DEVELOPMENTAL DYNAMICS 235:2353–2375, 2006

SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM

Recent Advances in Craniofacial

Morphogenesis
Yang Chai1* and Robert E. Maxson, Jr2

Craniofacial malformations are involved in three fourths of all congenital birth defects in humans, affecting
the development of head, face, or neck. Tremendous progress in the study of craniofacial development has
been made that places this field at the forefront of biomedical research. A concerted effort among
evolutionary and developmental biologists, human geneticists, and tissue engineers has revealed important
information on the molecular mechanisms that are crucial for the patterning and formation of craniofacial
structures. Here, we highlight recent advances in our understanding of evo– devo as it relates to
craniofacial morphogenesis, fate determination of cranial neural crest cells, and specific signaling
pathways in regulating tissue–tissue interactions during patterning of craniofacial apparatus and the
morphogenesis of tooth, mandible, and palate. Together, these findings will be beneficial for the
understanding, treatment, and prevention of human congenital malformations and establish the foundation
for craniofacial tissue regeneration. Developmental Dynamics 235:2353–2375, 2006. © 2006 Wiley-Liss, Inc.

Key words: cranial neural crest (CNC) cell; ectoderm; endoderm; evolution; mesoderm; mandible; palate; tooth
development

Accepted 30 March 2006

INTRODUCTION
Development of the craniofacial re-
gion is a complex process with many
features that reflect strong evolution-
ary forces controlling morphology. The
vertebrate craniofacial region houses
and protects the brain and provides
the scaffold on which the sensory and
feeding organs are located. The ability
to sense and devour prey is fundamen-
tal to animal survival. Variations in
craniofacial anatomy and function
provide the major driving force in evo-
lutionary adaptation.
One of the key features of craniofa-
cial development is the formation of
cranial neural crest (CNC) cells. The
specification, emigration and migra-
tion, proliferation, survival, and ulti-
mate fate determination of the CNC
play an important role in regulating
craniofacial development. Unlike the
trunk neural crest, CNC cells give rise
to an array of cell types during embry-
onic development. For example, CNC
cells form most of the hard tissues of
the head such as bone, cartilage, and
teeth, whereas hard tissues in the rest
of the body are formed from mesoderm
cells. Genetic disorders, environmen-
tal insults, or the combination of both
can alter the fate determination of
CNC cells and result in craniofacial
malformations. Significant progress
has been made in recent years toward
the understanding of how this impor-
tant population of pluripotent cells is
initially established in the early em-
bryo and of the molecular mechanisms
that mediate neural crest cell lineage
segregation, differentiation, and final
contribution to a particular tissue
type (Shah et al., 1996; LaBonne and
Bronner-Fraser, 1999; Chai et al.,
2003; Le Douarin et al., 2004).
The tissues of the head are com-
posed of cells from all three germ layer
origins: ectodermal, endodermal, and

1
Center for Craniofacial Molecular Biology School of Dentistry University of Southern California, Los Angeles, California
2
Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center and Hospital, Keck School of Medicine,
University of Southern California, Los Angeles, California
Grant sponsor: National Institute of Dental and Craniofacial Research; Grant sponsor: NIH; Grant numbers: DE 014078; DE012711;
DE017007; DE12941; DE12450; Grant sponsor: March of Dimes Birth Defects Foundation.
*Correspondence to: Yang Chai, Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar
Street, CSA 103, Los Angeles, CA 90033. E-mail: ychai@usc.edu
DOI 10.1002/dvdy.20833
Published online 5 May 2006 in Wiley InterScience (www.interscience.wiley.com).

© 2006 Wiley-Liss, Inc.

