REGULATION OF MANDIBULAR GROWTrH

AND MORPHOGENESIS
Mina Mina
Department of Pediatric Dentistry, School of Dental Medicine, University of Connecticut Health (enter, Farmington, al 06030; Mina@nsoL.uchc.edu

ABSTRACT: The development of the vertebrate face is a dynamic process that starts with the formation of facial
processes/prominences. Facial processes are small buds made up of mesenchymal masses enclosed by an epithelial layer that
surround the primitive mouth. The 2 maxillary processes, the 2 lateral nasal processes, and the frontonasal processes form the
upper jaw. The lower jaw is formed by the 2 mandibular processes. Although the question of the embryonic origin of facial struc-
tures has received considerable attention, the mechanisms that control differential growth of the facial processes and patterning
of skeletal tissues within these structures have been difficult to study and still are not well-understood. This has been partially
due to the lack of readily identifiable morphologically discrete regions in the developing face that regulate patterning of the face.
Nonetheless, in recent years there has been significant progress in the understanding of the signaling network controlling the pat-
terning and development of the face (for review, see Richman et al., 1991; Francis-West et al., 1998). This review focuses on cur-
rent understanding of the processes and signaling molecules that are involved in the formation of the mandibular arch.
Key words. Mandibular processes, branchial arches, signaling molecules, epithelial-mesenchymal interactions, transcription
factors, secreted factors, chick, mouse, growth, patterning, morphogenesis.

Introduction fate-mapping (Lumsden et al., 1991; Serbedzija et al., 1992; Couly

During normal development, the lower jaw is formed from
the mandibular component of the first branchial arch. The
mandibular processes are bilaterally symmetrical structures
located below the future mouth cavity and composed of mes-
enchymal tissue enclosed by an epithelial layer of ectodermal
and endodermal origin. After their initial formation, these
processes undergo considerable outgrowth along all 3 axes,
merge in the midline region, and give rise to the elongated tri-
angular-shaped lower jaw. In addition to merging in the midline
region, mandibular processes also fuse/merge at the lateral cor-
ners with the maxillary process and at their lower borders with
the hyoid process.
The mesenchyme of the mandibular processes, as in other
facial processes, is derived from cranial neural crest cells (CNCC)
and paraxial mesoderm. CNCC give rise to the majority of the
craniofacial skeleton (for reviews, see Le Douarin et al., 1994;
Osumi-Yamashita et al., 1997; Schilling, 1997). On the other hand,
the paraxial mesoderm forms the craniofacial muscles along
with some skeletal elements in the skull and vascular tissues
(Noden, 1983b, 1988; Couly et al., 1992, 1993). The skeletogenic
potential of neural crest cells (NCC) is restricted to the cephalic
region down to the level of somite 5. In addition to giving rise to
skeletal structures, CNCC give rise to the peripheral nervous
system and connective tissues associated with the facial muscles
(Noden, 1986, 1988; Couly et al., 1992; Kontges and Lumsden,
1996; Osumi-Yamashita et al., 1997).
The fate and final derivatives of CNCC cells have been
extensively analyzed in avian and mammalian embryos (for
reviews, see Le Douarin et al., 1994,1997; Osumi-Yamashita et al.,
1997; Teillet et al., 1999). In addition, after the identification of
rhombomeres (R), a series of segmented structures in the hind-
brain, an elegant scheme of the migratory routes and derivatives
of CNCC has been generated through short-term and long-term
et al.,
1993, 1996, 1998; Kontges and Lumsden, 1996).
The results of these studies indicate that, in contrast to the
NCC of the rest of the body, the hindbrain CNCC migrate in 3
non-mixing separate streams from RI and R2, from R4 and from
R6 (Lumsden et al., 1991; Osumi-Yamashita et al., 1997). The for-
mation of 3 separated streams of migrating cells is due to lack of
emigration and migration of CNCC from R3 and R5 (Lumsden
et al., 1991; Couly et al., 1996). The lack of emigration of CNCC
from R3 and R5 is mainly due to apoptotic elimination of NCC
and is regulated by molecules such as BMP-4 and Msx2 and by
interactions with neighboring rhombomeres (Graham et al.,
1996; Maden et al., 1997; Takahashi et al., 1998). More recent
studies indicate that crest depletion from R3 and R5 is not
absolute and that a small number of NCC from R3 and R5 sur-
vive the apoptotic elimination and join migratory cells in adja-
cent rhombomeres (Sechrist et al., 1993; Birgbauer et al., 1995;
Kontges and Lumsden, 1996).
Short-survival fate-mapping of CNCC in chick embryos
showed that CNCC originating from R4 and R6 populate the
second and third branchial arches, respectively (Lumsden et al.,
1991; Couly et al., 1996, 1998). A mixed population of CNCC
from the posterior mesencephalon, Rl, and R2 (Lumsden et al.,
1991) colonizes the first branchial arch (maxillary and mandibu-
lar processes). Long-survival fate-mapping with use of the quail
marker extended these observations and showed the com-
pound nature of the mandibular skeleton in which CNCC origi-
nating from multiple rhombomeres (Rl, R2, and R4) participate
in the formation of the mandibular arch skeleton (Couly et al.,
1996; Kontges and Lumsden, 1996). Interestingly, the mandibu-
lar arch skeleton displays an organization that precisely reflects
the rostrocaudal order of crest emigration. The proximal ele-
ments (the major part of Meckel's cartilage, and dentary and
splenial bones) are derived from the midbrain crest. The caudal

276Grit
276 Rev Oral Blot Med
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12(4)276-300 (2001)

12(4):276-300 (2001),
skeletal structures of the mandibular arch (elements around the
lower jaw joints and close to the middle ear structures, such as
the articular and its internal process, angular, suprangular, and
quadrate) are formed mainly by NCC from Rl and R2. Kontges
and Lumsden (1996) showed that the most caudal element of
the lower jaw in chick embryos, the retroarticular process of the
Meckel's cartilage, is derived from CNCC originating from R4
and a small number of NCC from R3 and R5 that survive apop-
tosis. On the other hand, Couly et al. (1996) found no contribu-
tion of R4 to the caudal skeletal elements. More recent labeling
studies in chick (Sohal et al., 1999) and mouse (Chai et al., 2000)
embryos showed that, in addition to the CNCC, Meckel's carti-
lage is populated by non-CNCC. Studies in chick embryos
showed that cells emigrating from the ventral region of the
neural tube (VENT) also make a significant contribution to
Meckel's cartilage (Sohal et al., 1999).
In addition, Kontges and Lumsden (1996) showed that the
same groups of CNCC also give rise to the connective tissue of
individual mandibular muscles and their respective attach-
ment sites to the skeletal elements in the mandibular arch and
the cranial base. While the CNCC from the posterior mesen-
cephalon and RI and R2 mix together, there is no mixing
between this population and those originating from R4 that
give rise to the retroarticular process and connective tissue of
the hyoid arch muscle (Kontges and Lumsden, 1996).
Cell transplantation and DiI labeling experiments also
showed similarities between the axial level of origin for the
paraxial mesoderm and the CNCC populating each branchial
arch (Noden, 1983b, 1986, 1988; Trainor et al., 1994; Trainor and
Tam, 1995; Hacker and Guthrie, 1998). These studies in mouse
and chick embryos showed that cranial paraxial mesoderm
migrates in overlapping streams into the nearest branchial
arch. Mesoderm originating from somitomeres III and IV enters
the developing mandibular and hyoid arch and colonizes their
core, a region known to correspond to the muscle mass where
they express myogenic markers such as Myf5, MyoD, and myo-
genin (Trainor and Tam, 1995; Hacker and Guthrie, 1998).
In contrast to the regions occupied by paraxial mes-
enchyme, the migrating CNCC colonize the periphery of each
branchial arch (Lumsden et al., 1991; Trainor and Tam, 1995;
Hacker and Guthrie, 1998). In the branchial arches, unlike
other regions in the face, there is a distinct segregation of these
2 cell populations (Trainor and Tam, 1995).
For many years, it has been thought that patterning of the
skeletal elements in the mandibular arch is the result of intrin-
sic morphogenetic specification in CNCC. This possibility was
suggested by the results of a series of hetero- and isotopic graft-
ing experiments in amphibian and avian embryos (Horstadius
and Sellman, 1946; Wagner, 1949; Noden, 1983a, 1988). The
quail-chick transplantation experiments demonstrated that
when presumptive first-arch CNCC (anterior region of the
hindbrain Rl and R2 and mesencephalic CNCC) are trans-
planted into the presumptive second-arch region (R4/R6), the
grafted CNCC migrate ventrally to the second arch but subse-
quently give rise to a subset of first-arch-specific structures and
form a partially duplicated mandibular arch skeleton (Noden,
1983a; Couly et al., 1998). One of the main conclusions drawn
from these experiments is that, in contrast to trunk NCC,
CNCC are morphogenetically pre-specified, and the patterns of
the CNCC-derived skeleton are imprinted in the pre-migratory
CNCC while they are still within the neural tube.
However, the concept of morphogenetic specification of cra-
2001)
12(4) 276-300 (2001)
12(4):276-300
Crit Rev Oral Biol Med
nial NCC has remained controversial for many years, because no
other subpopulation of pre-migratory NCC other than those giv-
ing rise to the mandibular arch showed such pre-specification in
the patterns of their derivatives. In addition, it was shown that
transplantation of mesencephalic NCC (which normally give
rise to skeletal elements in the frontonasal mass and maxillary
and peri-ocular region) into the presumptive second- and third-
arch NCC also gave rise to a duplicated subset of first-arch-spe-
cific structures (Noden, 1983a). Furthermore, these observations
are in contrast to the outcome of transplantation of the pre-
sumptive first-arch NCC into the rostral or more caudal regions
which gave rise to normal head skeletal structures and did not
form mandibular arch structures (Noden, 1978, 1986).
The first family of genes suggested to be involved in pre-
specification of CNCC and branchial arch development was
the Hox genes (for reviews, see Hunt et al., 1991a,b; Krumlauf,
1994; Schilling, 1997; Morrison, 1998). Analysis of the patterns
of expression of Hox genes in mouse embryos showed that 3
members of Hox A-D clusters are expressed in the hindbrain
region before the formation of rhombomeres, and that later
their patterns of expression respected rhombomeric bound-
aries (Krumlauf, 1994; Wilkinson, 1995). In general, with the
exception of paralogous group 1 and Hoxa2, the paralogous
groups have similar anterior boundaries within the hind-
brain. These overlapping patterns of Hox expression have
been called the Hox codes.
In addition, it was shown that at least part of the overlap-
ping patterns of Hox expression (Hox code) in each rhom-
bomere is maintained in migratory CNCC as they populate the
branchial arches, so that the branchial arches end up with a
distinct pattern of Hox expression that closely resembles the
Hox code of the rhombomeres from which they originated
(Hunt et al., 1991a,b; Krumlauf, 1994; Wilkinson, 1995). Thus,
CNCC-derived mesenchyme of the third branchial arch (simi-
lar to R6) expresses the second and third paralogous groups,
and the second branchial arch mesenchyme that is colonized
by CNCC from R4 expresses only the second paralogous
group. These observations provide strong support for the
involvement of a combinatorial Hox code in the pre-specifica-
tion of CNCC giving rise to the skeletal elements of the more
caudal branchial arches (2nd and 3rd arches). Direct evidence
for roles of Hox genes in regulating the patterning of the
CNCC-derived skeleton of the caudal branchial arches comes
from skeletal abnormalities in the third and fourth branchial
arches in knock-out mice lacking Hoxa3 (Chisaka and
Capecchi, 1991; Manley and Capecchi, 1995, 1998). of the Hox
However, with the exception of Hoxa2, none
genes is expressed rostral to R3 (Wilkinson et al., 1989; Hunt et
al., 1991b; Krumlauf, 1994; Prince and Lumsden, 1994; Richman,
1995). Although Hoxa2 is expressed up to the R1/R2 boundary,
the most anterior Hoxa2-expressing CNCC populate the hyoid
arch but not the first arch. Therefore, the first branchial arch that
was shown previously to be morphogenetically specified
(Noden, 1983a) is devoid of any Hox gene expression.
The lack of Hox expression in the most rostral regions of
the developing neural tube and in the CNCC of the first
branchial arch suggested that the primary characteristic dis-
tinguishing the CNCC populating the first branchial arch from
more caudal CNCC may be the absence of Hox gene products
(Gendron-Maguire al., et 1993; Rijli al., 1993, 1998).
et
A predication of this model is that removal of Hoxa2 func-
tion from the CNCC populating the second branchial arch
BioI Med
277
 would lead to re-specification of second-arch CNCC identity
into that of the first arch. In fact, in Hoxa2 mutants, morphogen-
esis of the first and second branchial arches is altered (Rijli et al.,
1993; Gendron-Maguire et al., 1993). Hoxa2 mutants lack all of
the skeletal elements of the second branchial arch and contain a
mirror-image duplicated set of caudal mandibular skeletal ele-
ments, including a truncated Meckel's cartilage, incus, malleus,
tympanic and squamosal bones, and duplicated external audi-
tory meatus (Gendron-Maguire et al., 1993; Rijli et al., 1993).
