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First published online 16 January 2008
doi: 10.1242/dev.012708
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1 Department of Physiological Chemistry and Metabolism, Graduate School of
Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan.
2 Tsukuba Safety Assessment Laboratories, Banyu Pharmaceutical Company Limited,
3 Okubo, Tsukuba, Ibaraki 300-2611, Japan.
3 Department of Developmental Medical Technology (Sankyo), Graduate School of
Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan.
4 Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366,
69120, Heidelberg, Germany.
* Author for correspondence (e-mail: kuri-tky{at}umin.ac.jp)
Accepted 15 November 2007
 |
SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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q/G11-deficient
mice, indicate that the dorsoventral axis patterning of pharyngeal arches is
regulated by the Ednra-selective, Gq/G11-dependent
signaling, while the formation of the distal pharyngeal region is under the
control of a Gq/G11-independent signaling, which can be
substituted by Ednrb. This RMCE-mediated knock-in system can serve as a useful
tool for studies on gene functions in craniofacial development.
Key words: Endothelin, G protein-coupled receptor, Pharyngeal arch, Neural Crest, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Le Douarin and Kalcheim, 1999;
Noden and Trainor, 2005). The
pharyngeal arches, the segmental structures of the embryonic head covered by
ectoderm- and endoderm-derived epithelium, are populated with neural crest
cells from the hindbrain and caudal midbrain, where they surround a mesodermal
core. Within each pharyngeal arch, complex cellular interactions are thought
to determine their regional identity and cell fates
(Le Douarin and Kalcheim,
1999; Noden and Trainor,
2005).
In the two anterior pharyngeal arches, the first and second arches,
dorsoventral axis patterning predominates the appropriate formation of
chondrocranial elements and associated dermal bones
(Kontges and Lumsden, 1996;
Le Douarin and Kalcheim,
1999). The first pharyngeal arch is subdivided along the
dorsoventral axis into the maxillary and mandibular arches, which give rise to
the upper and lower jaws, respectively. Recently, molecules involved in the
determination of their identities have been explored and the Dlx
genes, vertebrate Distal-less homologs, have been thought to play a
key role (Depew et al., 2005;
Merlo et al., 2000). Among the
six known Dlx genes, Dlx5 and Dlx6 are expressed in
the ventral part within the anterior pharyngeal arches
(Depew et al., 2005;
Merlo et al., 2000).
Dlx5/Dlx6 double null-mutant mice demonstrate homeotic
transformation of the lower jaw into an upper jaw, indicating that
Dlx5 and Dlx6 are major determinants of the mandibular
identity (Beverdam et al.,
2002; Depew et al.,
2002).
The endothelin (Edn) system, composed of three peptide ligands (Edn1, Edn2
and Edn3) and their two G-protein-coupled receptors [endothelin type A
receptor (Ednra) and type B receptor (Ednrb)], is involved in diverse
biological events (Kedzierski and
Yanagisawa, 2001; Kurihara et
al., 1999; Masaki,
2004). These receptors activate an overlapping set of G proteins
(e.g. Gq/G11), leading to various intracellular
responses such as activation of phospholipase C, increase in intracellular
calcium and induction of early responsive genes
(Kedzierski and Yanagisawa,
2001). During embryogenesis, the Edn1-Ednra axis regulates
craniofacial and cardiovascular morphogenesis, whereas the Edn3-Ednrb axis
contributes to melanocyte and enteric neuron development
(Kedzierski and Yanagisawa,
2001; Kurihara et al.,
1999; Masaki,
2004).