2354 CHAI AND MAXSON
mesenchymal. As seen in the develop-
ment of many organs, craniofacial
morphogenesis depends upon continu-
ous and reciprocal tissue–tissue inter-
actions, with tooth, palate, and mandi-
ble development as classic examples.
Research on the development of the
head requires a thorough understand-
ing of normal morphology, cell move-
ment, cell signaling, gene/gene interac-
tion, and transcriptional regulation in
time and space.
Investigation of craniofacial devel-
opment uses different animal species
as models as with other areas of re-
search in developmental biology.
Studies in mice combine the power of
genetics and genome manipulation
together with in vitro organ culture
techniques, leading to great progress
in recent years. Avian embryos
(chicken and quail) are easily acces-
sible and are used for grafting/
transplantation experiments. Ze-
brafish craniofacial developmental
studies have emerged more recently,
and mutant screens have led to the
identification of new cell signaling
interactions (Trainor and Krumlauf,
2000; Yelick and Schilling, 2002).
Central to these models and tech-
niques lies morphology. The head is
a very complex structure, and a de-
tailed analysis and appreciation of
morphology are essential to under-
standing the mechanism of craniofacial
malformations. Ultimately, “molecular
morphology,” the combination of classic
morphology and molecular biology, pro-
vides the basis for understanding the
evolutionary changes in head structure
and formation that in turn helps to clar-
ify key developmental principals as well
as the mechanism of craniofacial mal-
formations.
Mouse models are extremely valu-
able in our effort to gain a better
understanding of human craniofa-
cial birth defects. The remarkable
progress being made in the human
genome, in parallel with exquisite
functional genomic investigations in
various mouse models, has resulted
in the discovery of morphoregulatory
genes that determine craniofacial
morphogenesis. For example, we
have uncovered genes that are criti-
cal for determining cranial– caudal
axis, dorsal–ventral patterning,
left–right symmetry and segmenta-
tion in early forming neurulation as
well as branchial arches. More re-
cently, many animal models with
specific craniofacial malformations
have facilitated human genetic link-
age analysis, in which a genetic de-
fect has been identified as the cause
of congenital malformation(s) (Thya-
garajan et al., 2003; Murray and
Schutte, 2004). Overall, genetically
mutated mouse models highlight the
enormous challenges of uncovering
complex genetic mechanisms under-
lying craniofacial development and
malformations.
Progress and understanding of the
key control processes of head develop-
ment have advanced to an extent
where developmental biologists are
interacting with tissue engineers to
devise cell-based approaches to treat
clinical malformations in humans.
The prospect of harnessing develop-
mental processes to repair or replace
damaged or diseased craniofacial tis-
sues and organs is an exciting new
area that links basic animal research
with human genetics and provides
great promise in our effort to reduce
the pain and suffering associated with
craniofacial malformations.
In this review, we highlight some
recent advances in our understanding
of evo– devo (evolutionary– develop-
ment) as it relates to craniofacial de-
velopment, the fate determination of
cranial neural crest cells, craniofacial
patterning and organogenesis. We
also review new discoveries on the de-
velopment of craniofacial bones, sig-
naling interactions, and specificity in
craniofacial morphogenesis. Finally,
we will discuss how research advance-
ments will be beneficial for the under-
standing, treatment, and prevention
of human congenital malformations
and prospectus of tissue engineering.
EVOLUTION
Perhaps no other anatomical feature
more closely epitomizes vertebrates
than the head. Comprising paired sen-
sory elements, a muscularized masti-
catory apparatus, and a cartilaginous
or bony braincase, the head develops
from complex and massive movements
of mesenchymal cells derived from
mesoderm and neural crest. These
cells interact with each other and with
a variety of craniofacial epithelia to
produce the intricate structures of the
face, skull, teeth, and jaw. How this
trajectory of inductive interactions oc-
curs in molecular terms is a key ques-
tion in developmental biology; how it
has been modified during evolution to
produce the stunning variety of
craniofacial structures in vertebrates
is of major interest in evolutionary bi-
ology.
We begin our review with a brief
consideration of some current issues
concerning the evolution of the head
(for comprehensive treatments, see
Santagati and Rijli, 2003; Kuratani,
2004, 2005; Northcutt, 2005; Morriss-
Kay and Wilkie, 2005; Depew et al.,
2005). We focus on recent findings
that pertain to two broad questions:
First, how did the head evolve in the
chordate ancestor of vertebrates? Sec-
ond, how, over shorter evolutionary
periods, did modifications of the head
developmental program produce the
evolutionary novelties that made pos-
sible the huge array of craniofacial
morphologies evident in vertebrates?
Although the details of how the
head first evolved are as elusive as
fossils of the early chordates that once
lived in pre-Cambrian seas, there is
broad agreement with a scenario put
forward by Northcutt and Gans (1983)
and modified recently by Northcutt
(2005). Their New Head hypothesis
postulates that the ancestral verte-
brate was an animal similar to the
modern day cephalochordate Am-
phioxus. Like Amphioxus, this animal
was a filter feeder that lacked pharyn-
geal arch muscles to move water
through the gills. Also lacking was a
braincase and the structures charac-
teristic of the rostral head of verte-
brates, including olfactory bulbs and
telencephalic vesicles.
According to the New Head hypoth-
esis, this stem organism underwent
an evolutionary transition from filter
feeding to active predation (Fig. 1).
Underlying this transition were sev-
eral key innovations, including the de-
velopment of muscularized jaws and
gill arches, a nerve plexus that en-
abled the animal to detect and capture
prey, and cartilaginous and skeletal
elements that provided a fixed spatial
organization for this plexus. In princi-
ple, these new structures could be pro-
duced in two different ways: the ante-
rior portion of the trunk could be
restructured, or a section could be