However, the duplicated structures in the Hoxa2 mutants did
not include more proximal structures, such as the proximal por-
tion of Meckel's cartilage, dentary, maxilla, palatine, jugal
bones, and teeth. The phenotype of the Hoxa2 mutant is similar
if not identical to the phenotype of the chick embryo after trans-
plantation of the presumptive first-arch CNCC into the second-
arch region (Noden, 1983a).
Based on the long-term fate-mapping studies in chick
embryos and established homologies between mammals and
birds (Kontges and Lumsden, 1996), it was concluded that the
absence of Hoxa2 results in specific homeotic transformation in
which the R4-derived mesenchymal cells in the hyoid arch
acquired the identity of the caudal region of mandibular mes-
enchyme derived from Rl and R2, but not the identity of CNCC
mesenchyme derived mainly from the midbrain (Gendron-
Maguire et al., 1993; Rijli et al., 1993, 1998; Kontges and Lumsden,
1996). The phenotypic abnormalities in Hoxa2 mutants suggest-
ed essential roles for Hoxa2 in the morphogenetic identity of the
second branchial arch skeletal elements consistent with postulat-
ed roles of Hox codes in patterning of the CNCC-skeletal deriva-
tives of the more caudal branchial arch.
One of the predications of the CNCC pre-specification
model, in which patterning information is imprinted by the
presence absence of Hox expression in pre-migratory CNCC
or
while still in the neural tube, is that experimental manipula-
tions of rhombomeres should result in colonization of the
branchial arch with CNCC expressing inappropriate Hox codes
and, thus, should give rise to inappropriate skeletal elements.
Although the original grafting experiments of Noden (1983a)
support this model, findings of more recent transplantation
studies in chick embryos have provided conflicting results. The
differences in the results appear to be related to the axial level
of origin of crest cell, the type of experimental manipulation,
and the time intervals in which Hox expression in the branchial
arches has been examined.
Studies in which the neural tube and pre-migratory
CNCC are re-located within the hindbrain and patterns of Hox
expression in the branchial arches are examined early (one day
post-operatively) indicate that the grafted NCC populated the
branchial arches according to their new position but main-
tained the patterns of Hox gene expression appropriate for
their position of origin (Prince and Lumsden, 1994; Saldivar et
al., 1996, 1997; Couly et al., 1998). The stability of the Hox code
in crest cells in ectopic locations in some, but not all, cases was
correlated with skeletal abnormalities.
On the other hand, analysis of embryos in which crest cells
are re-routed by late deletion of adjacent crest cells and experi-
ments involving rotation of the entire hindbrain and its associ-
ated crest (R1-R7) indicates that the initial stable Hox code is
modified within the branchial arches 2 or 3 days after trans-
plantation (Hunt et al., 1995, 1998; Saldivar et al., 1997). In these
situations, the Hox code in CNCC in ectopic locations is modi-
fied, matches their new location within the branchial arches,
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CiCrit Rev
and gives rise to almost normal CNCC-derived skeletal com-
ponents. For example, Hunt et al. (1998) showed that when the
Hox-expressing R7 is placed in the Rl region, CNCC from the
grafted Hox-expressing R7 together with the host mesen-
cephalic crest cells migrate into the first branchial arch, become
Hox-negative (match their new position in the first branchial
arch), and give rise to normal first-arch skeletal components
with some additional ectopic first-arch structures. The results
of these studies suggest that the Hox code in the branchial arch-
es can be established independently of the hindbrain after
CNCC migration into the branchial arches.
The modification of Hox expression in the branchial arches
colonized with mixed populations of CNCC is thought to be
mediated by "community effects", where the Hox code
expressed by the majority of cells eventually predominates and
results in either gain or loss of Hox expression (Saldivar et al.,
1997; Hunt et al., 1998). Alternatively, it has been postulated that
alteration in Hox expression may be mediated by environmen-
tal influences emanating from adjacent tissues (i.e., neighboring
neural tube, mesoderm, and branchial arches). More recent
results suggest that a Hox code, rather than being involved in
pre-specification of CNCC, may be involved in the maintenance
of appropriate registration of different structures within each
branchial arch (Kontges and Lumsden, 1996; Hunt et al., 1998).
Despite the lack of expression of 3' members of the antenna-
pedia class of Hox genes, several other regulatory genes have
been shown to be expressed in presumptive CNCC in more ros-
tral regions of the brain and in distinct regions of CNCC-derived
mesenchyme of the first branchial arch, suggestive of their
involvement in the initial formation and morphogenesis of the
first branchial arch. These genes include members of Msx, Dlx,
Otx, Barx, Gsc, Pax, Hand, Pitx, and Prx genes. As transcription
factors, they achieve this function by regulating the expression of
other regulatory genes.
Outgrowth of the Mandibular Processes
Tissue recombination studies indicate that outgrowth of the
mandibular processes is dependent on epithelial-mesenchy-
mal interactions (Wedden, 1987; Richman and Tickle, 1989;
Hall and Coffin-Collins, 1990; Mina et al., 1994). Following
removal of the overlying epithelium, reduced outgrowth is
seen in the mandibular mesenchyme, suggesting that signals
derived from mandibular epithelium regulate growth of the
underlying mesenchyme. In addition, tissue recombination
studies in which epithelia and mesenchyme from different
facial processes were exchanged suggest similarities in the sig-
naling cascades regulating outgrowth of all the facial process-
es (Richman and Tickle, 1989). Outgrowth of the mandibular
mesenchyme is supported by epithelia covering other facial
processes, and mandibular epithelium supports the outgrowth
of the mesenchyme from other facial processes. Although
these recombination studies clearly indicated the mitogenic
roles of mandibular epithelium on the underlying mesen-
chyme, they did not identify any specific regions in the epithe-
lium or mesenchyme that may be involved in regulating the
outgrowth of the developing mandible. Furthermore, the
mandibular arch, similar to other facial processes, lacks clear-
ly identifiable morphological or functional regions.
Studies in the developing chick mandible suggest that a
population of cells in the medial region (see Fig. 1) plays a sig-
nificant role in the growth of the developing mandible. Dil
labeling studies indicated that cells in the medial region make
rlBoBiol Mede
12(4):276-300 (2001)
a greater contribution to the overall expansion of the develop-
ing mandible as compared with cells in the lateral region (A) Midline
(McGonnell et al., 1998). Cell proliferation studies showed a Medial (Proximal) 4 * Lateral (Caudal)
high percentage of S-phase-labeled cells in the medial region
(Mina et al., 1995; Barlow and Francis-West, 1997; McGonnell et Oral

al., 1998). Furthermore, when only the medial region of stage 24

mandibular arches is grown for 1 wk as a graft in the dorsal
surface of a chick wing, cartilage rods of substantial length
(60% of the length of the cartilage rods formed in the intact
mandible at stage 37) are formed (Richman and Tickle, 1989;
Mina et al., submitted manuscript).
Together, these observations suggest that the medial
region is a putative functional region involved in regulating
directional outgrowth of the developing mandible.
Interestingly, high levels of expression of many of the evolu-
tionary conserved signaling factors and transcription factors
are detected in the epithelium and mesenchyme of the medi-
al region (see below). Direct evidence for the involvement of
some of these genes in the outgrowth of mandibular process-
es comes from abnormalities in the developing mandibles in
homozygous null mutant mice lacking these genes (knock-
out mice). Furthermore, some of these genes have been
shown to be candidate genes causing human craniofacial dis-
orders affecting mandibular growth and morphogenesis.
Transcription Factors
in Mandibular Morphogenesis
Msx GENES
The vertebrate Msx gene family was originally isolated by
homology to the Drosophila Msh gene (muscle segment
homeobox gene) (Hill et al., 1989; Robert et al., 1989; Davidson,
1995) and consists of 3 members that are not positioned in a
single gene cluster. Studies in vertebrate embryos showed
that Msxl and Msx2 are expressed throughout the embryo,
including the rostral region of the developing head, suggest-
ing their involvement in craniofacial development (Davidson,
1995; Maas and Bei, 1997, and references therein). The most
interesting aspect of the vertebrate Msx family is their com-
plementary or overlapping patterns of expression in different
regions of the embryo where morphogenesis has been shown
to be dependent on inductive epithelial-mesenchymal inter-
actions (Davidson, 1995; Maas and Bei, 1997).
At early stages of development, the expression of Msxl
and Msx2 in the mandibular arch is limited to the mes-
enchyme in the medial region and is excluded from the mes-
enchyme in the lateral region (MacKenzie et al., 1991, 1992;
Nishikawa et al., 1994; Mina et al., 1995; Barlow and Francis-
West, 1997). In addition to its expression in the medial region,
Msxl is also expressed in the mesenchyme surrounding the
hyomandibular cleft. Tissue recombination and bead implan-
tation studies indicate that the expression of Msx genes in the
developing mandible and other facial processes is dependent
on signals derived from the overlying epithelium (Takahashi
et al., 1991; Brown et al., 1993, 1997; Jowett et al., 1993; Mina et
al., 1995; Chen et al., 1996; Bei and Maas, 1998; Wang et al.,
1998b). In the developing mandible at early stages, the
expression of Msxl in the medial region is correlated with
areas undergoing expansion which contain highly prolifera-
tive and undifferentiated mesenchyme cells (Mina et al.,
1995). In Msxl-deficient mice, the medial part of the
HyonmandiMar
cueft
(B()
CDP
Ibone
Figure 1. (A) Line drawings illustrating a frontal view of the develop-
ing mandibular arch at early stages of development, indicating thle
anatomical landmarks and terminologies used in this paper. The
medial (proximal region) is indicated by a hatched pattern. The lat-
eral region is indicated by stippling, and an arrow indicates the
region of the hyomandibular clef. (B) Line drawing illustratinq a lat-
eral view of the developing mandibular arch at late stages o devel-
opment, indicating the anatomical structures referred to in this study.
The mandibular arch structures derived from the posterior midbrain,
RI and R2, are indicated by a dotted pattern, and the skeletal ele-
ments of the hyaid arch (second branchial arch) that are derived
from R4 are idicated by hatched pattern. AP, articular process; CDP,
condylar process; CP, coronoid process; 1, incus; M, malleus; S,
stapes; Sq, squamosal. The letters in parentheses indicate avian
names for structures homologous in mammals in the middle ear
region and have been adapted from Kontges and Lumsden (1996).
(A) articular; (Pq) pterygoquadrate; (Q) quadrate.
mandible is truncated (Satokata and Maas, 1994).
On the other hand, the domain of Msx2 expression in
the lower medial region of the mandibular mesenchyme
(Wedden, 1991; Mina et al., 1995; Shen et al., 1997), similar
to its expression in many organs, is correlated with areas of
apoptosis (Graham et al., 1996; Macias et al., 1996; Maden et
al., 1997; Marazzi et al., 1997; Ferrari et al., 1998; Takahashi
et al., 1998). Studies in the developing chick mandible also
suggest that Msx genes may be involved in delineating the
non-chondrogenic region at the midline region (symph-
ysis). This possibility is supported by observations that, in
contrast to control explants, explants from chick mandibu-
lar arches treated with Msx2 antisense oligonucleotides
formed cartilage in the medial region, resulting in the

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fusion of the 2 bilateral rods of cartilage at the midline
(Mina et al., 1996). More recently, it was shown that over-
expression of Msx2 also inhibits chondrogenesis in organ-
cultured mouse mandibles (Semba et al., 2000).
Although lack of Msxl does not appear to disturb the
mandibular symphysis, Msxl/Msx2 knock-outs display severe
abnormalities in the developing mandible (Satokata et al., 2000).
In humans, mutation of one copy of MSX1 results in sin-
gle-tooth agenesis, and specific point mutations in the homeo-
domain in one copy of MSX2 result in Boston-type craniosyn-
ostosis (reviewed by Cohen, 2000).
DLX GENES
The Dlx genes constitute a highly conserved family of homeo-
box-containing genes, homologous to the Drosophila Distal-less
(DlI) gene that is required for many processes, including the for-
mation of the mouth (Cohen et al., 1989). In the mouse, there are
at least 6 Dlx genes arranged as pairs which are convergently
transcribed (Dlxl and Dlx2, Dlx3 and DIX7, Dlx5 and Dlx6) (see
review by Kraus and Lufkin, 1999, and references therein).
Members of the Dlx family are expressed in the craniofacial
region in both ectoderm and mesenchyme (Davideau et al., 1999;
Kraus and Lufkin, 1999, and other references therein). Among
these, DIX2, DIX3, and Dlx5 are expressed at the junction of the
neural plate and surface ectoderm, suggesting that they may be
expressed by pre-migratory and migratory CNCC cells
(Robinson and Mahon, 1994; Qiu et al., 1997; Yang et al., 1998;
Acampora et al., 1999; Depew et al., 1999).
In situ hybridization studies on E9.5-E1O mouse embryos
showed that Dlxl and Dlx2 are expressed in the mesenchyme of
both proximal and distal regions of all branchial arches, while
DIX3, DIX5, and DIx6 are expressed predominantly in mes-
enchyme of the distal regions (reviewed by Kraus and Lufkin,
1999). For example, in the first branchial arch, Dlxl and Dlx2 are
expressed in the maxillary processes (the proximally located
component) as well as in the mandibular processes (the distally
located component) and Dlx3, Dlx5, and Dlx6 are expressed
only in the mandibular processes. In the developing mandible,
Dlx genes are expressed in a lateral-to-medial gradient.