In craniofacial development, Edn1 is expressed in the epithelium
and mesodermal core of the pharyngeal arches, whereas Ednra is in
neural crest-derived ectomesenchyme
(Clouthier et al., 1998;
Kurihara et al., 1995;
Kurihara et al., 1994;
Maemura et al., 1996). Defects
in the Edn1/Ednra pathway results in the malformation of
pharyngeal-arch-derived craniofacial structures in mice
(Clouthier et al., 1998;
Kurihara et al., 1995;
Kurihara et al., 1994), rats
(Spence et al., 1999), birds
(Kempf et al., 1998) and fish
(Kimmel et al., 2003;
Miller et al., 2000). Homeotic
transformation of the mandibular arch with downregulation of
Dlx5/Dlx6 in Edn1-null embryos implicates the Edn1/Ednra
pathway in the dorsoventral axis patterning of the pharyngeal arch system as a
positive regulator of Dlx5/Dlx6 expression
(Kurihara et al., 1994;
Ozeki et al., 2004;
Ruest et al., 2004). Although
the Gq/G11-mediated signaling pathway has been suggested
to be involved in pharyngeal arch development
(Dettlaff-Swiercz et al.,
2005; Ivey et al.,
2003; Offermanns et al.,
1998), the intracellular signaling pathway coupling the Edn1-Ednra
system to the induction of Dlx5/Dlx6 remains unknown.
To investigate the intracellular signaling mechanism involving the
Edn1/Ednra pathway in craniofacial development, we choose a knock-in strategy
with a recombinase-mediated cassette exchange (RMCE) using the
Cre-lox system. This method enables an efficient exchange of a
chromosomal region flanked by incompatible mutant lox sequences for a
cassette located on a plasmid, and is highly advantageous in that it allows
the repetitive use of the same embryonic stem (ES) cell line to insert various
genes of interest into the identical recombinant allele
(Sorrell and Kolb, 2005). Here
we have established an Ednra knock-in system using RMCE. Knock-in of
the lacZ gene resulted in the visualization of
Ednra-expressing cells. Furthermore, knock-in of Ednra and
Ednrb revealed Ednra-selective and non-selective signaling pathways
operating in distinct regions. Ednrb knock-in mice, which
demonstrated homeotic transformation of the lower jaw into an upper jaw but
had relatively well-developed incisive alveolar bone and hyoid, resembled
neural-crest-specific Gq/G11-deficient
mice. Together with differences in distal pharyngeal-arch-derived structures
between these mice and Ednra-null mice, these results indicate that
Gq/G11-dependent and -independent Edn1/Ednra pathways,
which may correspond to Ednra-selective and non-selective signaling,
respectively, are used in different contexts during pharyngeal arch
development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The RMCE-mediated knock-in vector was made in a p66-2272 plasmid
(Araki et al., 2002). The
PGK-puromycin resistance gene cassette (Puro) was flanked by
Flp recombinase target (FRT) sequences
(Schlake and Bode, 1994) and
placed between lox66 and lox2272 with multiple cloning
sites. For the knock-in of lacZ, the lacZ gene with an SV40
large T antigen-derived nuclear localization signal was introduced into the
multicloning site placed between lox66 and the FRT-flanked
Puro using appropriate restriction enzymes. For the knock-in of
Ednra and Ednrb, PCR-amplified fragments encoding the open
reading frame of mouse Ednra and Ednrb cDNA were introduced
into the knock-in vector in the same way.
Homologous recombination and RMCE in ES cells
The targeting vector was linearized and electroporated into B6129F1-derived
ES cell line ATOM1 (Amano et al., unpublished). Clones surviving
positive-negative selection with neomycin and FIAU were screened for
homologous recombination with genomic PCR using diagnostic primers. Correct
recombination was confirmed by Southern blotting using the probes indicated in
Fig. 1. For RMCE, the ES cell
line in which the initial recombination was successfully achieved was used
repeatedly. ES cells were infected with AxCANCre, recombinant adenovirus
expressing the recombinase Cre tagged with a nuclear localization signal under
the control of the CAG promoter (Kanegae
et al., 1995). Forty-eight hours later, ES cells were
electroporated with the knock-in vector and seeded onto multidrug-resistant
embryonic fibroblasts derived from the DR4 mouse strain
(Tucker et al., 1997). Clones
selected with 1 µg/ml puromycin were picked up and genotyped by PCR to
identify RMCE-mediated recombination.
