some cell types
(McFadden et al., 2002), their
nonoverlapping expression in this region may account for the craniofacial
phenotype in BAenh/ embryos.
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Dlx6 expression is unaffected in
BAenh/ embryos
Our previous studies showed that Dlx6 is an ETA-dependent branchial arch
enhancer-binding factor and that the Dlx6 binding sites are essential for
activity of this dHAND enhancer
(Charité et al., 2001).
To test whether Dlx6 is regulated independently of dHAND in the
branchial arches, we examined Dlx6 expression in
BAenh/ embryos. At E10.5, Dlx6
expression was observed in the ventral aspect of branchial arches 1 and 2 of
wild-type embryos, excluding the most ventral portions
(Fig. 6H,
Fig. 7G). This expression
pattern was maintained in the branchial arches of
BAenh/ embryos
(Fig. 6I,
Fig. 7G). Most importantly,
ventrolateral expression of Dlx6, which was down-regulated in
EdnrA/ mutant embryos, was unaffected in
BAenh/ embryos. It is interesting that
Dlx6 expression was not observed in the most ventral portion of
branchial arches 1 and 2 of wild-type or
BAenh/ embryos, where dHAND
expression persisted in BAenh/ embryos. This
suggests that transcription factor(s) other than Dlx6 may control
dHAND expression in this region of the branchial arches.
Effect of the dHAND branchial arch enhancer on expression of
other transcription factors in the branchial arches
Several transcription factors, including Gsc, Dlx2 and Dlx3, are
down-regulated or absent in ectomesenchymal cells of branchial arches in
EndrA/ embryos
(Clouthier et al., 2000). In
addition, activity of the 208 bp dHAND branchial arch enhancer is
entirely dependent on ETA-mediated signals
(Charité et al., 2001
).
To determine whether these factors are dependent on dHAND, we examined their
expression in BAenh/ mutants by whole-mount
in situ hybridization. We chose panels of transcription factors whose
expression patterns in the branchial arches overlapped spatially or temporally
with that of dHAND, and/or that depend on ET-1/ETA mediated signals.
We first examined dHAND expression in
BAenh/ mutants, and confirmed that
dHAND was absent in the ventrolateral portions of the first and
second branchial arches, while eHAND expression was not affected in
the mutant embryos (Fig.
7A,B).
Msx1 and Msx2 are homeobox transcription factors regarded as general
repressors of transcription in developing branchial arches, and are required
for normal growth and development of branchial arch-derived structures
(Satokata et al., 2000;
Satokata and Maas, 1994
;
Takahashi et al., 2001
). Msx1
was previously reported to be down-regulated in branchial arches of
dHAND null embryos in an ET-1-independent manner
(Thomas et al., 1998
). The
expression domains of Msx1 and Msx2 are overlapping and are
detected in the ventrolateral aspects of the maxillary and mandibular arches
of wild-type embryos at E10.5 (Fig.
7C,D). Surprisingly, Msx1 was not down-regulated in the
branchial arches of the BAenh/ mutants. Most
likely, the residual expression of dHAND in the ventral portion of
branchial arches 1 and 2 is sufficient to induce expression of Msx1
in the anterior branchial arches. Msx2 expression was largely
unchanged in branchial arch 1 of BAenh/
mutants, except that the expression domain appeared to be shifted slightly
ventrally (Fig. 7D, ventral
view).
As we previously reported, Gsc is expressed in the ventral aspect of the
posterior half of the first mandibular arch and the anterior half of the
second arch at E10.5, and is severely down-regulated in
EdnrA/ embryos
(Clouthier et al., 1998).
Inactivation of Gsc in mice results in defects of most of the facial
region, suggesting its role in epithelial-mesenchymal interactions
(Yamada et al., 1995
). As
Fig. 7E shows, the Gsc
expression domain was maintained in BAenh/
mutant embryos.
There are six Dlx genes in mice; Dlx1/Dlx2, Dlx7/Dlx3 and
Dlx5/Dlx6 are organized as physically linked pairs
(Stock et al., 1996).
Dlx1 and Dlx2 are involved in development of derivatives of
the maxillary primordia (Qiu et al.,
1997
; Qiu et al.,
1995
) and Dlx5 and Dlx6 have redundant roles in
the development of the mandibular primordia
(Acampora et al., 1999
;
Depew et al., 1999
;
Robledo et al., 2002
), whereas
the roles of Dlx3 and Dlx7 in craniofacial development have
not been elucidated. Although expression of Dlx2 in the second
branchial arch and Dlx3 in the mandibular and second branchial arch
are almost undetectable in EndrA/ embryos,
we did not observe any changes in Dlx2 or Dlx3 expression in
BAenh/ mutants compared with wild-type
embryos (data not shown). As Fig. 7F and
G show, Dlx5 and Dlx6 are robustly expressed in
the ventral aspects of the mandibular and second branchial arches of wild-type
and homozygous BAenh mutants.