added onto the trunk de novo. The
latter possibility—that the head is a
neomorph—is a central proposal of
the New Head hypothesis. Also key to
this hypothesis is the idea that the cell
type most critical for the evolution of
the new head was the neural crest.
The neural crest is induced by an in-
teraction between cells of the neural
plate and adjacent surface ectoderm
(reviewed by Meulemans and Bronner-
Fraser, 2004). Nascent neural crest
cells delaminate from the developing
neural tube, take on a mesenchymal
character and migrate to distant sites
in the developing embryo. In the cranio-
facial region, they give rise to a wide
variety of cell types and tissues, includ-
ing intramembranous bone, cartilage,
muscle, and nerves (Creuzet et al.,
2005; Noden and Trainor, 2005). North-
cutt and Gans (1983) proposed that the
neural crest emerged in an early verte-
brate ancestor and served as a key
source of evolutionary novelty that
made the New Head possible. They
based this view on the apparent unique-
ness of neural crest to vertebrates and
on the ability of neural crest both to
contribute to structures that form the
new head as well as to serve as a source
of patterning information (Noden, 1978;
Couly et al., 1993; Jiang et al., 2002;
Schneider and Helms, 2003).
That the fate of the neural crest
includes elements of the rostral most
portion of the new head and that the
neural crest has a key role in pat-
terning these tissues are clear
(Couly et al., 1993; Jiang et al., 2002;
Gross and Hanken, 2005; Evans and
Noden, 2006). What is less clear is
whether the neural crest is unique to
vertebrates. Efforts to identify neu-
ral crest-like cell populations in ex-
tant nonvertebrate chordates have
produced mixed results. Yu et al.
(2002) found that amphioxus has
cells that express an amphioxus ho-
mologue of the crest marker FoxD3.
AmphiFoxD is expressed in the an-
terior neural plate but not in cells
bordering the neural plate, as is the
case for FoxD3 in vertebrates. Am-
phiFoxD is also expressed in axial
and paraxial mesoderm. Yu and co-
workers speculate that an amphi-
FoxD congener in a common ances-
tor of vertebrates and amphioxus
had a role in the development of the
mesoderm, and subsequently, during
RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2355
early vertebrate evolution took on a
function in the early neural crest.
On the other hand, recent work
from Jeffery’s group has shown that a
urochordate (ascidian) possesses cells
that exhibit neural crest-like behavior
(Jeffery et al., 2004). These cells mi-
grate from the neural tube and ex-
press the neural crest markers hnk
and Zic1. Ultimately they give rise to
pigment cells, leading Jeffery and co-
workers to propose that the neural
crest first arose as pigment cell pre-
cursors in a common ancestor of ver-
tebrates and ascidians, and later ac-
quired additional fates.
Whether such neural crest-like cells
exist in other nonvertebrate chordate
groups remains to be determined. Re-
sults to date show that, although cells
with neural crest-like properties are
present in some nonvertebrates, cells
possessing the full spectrum of neural
crest behavior are unique to verte-
brates. Thus, the idea that the neural
crest is a major source of New Head
structures remains intact (Northcutt,
2005).
Evolutionary Novelty in the
Craniofacial Complex
The first vertebrates exhibiting a
“New Head” were probably similar to
modern day jawless fishes (ag-
nathans; Fig. 1). These organisms,
which include lampreys and hagfish,
are similar to higher vertebrates in
their embryology, but lack jaws, pos-
sessing instead a filter-feeding appa-
ratus (Kuratani et al., 2001). A major
question is what were the sources of
evolutionary novelty that made possi-
ble changes that led to more complex
craniofacial morphologies of later ver-
tebrate groups? One approach to this
problem has been to carry out compar-
ative embryological studies on the
lamprey.
Kuratani and colleagues have ex-
amined marker gene expression in the
Japanese lamprey, comparing expres-
sion patterns with those seen in gna-
thostomes (Shigetani et al., 2002). A
generally accepted view of jaw evolu-
tion is that the mandibular arch, the
most rostral of the pharyngeal arches,
was modified to produce the mandible
and maxilla. In ancient vertebrates,
the arches were morphologically sim-
ilar. The lamprey lacks a jaw but has
upper and lower lips with distinct
morphologies. These authors exam-
ined the expression of Dlx and Msx
homeobox genes, which, in amniotes,
are expressed in neural crest-derived
mesenchymal cells along the future
proximal– distal axis of the mandibu-
lar arch. Additional markers with re-
gion-specific expression included Fgf8
and Bmp4.
The expression patterns of these
genes in lamprey embryos appeared
at first to support the view that lam-
prey larval lips and gnathostome jaws
are homologous. However, DiI label-
ing of neural crest populations sug-
gested a more complex picture. Neural
crest cells contributing to the upper
and lower lips are derived from fore-
brain and midbrain crest populations,
unlike the situation in gnathostomes
in which mandibular arch crest de-
rives from more posterior populations.
The lamprey Dlx1 homologue is ex-
pressed in different regions of the neu-
ral crest compared with gnathos-
tomes. This shift in expression is
correlated with a shift in expression of
Fgf8, an upstream regulator of Dlx1 in
both lamprey and gnathostomes, in
the epidermis. This shift is seen as the
cause of a corresponding shift in the
fate of subpopulations of neural crest.
An evolutionary change such as this
one in a jawless gnathostome ancestor
could have led to the emergence of the
jaw. If this scenario is correct, then
the gnathostome jaw is an evolution-
ary innovation resulting from a
change in the topographic location of
an epithelial–mesenchymal interac-
tion. This change resulted in a shift in
fate of neural crest cells and, thus, to
the innovation that became the verte-
brate jaw.
Recent work from the Tabin and
Chuong labs has identified potential
sources of evolutionary novelty in
the epithelial–mesenchymal interac-
tions that pattern the avian beak
(Abzhanov et al., 2004; Wu et al.,
2004). This work suggests that evo-
lutionary changes in beak morphol-
ogy in birds may be caused by simi-
lar shifts in epithelial–mesenchymal
signaling. Darwin’s finches, in the
course of their radiation into various
ecological niches in the Galapagos,
evolved diverse beak morphologies.
By examining the expression of var-
ious growth factors among Galapa-
2356 CHAI AND MAXSON