Similarly, in the second (hyoid) arch, Dlxl and DIx2 are
expressed throughout the proximo-distal axis, while DX3, Dlx5,
and Dlx6 are expressed only in the more distal (close to the mid-
line) region of the second arch. These patterns of expression
suggested that Dlx genes play essential roles in proximo-distal
patterning of the branchial arches (Qiu et al., 1997).
This possibility is supported by phenotypic abnormalities in
mice lacking Dlxl, Dlx2, Dlxl/Dlx2, and DIx5. Mice lacking Dlxl,
Dlx2, and Dlxl/Dlx2 exhibited abnormalities similar, but not
identical, to those of maxillary and proximal (caudal) hyoid arch-
derived structures (Qiu et al., 1995, 1997; Thomas et al., 1997).
However, in these mutants, the skeletal components of the
mandibular arch and distal hyoid arch appeared to be normal
(Qiu et al., 1995, 1997; Thomas et al., 1997). On the other hand,
one of the most noticeable abnormalities in the mice homozy-
gous for a targeted deletion of Dlx5 is in the developing
mandible (Acampora et al., 1995; Depew et al., 1999). At early
stages (E13-E14), the mandibular arch and the Meckel's cartilage
of Dlx5 mutants are shorter than those in wild-type embryos
and exhibit abnormalities in the caudal region of Meckel's carti-
lage. At its caudal end, Meckel's cartilage in DIx5 mutants is
bifurcated and gives rise to an ectopic novel cartilage, which
becomes surrounded by bone later in development. At birth, the
280 Crit
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mandibles of Dlx5 mutant mice are shortened, lack the coronoid
process, and contain misshapen condylar and angular processes.
The phenotypic abnormalities in the mandibular arches of Dlx5
mutants suggest essential (non-redundant) roles of Dlx5 for
proper development of mandibular processes and the skeleton
of the caudal region of the mandibular arch. Further analyses of
the branchial arches in Dlx5 mutants indicate that the absence of
Dlx5 did not affect cell proliferation or apoptosis but expanded
the territory of the proliferating cells within the first branchial
arch (Acampora et al., 1999). In situ hybridization analysis indi-
cated decreases of Goosecoid (Gsc) expression in the mesenchyme
of the frontonasal processes and in the mandibular and hyoid
mesenchyme of the Dlx5 mutants (Depew et al., 1999).
Unlike other members of the Dlx family, Dlx5 and Dlx6 are
also expressed in developing bones, cartilage, and teeth, sug-
gesting that the Dlx5 and Dlx6 genes may play roles in the
multi-step process of skeletal differentiation and/or morpho-
genesis (Zhao et al., 1994a; Ferrari et al., 1995; Newberry et al.,
1998; Acampora et al., 1999; Davideau et al., 1999; Depew et al.,
1999). Dlx5 is also expressed at specific stages of osteoblast dif-
ferentiation in vitro and could repress osteocalcin gene expres-
sion (Ryoo et al., 1997). Interestingly, lack of Dlx5 results in
hypo-mineralization of calvaria (Acampora et al., 1999;
Depew et al., 1999) and expression of osteocalcin in the perios-
teum at birth (Acampora et al., 1999).
In humans, there is evidence implicating DLX5 and DLX6
genes as candidate genes for ectrodactyly (split hand/foot mal-
formations, SHFM1) (Scherer et al., 1994; Crackower et al., 1996).
Often, these patients also have cleft palate and deafness
(Ignatius et al., 1996). A frame-shift mutation in DLX3 is also
associated with taurodontism and enamel hypoplasia in
humans with Tricho-dento-osseous syndrome (Price et al., 1998).
OTX- GENES
Another family of evolutionary conserved homeodomain fac-
tors with critical regulatory roles in the determination of head
structures during development is the Otx genes, vertebrate
homologs to Orthodenticle in Drosophila (for review, see Bally-
Cuif and Boncinelli, 1997; Simeone, 1998; Acampora et al., 2000).
The overlapping patterns of expression of Otx and Emx genes in
the rostral region of the developing brain, together with func-
tional studies, have suggested that these genes, analogous to the
Hox code for hindbrain development, provide a combinatorial
code for rostral brain development (for reviews, see Bally-Cuif
and Boncinelli, 1997; Simeone, 1998; Acampora et al., 2000). Otx2
homozygous null mutants die early in embryogenesis and fail
to develop structures anterior to R3 (Acampora et al., 1995;
Matsuo et al., 1995; Ang et al., 1996). The phenotypic abnormali-
ties in Otxl homozygous mutants indicated its essential role in
the formation of cortex in the adult brain (Suda et al., 1996;
Acampora et al., 1996, 1998).
In addition to its expression in the rostral region of the
developing brain, Otx2 is also expressed in neuroectoderm
from the forebrain up to the mid-hind-brain isthmus (Matsuo
et al., 1995; Ang et al., 1996). Interestingly, Otx2 heterozygote
animals exhibit otocephaly and abnormalities in midbrain
crest derivatives of the first arch, suggesting a role for Otx2 in
pre-migratory CNCC originating from the midbrain region
(Matsuo et al., 1995). In Otx2+/- mice, the elements that were
shown to be derived solely from midbrain crest (distal
Meckel's cartilage, dentary, maxilla, and palatine) (Kontges
and Lumsden, 1996) are lacking or severely reduced, suggest-
Biol
Med
12(4):276-300 (2001)
ing that defects in these animals may be due to deficiencies in
CNNC derived from the midbrain region. However, in Otx2+/-
mice, the hindbrain-derived, first-arch elements (malleus,
incus, and pterygoid) are almost unaffected. Analysis of the
knock-in mice in which Otx2 was replaced with Otxl showed
that most Otx2 functions, including the Otx2 haploinsufficien-
cy causing the otocephalic phenotype, are replaceable with
those of Otxl (Suda et al., 1999).
GOOSECOID
Goosecoid (Gsc) encodes another evolutionary conserved paired-
like homeoprotein that acts as a transcription factor with essen-
tial roles in head development (for review, see Bally-Cuif and
Boncinelli, 1997). In vertebrates, Gsc is expressed transiently at
the rostral end of the developing brain and then re-appears at
E9.5-E10.5 in many sites, including the mesenchyme of the
branchial arches (Gaunt et al., 1993; Rivera-Perez et al., 1995,
1999; Yamada et al., 1995, 1997). In the mandibular arch, Gsc is
strongly expressed in the mesenchyme in the region of the
hyomandibular cleft.
Gsc null mutants die soon after birth, with rib cage malfor-
mations and multiple craniofacial defects, including abnormali-
ties in the mandibular arch and middle ear structures (Rivera-
Perez et al., 1995, 1999; Yamada et al., 1995,1997). In the mandibles
of these mutants, although the condylar process appears normal,
the coronoid and angular processes are severely reduced in size,
and the mandible is shortened in length. Furthermore, Meckel's
cartilage is not enclosed by the mandibular bones but is embed-
ded in a novel groove that extends along the entire length of the
mandible. Recent studies showed that abnormalities in the
mandible of Gsc null mutants are due to the absence of cells des-
tined to express Gsc in these mutants, suggesting the essential
roles of Gsc in the initial proliferation and/or survival of Gsc-
expressing cells (Rivera-Perez et al., 1999).
PITX GENES
Pitxl (Ptxl /POTX) and Pitx2 (RIEG, Otx2, Otlx2, Brxl, Arpl) are
2 members of a vertebrate multigene family with overlapping
and distinctive patterns of expression during embryogenesis (for
reviews, see Drouin et al., 1998; Gage et al., 1999; Meijlink et al.,
1999, and references therein). Pitxl was originally identified as a
factor interacting with the pituitary-specific transcription factor
Pit-i and POMC promoter (see reviews). Pitxl is expressed in the
pituitary gland throughout its development, in the lateral plate
mesoderm of the caudal half of the embryo which leads to its
expression exclusively in the hindlimb and not the forelimb, in
the first branchial arch and its derivatives, and in oral ectoderm
(Szeto et al., 1996; Lanctot et al., 1997; Meijlink et al., 1999). The pat-
terns of expression suggest that Pitxl is a critical transcription fac-
tor involved in specification of the hindlimb and development of
the pituitary gland and structures derived from the first branchial
arch. This possibility was supported by phenotypic abnormalities
in null mutants for Pitxl and mis-expression of Pitxl in the chick
wing bud (Lanctot et al., 1999; Logan and Tabin, 1999; Szeto et al.,
1999). Pitxl null mutants die immediately or shortly after birth
and are readily recognizable by their shortened mandibular arch.
These mutants exhibit abnormalities in the limb and in the deriv-
atives of the first branchial arch, including cleft palate, signifi-
cantly shortened tongue and mandible, a novel bone surround-
ing Meckel's cartilage, and lack of gonial bones (Lanctot et al.,
1999; Szeto et al., 1999). In situ hybridization analysis indicated
that the expression of several markers expressed early in the first
12(4) 276 300 (2001)
12(4)1:276-300 Crit
Crlf Rev Oral Biol Med
Rev Oral BiolMed
branchial arch-including Msxl, Msx2, Gsc, Shh, Bmp2/4, Wnt5a,
and Pitx2-is unaltered in Pitxl null mutant mice (Szeto et al.,
1999), suggesting that defects in the craniofacial structures simi-
lar to those observed in the hindlimb may be due to defects in
proliferation and/or abnormal chondrogenesis of mesenchymal
cells (Lanctot et al., 1999; Szeto et al., 1999). Interestingly, it has
been suggested that, in humans, mutant PITX1 alleles might be
responsible for a subset of patients with Treacher-Collins syn-
drome (Crawford et al., 1997).
Pitx2/RIEG was initially identified by positional cloning of the
gene responsible for Rieger Syndrome in humans (Semina et al.,
1996; Gage and Camper, 1999; Meijlink et al., 1999). This autosomal-
dominant haploinsufficiency syndrome is characterized by anteri-
or chamber ocular abnormalities and other features of various
degrees of severity, including dental hypodontia, umbilical abnor-
malities, cardiac defects, and mild craniofacial dysmorphism.
Pitx2 is expressed in many tissues during development,
including the craniofacial mesenchyme and epithelium of the
first and second branchial arches (see review by Meijlink et al.,
1999, and references therein). Experimental evidence indicated a
role for Pitx2 downstream of sonic hedgehog and nodal in a genetic
pathway regulating laterality of heart, gut, and other asymmetric
organs (see review by Meijlink et al., 1999, and references therein).
The direct role of Pitx2 in left-right determination and devel-
opment of other structures, including the development of the
mandibular and maxillary processes, is provided by abnormalities
in mice deficient for Pitx2 (Gage et al., 1999; Lin et al., 1999; Lu et al.,
1999b). Pitx2 mutant mice die at around E14-15 and exhibit lung
isomerization and defects in cardiac positioning and pituitary
development (Lin et al., 1999; Lu et al., 1999b). In addition, Pitx2 null
mutants exhibited cleft palate and abnormalities in the maxillary
and mandibular arches, including severely hypoplastic mandible
and arrested tooth buds (Lin et al., 1999; Lu et al., 1999b). In situ
hybridization analysis indicated the absence of Fgf8 and altered
domains of expression of Bmp4, Msxl, and Msx2 in the developing
facial processes in Pitx2 null mutants, suggesting that the facial
abnormalities may be mediated by changes in the patterns of
expression of these genes (Lin et al., 1999; Lu et al., 1999b).
PAX GENES
Pax genes encode a family of transcription factors characterized
by an evolutionary conserved paired box domain, a 384-bp DNA-
binding motif that regulates embryonic development by the con-
trol of target genes (for reviews, see Balling et al., 1996; Dahl et al.,
1997; Peters et al., 1998a; Mansouri et al., 1999, and references
therein). In mammals, the Pax gene family consists of 9 members
that, based on the presence or absence of structural motifs, are
divided into 4 subgroups (see above reviews). Genes within an
individual group show a very high degree of sequence similarity
within the paired domain and exhibit similar patterns of expres-
sion during embryogenesis (Dahl et al., 1997). The roles and func-
tions of Pax genes in embryonic development have been eluci-
dated by analysis of naturally occurring mouse mutants, targeted
inactivation of several Pax genes in mice, and human syndromes
(see above reviews). A common feature of all Pax mutants is
reduction in size and malformation or loss of specific organs.
Among all Pax genes, members of group I (Paxl, Pax 9),
group III (Pax3, Pax 7), and group IV (Pax 6) are expressed in the
developing facial processes (Peters et al., 1998a; Mansouri et al.,
1999). Analyses of various mutants have indicated essential roles
for Pax3, Pax6, and Pax7 in the development of CNCC-derived
structures in the upper face (for reviews, see Peters et al., 1998a;
281
281
Mansouri et al., 1999; Francis-West et al., 1998). However, in these
mutants, no abnormalities were observed in CNCC-derived
structures of the lower face. On the other hand, phenotypic
abnormalities in the Pax9 null mutant (Peters et al., 1998a,b) indi-
cate essential roles for Pax9 for the lower face. Pax9-deficient
mice lacked structures derived from pharyngeal pouches, such
as thymus and parathyroid glands. In the developing mandible,
in addition to abnormalities in the developing teeth, the alveolar
ridge and the coronoid process are absent in Pax9 null mutants.