Mutant mice
Targeted ES clones were injected into ICR blastocysts to generate germline
chimeras. For excision of the FRT-flanked Puro, the Flp
recombinase-expression plasmid pCAGGS-FLPe (Gene Bridges, Dresden, Germany)
was injected into the male pronuclei of fertilized eggs having the knocked-in
allele. Sequential recombination events were verified by PCR with specific
primers, the sequences of which are available on request. Mutant mice were
intercrossed with ICR mice and F2 to F5 offspring were subjected to analysis.
P0-Cre-/+;G
flox/floxq;G
-/-11 mice were described previously
(Dettlaff-Swiercz et al.,
2005). All the animal experiments were performed in accordance
with the guidelines of the University of Tokyo Animal Care and Use
Committee.
RT-PCR
Expression of knocked-in genes was confirmed by RT-PCR. Primers p
(5'-CTGATCCACCGGACCATCGCTGGA-3') and q
(5'-TACCGTTCGTATAATGTATGCTATACGAACGGTA-3') were designed to detect
transcripts from both Ednra and Ednrb knocked-in alleles as
a 284 bp band. The combination of primers p and r
(5'-TCAATGACCACGTAGATAAGGT-3') was designed to detect both
endogenous and knocked-in Ednra transcripts as 753- and 659 bp bands,
respectively. Primer s (5'-GAGAAGTGACAGCGTGGCTT-3'), with primer
p, was designed to detect specifically knocked-in Ednrb transcripts
as a 477 bp product. The housekeeping gene GAPDH served as internal
control.
Skeletal staining
Alizarin Red/Alcian Blue staining was performed, as previously described
(McLeod, 1980).
β-Galactosidase staining
lacZ expression was detected by staining with X-gal
(5-bromo-4-chloro-3-indoyl β-D-galactoside) for β-galactosidase
activity. Whole-mount staining was performed as previously described
(Nagy et al., 2003) with minor
modifications. For sections, samples were embedded in OCT compound,
cryosectioned and subjected to X-gal staining. Some sections were
counterstained with 1% Orange G (Sigma).
In situ hybridization
Whole-mount in situ hybridization was performed as described previously
(Wilkinson, 1992). Probes for
Hand2 (Srivastava et al.,
1995) and goosecoid (Yamada et
al., 1995) were generously provided by D. Srivastava (University
of California, San Francisco, CA) and G. Yamada (Kumamoto University,
Kumamoto, Japan), respectively. Other probes were prepared by RT-PCR as
described (Ozeki et al.,
2004).
Apoptosis analysis
For detection of apoptotic cells, terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) staining was performed on
14 µm consecutive frozen sections of embryonic day 10.5 (E10.5) embryos
using a DeadEnd Labeling kit (Promega) with diaminobenzidine as substrate, as
previously described (Abe et al.,
2007).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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lacZ-labeling of Ednra-expressing cells during embryonic development
To analyze the expression patterns of Ednra during
postimplantation development, we performed β-galactosidase staining on
Ednra-lacZ knock-in embryos. lacZ expression patterns were
identical between EdnralacZ2FRT/+ and
EdnralacZ1FRT/+ embryos, indicating that the presence of
Puro in the mutant allele did not significantly affect the expression
of knocked-in genes. Thereafter, pictures of
EdnralacZ2FRT/+ embryos stained for β-galactosidase
activity are shown.
At E8.25 to 8.5, lacZ expression was observed in the head
mesenchyme at the hindbrain level (Fig.
2A-C). High magnification of transverse sections at E8.5 revealed
lacZ expression in migratory neural crest cells delaminating from the
dorsal neuroepithelium (Fig.
2D). By contrast, lacZ expression was undetectable in the
neuroepithelium, including the premigratory neural crest, surface ectoderm,
foregut endoderm and vascular endothelium
(Fig. 2C,D). At E9.0,
lacZ was highly expressed in the head and pharyngeal arch regions, in
the heart, and in the ventral half of the trunk
(Fig. 2E). In the first to
third arches, lacZ expression was detected in neural-crest-derived
ectomesenchymal cells, whereas the pharyngeal ectoderm and endoderm, vascular
endothelium and many cells in the core mesenchyme were lacking lacZ
expression (Fig. 2F).
lacZ expression was also extensively observed in the head mesenchyme
adjacent to the neuroepithelium (Fig.