Fig. 1. The New Head and the evolution of the jaw. Schematics of the oral region of Amphioxus, a
lamprey (jawless vertebrate), and a gnathostome (jawed vertebrate) are shown with their phylogenetic
relationships (redrawn from Shigetani et al., 2002, and Holland et al., 2004). As proposed by Shigetani
et al. (2002), the neural crest-derived mesenchyme of lampreys and gnathostomes is subdivided into
postoptic (po) and mandibular arch (ma) subdomains. Growth factors are secreted by the epidermis (red
line). In the lamprey, these induce homeobox genes (brown and blue) over the entire length of the neural
crest-derived mesenchyme, whereas in the gnathostome, such signals are effective only in the man-
dibular component. Thus, it is proposed that a shift in epithelial–mesenchymal interactions results in the
evolutionary emergence of the jaw. Ulp, upper lip; llp, lower lip; prc, prechordal cranium.

Fig. 2. Neural crest boundary relationships in

skull vaults of mouse, chicken, and frog. Skulls
are shown in the dorsal aspect. In the mouse,
the coronal suture (CS) marks a neural crest–
mesoderm boundary (blue line; neural crest an-
terior) (Jiang et al., 2002). Note also that the
frontal (F) and interparietal (IP) bones are neural
crest-derived (blue). In the chicken, the neural
crest–mesoderm boundary lies within the fron-
tal bone (F) and does not coincide with a suture
(Noden, 1975; Le Lievre, 1978; Noden and
Trainor, 2005; Evans and Noden, 2006). The
solid blue frontoparietal (FP) bone of the frog,
Xenopus, indicates that neural crest contributes
over its entire length (Gross and Hanken, 2005).
The extent to which mesoderm contributes to
this bone is not known. See Figure 3 for a
detailed depiction of neural crest- and meso-
derm-derived bones of the skull vault. P, pari-
etal bone.

gos finches, Tabin’s group found that

Fig. 3. Contribution of ectoderm, mesoderm, and endoderm during craniofacial development. the expression of Bmp4 in the mes-
A: Neural crest cells are formed at the junction of neural and surface ectoderm. These cells undergo
enchyme of the upper beak was
epithelial–mesenchymal transformation, become ectomesenchyme, and travel into multiple destina-
tions. B: Side view of an E9.5 mouse embryo shows unsegmented paraxial mesoderm in the head and closely associated with a particular
mesoderm-derived somites in the trunk. OP, optic vesicle; OV, otic vesicle. C: Transverse section of the morphology, a broad shape (Abzh-
developing first branchial arch that is covered by surface ectoderm. The core of the first arch contains anov et al., 2004). By misexpressing
cranial neural crest (CNC) -derived (blue) and paraxial mesoderm-derived (pink) cells. The pharyngeal bone morphogenetic protein-4
endoderm (yellow) lines the inner aspect of the branchial arch. D: Schematic drawing of an adult mouse
skull shows both the CNC- and paraxial mesoderm-derived elements (modified from Noden and
Trainor, 2005). Mesoderm-derived cells are in pink, and CNC-derived cells are in blue.