PRX GENES
Prxl (previously called Mhox) and Prx2 (previously called S8)
are closely related members of the paired-related family of
homeobox genes that are co-expressed in a variety of sites,
including the craniofacial mesenchyme (see review by
Meijlink et al., 1999, and references therein).
Studies in developing mice and chickens indicate that Prxl
and Prx2 are co-expressed in the CNCC-derived mesenchyme
of the frontonasal process, in the mesenchyme of the first and
second branchial arches, and in the pre-osteogenic areas. In the
developing mandible, at early stages of development, high lev-
els of expression of both genes are detected in the mesenchyme
of the medial region. In addition to the medial region, Prxl is
also expressed in the cells around the first branchial groove. As
development proceeds, the expression of both genes is down-
regulated in the mandibular process but maintained in the
maxillary and nasal processes (Lu et al., 1999a).
Although Prx2 null mutant mice show no obvious cranio-
facial and skeletal abnormalities (ten Berge et al., 1998), Prxl null
mutant mice show defects in skeletal elements derived from the
maxillary processes and the caudal part of the mandibular
processes, including hypoplastic coronoid, condylar, and angu-
lar processes and malformed malleus (Martin et al., 1995). In
addition, Meckel's cartilage in Prxl mutants displayed abnor-
mal sigmoidal morphology (Martin et al., 1995). More recent
studies (Lu et al., 1999a) indicate that, in the Prxl null mutants,
cells fated to express Prxl are initially present in the regions that
give rise to the defective structures, but disappear later, sug-
gesting that Prxl product may be required for the maintenance
(proliferation and/or survival) of specific subpopulations of
CNCC-derived mesenchymal cells in the branchial arches.
In contrast to single mutants, Prxl/Prx2 double-knock-out
mice exhibited severe craniofacial abnormalities, including
pointed snout, the absence of external ears, and severely short-
ened lower jaws (ten Berge et al., 1998; Lu et al., 1999a; Meijlink et
al., 1999). Double-mutant mice also had novel phenotypes, inclu-
ding abnormalities in the medial region of the developing
mandibles, lower incisors, and Meckel's cartilage (ten Berge et
al., 1998; Lu et al., 1999a; Meijlink et al., 1999). Approximately 8%
of the newborn double-mutants generated by ten Berge et al.
(1998) exhibited clefts in the mandible and tongue, whereas the
mandibular processes of the double-mutant mice generated by
Lu et al. (1999a) lacked the midline symphysis and were fused. In
these double-mutants, either a single incisor arrested in the bud
stage or no incisors were present. The arrested incisor tooth buds
showed decreases in the expression of Pax9 and patched (Lu et al.,
1999a; Meijlink et al., 1999). Furthermore, in these double-
mutants, most of Meckel's cartilage was absent (ten Berge et al.,
1998; Lu et al., 1999a; Meijlink et al., 1999). In situ hybridization
analysis showed that, in double-mutants, Sox9 (a marker of pre-
chondrogenic mesenchyme of the branchial arches) was
expressed normally in the area of the pre-chondrogenic conden-
282CrtRvOaBilMd1()7630(0)
282 Crit Rev Oral Biol Med
sation of Meckel's cartilage, suggesting that, in the absence of
Prxl and Prx-2, the pre-chondrogenic condensation of Meckel's
cartilage is initiated but is not maintained (Lu et al., 1999a). The
phenotypic abnormalities in Prxl and Prxl/Prx2 mutants indi-
cate redundant but essential roles for Prxl and Prx2 in the sig-
naling network regulating epithelial-mesenchymal interactions
that promote outgrowth and skeletogenesis in the lower jaw.
Other members of the paired-related family of homeobox
genes in vertebrates include Alx3, Alx4, and Cartl (Meijlink et
al., 1999). These genes are also expressed in the distal part of
the mandibular arch (Zhao et al., 1994b; Qu et al., 1997a,b; ten
Berge et al., 1998). However, no phenotypic abnormalities in
the developing mandible have been reported in mice lacking
these genes (Zhao et al., 1994b; Qu et al., 1997a). It is possible
that, similar to Prxl and Prx2, the absence of abnormalities in
the mandibular arch in these knock-outs may be due to func-
tional redundancies.
BARX GENES
Barxl and Barx2 are 2 members of the vertebrate Bar class of
homeobox-containing genes homologous to Drosophila BarHl
and BarH2 (Higashijima et al., 1992a,b; Kojima et al., 1991). Three
mouse and 2 chick homologues of the Barx genes have been iso-
lated (Tissier-Seta et al., 1995; Jones et al., 1997; Saito et al., 1998;
Barlow et al., 1999; Smith and Tabin, 1999). Tissue distribution
studies showed that these genes are expressed in many sites,
including the facial processes (Tucker et al., 1989b; Tissier-Seta et
al., 1995; Jones et al., 1997; Mitsiadis et al., 1998b; Barlow et al.,
1999; Smith and Tabin, 1999). Studies in developing mice
showed that, in the developing maxilla and mandible, Barxl is
expressed in the mesenchyme (Tissier-Seta et al., 1995; Jones et al.,
1997; Mitsiadis et al., 1998b; Tucker et al., 1998b), and Barx2 is
expressed in the overlying epithelium (Jones et al., 1997).
However, studies in chick embryos indicate that, unlike in the
mouse, Barxl is expressed in both epithelium and mesenchyme
of the maxillary and mandibular processes (Barlow et al., 1999).
Furthermore, in chick embryos, Barx2b-which is 80% and 61%
identical to mouse Barx2 and Barxl, respectively-is expressed
prominently in myogenic populations in the craniofacial region,
in the mesoderm of the branchial arches, and in areas of the
forming bones (Smith and Tabin, 1999). At stage 25, Barx2b is
expressed at the tips of the outgrowing maxilla and mandible
but disappears by stage 30 (Smith and Tabin, 1999).
In both the chick and mouse, the mesenchymal domain of
Barxl expression is restricted to the lateral region, where Fg,f8
is expressed by the overlying epithelium (Tucker et al., 1998b;
Barlow et al., 1999). The expression of Barxl is excluded from
the mesenchyme in the medial region in which Bmp4 is
expressed in the overlying epithelium (Tucker et al., 1998b;
Barlow et al., 1999). The expression of Barxl is also excluded
from the central core corresponding to the regions forming
Meckel's cartilage and the muscle of the mandibular process
(Tissier-Seta et al., 1995; Barlow et al., 1999).
These patterns of expression suggest the involvement of
signals derived from the overlying epithelium (Fibroblast
growth factor-8 and Bone Morphogenetic proteins) in regulat-
ing the spatial patterns of Barxl expression in the mandibular
mesenchyme. In fact, studies in mouse mandibles indicate that
beads soaked in FGF8 can induce/maintain expression of Barxl
in the lateral mandibular mesenchyme (Tucker et al., 1998b). On
the other hand, beads soaked in BMP-4 inhibited expression of
Barxl in the lateral mandibular mesenchyme expression
12(4):276-300 (2001)
(Tucker et al., 1998b). Inhibition of BMP4 signaling by applica-
tion of Noggin protein during the early stages of mandibular
development resulted in ectopic expression of Barxl in the mes-
enchyme in the medial region (Tucker et al., 1998b). Similar
antagonistic interactions between BMP and FGF signaling also
restrict expression of Barxl to the maxillary mesenchyme in the
posterior region (Barlow et al., 1999), suggestive of the involve-
ment of Barxl in patterning of the facial processes.
HAND GENES
dHAND and eHAND (Cserjesi et al., 1995; Srivastava et al.,
1997; Thomas et al., 1998), 2 members of the bHLH (basic helix-
loop-helix) family of transcription factors, are co-expressed in
many regions, including the medial region of the developing
mandible. Deletion of the dHAND gene in mice resulted in
embryonic death at Ell secondary to cardiac failure and many
abnormalities, including severely hypoplastic first and second
branchial arches (Srivastava et al., 1997; Thomas et al., 1998).
Molecular analyses indicated that although Prxl, Dlx2, eHand,
and Msx2 were expressed at normal levels, Msxl expression
was not detectable in the medial region of the developing
mandible of E9.5 dHAND null mutants (Thomas et al., 1998).
These results also indicate that the hypoplastic mandible in the
dHand-/- mutant is not due to defects in the migration of neur-
al crest cells into the branchial arch and occurs secondary to
programmed cell death. The unchanged patterns of expression
of eHAND in the dHAND mutant suggest that eHAND is
unable to compensate fully for dHAND in the branchial arch
and that these genes may have some unique roles in the devel-
opment of the developing mandible (Thomas et al., 1998).
OTHER TRANSCRIPTION FACTORS
Other transcription factors that have been shown to have a role
in facial development are twist, ski, and AP-2, Tbx2, Tbx3, and
TCOF1/Freacle. Twist is expressed at high levels in the mes-
enchyme of the first branchial arch, and twist null mutants
exhibit abnormalities in branchial arch formation (Chen and
Behringer, 1995; Gitelman, 1997). AP-2 is expressed in the cra-
nial neural crest, and mice lacking Ap-2 had severe craniofacial
abnormalities (for review, see Morriss-Kay, 1996; also see Shen
et al., 1997; Zhang et al., 1996). However, in Ap-2 knock-out,
similar to ski knock-out, the mandibular arch skeleton is less
affected than the skeleton of the upper face (reviewed by
Morriss-Kay, 1996; Berk et al., 1997). Tbx2 and Tbx3 are 2 mem-
bers of the T-box family that are expressed in the mesenchyme
of the maxillary and mandibular processes (Chapman et al.,
1996; Gibson-Brown et al., 1998a,b; Isaac et al., 1998).
TCOF1/treacle was initially identified as the gene responsi-
ble for Treacher-Collins syndrome, the most common autoso-
mal-dominant inherited human mandibulofacial disorder
characterized by cleft palate and abnormalities in the ear and
of some skeletal structures derived from the first arch
(Gladwin et al., 1996; Dixon et al., 1997a,b; Wise et al., 1997;
Winokur and Shiang, 1998). The murine homologue of the
TCOF1 gene has been isolated (Dixon et al., 1997b; Paznekas et
al., 1997) and shown to be expressed in a wide variety of
embryonic and adult tissues of the mouse, including the
developing branchial arches at the time of critical morpho-
genetic events. The phenotypic abnormalities resulting from
the targeted inactivation of TCOF1 should reveal its role in
Treacher-Collins syndrome.
12(4) 276 300 (2001
12(4):276-300 2001))
Crit Rev C'ral
Grit Rev
Secreted Factors in Mandibular Morphogenesis
Secreted factors involved in mandibular outgrowth and mor-
phogenesis include members of the fibroblast growth factor
(FGFs), transforming growth factor beta (TGF3), and hedge-
hog families, Wnt genes, endothelin pathway, notch pathway,
and epidermal growth factor.
BONE MORPHOGENETIC PROTEINS (BMPS)
The transforming growth factor-beta (TGFI) superfamily is
composed of more than 30 members and has been divided into
several subgroups based on the extent of homology in the
mature ligand (for review, see Hogan, 1996; Wozney, 1998). The
largest subgroup constitutes the bone morphogenetic proteins
(BMPs). In mammals, there are at least 12 BMPs that have been
implicated in the development of almost all vertebrate organs
and are thought to be important mediators of inductive tissue
interactions during embryogenesis (Hogan, 1996; Wozney,
1998). The actions of TGF3s are mediated by the TGF3 recep-
tors. These serine/threonine receptor kinases fall into 2 classes,
type I and type II receptors (for review, see Kawabata et al.,
1998; Massague, 1998; Raftery and Sutherland, 1999).
Ligand binding leads to the formation of heterodimers of
these 2 receptor types, phosphorylation of the type I receptor by
the type II receptor, and subsequent propagation of the intracel-
lular signal. Seven type I or activin-like kinases (ALKs) and 5
type II receptors have been identified in vertebrates (Massague,
1998). Among these, BMPR1A (ALK-3), BMPR1B (ALK-6), and
ActR 1 (ALK-2) appear to be essential for BMP signals (Hogan,
1996; Massague, 1998). Different BMP ligands are capable of
activating common signaling pathways (for review, see
Massagu6, 1998). Ligands in the TGFI superfamily are active as
dimers, and the subunit composition of these molecules can
dramatically affect signaling activity. Although BMP het-
erodimers have yet to be identified in vivo, BMP heterodimers
made in vitro (BMP-4/BMP-7 and BMP-2/BMP-7) show more
potent biological activity than BMP homodimers (Aono et al.,
1995; Israel et al., 1996; Suzuki et al., 1997; Tsuji et al., 1998).
In addition, Smads are essential for TGFJ-related signal
transduction. Smad-1, Smad-5, and Smad-8 proteins (reviewed
by Kretzschmar and Massague, 1998; Miyazono, 1999) are
BMP receptor substrates, and Smad 2 and Smad 3 are sub-
strates for the related TGF3 and activin receptors in vertebrate
(Kretzschmar and Massague, 1998; Miyazono, 1999).