2F). In the cardiac outflow region, cardiac neural crest cells
surrounding the second and third pharyngeal arch arteries and colonizing
between the foregut and the aortic sac showed intense lacZ expression
(Fig. 2G). These lacZ
expression patterns appeared to recapitulate endogenous Ednra
expression revealed by in situ hybridization in whole mounts
(Fig. 2H) and in sections
(Clouthier et al., 1998;
Yanagisawa et al., 1998).
By comparison, in situ hybridization of E9.0 embryos for Crabp1, a
neural crest marker (Ruberte et al.,
1992), revealed its expression in the streams of migratory neural
crest cells, which overlapped with Ednra-lacZ expression
(Fig. 2I,L). However,
Ednra-lacZ expression was also found in Crabp1-negative
mesenchymal regions ventromedial to the neural crest streams
(Fig. 2K). The expression
pattern of Snail1, which is expressed in neural-crest- and
mesoderm-derived head mesenchyme before E10.5
(Nieto et al., 1992;
Smith et al., 1992), is more
similar to that of Ednra-lacZ
(Fig. 2J,M). These results
suggest that lacZ-expressing mesenchymal cells are likely to
originate from both neural crest and mesoderm.
At E10.0, lacZ expression was observed throughout the head mesenchyme adjacent to the neural tube (Fig. 3A,B). Trigeminal ganglia, which originate in neural crest cells and placode cells, were highly populated with lacZ-positive cells (Fig. 3B). In the pharyngeal arches, lacZ expression was mainly located in ectomesenchyme underlying the epithelium (Fig. 3A). The distribution of lacZ expression within the wall of the aortic sac and pharyngeal arch arteries corresponded to cardiac neural crest cell population (Fig. 3C). At E12.5, lacZ expression was found in ectomesenchyme underlying the oral epithelium in the lower and upper jaws (Fig. 3D,G). In developing tooth buds, lacZ expression was present in mesenchyme surrounding the endoderm-derived dental lamina (Fig. 3E). In the precartilage primordium of Meckel's cartilage, lacZ expression was undetectable in the bilateral rod portion (Fig. 3F), but was intensely present in the rostral process (Fig. 3G,H).
Craniofacial defects of Ednra-null mice
As expected, Ednraneo/neo, EdnralacZ/lacZ
and Ednraneo/lacZ mice demonstrated perinatal lethality
and craniofacial abnormalities, which were almost identical to the phenotype
of mice lacking Edn1 or Ednra
(Ozeki et al., 2004;
Ruest et al., 2004). In these
mutants, the lower jaw appeared as a mirror image of the upper jaw with arrays
of vibrissae (Fig. 4A,B).
Skeletal analysis revealed transformation of mandibular arch-derived
structures into maxillary arch-derived elements in mutants
(Fig. 4C-F). Most of the
dentary was replaced with an ectopic bone, which appeared as a mirror image
duplication of the ipsilateral maxilla
(Fig. 4G,H), the morphology of
which was almost identical to that of wild-type maxillae
(Fig. 4I). In addition, a
second set of jugal and palatine bones was observed in mutants
(Fig. 4E,F). In the most distal
portion, alveolar bone was formed around a tiny rostral process of Meckel's
cartilage and surrounded a lower incisor, although its growth was variable
among individuals (Fig. 4F,G).
Zygomatic processes of the squamosal were malformed and often absent
(Fig. 4F). In addition,
ectotympanic and gonial bones were lost, whereas the pterygoid process, ala
temporalis and lamina obturans were duplicated in mutants
(Fig. 4J,K). The malleus and
incus were malformed, giving an ectopic structure extending to the squamosal
(Fig. 4L,M).