(Bmp4) in chick embryos, a similar
broad shape could be produced.
The Chuong group (Wu et al.,
2004) addressed a related question
by examining the basis of morpho-
logical differences between the
shapes of chicken and duck beaks.
Ducks have two growth zones in the
frontonasal mass, whereas chickens
have only one. This difference ac-
counts for the difference in beak
morphology between these two birds.
Bmp4 is expressed in the growth
zones. By manipulating the location
of Bmp4 expression, Chuong’s group
was able to modulate the shape of
the beak. Together, these studies on
beak development demonstrate that
subtle changes in the topography of
Bmp signaling within the developing
beak can account for dramatic evolu-
tionary shifts in beak morphology.
Schneider and Helms (2003) ad-
dressed the role of the neural crest in
beak patterning and the evolution of
beak shape. These authors exchanged
neural crest cells fated to contributed
to the beak between quail and chicks,
which exhibit distinct beak morpholo-
gies. They sought to test the tripartite
hypothesis that neural crest cells con-
tain patterning information for beak
morphology, that such cells possess an
autonomous program by which they
express this information, and that
such cells influence the fates of non-
neural crest cells.
Reciprocal transplantation experi-
ments confirmed each of these points.
Thus, for example, quail neural crest
cells, when transplanted into duck
embryos gave rise to beaks like those
found in quail. That donor neural
crest cells pattern host structures was
shown by an examination of egg tooth
morphology. Duck and quail egg teeth,
which are derived from epidermis, not
neural crest, exhibit distinct morphol-
ogies. The results showed that the mor-
phology was characteristic of the trans-
planted tissue. Transplanted neural
crest cells express molecular markers
characteristic of the donor species, con-
firming the idea that such cells express
an autonomous molecular program. It
is not hard to envisage that evolution-
ary shifts in neural crest populations
could produce profound morphological
changes.
RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2357
Tissue Boundaries in
Cranial Evolution
The finding that the neural crest can
provide patterning information sug-
gests that evolutionary shifts in neu-
ral crest–non-neural crest boundaries
could be an additional source of nov-
elty (Fig. 2). Recent studies have ad-
dressed the significance of boundaries
in the development and evolution of
the skull vault (Jiang et al., 2002;
Merrill et al., 2006; Evans and Noden,
2006).
As discussed above, the skull vault
develops from populations of mesen-
chymal cells with distinct embryolog-
ical origins, neural crest, and head
mesoderm. The extent of the contribu-
tion of neural crest to calvarial bones
of birds has been controversial. Sev-
eral studies using quail– chick chime-
ras or retroviral infection to produce
fate maps (Noden, 1975; Le Lievre,
1978; Evans and Noden, 2006) sug-
gest that the neural crest–non-neural
crest boundary lies within the frontal
bone (reviewed in Noden and Trainor,
2005). The frontal bones develop after
the fusion of two intramembranous
centers (Jollie, 1981; discussed in No-
den and Trainor, 2005). The more ros-
tral of these is of neural crest origin,
the caudal of mesoderm origin. Thus,
the boundary lies at the interface of
these two ossification centers. Later
studies, however, have suggested that
the entire chick cranial vault, includ-
ing the frontal and parietal bones, is of
neural crest origin (Couly et al., 1993).
Similarly, in Xenopus, dye-marking
experiments have shown that crest
contributes to the full length of the
frontoparietal bone, the major bone of
the skull vault in anuran amphibians
(Gross and Hanken, 2005). In mouse,
in contrast, the frontal bone appears
to be derived entirely from neural
crest, whereas the parietal bone is de-
rived from mesoderm (Jiang et al.,
2002; Ishii et al., 2003). Thus, in the
mouse, the interface between the fron-
tal and parietal bones—the coronal
suture—is a boundary between neural
crest and mesoderm (Merrill et al.,
2006).
If we make the assumption that the
boundary in the chick is within the
frontal bone (Noden, 1975; Le Lievre,