Tissue distribution studies showed that multiple Bmps
(Bmp-2, -4, -5, and -7) are expressed in the epithelium of the
mandibular arch (Francis-West et al., 1994; Wall and Hogan,
1995; Aberg et al., 1997; Barlow and Francis-West, 1997; Tucker
et al., 1998a; Wang et al., 1998b, 1999; Solloway and Robertson,
1999). At early stages, expression of Bmp-4 and Bmp-5 is
restricted to the epithelium of the medial region overlying the
Msxl and Msx2 expressing mesenchyme. In contrast to the
restricted patterns of expression of these Bmps, Bmp-2 and
Bmp-7 are expressed in a broader domain throughout the
epithelium of the branchial arches. Furthermore, similar to the
effects of mandibular epithelium in tissue recombination
experiments, agarose beads soaked in BMP-2, -4, or -7 are able
to maintain cell proliferation and Msx expression in the medi-
al mandibular mesenchyme (Barlow and Francis-West, 1997;
Wang et al., 1998b, 1999). When placed laterally, BMPs induced
cell proliferation and ectopic expression of Msx expression in
the lateral mandibular mesenchyme, where Msx genes are not
283
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283
normally expressed (Barlow and Francis-West, 1997; Wang et
al., 1998b, 1999). However, unlike the mandibular epithelium,
laterally or medially placed BMPs did not support the elonga-
tion of stage 23 chick mandibular mesenchyme and Meckel's
cartilage (Wang et al., 1999).
Further insight into the roles of BMPs in outgrowth and
morphogenesis of the developing mandible comes from stud-
ies in which beads soaked in BMPs were implanted in vivo at
different stages into different regions of the developing chick
mandible (Barlow and Francis-West, 1997; Ekanayake and Hall,
1997; Wang et al., 1999; Mina et al., submitted manuscript).
Although application of BMP-2, BMP-4, and BMP-7 for 7 to 10
days to the lateral region of a stage 23 chick mandible did not
affect the growth of the mandibular mesenchyme, it did induce
changes in the morphology and patterns of the skeletal ele-
ments in the caudal region (jaw joint) of mandibles on the
implanted side. In contrast to their effects at stage 23, in vivo
implantation of beads soaked in similar concentrations of
BMPs into the lateral region of stage 20/22 mandibles exerted
negative effects, resulting in reduced growth of the mandibular
process and complete loss of Meckel's cartilage and its articu-
lating elements on the implanted side (Barlow and Francis-
West, 1997; Ekanayake and Hall, 1997; Mina et al., submitted
manuscript). On the other hand, implantation of beads soaked
in BMPs into a more medial position (closer to the Msx-
expressing mesenchyme in the medial region) of stage 20/22
mandibles induced the formation of an additional outgrowth
from the body of Meckel's cartilage (Barlow and Francis-West,
1997, Mina et al., submitted manuscript).
Analysis of these data indicates that chick mandibular
mesenchyme from different regions (lateral vs. medial) at dif-
ferent stages (20 vs. 23) responds differently to BMPs. In con-
trast to the observations in the chick mandible, a recent study
showed that application of beads soaked in 100 ng/pL for 6
days to the lateral region of organ-cultured mouse mandibles
(E10) induced ectopic cartilage (Semba et al., 2000). However,
BMP beads applied to the medial region did not induce ectopic
cartilage (Semba et al., 2000).
The overlapping patterns of expression of Bmps in man-
dibular epithelium and the similarities between the effects of
BMPs on the mandibular mesenchyme suggest cooperative or
redundant roles for BMPs in signaling cascades mediating epi-
thelial-mesenchymal interactions regulating outgrowth and
morphogenesis of the developing mandible. However, the
roles of BMPs in the outgrowth of the mandibular arch remain
unclear.
On the one hand, the formation of an ectopic outgrowth
from the body of Meckel's cartilage after implantation of BMP-
beads to the medial region at stage 20/22 suggests that BMPs,
through their interactions with target genes such as Msxl and
Msx2, may be involved in the outgrowth and morphogenesis
of the tip of the developing mandible. The phenotypic abnor-
malities in the Bmp-5/Bmp-7 double-knock-out (Solloway and
Robertson, 1999) provide direct evidence for the involvement
of BMPs in outgrowth of the mandibular arch. Previous stud-
ies demonstrated that loss of either Bmp5 or Bmp7 has no
apparent effect on the development of the mandibular arch
(Kingsley et al., 1992; King et al., 1994; Dudley et al., 1995; Luo
et al., 1995; Dudley and Robertson, 1997). However, unlike the
single null mutants, Bmp-5/Bmp-7 compound mutants display
abnormalities in many organs in which Bmp5 and Bmp7 are co-
expressed, including the first branchial arch (Solloway and
284 (nt Rev C)ral
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Robertson, 1999). The first branchial arches in double-mutants
were smaller than those in the wild-type, and displayed
increased apoptosis in the surface ectoderm at the base of the
arch. Analysis of the patterns of expression of twist and AP2
showed defects in the mesodermally derived tissues, suggest-
ing that Bmp5 and Bmp7 are necessary for the maintenance/
survival of mesodermally derived cells in the first branchial
arch (Solloway and Robertson, 1999).
However, the negative effects of BMPs on the outgrowth
of the stage 20/22 chick mandibular arch, and their inability to
support outgrowth of stage 23 chick mandibular mesenchyme
(Barlow and Francis-West, 1997; Ekanayake and Hall, 1997;
Wang et al., 1999), suggest that BMPs, similar to their roles in
outgrowth of the developing limb (Pizette and Niswander,
1999), may in fact be involved in limiting the outgrowth of the
mandibular process, and that other growth factors may act
cooperatively with BMPs to promote the directed outgrowth
of the mandibular mesenchyme.
Furthermore, since all BMPs also induced cell death in the
lateral mandibular mesenchyme (Ekanayake and Hall, 1997;
Barlow and Francis-West, 1998; Wang et al., 1999), and BMP-2
inhibited chondrogenesis of the mandibular mesenchyme in
vitro (Ekanayake and Hall, 1997), it is possible that deficiencies
in the outgrowth of the mandibular arch may be either due to
cell death and/or secondary to the inhibitory effects of BMPs
on chondrogenesis and elongation of Meckel's cartilage that
are thought to regulate the growth of the mandible
(Ekanayake and Hall, 1997). Essential roles for members of the
TGF3 family in mandibular morphogenesis are also support-
ed by phenotypic abnormalities in Activin /3A and ActRcII
knock-outs (Matzuk et al., 1995a,b,c).
FAMILY OF FIBROBLAST GROWTH FACTORS (FGFs)
The FGFs consist of a family of 18 low-molecular-weight
polypeptide growth factors (for reviews, see Naski and Omitz,
1998; Szebenyi and Fallon, 1999). The actions of FGFs are medi-
ated by FGFRs, a family of 4 transmembrane receptors with lig-
and-induced tyrosine kinase activity (for reviews, see Szebenyi
and Fallon, 1999; Klint and Claesson-Welsh, 1999). The FGFRs
are similar in their overall structural organization and consist of
an extracellular ligand-binding domain, a single transmem-
brane domain, and an intracellular tyrosine kinase domain. The
extracellular ligand-binding domain contains 3 immuno-
globulin-like (Ig-like) domains, a heparin-binding domain, and
a stretch of 7 conserved acidic amino acids (acidic box).
Alternative splicing of the FGF receptor mRNAs generates sev-
eral different variants of the receptors that are either soluble or
membrane-anchored and have different ligand-binding proper-
ties. The functionally critical splicing event results in the differ-
ential exon usage in the region coding the carboxyl-terminal
half of the third Ig-like domain. The third Ig-like domain in all
but FGFR-4 is encoded by 3 exons designated as A-C that are
alternatively spliced. Receptors containing the region encoded
by exon A are secreted and may interfere with FGF signal trans-
duction. Receptors containing the region encoded by exon B or
C differ in their ligand-binding properties and tissue distribu-
tion. In general, the B form appears to be expressed in epithelial
cells and the C form in mesenchymal cells.
There is compelling experimental evidence that interac-
tions between the members of the FGF and FGFR families
have important roles in mediating epithelial-mesenchymal
interactions during embryogenesis (for reviews, see Burke et
(2001)
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al., 1998; Martin, 1998; Hogan, 1999).
Similar to the Bmps, several Fgfs, including Fgf-2, Fgf-4, Fgf-
8, and Fgf-9, are expressed in facial processes and the mandibu-
lar arch (Niswander and Martin, 1992; Heikinheimo et al., 1994;
Crossley and Martin, 1995; Mahmood et al., 1995; Wall and
Hogan, 1995; Barlow and Francis-West, 1997; Richman et al.,
1997; McGonnell et al., 1998; Kettunen and Thesleff, 1998;
Kettunen et al., 1998; Colvin et al., 1999). Whereas Fg,f-2 and Fgf-
9 are expressed throughout the mandibular epithelium, Fgf-8
and Fgf-4 are expressed in restricted regions. Although Fgf-1,
Fgf-5, Fgf-12, and Fgf-10 are expressed in mouse craniofacial
mesenchyme, detailed expression patterns for these genes have
not yet been reported (Hebert et al., 1990; Haub and Goldfarb,
1991; Hartung et al., 1997). Fgf-13, Fgf-17, and Fgf-18 are not
expressed in the facial processes of mouse embryos (Hartung et
al., 1997; Maruoka et al., 1998).
Fgr genes are also expressed in the developing mandible
(Orr-Urtreger et al., 1991, 1993; Peters et al., 1992, 1993; Wilke et
al., 1997; Kettunen et al., 1998). Fgfr2b (KGFR) is expressed in the
mandibular epithelium, and Fgfrlc, Fg2c, both isoforms of
Fgfr3, and Fgfr4 in the mandibular mesenchyme. At early stages,
both isoforms of Fffrl (flg, cek-1), Fgfr3 (cek-2), and Fgr2c (bek,
cek-3) are expressed uniformly throughout the mandibular mes-
enchyme. However, at later stages of development, Fgfr-2c and
Fgfr3 are no longer expressed uniformly and are expressed in
restricted regions (Orr-Urtreger et al., 1991; Wilke et al., 1997;
Mina et al., submitted manuscript). High levels of expression of
both genes are seen in the mesenchyme in the lower border of
the mandible that surrounds the hyomandibular cleft.
Expression of Fgfr2c but not Fgfr3 also extends to the mes-
enchyme in the medial region. At the time of initiation of chon-
drogenesis, Fgfr2c and Ffr-3 are expressed in the condensing
mesenchyme of Meckel's cartilage, and Fe4 is expressed in the
developing muscles (Kettunen et al., 1998).
Similar to BMPs, FGFs can also mimic many effects of
mandibular epithelium on the mandibular mesenchyme in
vitro. In the absence of mandibular epithelium, FGF-2 and
FGF-4 can maintain expression of Msxi and Msx2 in the mes-
enchyme of the medial region (Mina et al., 1999, submitted
manuscript). In vitro studies indicate that, in contrast to BMPs,
in the absence of mandibular epithelium, locally applied FGFs
can partially or completely support outgrowth of the
mandibular mesenchyme and Meckel's cartilage (Richman et
al., 1997; Mina et al., 1999, submitted manuscript). Application
of beads soaked in recombinant FGF-2 and FGF-4 to the medi-
al (but not lateral) region of isolated stage 24 mandibular mes-
enchyme stimulated increases in the length of Meckel's carti-
lage. The cartilage rods formed in FGF-treated grafts were 60%
or almost the same length as those formed in homotypic
recombinations. Taken together, these observations suggest
essential roles for FGFs/FGFR2c (the only receptor with high
levels of expression in the mesenchyme in this region) in the
mediation of epithelial-mesenchymal interactions in the medi-
al region of the developing mandible.
Based on their patterns of expression in vivo, FGF-2, FGF-4,
and FGF-9 (but not FGF-8) are likely endogenous candidates for
mediating the growth-promoting effects of mandibular epithe-
lium from the medial region. Furthermore, it has been shown
that Fgfr2c binds with high affinity and transmits mitogenic sig-
nals from FGF-1, -2, -4, and -9 (for review, see Szebenyi and
Fallon, 1999).
However, the phenotypes of Fgfr2 null mutants suggest
2001)
12)4) 276-300 (2001)
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that Fgfr2c may not have an essential role in the initial forma-
tion of mandibular processes (Arman et al., 1998; Xu et al.,
1998). Despite the lack of detailed descriptions of the craniofa-
cial structure, apparently a normal mandibular process forms
in E10 mice that express a hypomorphic allele of Fgfr2 (Xu et al.,
1998). On the other hand, transgenic expression of the extracel-
lular ligand-binding domain of Fgfr2b (KGER) (the isoform that
is expressed in the epithelium of facial processes), which acts as
a dominant-negative receptor of FGFR2b, resulted in the devel-
opment of smaller faces, suggestive of the involvement of
FGFR2b in craniofacial development (Celli et al., 1998).