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Knock-in of Ednra cDNA rescued the Ednra-null phenotype
Recapitulation of Ednra expression by the RMCE-mediated knock-in
of lacZ may justify the application of this system for the functional
analysis of the Edn1/Ednra signaling mechanism in embryonic development. To
confirm this, we introduced Ednra cDNA into the
Ednraneo allele, expecting rescue of the
Ednra-null phenotype by restoring Ednra expression
(Fig. 5A). The efficiency of
RMCE-mediated knock-in in ES cells was 44% (41 in 94 surviving clones). Four
clones generated germline chimeras following blastocyst injection. Chimeric
and heterozygous mice carrying the Ednra knock-in allele
(EdnraA) were intercrossed with
EdnralacZ/+ mice to obtain EdnralacZ/A
offspring.
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Knock-in of Ednrb cDNA could only partially rescue the Ednra-null phenotype
Previous studies have demonstrated that Edn1 can bind to both Ednra and
Ednrb receptors with similar affinities and activate common signaling
pathways, including Gq/G11-dependent signals in many
cells (Cramer et al., 2001;
Jouneaux et al., 1994;
Kedzierski and Yanagisawa,
2001; Masaki et al.,
1999; Takigawa et al.,
1995). If Ednra and Ednrb are interchangeable in the context of
pharyngeal arch development, Ednrb knock-in is expected to rescue the
Ednra-null phenotype. To test this possibility, we introduced Ednrb
cDNA into the Ednraneo allele in the same procedure as
Ednra knock-in (Fig.
5A). Among 12 knock-in clones obtained through the screening, two
clones were used to generate germline chimeras. Resultant heterozygous mice
with the Ednrb-knock-in allele (EdnraB) were
intercrossed with Ednraneo/+ or
EdnralacZ/+ mice. The expression levels of Ednrb
from the single knock-in allele were comparable to those of Ednra as
estimated by RT-PCR analysis of E9.5 mandibular arches
(Fig. 5A).
Unexpectedly, all the Ednraneo/B and EdnralacZ/B mice died at birth and demonstrated craniofacial abnormalities similar to Ednra-null mice. In E18.5 EdnralacZ/B mice, the appearance of the lower jaw was a mirror image of the upper jaw with vibrissae (Fig. 5C). Skeletal analysis of EdnralacZ/B mice demonstrated a duplication of the upper jaw elements in the lower jaw region (Fig. 5E). Similar abnormalities were manifested in EdnraB/B mice, in which both of the two Ednra alleles were replaced with knocked-in Ednrb cDNA (data not shown).
The skeletal phenotype of EdnralacZ/B and EdnraB/B mice, however, was different from that of Ednra-null mice in some respects. First, whereas the distal portion of the Ednra-null mandible was variable and often severely hypoplastic, failing to fuse at the midline (Fig. 4F,G), all the EdnralacZ/B (n=7) and EdnraB/B (n=9) mice examined had relatively well-developed incisive alveolus, the appearance of which was similar to that of the normal dentary (Fig. 5E,F). Second, the hyoid in some Ednrb-knock-in mice was fused only unilaterally to the basisphenoid (in one of seven EdnralacZ/B mice and four of nine EdnraB/B mice) or separated from the basisphenoid (in three of nine EdnraB/B mice), whereas all the Ednraneo/neo mice compared (n=10) showed bilateral hyoid-basisphenoid fusion. The hyoid in EdnralacZ/B and EdnraB/B mice, whether it was fused to or separated from the basisphenoid, had nearly normal appearance compared with that of Ednra-null mice, although it typically had an extended ossification center and connected to the stapes though a transformed lesser horn as in Ednraneo/neo mice (Fig. 5G). Thirdly, ectopic cartilage appeared between the malleal/incal region and the extended basitrabecular process and/or ala temporalis in four of seven EdnralacZ/B mice and six of nine EdnraB/B mice (Fig. 5H), whereas similar cartilage was observed in none of ten Ednraneo/neo mice (Fig. 4L). The first two differences may indicate that Ednrb can partially rescue the Ednra-null phenotype in the distal portion of the pharyngeal arch structures.
