1 Department of Molecular, Cellular and Craniofacial Biology and the Birth
Defects Center, University of Louisville, Louisville, KY 40292, USA
2 Department of Cell and Developmental Biology, University of Michigan Medical
School, Ann Arbor, MI 48109, USA
3 UMR5166 CNRS/MNHN Evolution des Régulations Endocriniennes, 7 rue
Cuvier, 75005 Paris, France
* Author for correspondence (e-mail: clouthier{at}louisville.edu)
Accepted 7 June 2004
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SUMMARY |
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Key words: Mouse, Bone, Mandible, Patterning, Neural crest cell, Homeobox gene
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Introduction |
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Although the development of more caudal NC cell populations is partially
regulated by Hox genes (Hunt et al.,
1991; Prince and Lumsden,
1994
), CNC cells within the first mandibular arch do not express
Hox genes, a crucial aspect for proper first arch patterning
(Couly et al., 1998
;
Creuzet et al., 2002
).
Mandibular arch CNC cells were initially believed to carry programming
information with them from the midbrain/hindbrain region
(Noden, 1983
). However, it
currently appears that environment signals provide patterning information to
CNC cells. These signals may begin very early in development, as foregut
endoderm in the chick is crucial for proper patterning of mandibular arch
derivatives, including both size and polarity of structures along the
embryonic axis (Couly et al.,
2002
). Similarly, ventral cartilage development (including
Meckel's cartilage) is disrupted in the zebrafish cas mutant, which
contain very little endoderm (David et
al., 2002
). One potential mediator of endoderm signaling appears
to be fgf3, though numerous other molecules are probably involved. In
addition to this early requirement, various other secreted molecules from the
surrounding ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm can
influence CNC cell development once the CNC cells arrive in the mandibular
arch (Cobourne and Sharpe,
2003
; Graham and Smith,
2001
; Jernvall and Thesleff,
2000
; Schilling and Kimmel,
1997
; Trainor and Krumlauf,
2000
).
One factor involved in CNC cell development is endothelin 1 (Edn1), a 21
amino acid peptide secreted by pharyngeal arch ectoderm, core paraxial
mesoderm and pharyngeal pouch endoderm
(Clouthier et al., 1998;
Maemura et al., 1996
;
Yanagisawa et al., 1998b
).
Edn1 binds to the endothelin A receptor (Ednra) found on cephalic and cardiac
NC cells. Targeted inactivation of Edn1
(Kurihara et al., 1994
),
endothelin converting enzyme 1 (Ece1; the enzyme that cleaves Edn1
from an inactive to active peptide)
(Yanagisawa et al., 1998a
) or
Ednra (Clouthier et al.,
1998
) in the mouse results in severe craniofacial and
cardiovascular defects. This is due in part to aberrant expression of genes
involved in post-migratory NC cell development
(Clouthier et al., 1998
;
Clouthier et al., 2000
;
Ivey et al., 2003
;
Thomas et al., 1998
). Ednra
signaling during CNC cell development appears conserved among vertebrates, as
pharmacological antagonism of Ednra in the rat
(Spence et al., 1999
) or chick
(Kempf et al., 1998
) results
in similar craniofacial defects as those observed in
Ednra/ mice. Similarly, an edn1
mutation in zebrafish, termed sucker or suc/et1, results in
disruption of most cartilages of the ventral (distal) jaw
(Kimmel et al., 2003
;
Miller and Kimmel, 2001
;
Miller et al., 2000
).
The distal-less homeobox gene family member Dlx6 is a downstream
effector of Ednra signaling in the mouse
(Charité et al., 2001),
which in turn induces expression of the bHLH transcription factor dHAND/Hand2
(Charité et al., 2001
;
Yanagisawa et al., 2003
). Not
surprisingly, Hand2 is one of several mandibular arch genes whose expression
is disrupted in Dlx5/Dlx6/ mouse embryos
(Beverdam et al., 2002
;
Depew et al., 2002
). In
addition, maxillary first arch gene expression expands into the mandibular
arch. In term Dlx5/Dlx6/ embryos, most
mandibular arch-derived bone and cartilage are missing, instead replaced with
structures that appear to be mirror image duplications of maxillary
structures. These findings suggest that Dlx5 and Dlx6 provide a `mandibular
identity' to the mandibular arch NCCs.