Alx3, a homeobox gene related to Drosophila aristaless,
is expressed in the neural crest-derived ectomesenchyme in the first and
second branchial arches (ten Berge et al.,
1998). There was no difference in the expression of Alx3
between the wild-type and the BAenh/ embryos
(data not shown).
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DISCUSSION |
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Interestingly, the craniofacial defects observed in
BAenh/ mutants are milder than those in
ET-1/ or
EndrA/ mice. The primordium of Meckel's
cartilage was observed in BAenh/ mutants at
E14.5, and all of the bones and cartilages from the mandibular and hyoid
arches were present, though truncated or malformed, indicating that
ventrolateral expression of dHAND is not required for specification
of the cell lineages that contribute to these structures. Rather, dHAND may be
involved in differentiation events in these cell lineages such as regulating
the genes required for proper condensation and differentiation of cartilages,
namely Bmp2, Bmp4 and Fgf2
(Sarkar et al., 2001).
Alternatively, maintenance of dHAND expression may be required for
continuous proliferation of mesenchymal cells or maintenance of a local
concentration of a survival factor such as FGF8
(Schneider et al., 2001
), as
suggested in the branchial arches of dHAND null embryos
(Thomas et al., 1998
).
It is interesting to note that dHAND has also been shown to regulate
patterning of zeugopods and digits of the limbs
(Charité et al., 2000).
Misexpression of dHAND in the anterior region of the limb bud results in
preaxial polydactyly and repatterning of posterior skeletal elements.
Conversely, forced expression of dHAND mutant proteins that fail to bind DNA
or activate transcription results in truncation of the zeugopods
(McFadden et al., 2002
). The
limb patterning activity of dHAND has been attributed to its ability to induce
ectopic expression of sonic hedgehog
(Charité et al., 2000
),
a morphogen that establishes anteroposterior polarity in the developing limbs
(Laufer et al., 1994
).
However, the downstream effectors of dHAND activity in zeugopod outgrowth have
not been identified. It is not unreasonable to speculate that the same sets of
effector genes might mediate the activity of dHAND in the growth of
craniofacial and limb skeletal structures.
Msx1 was previously shown to be down-regulated in branchial arch 1
of dHAND null embryos (Thomas et
al., 1998), whereas Msx-1 expression was not affected in
EndrA/ null embryos (D. E. C., unpublished
observation). In our BAenh/ homozygous
mutants, we did not observe a significant change in Msx1 expression
in the area where dHAND expression was abolished. This finding could
be explained if the remaining ventral (distal) expression of dHAND
induced a soluble factor(s) to maintain expression of Msx1 in
adjacent cells within the branchial arches. Loss-of-function and
gain-of-function of Msx2 have been reported to cause various
craniofacial disorders in humans and mice, suggesting that gene dosage of
Msx2 influences chondrogenesis and osteogenesis in vivo
(Liu et al., 1995
;
Satokata et al., 2000
;
Wilkie et al., 2000
). In the
developing mandibular process, BMP4 induces expression of Sox9 and Msx2, which
function as positive and a negative regulators of chondrogenesis, respectively
(Semba et al., 2000
). While
the possibility that dHAND is involved in BMP4 signaling in the developing
branchial arches remains to be investigated, it is interesting to note that
BMP4 is sufficient to induce dHAND expression in post-migratory
neural crest cells during terminal differentiation to become sympathetic
neurons (Howard et al.,
2000
).
A Dlx6-dependent enhancer controls dHAND expression in the
ventrolateral, but not distal, portions of branchial arches 1 and 2
Mice homozygous for the deleted enhancer failed to express dHAND
in the ventrolateral portion of branchial arches 1 and 2. However,
dHAND expression in the ventral (distal) portion was unaffected in
the mutants. Since dHAND expression is abolished throughout the
branchial arches except in the ventral most tip in ET-1 and
EndrA/ mutant mice, these results strongly
suggest the involvement of at least two distinct ETA-dependent enhancers in
the control of dHAND expression in developing branchial arches 1 and
2 in vivo.
Mesenchymal cells in the dorsal and ventral (distal) regions of the
anterior branchial arches appear to be independently specified
(Miller et al., 2000). Mosaic
analysis in zebrafish showed that ventral postmigratory neural crest cells
adopt a ventral fate when they interact with ventral paraxial mesoderm, which
expresses suc/ET-1. These ventral mesenchymal cells were
shown to express dHAND, msxE, Dlx3 and EphA3 in a
suc/ET-1 dependent manner
(Miller et al., 2000
). The
complexity of dHAND expression revealed in the present study clearly
points to the involvement of multiple spatially restricted neural crest
enhancers in the control dHAND expression. Once the distal branchial
arch enhancer for dHAND is identified, it will be interesting to
determine if it shares sequence homology with enhancers that regulate distal
arch expression of other genes such as Msx1 and eHAND.