It has recently been reported that apoptotic cells are increased and extend
distally in the mandibular arch mesenchyme of E9.5-10.5 Ednra-null
embryos (Abe et al., 2007). To
test whether knocked-in Ednrb could prevent increased apoptosis, we
performed TUNEL staining on sections of E10.5 embryos. Consistently with the
previous report, TUNEL-positive cells extended from the proximal to the distal
mandibular region in E10.5 Ednraneo/neo embryos
(n=5), whereas TUNEL-positive cells were confined to the proximal
region in wild-type embryos (n=6)
(Fig. 6A,B). Similarly,
Ednraneo/B embryos also showed apparently increased
numbers of TUNEL-positive cells extending into the distal mandibular region
(n=4) (Fig. 6C).
|
;
Clouthier et al., 2000;
Ozeki et al., 2004). The
expression of Hand2 and Goosecoid was also downregulated in
EdnraB/B pharyngeal arches
(Fig. 7I-L), as in
Edn1-/- and Ednra-/- embryos
(Clouthier et al., 1998;
Thomas et al., 1998) as well
as in Dlx5-/-;Dlx6-/- embryos
(Charite et al., 2001;
Depew et al., 1999;
Depew et al., 2002). Taken
together with morphological and apoptosis analysis, these results indicate
that Ednrb cannot substitute for Ednra in pharyngeal development, although it
may partially rescue the Ednra-null phenotype.
Comparison of craniofacial structures between Ednra-modified mice and neural-crest-specific Gq/G11-deficient mice
Offermanns's group has demonstrated that
P0-Cre-/+;G
flox/floxq;G
-/-11 mice lacking
Gq/G11 in a neural-crest-specific manner
show craniofacial defects similar to those in Edn1- or
Ednra-deficient mice
(Dettlaff-Swiercz et al.,
2005). However, detailed description in terms of this similarity
has not yet been reported. To clarify the relationship between Edn receptors
and Gq/G11 signaling in
neural-crest-derived ectomesenchyme, we revisited the craniofacial phenotype
of neural-crest-specific Gq/G11-deficient
mice.
|
q/G11-deficient mice
had duplicated palatine, pterygoid and lamina obturans
(Fig. 8A-D), as observed in
Ednra-null (Fig. 4D,F)
and Ednrb-knock-in (Fig.
5E, Fig. 8E) mice.
Unlike Ednra-null mice, however, the incisive alveolar bone in the
distal mandibular region was relatively well developed in all the
Gq/G11-deficient mice examined
(n=8) (Fig. 8B,D,F).
This morphological feature is reminiscent of the Ednrb-knock-in
phenotype (Fig. 8E,G) rather
than the Ednra-null one (Fig.
8H). The basitrabecular process was abnormally extended to the ear
region in all the cases (Fig.
8B,D). Furthermore, the hyoid of
Gq/G11-deficient mice was not fused to the
basisphenoid in all the cases (Fig.
8I,J). Instead, the body has an extended ossification center and
is fused with the lesser horn and often with the superior horn of the thyroid
(Fig. 8I,J), as seen in
Ednrb-knock-in mice (Fig.
5G). These similarities suggest that
Gq/G11 mainly mediate Ednra-selective
signaling in neural-crest-derived mesenchyme during pharyngeal arch
development, but the Ednrb-replaceable signaling is likely to be mediated by
the Gq/G11-independent pathway. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Liu et al., 2006;
Toledo et al., 2006) or the
Flp-FRT system (Cesari et al.,
2004; Roebroek et al.,
2006). In our RMCE procedure, we replaced a 1.0 kb genomic
sequence with knock-in sequences containing mutant lox sites and an
FRT-flanked Puro. The expression of knocked-in lacZ
appeared to recapitulate endogenous Ednra expression irrespective of
the presence of Puro, and the knock-in of Ednra cDNA could
normalize craniofacial defects caused by Ednra-null mutation.
Furthermore, no additional abnormalities were evident in any knock-in mice
after RMCE procedure at least in terms of embryonic development, viability and
fertility. These results support the relevance of our RMCE-mediated knock-in
procedure for analysis of gene function in craniofacial development.
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|
; Ozeki et al.,
2004; Ruest et al.,
2004). The crucial window for this Edn1-Ednra interaction in
pharyngeal arch development is around E8.5 to 9.0, when regional identities
start to be specified within the anterior pharyngeal arches
(Fukuhara et al., 2004).