Until recently, the roles of FGF-8 in mandibular morphogen-
esis were not fully understood. Previous studies in mouse
embryos indicate that Fgf8 is expressed in regions of the embryos
where epithelium is known to direct outgrowth (Crossley and
Martin, 1995). Studies in the chick face also showed correlations
of the patterns of expression of Fgf-8 in the epithelium of devel-
oping facial processes with areas where mesenchyme undergoes
the greatest expansion and which express high levels of Msxl
(McGonnell et al., 1998). However, unlike its patterns of expres-
sion in other facial processes, Fgf8 is expressed in the epithelium
in the lateral but not the medial region of the developing
mandible. Studies in mice suggest that FGF-8 may be involved in
the establishment of the oral-aboral polarity of the developing
mandible (Tucker et al., 1999). A recent study in which Fgf8 was
inactivated in the epithelium of the first branchial arch of trans-
genic mice by means of Cre/lox technology has provided direct
evidence for roles of FGF8 in the development and morphogene-
sis of the lateral (but not medial) regions of the developing
mandible (Trumpp et al., 1999). This study provided direct evi-
dence that the mandibular process is specified by at least 2 dis-
tinct functional regions: 2 large lateral (proximal) regions, where
morphogenesis is dependent on and controlled by FGF-8, and a
small medial region, in which morphogenesis is independent of
FGF-8 and is controlled by other signaling molecules. In these
mutants, the symphysial portion of Meckel's cartilage and the
mandibular incisors and their associated bones were invariably
present. On the other hand, with the exception of the presence of
a vestigial malleus, the skeletal elements in the lateral regions of
the developing mandible were absent (Trumpp et al., 1999).
Molecular analyses indicated that Fgf8;Nes-cre mutants fail to
express Lhx6 and, for the most part, Barxl. In addition, although
ET-1 expression was not detected in most of the epithelium of the
developing mandible, it was detected in the epithelium of the
hyomandibular cleft. In these mutants, expression of Gsc was
seen only in a restricted region in which cells expressing Barxl
and ET-1 were found. These observations indicate that expression
of Lhx6, Barxl, Etl, and Gsc in the mandibular arch are depen-
dent, directly or indirectly, on FGF8 signaling. However, expres-
sion of Dlxl, Dlx2, Dlx5, and Lhx7 in the mesenchyme of the lat-
eral regions and Pitxl in the mandibular epithelium was
unchanged. Although Pax9 was not detected in the molar region
of the mutant embryos, it was detected in the regions of pre-
sumptive mandibular incisors. The absence of abnormalities in
the small medial region and the unaltered patterns of expression
of Bmp-4, dHand, eHand, Msxl, and Msx2 (genes that are normal-
ly expressed in the medial region) indicate that morphogenesis of
the small medial region depends on signals other than FGF8
(Trumpp et al., 1999). The similarities between the phenotypic
abnormalities in the Fgf8;Nes-cre mutants and abnormalities in
the human first-arch syndromes characterized by agnathia/
micrognathia raise the possibility that mutations in Fgf8 or in
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genes directly upstream or downstream of it might cause some of
these human syndromes (Trumpp et al., 1999).
Roles for FGFs and FGFRs in craniofacial development are
also supported by abnormalities in human syndromes associ-
ated with mutations in FGFRs (for reviews, see Muenke and
Schell, 1995; Yamaguchi and Rossant, 1995; Webster and
Donoghue, 1997; Wilkie, 1997; Naski and Ornitz, 1998). In gen-
eral, the disorders caused by mutations in FGFRs can be clas-
sified into 2 groups: (1) dwarfing conditions and (2) conditions
with craniosynostosis. While dwarfing conditions have been
shown to be associated with mutations in FGFR3, the second
group has been associated with mutations in FGFR2.
HEDGEHOG FAMILY
The Hedgehog (Hh) family of secreted proteins is involved in
the regulation of a wide range of developmental processes
during vertebrate development (for reviews, see Ingham,
1995; Hammerschmidt et al., 1997).
There are 3 known mammalian homologs of Hh: sonic hedge-
hog (Shh), Indian hedgehog (11h), and desert hedgehog (Dhh).
Among these, Shh has been shown to be expressed in numerous
sites of epithelial-mesenchymal interactions (Bitgood and
McMahon, 1995) and is thought to play an important role in many
developmental processes, including dorso-ventral patterning of
the neural tube and somites, anterio-posterior patterning of the
limb buds, morphogenesis of the hindgut, and craniofacial devel-
opment (reviewed by Bumcrot and McMahon, 1995; Bronner-
Fraser and Fraser, 1997; Weed et al., 1997; Schneider et al., 1999).
Most of the details of components of the Hh signaling
pathway have been elucidated from studies in Drosophila. A
similar signaling cascade is also present in vertebrates (for
reviews, see Alcedo and Noll, 1997; Ingham, 1998; Johnson
and Scott, 1998; Murone et al., 1999). These studies indicate
that Hh signaling is transduced by a heterodimeric receptor
complex that includes 2 transmembrane proteins: Smoothened
(Smo), a member of the Frizzled (Fz) family of receptors, and
Patched (Ptc). Hedgehog proteins bind specifically with high
affinity to Ptc without any direct involvement of Smo.
However, Smo, which does not directly interact with Shh, is
the signaling subunit of the receptor complex.
The current model for the Hh signaling pathway sug-
gests that, in the absence of ligand, the Hh receptor complex
is inactive, because Ptc interacts with Smo, resulting in
repression of Smo signal-transducing activity. Binding of Hh
to Ptc causes a conformation change that alleviates this
repressive interaction and results in the release and activa-
tion of Smo from the complex (Murone et al., 1999). After its
activation, Smo activates the transcription of downstream
target genes via cubitus interruptus (Ci), a zinc-finger-con-
taining protein, which appears to act as the final component
in the Hh signaling pathway (Murone et al., 1999).
Homologous to Ci, in vertebrates there are 3 Gli genes: Gli
(Glil), Gli2, and Gli3 (for review, see Ingham, 1998).
The possible involvement of this pathway in mandibular
morphogenesis is suggested by the patterns of expression of
genes in the Hh signaling pathway in the developing mouse
mandible. Shh is expressed at low levels in the endoderm of
the hyomandibular cleft (Platt et al., 1997; Hardcastle et al.,
1998). At early stages, Gli2, Gli3, and Smo are expressed
throughout the mandibular mesenchyme (Walterhouse et al.,
1993; Hui et al., 1994; Hardcastle et al., 1998). Similar to their
patterns of expression in other tissues, Glil and Ptc are
286 Crit Rev
Crit Rev Oral Biol Med
expressed in the developing mandible in similar domains in
regions near the cells expressing Shh (Platt et al., 1997).
The phenotypic abnormalities in Gli2, Gli-3, and
Gli2/Gli3 double-mutants indicated that Gli2 and Gli3 have
specific as well as redundant functions in the development
of mandibular arch. Both Gli2 and Gli3 mutant mice die at
birth and have severe skeletal and facial abnormalities
(Vortkamp et al., 1991, 1992; Hui and Joyner, 1993; Mo et al.,
1997). Loss of Gli2 is associated with truncation of the prox-
imal regions of the maxilla and mandible, resulting in the
absence of maxillary and mandibular incisors (Mo et al.,
1997). On the other hand, Gli3 mutants displayed abnormal-
ities in the maxillary region but not in the mandible (Hui et
al., 1994; Mo et al., 1997). In contrast to the single mutant,
mandibles of Gli2-/Gli2;Gli3 -/+ mutants were severely short-
ened and had hypoplastic coronoid, condylar, angular, and
alveolar bones.
The roles of Shh in craniofacial development have also
been elucidated in more recent studies in chick embryos
(Helms et al., 1997; Ahlgren and Bronner-Fraser, 1999; Hu and
Helms, 1999). Ahlgren and Bronner-Fraser (1999) showed that
inhibition of Shh signaling after neural tube closure resulted in
a transient decrease in neural tube cell proliferation and an
extensive increase in cell death in the neural tube and neural
crest. These changes, in turn, resulted in decreased head size
and loss of branchial arch structures. The phenotypes
observed after reduction of Shh in chick embryos are similar to
those observed after cranial neural crest ablation.
Mice homozygous for a disruption of Shh die at birth with
defects in all tissues, including holoprosencephaly, which is
characterized by abnormal forebrain and facial development.
Although the branchial arches appear to be normal at E9.5, cra-
niofacial bones are severely affected in Shh null mutants and
are almost entirely absent (Chiang et al., 1996). Mice homozy-
gous for the Ptc deletion also die during embryogenesis and
have open and overgrown neural tubes (Goodrich et al., 1997).
Facial abnormalities in human syndromes associated with
mutations in the components of the Hh signaling pathway also
support the involvement of the Hh signaling pathway in
mandibular and craniofacial morphogenesis (for reviews, see
Roessler et al., 1996; Ming and Muenke, 1998; Ming et al., 1998).
In humans, haplo-insufficiency of Ptc results in Gorlin syn-
drome. Partial loss of Gli3 function results in Greig cephalopoly-
syndactyly syndrome (GCPS), and a point mutation in Shh
results in autosomal-dominant holoprosencephaly, one of the
most common developmental defects of the forebrain and face.
WNT GENES
The Wnt gene family also constitutes another group of signal-
ing factors with important roles in many developmental
processes, including specifications of the dorso-ventral axis of
the developing limb (for reviews, see Johnson and Tabin, 1997;
Sokol, 1999) and modulation of chondrocyte differentiation in
the limb and face (Rudnicki and Brown, 1997; Kawakami et al.,
1999). This family consists of at least 15 developmentally reg-
ulated genes that encode cysteine-rich glycoproteins (Cadigan
and Nusse, 1997; Wodarz and Nusse, 1988).
Studies in mice indicate that Wnt5a, -1Qa, -1Ob, and -11 (but
not Wnt-1, and Wnt -2) are expressed in the facial mesenchyme
(Gavin et al., 1990; Dealy et al., 1993; Parr and McMahon, 1995;
Takada et al.., 1994; Tanda et al., 1995; Christiansen et al., 1995;
Wang and Shackleford, 1996; Yamaguchi et al., 1999).
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Expression of Wnt5a in the facial processes is best-document-
ed. Wnt5a expression is first detected at the time of formation
of the facial processes (Takada et al., 1994; Yamaguchi et al.,
1999), and later in a graded fashion (being highest at the tips
of the processes) in the frontonasal mass, and in maxillary and
mandibular processes (Yamaguchi et al., 1999). The graded
pattern of expression of Wnt-5a in the facial processes is simi-
lar to its pattern of expression in the developing limb, in which
high levels of Wnt5a are expressed in the Apical Ectodermal
Ridge (AER) and mesenchymal cells underneath the AER
(Dealy et al., 1993; Kawakami et al., 1999; Yamaguchi et al.,
1999). These patterns of expression suggest a conserved role
for Wnt5a in the morphogenesis of many structures whose
development requires extension from the primary body axis
(Yamaguchi et al., 1999). This possibility is supported by phe-
notypes of the homozygous Wnt5a null mutants which have
morphological defects in outgrowing tissues, including limb,
maxilla, and mandible (Yamaguchi et al., 1999).
ENDOTHELIN PATHWAY
Ligands of the endothelin pathway consist of 3 structurally
related small peptide ligands, called ET-1, ET-2, and ET-3, that
bind to 2 of the G-protein-coupled endothelin receptors, the
endothelin-A receptor (ETA) and the endothelin-B receptor
(ETB) (for a recent review, see Kurihara et al., 1999, and refer-
ences therein). The endothelin pathway also includes 2
isozymes of endothelin-converting enzyme (ECE), ECE-1 and
ECE-2 (Kurihara et al., 1999, and references therein).
In situ hybridization analysis indicates that the ETA recep-
tor is expressed by migratory CNNC originating from the hind-
brain and ECE-1 in the head mesenchyme (reviewed by
Kurihara et al., 1999). In the branchial arches, the spatial pat-
terns of ET-1 and ETA expression are complementary, in that
ETA is expressed by the CNCC-derived mesenchymal cells of
branchial arches and the ET-1 ligand is expressed in the surface
epithelium of arches 1, 2, and 3 and in the paraxial mesodermal
core of the first branchial arch (Barni et al., 1998; Clouthier et al.,
1998, 2000; Kurihara et al., 1999). ECE-1 is expressed in both the
mesenchyme and surface epithelium of branchial arches
(Yanagisawa et al., 1998).
Several components of the endothelin pathway have been
inactivated in mice by homologous recombination (Baynash et
al., 1994; Hosoda et al., 1994; Kurihara et al., 1994; Clouthier et al.,
1998; Yanagisawa et al., 1998). These studies indicate that 2
endothelin-mediated cell-cell signaling pathways (ET-1/ETA
and ET-3/ETB) are independently involved in the development
of 4 distinct groups of NCC-derived tissues (for review, see
Kurihara et al., 1999). Mice deficient in ET-3 and ETB exhibited
similar abnormalities and revealed essential roles for ET-3/ETB-
mediated signaling in the development of epidermal and
choroidal melanocytes and enteric neurons (Baynash et al., 1994;
Hosoda et al., 1994). On the other hand, mice deficient in ETA
and ET-1 showed striking craniofacial and cardiovascular
abnormalities that were virtually identical to each other and to
those observed in mice deficient for ECE-1 (Kurihara et al., 1994,
1999; Clouthier et al., 1998, 2000; Yanagisawa et al., 1998).