As Dlx6 is a downstream effector of Ednra signaling, we have re-examined the development of the lower jaw in Ednra/ embryos and followed the fate of specific populations of mandibular mesenchymal cells during this developmental process. We find that most structures of the lower jaw undergo a homeotic transformation into maxillary-like structures, with these changes reflected in earlier disruption of mandibular arch gene expression. However, normal gene expression is partially maintained in a distal mandibular arch domain that appears to be later involved in lower incisor development. This suggest that although Ednra signaling is crucial for patterning most of the CNC-derived mesenchyme and surrounding epithelium of the mandibular arch by initiating a Dlx/Hand2 gene expression pathway, a region of the distal arch appears to be patterned by Ednra-independent mechanisms.
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Materials and methods |
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Skeletal analysis
To analyze bone and cartilage development, E18.5 embryos were stained as
previously described (McLeod,
1980). Briefly, E18.5 embryos were collected, skinned and
eviscerated. The skeletons were then fixed in 95% ethanol for 3 days followed
by 100% acetone for 2 days. Embryos were then stained for 5 days in 0.015%
Alcian Blue (stock solution: 0.3% in 70% ethanol) and 0.005% Alizarin Red
(stock solution: 0.1% in 95% ethanol) in 70% ethanol/5% glacial acetic acid at
37°C with periodic rotation. After staining, embryos were cleared in 1%
potassium hydroxide and successive immersions of 1% potassium hydroxide in
20%, 50% and 80% glycerol. Skeletons were photographed with an Olympus DP11
digital camera mounted on an Olympus SZX12 stereomicroscope.
ß-Galactosidase staining
To examine ß-gal staining in whole embryos, E10.5 and E16.5
Ednra+/+;R26R; Hand2-Cre and
Ednra/;R26R;Hand2-Cre embryos were
collected and fixed for 1 hour in 4% paraformaldehyde. Embryo staining and
photography was performed as previously described
(Ruest et al., 2003). Stained
E16.5 embryos were cleared for 1.5 to 2 hours in benzyl benzoate:benzyl
alcohol (1:2) with rotational mixing and then photographed.
To analyze ß-gal staining in embryo sections, E16.5
Ednra+/+;R26R;Hand2-Cre and
Ednra/;R26R;Hand2-Cre embryos were
collected, snap-frozen in OCT freezing media in a dry ice/ethanol bath,
sectioned and stained as previously described
(Ruest et al., 2003). Sections
were counterstained with nuclear Fast Red and coverslipped in DPX mounting
media (BDH).
In situ hybridization analysis
Gene expression in whole mount was analyzed using digoxigenin-labeled RNA
riboprobes against Bmp4 (Furuta
and Hogan, 1998), Dlx1
(McGuinness et al., 1995
),
Dlx2 (Robinson and Mahon,
1994
), Dlx5 (Liu et
al., 1997
), Dlx6
(Charité et al., 2001
),
Hand2 (Srivastava et al.,
1997
), Gata3 (George
et al., 1994
), Msx1
(Thomas et al., 1998
),
Twist (Chen and Behringer,
1995
) and Wnt5a
(Yamaguchi et al., 2000
) as
previously described (Clouthier et al.,
1998
). For sectional in situ hybridization analysis, E16.5
Ednra+/+ and Ednra/
embryos were embedded in OCT, sectioned at 14 µm onto plus-coated slides
and hybridized at 65°C with a digoxigenin-labeled RNA riboprobes against
Hand2. After color development, slides were dehydrated, coverslipped
and photographed. For all in situ hybridization analyses, a minimum of three
embryos of each genotype were examined per probe.
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Results |
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|
|
On ventral view, the bone in the lower jaw in
Ednra/ embryos appeared to be a mirror image
duplication of the maxilla [Fig.