Consistently, Ednra-lacZ expression was detectable just
before this stage (E8.25; 
6-somite stage) in the head mesenchyme
including migratory neural crest cells delaminating from the dorsal
neuroepithelium. By contrast, premigratory neural crest cells in the neural
plate did not express lacZ, suggesting that the induction of
Ednra expression may be coupled with epithelial-mesenchymal
transition. Thereafter, Ednra-expressing neural crest cells migrating
into the ventral region of the anterior pharyngeal arches are supposed to
receive the Edn1 signal, leading to the upregulation of
Dlx5/Dlx6 expression and the specification of a ventral
identity.
In addition to the expression in neural-crest-derived mesenchymal cells,
Ednra-lacZ was likely to be expressed in the
mesoderm-derived head mesenchyme. This is further supported by the difference
in the pattern of lacZ expression between the
Ednra-lacZ mice and other mice in which neural crest cells
are specifically marked. Protein 0 (P0)-Cre
transgenic mice harboring a conditional lacZ allele, for example,
demonstrated lacZ expression broadly in neural-crest derivatives
(Yamauchi et al., 1999). The
Wnt1-Cre transgene also directed the expression of a
conditional lacZ allele specifically to neural crest cells in mice
(Chai et al., 2000). In both
cases, the head mesenchyme adjacent to the neural tube was largely
lacZ-negative. In fact, the head mesenchyme originated from both the
cranial paraxial mesoderm and neural crest
(Trainor and Tam, 1995). Thus,
Ednra appears to be extensively expressed in mesoderm-derived
mesenchyme (except for the pharyngeal core mesoderm and vascular endothelium)
as well as in neural-crest-derived ectomesenchyme in the craniofacial
region.
At later stages, Ednra-lacZ expression was observed in
many cranial/cardiac neural crest derivatives. In the Meckel's cartilage
primordium, which is also derived from neural crest cells, lacZ
expression was detected only in the rostral process, a distalmost portion, at
E12.5. At E12.5 to 13.5, the rostral process is rich in proliferative,
undifferentiated cells, while cells start to differentiate into chondrocytes
in the bilateral rod portion (Ramaesh and
Bard, 2003). Thus, Ednra expression in neural-crest
derivatives may be stage- and/or lineage-dependent.
Diversity of Edn receptor signaling in pharyngeal arch development
Ednra and Ednrb share common ligand affinities and downstream signaling
pathways. In particular, the Gq/G11-mediated pathway,
which is assumed to be responsible for Edn1/Ednra-dependent craniofacial
development, is also activated by Ednrb stimulation in various cell types
(Cramer et al., 2001;
Jouneaux et al., 1994;
Masaki et al., 1999;
Takigawa et al., 1995).
However, knock-in of Ednrb failed to rescue homeotic transformation
of the lower jaw into an upper jaw-like structure in Ednra-null mice,
although knocked-in Ednrb appeared to be expressed at levels similar
to those of knocked-in Ednra. This result indicates that Ednrb cannot
restore Ednra function in the specification of mandibular identity in
pharyngeal arch development.
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|
;
Takigawa et al., 1995). It is
also possible that Ednrb may not associate with Gq/G11
in cranial neural crest cells in a physiological context. In trunk neural
crest development giving rise to the enteric nerve system, the Edn3/Ednrb
signal is likely to be mediated by inhibition of protein kinase A
(Barlow et al., 2003), which
appears to be independent of Gq/G11. Thus, Ednra and
Ednrb may couple to different G proteins, leading to activation of distinct
signaling pathways. Another possibility is that difference in receptor
desensitization or differences in the potency and/or efficacy of the receptor
agonists with regard to receptor activation might cause the failure of rescue
by Ednrb. Further studies are required to test these
possibilities.