Our results have led us to define two distinct ET-1/ETA-dependent
dHAND sub-domains in the anterior branchial arches. One is a
Dlx6-dependent ventrolateral domain controlled by a 208 bp proximal
branchial arch enhancer. This dHAND expression domain overlaps with
the ventrolateral portion of the Dlx6 expression domain, which
depends on ETA-mediated signals
(Charité et al., 2001).
The other is a ventral domain controlled by a putative distal dHAND
branchial arch enhancer(s). A recent finding that the expression of
dHAND is abolished in the branchial arches of Dlx5/Dlx6
double mutant embryos, which exhibit homeotic transformation of the lower jaw
into an upper jaw, suggests that the ventral domain of
dHAND-expressing cells is controlled by a putative enhancer that is
potentially regulated by the combination of these two genes (Beverdam, 2002;
Depew, 2002). It is plausible that the loss of both Dlx5 and
Dlx6 in mandibular arch mesenchyme may affect epithelial expression
of soluble factors critical for branchial arch development such as BMP7
(Depew, 2002) and ET-1. A third sub-domain of dHAND seems to exist in
the most ventral region of the branchial arch, which is independent of
ET-1/ETA-mediated signals (Fig.
8). Examination of dHAND expression in Dlx6 null
embryos may further define dHAND sub-domains required for development
of the anterior branchial arches.
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Multiple and parallel signaling pathways involved in craniofacial
development
Craniofacial development is a complex process regulated by a plethora of
transcription factors and signaling molecules that comprise multiple
independent signaling pathways
(Francis-West et al., 1998).
One molecule that appears to initiate or maintain one or more of these
pathways is ETA, expressed by migratory and post-migratory neural crest cells
and their derivatives. We have previously shown that the expression of at
least several transcription factors involved in facial patterning is disrupted
in EndrA/ mutant embryos. Among those
transcription factors, the interrelationship of ETA signaling and
Distalless function appears to be tightly linked. The expression of
Dlx3, Dlx5 and Dlx6 are all partially or completely
disrupted in the EndrA/ mutant embryos,
while Dlx1 and Dlx2 are largely unaffected. This suggests
that ETA signaling is required in a similar manner by linked pairs of
Dlx genes. Interestingly, disruption of Dlx5 expression in
ETA mutant embryos is only observed in the proximal domains, similar to the
pattern observed for Dlx6 (D. E. C., unpublished observation).
However, Dlx5 and Dlx6 are likely to be involved in
different signaling pathways in facial morphogenesis, as Dlx5 induces
Gsc expression, while Dlx6 induces dHAND expression. Mice
homozygous for both Dlx5 and Dlx6 develop craniofacial and
ear defects, including the failure of Meckel's cartilage, mandible, and
calvaria formation, all of which are more severe than those observed in
Dlx5 mutant mice or EndrA/ mutant
mice (Robledo et al., 2002
).
These findings clearly illustrate the similar but non- redundant roles of
Dlx genes in facial morphogenesis.
dHAND and its branchial arch enhancer as potential targets
for mutations in cleft palate syndromes
Cleft palate has a multifactorial etiology and has been associated with
abnormal expression of numerous signaling molecules and transcription factors
(Ferguson, 1994). Palate
formation involves a complex series of steps that include growth of the
palatal shelves, palatal elevation and fusion, and disappearance of the
midline epithelial seam (Ferguson,
1988
). The role of dHAND in the formation of palatine shelves may
not be mediated by a direct effect on palatine bone formation. Rather, dHAND
may be required for the elevation of the palatine shelves by sustaining
concurrent growth of the mandibular bone, as dHAND-positive cells do
not populate the palatine bones as judged from lineage analysis using
dHAND-Cre; R26R indicator mice (Clouthier et al.,
unpublished observation). This hypothesis is supported by the observation that
a relationship between growth retardation of Meckel's cartilage coupled with
relative macroglossia and malformation of the secondary palate is a critical
determinant in the development of cleft palate in mice homozygous for a
semi-dominant Col2a1 mutation (Ricks, 2002).
The T-box gene TBX 22 has been reported to be responsible for CPX
(X-linked cleft palate and tongue-tie) syndrome
(Braybrook et al., 2001), and a
significant linkage-disequilibrium has been found between non-syndromatic
cleft lip with or without cleft palate (CL/P) and the Msx1 and
TGFß3 genes, and between Cleft Palate Only (CPO) and
Msx1 (Lidral et al.,
1998
). In addition, craniofacial defects often accompany
congenital heart defects as seen in the DiGeorge and Holt-Oram syndromes
(Murray, 2001
). The expression
of dHAND during craniofacial as well as cardiovascular development
suggests that mutations in the 5' regulatory region of the
dHAND gene could also be associated with cleft palate.
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ACKNOWLEDGMENTS |
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