1F,H; termed pseudo-maxilla (mx*)], although it was significantly
smaller than the real maxilla (Fig.
1E,G). Similar to the pseudo-maxilla observed in
Dlx5/Dlx6/ embryos
(Beverdam et al., 2002;
Depew et al., 2002
), this
pseudo-maxilla contained foramina and a second set of palatine bones (pt*;
Fig. 1F,H,J). These bones
projected towards each other and elevated as though forming palatal shelves
(Fig. 1J), similar to those
observed in wild-type embryos (Fig.
1I), though some variation in the extent of apposition was
observed among mutant embryos. As observed for other bones, both the original
and pseudo-palatines bones in Ednra/ embryos
were smaller than the palatine bones of wild-type embryos, unlike relatively
normal sized structures and pseudo-structures found in
Dlx5/Dlx6/ embryos. These pseudo-palatine
bones were fused with other aberrant membranous bones that appeared to be
duplications of the pterygoid bones (data not shown). Depew et al.
(Depew et al., 2002
) have
hypothesized that these ectopic bones arise from mesenchyme that normally
forms the tympanic and gonial bones, structures also absent in
Ednra/ embryos
(Fig. 1D), though we will limit
our further analysis to more distal facial structures. Likewise, although we
will not discuss defects in second arch-derived elements (such as the hyoid),
these structures appear to undergo changes in shape and/or size rather than to
undergo any homeotic change (Fig.
1D and data not shown)
In the distal mandible, incisors were present in most mutant embryos
examined but were set only in a small amount of alveolar bone and residual
cartilage (Fig. 1D,H,J; insets
in Fig. 1D,F). The body of
Meckel's cartilage was absent, unlike the apparent transformation of Meckel's
cartilage into a pseudo-lamina obturans observed in
Dlx5/Dlx6/ embryos
(Beverdam et al., 2002;
Depew et al., 2002
).
Furthermore, the rostral process of Meckel's cartilage was hypoplastic at
E14.5 (data not shown) and E18.5 (Fig.
1D,H,J; insets in Fig.
1D,F), in contrast to more extensive cartilage found in
Dlx5/Dlx6/ embryos, suggesting a loss of
precursor cells in the absence of Ednra signaling.
Duplication of the alisphenoid bones was also observed in Ednra/ embryos. The ala temporalis region of the alisphenoid, consisting of two cartilage processes that normally fuse to the lamina obturans (Fig. 1K, lower structure), was composed of four processes in Ednra/ embryos (Fig. 1K, upper structure). The extra processes attached to a structure whose shape suggested it was a duplicated lamina obturans (lo*) (Fig. 1L, upper structure), though this structure was smaller than the normal lamina obturans. Taken together, it appears that most structures derived from the mandibular (distal) arch appeared to have undergone a transformation into maxillary (proximal) structures.
Gene expression boundaries in the mandibular arch
Ednra signaling is crucial for proper expression of transcription factors
involved in mandibular arch development
(Clouthier et al., 1998;
Clouthier et al., 2000
;
Park et al., 2004
;
Thomas et al., 1998
). Similar
changes are observed in suc/et1 zebrafish mutants, along with
expansion of more dorsal (proximal) first arch gene expression into the
ventral (distal) arch, suggesting a loss of boundary identity
(Miller et al., 2003
). As
similar boundary changes are observed in
Dlx5/Dlx6/ mouse embryos
(Beverdam et al., 2002
;
Depew et al., 2002
), we
examined the expression of multiple genes expressed in different regions of
the first pharyngeal arch in both wild-type and
Ednra/ embryos.
Wnt5a expression, observed in the proximal portion of the
mandibular arch of E10.5 wild-type embryos
(Fig. 2A), spread over the
caudal half of the mandibular arch in Ednra/
embryos (arrows in Fig. 2B).
Similarly, Dlx1 expression in
Ednra/ embryos extended further distally
along the rostral half of the mandibular arch compared with wild-type embryos
(compare yellow lines in Fig.