In ET-1, ETA, and ECE-1 null mutants, numerous struc-
tures derived from branchial arches 1, 2, and 3 were affected
(Kurihara et al., 1994, 1999; Clouthier et al., 1998; Yanagisawa et
al., 1998). The most striking phenotype in all 3 mutants was the
poorly formed mandible that sometimes lacked the midline
fusion. The mutant mandibles also exhibited complete absence
112(4) 276-300 ((200h
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of Meckel's cartilage. The mandibular bone was hypoplastic
and highly disorganized, with abnormal articulation to the
maxillary arch and the base of the skull. Furthermore, the
tongue was severely hypoplastic. Teeth were intact but embed-
ded in loose mesenchyme instead of mandibular bone.
The defects observed in ETA-deficient embryos suggest that
the mutation disrupts the development of CNCC arising from
the posterior midbrain as well as Ri, R2, R4, and R6. This sug-
gests that ET-1 /ETA signaling may be required for cranial NCC
migration and/or proliferation (Clouthier et al., 1998;
Yanagisawa et al., 1998). While migration of neural crest cells into
the arches of ETA-mutant embryos appears unaffected, the
absence of ETA signaling appears to affect the proliferation/dif-
ferentiation of post-migratory crest cells and some apoptosis in
the branchial arch mesenchyme. In situ hybridization analysis of
E9.5 and E10.5 ETA-/- null mutants indicated either the absence
or a significant reduction in the expression of several transcrip-
tion factors, including Gsc, Dlx2, Dlx3, dHAND, and eHAND in
the post-migratory ectomesenchymal cells of the first and second
branchial arches. Interestingly, while Barxl expression was
absent in the second branchial arch, its expression was unaffect-
ed in the first branchial arch of E10.5 ETA-/- embryos (Clouthier
et al., 1998, 2000). On the other hand, in these mutants, the
expression of other transcription factors-including Prxl, Hoxa2,
CRABP1, and Ufdl-was unaffected, suggesting that these fac-
tors either act upstream of ETA signaling or belong to different
or parallel genetic pathways involved in mandibular develop-
ment (Clouthier et al., 2000). Analyses of ET1-/- null mutants
showed that dHand and eHand are down-regulated in the
branchial arches of these mutants (Thomas et al., 1998).
The phenotype of ETA null mutant mice resembles the
human conditions collectively termed CATCH 22 or velocar-
diofacial syndrome, and includes severe craniofacial deformi-
ties and defects in the cardiovascular outflow tract (Wilson et
al., 1993; Clouthier et al., 1998). However, the patterns of expres-
sion of Ufdl, a gene shown to be deleted in CATCH 22 patients,
was unchanged in ETA-/- null mutants (Clouthier et al., 2000).
NOTCH SIGNALING PATHWAY
The Notch signaling pathway is an evolutionary conserved
pathway with essential roles in mammalian embryonic devel-
opment (for reviews, see Gridley, 1997; Greenwald, 1998;
Weinmaster, 1998; Artavanis-Tsakonas et al., 1999). This path-
way is characterized by interactions of the receptor Notch with
the Delta and Serrate/Jagged families of ligands. Studies in
Drosophila showed that the Notch gene encodes large trans-
membrane receptors that interact with membrane-bound lig-
ands encoded by Delta and Serrate genes (Greenwald, 1998;
Artavanis-Tsakonas et al., 1999; Gray et al., 1999). Genes
homologous to members of the Notch signaling pathway have
been cloned in mammals and include the 4 genes encoding the
ligands: Jagged-1 (Serrate-1), Jagged 2 (Serrate 2), Delta-like 1
(Dlll), and Delta-like 3 (D113) (Bettenhausen et al., 1995;
Lindsell et al., 1995; Shawber et al., 1996; Dunwoodie et al.,
1997; Gray et al., 1999).
The importance of the Notch signaling pathway in develop-
ment is also implicated by the findings that 3 human diseases-
T-cell acute lymphoblastic leukemia/lymphoma, the Alagille
syndrome, and CADASIL (Cerebral Autosomal Dominant
Arteriopathy with subcortical Infarcts and Leukoencepha-
lopathy)-are associated with mutations in genes encoding
Notch-1, Jagged-1, and Notch-3, respectively (for reviews, see
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Gridley, 1997; Joutel and Tournier-Lasserve, 1998).
Among these, Alagille syndrome exhibits autosomal-domi-
nant inheritance and is one of the major forms of chronic liver
disease in childhood (Krantz et al., 1997). Accompanying features
of this syndrome include a characteristic facial appearance,
including a pointed mandible (Krantz et al., 1997). In the majori-
ty of patients, mutations in JAG1 result in the formation of trun-
cated protein, suggesting that Alagille syndrome is due to the
haploinsufficiency of JAG1 (Li et al., 1997; Oda et al., 1997; Krantz
et al., 1998; Yuan et al., 1998). However, studies in Drosophila sup-
port alternative dominant-negative roles for truncated forms of
Serrate proteins (Hukriede et al., 1997; Sun and Artavanis-
Tsakonas, 1997).
Studies in developing mice indicate that, whereas Jaggedl
(Serratel) is expressed in the maxillary and mandibular mes-
enchyme, Jagged2 (similar to Notchl) is expressed in the epithe-
lium of the branchial arches (Mitsiadis et al., 1995, 1997, 1998a;
Shawber et al., 1996; Sidow et al., 1997; Valsecchi et al., 1997; Jiang
et al., 1998). Mice homozygous for Jaggedl null mutations die
early in embryogenesis, and mice heterozygous for the Jaggedl
null allele exhibit abnormalities in the eye but not in the cranio-
facial region (Xue et al., 1999). Null mutants of Jagged2 (Jiang et
al., 1998) and expression of a hypomorphic allele of Jagged2
(Sidow et al., 1997) exhibit abnormalities in many Jagged2
(Serrate2)-expressing tissues, including defects in the craniofa-
cial region. However, abnormalities in the developing mandible
have not been reported.
EPIDERMAL GROWTH FACTOR (EGF)
The EGF-superfamily has been implicated in inductive interac-
tions in the first branchial arch, including those involved in
odontogenesis, bone and cartilage formation, and outgrowth of
the maxillary and mandibular arches. EGF and EGFR are
expressed in the developing mandible (Partanen et al., 1985;
Partanen and Thesleff, 1987; Snead et al., 1989; Kronmiller et al.,
1991; Shum et al., 1993). Treatment of explants of mouse
mandibular arches with EGF antisense oligonucleotides results
in the formation of bilaterally symmetrical Fussilli-formed dys-
morphic Meckel's cartilage, and inhibition of EGFR kinase
activity caused overall retardation in mandibular growth and
the complete absence of Meckel's cartilage (Shum et al., 1993). In
addition, it has been shown that EGF can substitute for the mito-
genic effects of mandibular epithelium on the underlying mes-
enchyme, suggesting a role for EGF and EGF-like protein in
epithelial-mesenchymal interaction involved in the outgrowth
of the developing mandible (Coffin-Collins and Hall, 1989; Hall
and Coffin-Collins, 1990). A recent study by Nonaka et al. (1999)
showed that exogenous EGF inhibited the BMP-4-induced
ectopic cartilage in organ-cultured mouse and chick mandibles.
It has also been shown that application of EGF-releasing
beads to mouse and chick mandibular mesenchyme induces a
translucent zone and extensive cell proliferation, and prevents
apoptosis (Vaahtokari et al., 1996; Wang et a!., 1998a, 1999).
Although EGF beads are not able to induce expression of Msxl,
Msx2, and Bmp4 in these mesenchymes, they were able to mod-
ulate the effects of BMPs on the expression of Msxl and Msx2
(Wang et al., 1998a, 1999). These observations suggest the pos-
sibility that EGF, by modulating the effects of BMPs on the
expression of Msxl and Msx2 in mandibular mesenchyme, may
be involved in mediating inductive interactions during the ini-
tiation phase of odontogenesis and during outgrowth of the
mandibular arch (Wang et al., 1999). This possibility is support-
288288CrtRvOaBilMd1(:7600(01 Crit Rev Oral Biol Med
ed by observations showing that mitogenic growth factors that
signal through the Erk-MAP kinases pathway, such as EGF, can
antagonize the BMP-induced responses by phosphorylation of
Smad-1 at multiple serines in the linker region (Kretzschmar et
al., 1997). Although Erk-mediated phosphorylation of Smad-1
does not directly interfere with the association of Smad-1 with
Smad-4, it prevents the translocation of Smad-I to the nucleus,
thereby inhibiting BMP-4 signal transduction (Kretzschmar et
al., 1997; Attisano and Wrana, 1998; Kretzschmar and
Massague, 1998). This possibility is supported by the recent
observations in micromass cultures (Nonaka et al., 1999) that
showed that the addition of EGF inhibited BMP-induced
Smadl nuclear translocation and accumulation.
The roles of EGF/EGFrs in craniofacial morphogenesis
were confirmed by craniofacial abnormalities in Egfr null
mutants (Miettinen et al., 1999). The abnormalities in Egfr null
mutants included a pointed snout, high incidence of cleft
palate, short mandibles, and poorly developed Meckel's carti-
lage (Miettinen et al., 1999). Furthermore, in Egfr null mutants,
there were decreases in the amount of matrix metallopro-
teinases (MMPs). Inactivation of MMPs in explanted wild-
type mandibles resulted in defects similar to those in Egfr null
mutants, suggesting that MMPs may act downstream of the
EGF/EGFR signaling pathway (Miettinen et al., 1999).
PLATELET-DERIVED GROWTH FACTORS (PDGFs)
Mice homozygous for a targeted deletion of the PDGFot recep-
tor that is expressed by CNNC-derived mesenchyme of the
branchial arches (Orr-Urterger and Lonai, 1992; Schatteman et
al., 1992; Chai et al., 1998) and can bind to all PDGF (for
review, see Westermark et al., 1989) exhibit significant defects
in the skull, most noticeable in the nasal bone (Soriano, 1997).
However, PDGFRot mutants have relatively normal maxillary
and mandibular arches (Soriano, 1997). Increased apoptosis
observed along the pathway of CNNC migration and mes-
enchyme of the branchial arches in these mutants suggested
roles for PDGFs and PDGFRo- in the survival of non-neuronal
derivatives of CNNC (Soriano, 1997).
MERGING OF THE TWO MANDIBULAR PROCESSES
Another essential feature of facial morphogenesis is the union
of the facial prominences/processes that occurs either by
fusion or by merging of the adjacent processes (for review, see
Ten Cate, 1998). In general, the process of fusion seen in the
palatal processes involves initial contact and subsequent
fusion of the surface epithelia of the neighboring processes,
removal of the epithelial cells from the fusion region, and
merging of the mesenchymal cells of the neighboring process-
es (reviewed by Shuler, 1995, and references therein). On the
other hand, union of other facial processes, such as the
mandibular processes, appears to involve the merger of 2
incompletely separated processes. During this merger, the fur-
row between these processes is eliminated (Ten Cate, 1998).
The mechanisms for removal of the epithelial cells that results
in mesenchymal continuity in facial processes are thought to
be mediated by either programmed cell death (apoptosis),
epithelial-mesenchymal transformation, or migration to adja-
cent epithelia (Shuler, 1995). Although in many regions of the
developing face, cell death occurs in the epithelium at sites of
fusion, analysis of available datas suggest that union of the 2
mandibular processes does not involve epithelial cell death or
epithelial-mesenchymal transformation. Rather, it involves
1 2(4):276-300 (2001 )
epithelial cell migration (Shuler et al., 1992; Shuler, 1995; Chai thelial-mesenchymal interactions. Based on patterns of gene
et al., 1997; McGonnell et al., 1998). expression and on functional studies, the mandibular arch can be
Studies in the mouse embryo indicate that merger of the 2 divided into 3 overlapping functional regions (Fig. 1A): a small
mandibular processes starts at E9.5 and is completed within 24 medial region, 2 large lateral regions, and the regions of the hyo-
to 36 hrs at Ell (Chai et al., 1997). During this process, the mandibular clefts located in the lower border of the lateral
epithelium covering the tips of the mandibular processes con- regions. Available evidence suggests that each of these regions
sists of 2 cell layers that adhere to each other and subsequent- contains signaling centers. Signaling centers are defined as
ly fuse to form an epithelial seam about 3 or 4 cell layers thick. groups of cells that regulate the behavior of the surrounding cells
Disappearance of the epithelial seam in the midline is first by producing positive and negative intercellular signaling mole-
seen at E10.5 and is completed at Ell, at which time the mes- cules, including FGFs, BMPs, Hedgehogs, Wnts, and EGFs
enchymal cells of 2 mandibular processes mix together and (Hogan, 1996).
become continuous. In contrast to the region of merging in the The 3 functional regions can be characterized by restricted and
midline, fusion/merging of the mandibular process to the region-specific expression of many of the evolutionary conserved
maxillary process and hyoid process involves epithelial apop- signaling factors and transcription factors. During recent years,
tosis (McGonnell et al., 1998). Interestingly, mesenchymal cells it has also become clear that the restricted patterns of expression
around these sites undergo expansion and bi-directional cell of many regulatory molecules in the mesenchyme of these
movements that result in a mixing of the mesenchymal cells regions (e.g., Pax9, Barxl, Msxl, Msx2, Gsc) are established by
between the 2 mandibular process-
es and mandibular processes and
neighboring processes (McGonnell
et al., 1998).