By contrast, there were some differences in craniofacial defects between
Ednra-null and Ednrb knock-in mice. Unlike
Ednra-null mice, Ednrb knock-in mice had a relatively
well-developed incisive alveolar bone of the mandible and, in some cases, the
hyoid separated from the basisphenoid as in normal mice. Extended
basitrabecular process with connection to ectopic cartilage was often observed
in Ednrb knock-in mice. The first two differences suggest that Ednrb
may partially replace Ednra function in the development of distal structures
in the mandibular and hyoid arches. Considering the regional heterogeneity in
Ednra expression within the Meckel's primordium, the Edn1/Ednra
signal may be also required for the formation of the distal region later than
the stage of dorsoventral specification. Interestingly, craniofacial defects
of neural-crest-specific Gq/G11-deficient
mice were very similar to those of Ednrb knock-in mice rather than
Ednra-null mice. This similarity suggests that aspects of the
Edn1/Ednra signaling that Ednrb can substitute may be mediated by a
Gq/G11-independent pathway. Thus, Edn1/Ednra may
activate different G proteins in different contexts during pharyngeal arch
development. Alternatively, Edn receptor functions in cells other than the
neural-crest derivatives may also be required for pharyngeal arch development.
The mechanism underlying the selectivity of the Edn receptors in terms of
G-protein coupling in cranial neural crest cells in different contexts should
be clarified in further investigations.
Regionality within the mandible in terms of the requirement for Edn1/Ednra-Gq/G11 signaling
The present study showed that the proximal portion of the dentary underwent
homeotic transformation into upper jaw elements, whereas the distal portion
maintained its mandibular identity in Ednra-null,
Ednrb-knock-in and Gq/G11-null
mice. Notably, the ectopic bone replacing the dentary in mutant mice resembled
the maxilla, leaving incisive alveolar bone with mandibular identity in the
distal portion. Thus, the proximal region of the dentary requires the
Edn1/Ednra to Gq/G11 signaling for establishing its
mandibular identity, and this signaling cannot be replaced by Ednrb. Dlx5 and
Dlx6 are likely to be activated by this pathway, because the expression of
Dlx5, Dlx6 and their downstream genes was downregulated in the
mandible of Ednrb-knock-in mice as well as
Gq/G11-deficient mice
(Ivey et al., 2003). Increased
apoptosis extending to the distal mandibular arch may also be related to
defects in this pathway, although apoptosis in
Gq/G11-deficient mice has not yet been
analyzed. By contrast, this signaling pathway is dispensable for the
mandibular identity of the distal incisive alveolus.
Gq/G11-independent Ednra signaling, which can be
replaced by Ednrb knock-in, may contribute to the growth of this
portion. It has previously been shown that neural-crest-specific
G12/G13-deficient mice have no
craniofacial defects, making it unlikely that G12/G13
are involved (Dettlaff-Swiercz et al.,
2005). Thus, other types of trimeric G proteins such as
Gi/Go may be involved in this pathway. The possible dual
roles of the Edn1/Ednra signaling in pharyngeal arch development are
summarized in Fig. 9.
The incisive alveolus appears to be equivalent to the premaxilla in the
upper jaw, as extrapolated from the conjunction to the maxillary(-like) bone
in Ednra-null mutants (Fig.
4G,H). This portion and the premaxilla are missing in
Dlx5/6-/- mice, indicating that these structures are
dependent on Dlx5 and Dlx6 (Beverdam et
al., 2002; Depew et al.,
2002). Residual Dlx5 and Dlx6, independent of
the Edn1/Ednra to Gq/G11 signaling, may be responsible
for the formation of these distal structures.
Usefulness of the present RMCE-mediated knock-in system in studies on craniofacial development
The present RMCE-mediated knock-in procedure enabled us to introduce a
series of gene cassettes to be expressed in a spatiotemporal pattern similar
to the endogenous Ednra. The efficiency was much higher than that of
conventional homologous recombination in ES cells. Using this procedure, we
could examine the expression pattern and function of Ednra in craniofacial
development. In particular, differences in Ednra and Ednrb
knock-in have provided a clue to further analysis of the receptor domain
function. Furthermore, this system can be broadly applicable for studies on
gene function, including cell fate determination, differentiation, regional
specification and so on. These applications will largely contribute to our
understanding of the molecular mechanisms that regulate craniofacial
development.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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