2C,D). Expression changes were also observed for Msx1 and
Twist, two transcription factors involved in multiple aspects of
lower jaw development (Chen and Behringer,
1995; Han et al.,
2003
; Satokata and Maas,
1994
; Soo et al.,
2002
). In E10.5 wild-type mouse embryos, Msx1 expression
covered the distal half of the mandibular arch, while in
Ednra/ embryos, expression appeared to
slightly expand more proximally (Fig.
2E,F). Likewise, Twist expression expanded distally in
Ednra/ embryos compared with the pattern
observed in wild-type embryos (broken yellow lines in
Fig. 2G,H).
|
Hand2 expression in Ednra/ embryos
As Hand2 expression is downregulated in both
Ednra/ and
Dlx5/Dlx6/ embryos
(Beverdam et al., 2002;
Clouthier et al., 2000
;
Depew et al., 2002
) and both
embryos show homeotic changes in lower jaw structures, we closely examined
expression of Hand2, Dlx5 and Dlx6 in the developing
mandible of Ednra/ embryos. In contrast to
the arch expression in wild-type embryos
(Fig. 2M,O), Dlx5 and
Dlx6 expression in Ednra/ embryos
was downregulated throughout the mandibular arch mesenchyme
(Fig. 2N,P). As we have
previously shown (Clouthier et al.,
2000
), Hand2 expression was also absent in the mandibular
arch of Ednra/ embryos
(Fig. 2R), although a small
Hand2 expression domain could be detected within the distocaudal arch
(yellow arrows in Fig. 2R).
This domain, also present in Edn1/ embryos
(Thomas et al., 1998
),
correlated with the domain in which neither Dlx5 nor Dlx6 is
expressed (Fig. 2M,O). Sections
through this distal region in wild-type embryos illustrated that
Hand2 expression was confined to the mesenchyme, whereas in
Ednra/ embryos, Hand2 expression
was observed in both the mesenchyme and overlying epithelium (data not
shown).
To further examine this distal Hand2 domain, we took advantage of
a two-component genetic system that allows us to examine both active and fated
expression of Hand2 in the mandibular arch and its derivatives
(Ruest et al., 2003). This
system consists of a pharyngeal arch-specific Hand2 enhancer fused to a
Cre cDNA. When Hand2-Cre mice (previously referred to as
dHAND-Cre) are crossed with R26R mice
(Soriano, 1999
),
ß-galactosidase (ß-gal) activity is observed in all cells in which
Hand2 is or was expressed. In E10.5
Ednra+/+;R26R;Hand2-Cre embryos, ß-gal
staining was observed throughout most of the mandibular arch
(Fig. 3A,C). Analysis of both
sagittal (Fig. 3E) and frontal
(Fig. 3G) sections through the
mandibular arch confirmed that labeled cells were confined to the mesenchyme.
In Ednra/; R26R;Hand2-Cre embryos,
ß-gal stained cells were only observed in the distocaudal arch
(Fig. 3D). In sections through
this region, scattered labeled cells were observed in the arch mesenchyme,
with a higher contribution in the epithelium
(Fig. 3F,H).
|
GATA3 and distal Dlx5/Hand2 expression
The Hand2 enhancer driving Cre expression in
Hand2-Cre transgenic mice is the only mandibular arch-specific
cis-regulatory element thus far identified for Hand2
(Charité et al., 2001;
McFadden et al., 2000
).
Although we have previously shown that this enhancer is regulated in part by
Dlx6 (Charité et al.,
2001
), our current findings indicate that other factors may
function in combination with Dlx6 to direct distal Hand2 expression.
We therefore examined the sequence of the arch enhancer using MatInspector, a
transcription factor binding site analysis program developed by Genomatix
(www.genomatix.de).
One site identified within the enhancer was the consensus-binding site for
GATA3 (nngaGATAanann), with an overall similarity of 0.831 (actual sequence:
aggaGATCagaga, with the underlined base pairs showing
the highest conservation in mathematical models) (data not shown). GATA3 is a
member of the GATA family of zinc-finger transcription factors
(George et al., 1994
;
Massari and Murre, 2000
).