Conclusions
This review attempts to provide an
overview of the current under-
standing of the putative regions
and signaling cascades involved in
mandibular outgrowth and mor-
phogenesis. Classic tissue recom-
bination studies have demonstrat-
ed that the epithelium is required
for outgrowth and correct skeleto-
genesis in the mandibular arch.
However, these studies did not
identify specific regions in the
epithelium or mesenchyme that
regulate the outgrowth of the
developing mandible.
As was discussed in this re-
view, insight into putative regions
and signaling molecules involved in
mandibular outgrowth and mor-
phogenesis has come through stud-
ies examining: patterns of expres-
sion of signaling molecules, func- Figure 2. Summary of multiple genetic pathways that are involved in mandibular morphogenesis.
tional studies examining the simi- The differences in t e signaling cascades in the medial vs. lateral regions of the developing mandible
larities and differences between the were shown by Trumpp et al. (1 999). Morphogenesis of the large lateral region is dependent on and
effects of signaling molecules and controlled by FGF8. In the lateral region, Fgf8 expression requires Pitx2 [indicated by (1),thesee Lin et
mandibular epithelium, abnormali-
al., 1999; Lu et al., 1 999b], and the expression of Pax9 and Lhx6 in the oral side and expres-
sion of Barxl in both the oral and aboral regions of the hyomandibular cleft is dependent on FGF8
ties in the developing mandible signaling, which also regulates the expression of ET-1 in the mandibular epithelium [indicated by (2),
caused by targeted inactivation of see Trumpp et al., 1999].
candidate molecules, and identifica- In the region of the hyomandibular cleft, the expression of Gsc and Dlx3 is dependent on ET-
tion of mutated genes associated 1 /ETA signaling [indicatedby (3) and (A), see Clouthier et al., 1998, 2000]. Gsc expression in this
with craniofacial abnormalities the signaling
region is also dependent on DIx5 [indicated by (5), see Depew et al., 1999]. However,factors
pathway regulating the expression of other signaling molecules and transcription in this
affecting the mandible in humans.
Collectively, these studies show that region has not been fully identified (indicated by .).
signaling factors involved in the Unlike the lateral region, morphogenesis of the small medial region depends on signals other
outgrowth and morphogenesis of than FGF8. In this region, the expression of dHand and Msxl is dependent on ET-1 /ETA signalingis
[indicated by (6), Thomas et al., 1998]. The expression of Msxl and Msx2 in the medial region
the mandibular arch are similar to also de encent on BMP and FGF signaling secreted by the epithelium of medial region [indicated by
those involved in morphogenesis of (7) and (8), see references in the text]. However, the hierarchy of the signaling molecules in this
many other organs in which mor- region and their roles in regulating other genes expressed in the medial region are still not fully
phogenesis is dependent on epi- understood (indicated by ?).

2001)
12(4) 276-300 (2001) Crit Rev Oral Biol Med
Oral Biol Med
289
12(4):276-300
stage-specific antagonistic interactions of BMP-4 and FGF-8 sig-
naling in the lateral and medial regions (Neubuser et al., 1997;
Thomas et al., 1998; Tucker et al., 1998b).
The small medial region (Fig. 1A), where the 2 mandibular
processes merge, is characterized by the restricted patterns of
expression of Bmp4 in the epithelium and Msxl, Msx2, HAND,
Bmp2, and Fgfr2 in the underlying mesenchyme. In addition, Dlx,
Prxl, Prx2, and Wnt5a genes are expressed at high levels in the
mesenchyme of this region. This region gives rise to the medial
region of the mandibular arch containing incisor teeth, the most
medially (proximally) located skeletal elements including the
symphysis that separates the 2 rods of Meckel's cartilage. Ex-
perimental evidence indicates that the medial region is derived
exclusively from CNCC originating from the midbrain (Fig. 1B),
contains highly proliferative mesenchymal cells that, for the most
part, lack in vitro chondrogenic potential, and makes a significant
contribution to the overall growth of the developing mandible.
Their proliferative activity and the expression of several genes in
the mesenchyme of the medial region are dependent on signals
derived from the overlying epithelium. The mesenchymal cells in
the lower medial region also contain apoptotic cells. Analysis of
the available data suggests that epithelial-mesenchymal interac-
tions in the medial region may be involved in directional out-
growth of the developing mandible, and that ET-1/ETA,
FGFs/FGFR2C, BMPs/BMPRs, EGF/EGFR, ET-11ETA, and Wnt5a,
through their interactions with downstream target genes (inclu-
ding Msx, Prx, and dHand) are a component of the signaling path-
way mediating the interactions in this signaling center (Fig. 2).
The possible involvement of the medial region in regulating
the directional outgrowth of the developing mandible is support-
ed by the expression of mitogens in this region and by abnormal-
ities in mandibular outgrowth in mice lacking regulatory genes
that are either specifically expressed in this region (e.g., dHand-/-
and Msx-1-/-; for more details, see text) or their expression
extends into the medial region (e.g., Prxl/Prx2 compound
mutants, ET-1-/-, ETA-/-, ETC-1-/-; for more details, see text).
The medial region may also be involved in inhibiting chon-
drogenesis at the midline (symphysis), thus preventing the 2
rods of Meckel's cartilage from fusing and allowing for contin-
ued growth and morphogenesis of the mandible and Meckel's
cartilage. The abnormalities in Prxl/Prx2 double-knock-outs,
and in mice lacking 3 members of the endothelin pathway
(ETA, ET-1, ECE-1), suggest essential roles for these genes in the
formation of midline structures in the developing mandible.
The 2 larger lateral regions (Fig. 1B), where chondrogen-
esis and osteogenesis are initiated in vivo, are derived from
CNNC originating from the midbrain and from R2-4 (Fig.
2A), and give rise to the regions containing the molar teeth,
most of the skeletal components of the mandibular arch
(including the joint elements), and the middle ear structures.
The lateral region is characterized by restricted expression of
Fgf8 in the epithelium and high levels of expression of a vari-
ety of genes, including Dlx genes, Barxl, Lhx-6/7, Pitxl, Pax
genes, and Wnt5a. Knock-outs of many of the genes expressed
in the lateral region also result in mandibular hypoplasia of
various degrees and/or skeletal abnormalities in the articu-
lating ends of the mandibular arch (coronoid, condylar, and
angular processes), indicating that the lateral regions also
contain signaling centers regulating mandibular morphogen-
esis (Fig. 2). The abnormalities in the lateral but not the medi-
al regions of the mandibles of the Fgf8 mutants provide direct
evidence that FGF-8 signaling, through interactions with
290
290 Crit
Crit Rev Oral Biol
Rev Oral Biol Med
downstream target genes including ET-1, Lhx6, and Barxl, is
involved in mediating the interactions that regulate survival,
outgrowth, and morphogenesis of this region (Fig. 2).
The hyomandibular cleft region separating the mandibular
and hyoid arches (Fig. 1A) gives rise to middle ear structures (for
review, see Mallo, 1998). In these regions, high levels of expres-
sion of many regulatory genes-including Shh, Fgfs, Ffr2c, Ff3,
Msxl, Gsc, Prxl, Et-1, Glil, and ptc-are detected in the epitheli-
um and mandibular mesenchyme surrounding these regions
(Mallo et al., 1998), suggestive of the intriguing possibility that
these additional signaling centers are involved in mandibular as
well as middle ear morphogenesis. This possibility is further sup-
ported by abnormalities in the developing mandible and middle
ear structures of homozygous null mice lacking these genes
(Mallo et al., 1998). Furthermore, the mirror-image duplication of
caudal mandibular arch skeletal elements in the Hoxa-2 null
mutants suggests that inductive signals from the region of the
hypomandibular cleft may be involved in the establishment of
the polarity of the skeletal elements derived from the caudal
region of the mandibular arch, including the middle ear struc-
tures (Rijli et al., 1993). Furthermore, branchial arches with normal
skeletal polarity develop in embryos in which short segments of
the cranial neural tube were rotated along the rostrocaudal axis or
dorsoventral axis (Noden, 1978, 1983a,b; Hall, 1982). This sug-
gests the existence of putative signaling centers (polarizing sig-
nals) in the local environment into which CNCC migrate and
which regulate the polarities of the skeletal tissues within the
mandible arch. Although these observations suggest that sig-
nal(s) from the hyomandibular cleft may be involved in pattern-
ing the skeletal elements within the mandible, the roles of the
hyomandibular cleft region in the development of the mandibu-
lar arch are poorly understood.
Experimental evidence suggests that expression of Gsc in
the ectomesenchyme of the hyomandibular cleft region is
dependent on FGF8 and ET-1/ETA signaling from the overlying
epithelium (Clouthier et al., 1998, 2000; Trumpp et al., 1999) (Fig.
2). These studies also indicate that FGF-8 acts upstream of ET-1
in this pathway (Trumpp et al., 1999). The abnormalities in the
mandibular arch in Gsc null mutants are similar to, although
less severe than, abnormalities in Dlx5 null mutants, suggesting
that these gene products may directly or indirectly interact and
function in the same pathway. In situ hybridization studies
showed decreased Gsc expression at E10.5 in Dlx5 - null
mutants (Depew et al., 1999).
The absence of abnormalities in the medial region in FGF8
mutants also provides clear evidence that, unlike the lateral
region, the small medial region depends on signals other than
FGF8 (Trumpp et al., 1999) (Fig. 2).
The similarities in the mandibular phenotypes in dHAND-
I-, ETA-I-, ETi-l-, Prxl/Prx2 double-knock-outs, and Msxl-/-
mutants also suggest that direct or indirect interactions
between products of these genes are involved in morphogene-
sis of the medial region. Molecular analyses of dHAND-/-,
ETA-!-, ET1-/- mutants indicate that the "ET-1-dHAND-Msxl
pathway" constitutes one of the genetic pathways regulating
mandibular outgrowth. This pathway suggests that ET-1/ETA
signaling is required for high levels of expression of dHAND in
the underlying mesenchyme, which, in turn, regulates expres-
sion of Msxl (but not Msx2) (Fig. 2). However, the unchanged
patterns of expression of Prxl in the mandibles of dHAND-/-
and ETI-1- mutants (Clouthier et al., 1998, 2000; Thomas et al.,
1998) indicate that Prxl is not regulated by ET-1 and either acts
Med
12(4):276-300
12(4):276-300 (2001)
upstream of dHAND or belongs to different or parallel genet-
ic pathways. Based on the patterns of expression of mRNAs,
BMP-4, FGF2, FGF4, and FGF9 are also likely candidates
involved in regulating morphogenesis of the medial region.
In addition to Bmps, ET-1, and Fgfs, mandibular epithelium
also expresses other regulatory genes, including Jagged2, Pitx2,
PDGFA, and Egf and Egfr. In situ hybridization analysis shows a
lack of Fgf8 in the mandibular epithelium of Pitx2 knock-outs,
placing Pitx2 upstream of FGF-8 (Fig. 2). Although the roles of
some of these gene products in the outgrowth and morphogene-
sis of the mandibular arch have been elucidated by phenotypic
abnormalities in knock-outs, with few exceptions, the effects of
many of these gene products on the expression of the target genes
in the mandibular mesenchyme have not been fully examined.
In summary, abnormalities in the developing mandibles of
various mouse mutants caused by targeted inactivation of indi-
vidual candidate molecules indicate that multiple factors form a
combinatorial code which regulates outgrowth and morphogen-
esis of the mandibular arch and its skeletal elements. In addition,
the similarities between the mandibular phenotypes of various
mouse mutants suggest that these gene products regulate
mandibular outgrowth (and morphogenesis) through a common
pathway by acting on similar populations of cells. These mutants
provide powerful tools for examining the hierarchy of signaling
molecules involved in mandibular morphogenesis. Furthermore,
the generations of compound mutants (mice bearing mutations
in different pairs of related genes) provide strong evidence for
shared and/or unique functions for these genes and are valuable
for placing them within the genetic pathways. Further insight
into the roles of these gene products will also come from pheno-
typic analysis of animals in which candidate genes are over-
expressed in the developing mandible. An essential requirement
for the performance of these studies in transgenic mice will be the
identification of promoter elements, which specifically direct
gene expression to the mandibular arch. Furthermore, accessibil-
ity of the developing face in the chick embryo for experimental
manipulations and infection by means of retrovirus vectors,
together with detailed analysis of chick mutants with abnormali-
ties in the lower jaw, will continue to provide invaluable infor-
mation in the area of craniofacial biology. Elucidating the molec-
ular pathways and mechanisms regulating development and
morphogenesis of the mandibular arch is fundamental to an
understanding of the pathogenesis of the numerous human con-
genital syndromes which manifest severe abnormalities of first
branchial arch derivatives, including first-arch syndromes,
Treacher-Collins syndrome, and Pierre Robin sequence.
Acknowledgments
The author thanks Drs. Edward Kollar, William Upholt, Dimitrios Velonis,
and Y.-H. Wang, for their helpful discussions and critical comments on the
manuscript. The work in the author's laboratory was supported by NIH
Grant DE08682
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