1978; Evans and Noden, 2006), then,
as pointed out by Noden and Trainor
(2005), the situation may not be sub-
stantially different from the mouse.
On the other hand, if, in the chicken,
crest indeed contributes to the entire
skull vault, as it apparently does in
Xenopus, then there may have been an
evolutionary shift in the neural crest–
mesoderm boundary in the ancestor of
birds and mammals (Gross and Han-
ken, 2005).
It is also possible that, as pointed
out by Gross and Hanken (2005), am-
phibians, birds, and mammals may
each exhibit distinct contributions of
neural crest to the skull vault. We
note as well that there remains some
uncertainty about the homology rela-
tionships between the frontal, pari-
etal, and frontoparietal bones of these
three taxa—an uncertainty that clearly
complicates the interpretation of neural
crest labeling studies.
In the mouse, the coronal suture is
located at the neural crest–mesoderm
interface and is a major growth center
of the skull vault. Boundaries between
dissimilar cell populations often serve
as important signaling centers that
function in growth control (reviewed
in Dahmann and Basler, 1999). The
location of the tissue boundary at the
coronal suture in mammals might rep-
resent an evolutionary innovation
that makes possible, for example, new
modes of mastication, locomotion, or
cranial growth. Further analysis of
contributions of neural crest to the
skull vault in extant vertebrates will
be required to illuminate this issue.
FATE DETERMINATION OF
CRANIAL NEURAL CREST
CELLS
The vertebrate neural crest is a pluri-
potent cell population derived from
the lateral ridges of the neural plate
during early stages of embryogenesis
(Fig. 3). The functions of neural crest
cells include coordination of various
visceral activities, such as in the pe-
ripheral nervous system; protection of
the body from external conditions,
such as by melanocytes; and partici-
pation in craniofacial skeletal devel-
opment (Le Douarin et al., 2004).
The formation of the neural crest is
a classic example of embryonic induc-
tion, in which tissue–tissue interac-
tions and the concerted action of sig-
naling pathways are critical for the
2358 CHAI AND MAXSON
induction of neural crest precursor
cells. After induction, neural crest
cells disperse from the dorsal surface
of the neural tube, undergo epithelial–
mesenchymal transformation, and mi-
grate extensively through the embryo,
giving rise to a wide variety of differ-
entiated cell types. The migration,
proliferation, and differentiation of
neural crest cells along multiple dis-
tinctive pathways have been studied
extensively in various animal models
(Bronner-Fraser, 1993; LaBonne and
Bronner-Fraser, 1999; Le Douarin et
al., 2004; Basch et al., 2004). Recent
studies suggest that fate determina-
tion of the neural crest is strongly in-
fluenced by environmental cues. CNC
cells interact with and are consequently
instructed by pharyngeal endoderm, ec-
toderm, and mesoderm before giving
rise to various types of tissues in the
craniofacial region.
During craniofacial development,
neural crest cells migrate ventrolater-
ally as they populate the craniofacial
region. The proliferative activity of
these crest cells produces the fronto-
nasal process and the discrete swell-
ings that demarcate each branchial
arch. As these ectodermally derived
cells migrate, they contribute exten-
sively to the formation of mesenchy-
mal structures in the head and neck.
Cell labeling studies have demon-
strated that neural crest cells arising
from rhombomeres 1–3 (r1–3) of ante-
rior hindbrain migrate into the first
branchial arch and, thereafter, reside
within the maxillary and mandibular
prominences (Osumi-Yamashita et
al., 1990; Serbedzija et al., 1992; Bron-
ner-Fraser, 1993; Selleck et al., 1993;
Lumsden and Krumlauf, 1996). The
migration of these rhombencephalic
crest cells is regulated by growth fac-
tor signaling pathways and their
downstream transcription factors be-
fore the CNC cells become committed
to several different tissue types such
as bone, cartilage, tooth, and cranial
nerve ganglia (Noden, 1983, 1991;
Lumsden, 1988; Graham and Lums-
den, 1993; Le Douarin et al., 1993;
Echelard et al., 1994; Imai et al., 1996;
Trainor and Krumlauf, 2000). Recent