Targeted inactivation of mouse Gata3 results in embryonic lethality
by E11.0 in part because of noradrenalin deficiency, although this lethality
can be rescued by feeding pregnant female mice a high catechol diet
(Lim et al., 2000
) (K.-C. Lim,
unpublished). At E16.5, rescued mutant embryos show hypoplasia of the
mandible, tongue and tooth primordia (Lim
et al., 2000
), suggesting a function for GATA3 in distal
mandibular arch development. We therefore examined Hand2 expression
in diet-rescued E10.5 Gata3/ embryos.
Although these embryos were found to have hypoplastic pharyngeal arches,
suggesting cell death or decreased proliferation, Hand2 expression
was still observed in the mandibular and second arches. However, this
expression was confined to the rostral half of each arch
(Fig. 4B,D), but the extent of
confinement was variable (data not shown). Although this could imply a direct
function for GATA3 in Hand2 expression, we also examined
Dlx5 expression in Gata3/ embryos,
as GATA3 could indirectly regulate Hand2 expression through Dlx5.
Similar to Hand2 expression, Dlx5 expression was also
downregulated in the caudal half of the mandibular arch of
Gata3/ embryos examined
(Fig. 4F,H).
|
Fate of Hand2 daughter cells in the mandibular arch cells in Ednra/ embryos
Hand2 expression in the distal mandibular arch in the absence of
Ednra signaling suggests that Hand2 may have an Ednra-independent role in
lower jaw development. To investigate this aspect, we again took advantage of
the R26R;Hand2-Cre mice to follow the fate of these distal cells. In
E16.5 Ednra+/+;R26R;Hand2-Cre embryos, the entire
lower jaw was composed of labeled cells
(Fig. 5A,C,E). By contrast,
labeled cells within the lower jaw of
Ednra/;R26R;Hand2-Cre embryos were
primarily observed in the cleft between the two poorly fused halves
(Fig. 5B,D), with labeled cells
also observed in the hypoplastic tongue and lower incisors
(Fig. 5F).
To better examine the spatial distribution of cells, frozen sections of littermate embryos were stained for ß-gal activity. In Ednra+/+;R26R;Hand2-Cre embryos, stained cells were observed throughout the lower jaw, including in the mandible, Meckel's cartilage and surrounding connective tissue (Fig. 5G; data not shown). Labeled cells were also present in the tongue and lower incisor dental pulp but were not observed in the dental lamina (asterisk in inset, Fig. 5G). In Ednra/;R26R;Hand2-Cre embryos, labeled cells were devoid from most of the lower jaw, although were present in the small amount of bone and cartilage that remained under the incisors (Fig. 5H). Labeled cells were also observed in the area of odontoblast formation (black arrows in inset), relatively evenly spaced with groups of unlabeled cells. Labeled cells were scattered in the remainder of the dental pulp, with the overall contribution lower than that observed in wild-type embryos. The dental lamina epithelium also contained scattered labeled cells (asterisk in inset, Fig. 5H), suggesting that the Hand2-Cre transgene was expressed at some point in oral epithelium of Ednra/ embryos.
Compared with ß-gal staining, endogenous Hand2 expression in E16.5 wild-type embryos was only observed in the odontoblast region and the mandibular bone. Hand2 expression in Ednra/ embryos was most prominent in the odontoblast region and shaft of the vibrissae (Fig. 5I). Expression was also present in the residual bone and cartilage (data not shown). Expression was not observed in the dental lamina of either embryo, indicating that the ß-gal cells observed in Ednra/;R26R;Hand2-Cre embryos was due to earlier mis-regulation of the endogenous gene and/or transgene. Furthermore, we did not observe either ß-gal staining or endogenous Hand2 expression in the upper incisors in either embryo. These findings illustrate that normal mandibular arch gene expression is partially maintained in the lower incisor region of Ednra/ embryos.
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Discussion |
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Ednra signaling may be additionally required for proliferation or survival
of CNC cells, as most duplicated structures appear smaller than their
maxillary counterparts and are smaller than those observed in
Dlx5/Dlx6/ embryos
(Beverdam et al., 2002;
Depew et al., 2002
). This
could reflect a loss of precursor cells, as we have previously shown that loss
of Ednra signaling causes both a decrease in proliferation and a fourfold
increase in apoptosis of mandibular arch ectomesenchyme
(Clouthier et al., 2000
).
However, it is also possible that Ednra signaling is required for later
osteogenic events, because in a mouse model of osteoblastic bone metastasis
using breast cancer cells lines, antagonism of Ednra receptors decreased
osteoblastic metastases (Yin et al.,
2003
). Analysis of Ednra function during bone development,
potentially using a conditional knockout of the Ednra gene
(Kedzierski et al., 2003
),
will be required to address this issue.
Aberrant gene expression and lower jaw transformation
Why loss of Ednra signaling leads to a homeotic transformation is not
known. However, our analysis of gene expression suggests that loss of Bmp4 may
be crucial for changes in arch development. Ectopic Bmp4 can induce epithelial
Dlx2 expression in mandibular explant cultures, while introduction of
ectopic noggin inhibits this expression
(Thomas et al., 2000). Loss of
Bmp4 in Ednra/ embryos could thus explain
why Dlx2 epithelial expression is lost. Furthermore, distal expansion
of the Dlx2 mesenchymal domain could also aberrantly affect arch
development, as Dlx2 can form heterodimers with Msx1
(Zhang et al., 1997
). Because
Msx1 appears to promote proliferation of CNC cells
(Han et al., 2003
), increased
Dlx2 in the distal arch could increase heterodimer formation, resulting in
decreased CNC cell proliferation and aberrant differentiation. It is not clear
why Dlx2 expression decreases in cultured mandibular arches treated
with the non-peptidic dual Ednra/Ednrb antagonist bosentan
(Park et al., 2004
). Although
Ednrb/ mice do not have facial defects at
birth (Hosoda et al., 1994
),
perhaps maintenance of mesenchymal Dlx2 expression requires
Edn1-mediated signaling from either Ednra or Ednrb, with the blockage of both
disrupting expression.
Continued Msx1 expression in the remainder of the mandibular arch
is somewhat surprising, considering that Msx1 expression is lost in
Hand2/ embryos
(Thomas et al., 1998). This
finding led to the hypothesis that Msx1 was downstream of an Edn1/Hand2
pathway (discussed below). However, more recent studies have illustrated that
Msx1 expression is normal in Edn1/
embryos (Ivey et al., 2003
).
Our results here also indicate that Msx1 expression is not dependent
on Ednra signaling. It is possible that the absence of Msx1 observed
in Hand2/ embryos could be due to apoptosis
of specific Msx1-expressing arch mesenchyme cells, as cell death is
widely observed in the first arch of Hand2/
embryos (Thomas et al., 1998
).
Alternatively, normal Msx1 expression may only require distal
Hand2 expression, hence explaining Msx1 expression in
Edn1/ and
Ednra/ embryos. Proof of this awaits
analysis of gene expression in Hand2 chimeric or conditional knockout
mice.
Hand2 as a prominent effector of Ednra signaling in the pharyngeal arches
Comparative analysis of developmental signaling pathways in multiple
species can point to common crucial mediators. One gene that lies downstream
of Edn1/Ednra signaling in the pharyngeal arches in both mouse and zebrafish
is the bHLH molecule Hand2. In the hand2 zebrafish mutant hands
off (han), most ventral (distal) arch cartilage is missing
(Miller et al., 2003). Loss of
hand2 disrupts ventral gene expression, though more narrowly than
observed in suc/et1 mutants. Furthermore, Hand2 appears to cooperate
with Edn1 in establishing domains in the first arch that demarcate both the
ventral arch and the joint region separating the upper and lower jaws.
Hand2/ mouse embryos die from vascular
failure by E10.5, preventing analysis of craniofacial bone/cartilage formation
(Srivastava et al., 1997
;
Thomas et al., 1998
;
Yamagishi et al., 2000
).