studies provide strong supportive evi-
dence that the CNC cells are develop-
mentally “plastic,” i.e., their fate is not
predetermined before they reach their
final destination; rather, these pro-
genitor cells must be instructed by sig-
nals from other tissues to generate skel-
etal elements of appropriate shape and
size in the craniofacial region. Tissues
that provide the instructive signaling
for CNC fate specification include, but
are not limited to, the pharyngeal
endoderm, the branchial arch ectoderm,
and the isthmic organizer at the mid-
brain– hindbrain boundary (Baker and
Bronner-Fraser, 2001; Trainor et al.,
2002; Couly et al., 2002; Le Douarin et
al., 2004).
The Pharyngeal Endoderm
Most of our knowledge about the
critical function of pharyngeal
endoderm in regulating the fate of
CNC cells and the patterning of mid-
dle and lower face derives from stud-
ies using chick, quail, or zebrafish
embryos as models. These embryos
allow relatively easy manipulation
compared with the mouse model.
Surgical removal of pharyngeal
endoderm during early stages of
chick embryogenesis resulted in de-
fects in facial bone and cartilage devel-
opment (Couly et al., 2002). During the
development of the first branchial arch,
pharyngeal endoderm is thought to
prepattern the orofacial epithelium,
which in turn will provide instructive
signals to pattern the CNC-derived
mesenchyme (Haworth et al., 2004).
In zebrafish studies, fibroblast growth
factor (FGF) signaling has been shown
to be critical for the development of
pharyngeal endoderm itself and for
the mediation of the endoderm to reg-
ulate facial skeletal morphogenesis
(Ruhin et al., 2003; Crump et al.,
2004; Helms et al., 2005). On the other
hand, a recent study has shown that
pharyngeal endoderm is not critical
for the normal development of the up-
per and lower face. Instead, it is the
ectoderm that is critical for providing
the instructive information for facial
morphogenesis (Aoki et al., 2002).
Furthermore, CNC cells also contain
intrinsic information that can affect
facial development, although this con-
tribution may depend upon the collec-
tive number of neural crest cells
present at a given time and position
(Schneider and Helms, 2003; Tucker
and Lumsden, 2004). Overall, there is
overwhelming evidence to support the
notion that the pharyngeal endoderm
has a critical role in regulating the
fate of CNC. Discovery of the critical
signaling pathway involved in this
regulatory process awaits the develop-
ment of mouse pharyngeal endoderm-
specific gene inactivation models.
Orofacial Ectoderm
Early patterning of the oral ectoderm is
independent of the neural crest (Veitch
et al., 1999). In the mouse model, the
branchial arch epithelium is correctly
patterned, despite the CNC migration
defect (Gavalas et al., 2001). After the
interaction with pharyngeal endoderm,
the orofacial ectoderm is roughly di-
vided into proximal and distal domains.
The establishment of the ectodermal
domain greatly influences the fate de-
termination of migrating CNC cells as
they populate the branchial arch. At
later stages of embryonic develop-
ment, CNC cells interact with and
provide instructive signals to the over-
lying epithelium and have an effect on
epithelial patterning (Miletich and
Sharpe, 2004). Tooth development is a
clear example of the consistent shift of
instructive signal between orofacial
ectoderm and the CNC-derived mes-
enchyme (see below for detailed dis-
cussion).
Concerted Action of Growth
and Transcription Factors in
Determining the Fate,
Expansion, and Survival of
CNC Cells
Recent studies have addressed the in-
fluence of growth factors on the fate of
multipotent progenitor cells, such as
the neural crest, during embryogene-
sis. It turns out that signaling centers,
such as the isthmic organizer, rely on
growth factor signaling in regulating
the fate of CNC cells. Specifically,
FGF8 signaling from the isthmic orga-
nizer can alter Hoxa2 expression and
consequently control branchial arch
patterning, demonstrating that neu-
ral crest cells are patterned by envi-
ronmental signals (Trainor et al.,
2002). Several growth factor signaling
pathways have been shown to be crit-
ical for CNC fate determination, of
which transforming growth factor-be-