However, misexpression of Hand2 in the chick limb bud results in
digit duplication and polydactyly
(Charité et al., 2000
;
Fernandez-Teran et al., 2000
;
McFadden et al., 2002
),
suggesting that the level of Hand2 (and the types of bHLH dimers they form)
may be crucial for specifying the identity of cell populations or establishing
gene expression boundaries within tissues
(Firulli, 2003
). Perhaps the
aberrant ectodermal Hand2 expression observed in
Ednra/ embryos is another example of loss of
expression boundaries within the arch. It is intriguing that the expression
domain of the bHLH molecule Twist expands into the distal arch of
Ednra/ embryos, as Twist can form
heterodimers with Hand2 (Firulli et al.,
2003
) and is required for expression of multiple transcription
factors involved in mandibular arch development
(Soo et al., 2002
); (see also
Fig. 6A). Determining how Hand2
might establish expression boundaries and the identity of its prospective
partners in this process will require a better understanding of the
biochemistry of Hand2 dimer formation in the pharyngeal arches.
Ednra independent regulation of Hand2 expression
Our results demonstrate that multiple mechanisms, potentially including
GATA3 (see below), regulate distal Hand2 expression in the absence of
Ednra signaling (Fig. 6B).
However, the limited number of Hand2 daughter cells in the mandibular arch of
Ednra/ embryos suggests that Ednra-dependent
and -independent mechanisms probably collaborate to fully induce Hand2
expression in wild-type embryos. Although Hand2 expression is also
lost in the pharyngeal arches of suc/et1 zebrafish, a
cluster of Hand2-positive cells remains in proximal arch one, roughly
corresponding to distocaudal arch one in the mouse
(Miller et al., 2003). This
indicates that Edn1/Ednra-dependent and -independent mechanisms regulating
Hand2 expression in the distal arch may be conserved between mouse
and zebrafish. In addition, Hand2 expression is observed in a small
domain in the second arch of Dlx5/Dlx6/
embryos, resembling a domain observed in suc/et1 zebrafish
(Miller et al., 2000
). The
absence of a second arch Hand2 domain in
Ednra/ embryos could indicate either that
these cells are lost in the absence of Ednra signaling or that regulation of
Hand2 expression has become more complex with evolution.
One potential regulator of distal Hand2 expression may be GATA3.
We have shown that loss of GATA3 partially disrupts Hand2 expression
in the caudal arch. Regulation of Hand2 function through GATA factors has been
previously described (McFadden et al.,
2000), suggesting this may be a common mechanism for regulating
Hand2 function. However, understanding regulation of gene expression based
solely on expression patterns can be difficult. Even though expression of both
Hand2 and Gata3 overlaps in the distal arch, Hand2
expression is lost only along the caudal half of the mandibular arch in
Gata3/ embryos. In addition, the loss occurs
in both distal and proximal regions of the Hand2 domain, even though
Gata3 expression is confined to the distal domain. It is clear that
multiple factors are involved in regulating these genes, with our results
simply providing an entry point into understanding these hierarchical
pathways. Defining the exact role of GATA3 in Ednra-dependent and independent
Hand2-mediated developmental processes, including odontogenesis, will require
a more thorough understanding of both the molecular and cellular changes
within the mandibular arch of Gata3 mutant embryos and the
relationship between GATA3 and Ednra (Lim
et al., 2000
). Furthermore, it will be important to determine if
other GATA factors are involved in regulating Hand2 or Dlx5
expression in the rostral arch, as GATA regulation of Hand2 is observed in
other developmental paradigms (McFadden et
al., 2000
). GATA2 can also bind to a core GATC consensus sequence
(as found in the Hand2 enhancer) (Ko and
Engel, 1993
), suggesting it as a potential candidate.
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ACKNOWLEDGMENTS |
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Footnotes |
---|
While this manuscript was in review, Ozeki et al.
(Ozeki et al., 2004) reported
similar homeotic changes in mandibular arch structures in
Edn1/ embryos.
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