ta/BMP (TGF-␤/BMP) signaling is a
classic example. Members of the
TGF-␤ superfamily of growth factors
are expressed at sites where neural
crest cells commit to form particular
cell types. TGF-␤ superfamily mem-
bers promote alternative fates for
trunk neural crest cells; BMP signal-
ing promotes neurogenesis by induc-
ing MASH1 expression, whereas
TGF-␤ signaling favors smooth mus-
cle differentiation (Shah et al., 1996).
In contrast, CNC cells react to TGF-␤
signaling differently. TGF-␤ controls
the differentiation of CNC cells to glial
cells, and TGF-␤, BMP, and Wnt to-
gether control chondrocyte differenti-
ation. Apparently, one of the major
differences between CNC and TNC is
the expression of Hox genes, which may
account for the differential responsive-
ness between CNC and TNC to the
same environmental cues (Abzhanov et
al., 2003). Furthermore, the function of
TGF-␤ in regulating neural crest cell
differentiation is sensitive to the TGF-␤
expression level, such that TGF-␤ may
promote alternative cell fates or induce
apoptosis (Hagedorn et al., 2000). Dur-
ing early mouse craniofacial develop-
ment, TGF-␤ subtypes are present in
the CNC-derived mesenchyme during
critical epithelial–mesenchymal inter-
actions related to the formation of var-
ious organs (Lumsden, 1984; Hall et al.,
1992; Chai et al., 1994; Lumsden and
Krumlauf, 1996; Chai et al., 2003). Tar-
geted null mutation of Tgfb2 or haplo-
insufficiency of Smad2 results in a wide
range of developmental defects, includ-
ing craniofacial malformations such as
small mandible, dysmorphic calvaria,
and cleft palate (Sanford et al., 1997;
Nomura and Li, 1998). Significantly,
many affected tissues have neural
crest-derived components and simulate
neural crest deficiencies; thus, TGF-␤
signaling may provide significant in-
structive information to specify the fate
of CNC during early craniofacial devel-
opment.
Members of the TGF-␤ superfamily
regulate the expression of transcription
factors to influence cell fate decisions
instructively during embryogenesis
(Shah et al., 1996; Dorsky et al., 2000).
For example, the expression patterns of
Tgfb2 and transcription factor Msx1
have significant overlaps during early
tooth and palate development when
CNC-derived cells become specified to
RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2359
form dental and palatal mesenchyme,
respectively, suggesting an epistatic re-
lationship between these two genes
(Ferguson, 1994; Ito et al., 2003). In the
palatal mesenchyme, overexpression of
TGF-␤ suppresses transcriptional activ-
ity of the Msx1 gene (Nugent and
Greene, 1998). We have shown recently
that compromised TGF-␤ signaling af-
fects the expression of Msx1, which in
turn controls the progression of the
CNC cell cycle by regulating cyclin D1
expression (Ito et al., 2003). In parallel,
in vitro studies also suggest that Msx1
gene expression maintains cyclin D1 ex-
pression and holds cells in an undiffer-
entiated state by promoting prolifera-
tion (Hu et al., 2001). In general, TGF-␤
signaling specificity in regulating down-
stream target gene expression is deter-
mined by the interaction with other fac-
tors (such as BMP, Wnt, FGF, and so
on) in a temporal- and spatial-specific
manner (Massague, 2000). The close in-
tertwining of TGF-␤ signaling with
other pathways appears to be a critical
component of specific cell fate determi-
nation mediated by TGF-␤ family mem-
bers.
In addition to regulating the fate of
CNC cells, the concerted action of
growth and transcription factors also
controls cell proliferation and death.
For example, BMP signaling controls
Msx gene expression by directly regu-
lating Msx2 promoter activity (Brug-
ger et al., 2004). There is also an ap-
parent feedback for BMP signaling by
the Msx genes, because loss of Msx1
and Msx2 results in altered Bmp4 ex-
pression in the CNC cells (Ishii et al.,
2005). Msx1 and Msx2 function redun-
dantly in regulating the survival and
proliferation of CNC cells during
craniofacial development. This regu-
lation is most likely carried out
through the control of cell cycle pro-
gression (Hu et al., 2001; Han et al.,
2003, Ishii et al., 2005). Recently,
studies in Xenopus have shown that a
Myc-mediated Id3 expression level is
critical for determining whether the
neural crest cells will proliferate or
die, thus determining the size of the
neural crest population (Kee and
Bronner-Fraser, 2005; Light et al.,
2005). It is currently unknown
whether Id3 plays a similar role dur-
ing mouse development.
Finally, it is important to address
the issue of prepatterning of premi-
gratory neural crest cells. Noden’s ex-
periment suggested that chick CNC
cells are predetermined according to
their rostrocaudal origin in the neural
tube (Noden, 1983). However, recent
work has demonstrated that adjacent
tissues, such as the isthmus, provide
instructive signals to regulate down-
stream target genes to determine the
fate of CNC cells (Trainor et al., 2002).
Furthermore, by providing critical
feedback to the surface ectoderm,
CNC can provide species-specific pat-
terning information during craniofa-
cial development, highlighting the im-
portance of tissue–tissue interaction
in regulating organogenesis (Schnei-
der and Helms, 2003; Helms et al.,
2005). In the mouse model, it has been
demonstrated elegantly that CNC
cells retain a remarkable degree of
plasticity, even after their migration
into the branchial arch (Zhao et al.,
2006). The second arch CNC cells
have a cell-autonomous requirement
for the Hoxa2 gene for their intrinsic
patterning program (Santagati et al.,
2005). In vitro studies have also begun
to address the plasticity of postmigra-
tory CNC cells (Zhao et al., 2006).
Taken together, it is clear that the
fate of mouse postmigratory CNC cells
is determined through the reciprocal
signaling between neural crest mesen-
chyme and the surrounding environ-
ment, where timing is an essential
component.
CRANIOFACIAL
PATTERNING AND TISSUE–
TISSUE INTERACTION
The principal goal of developmental bi-
ology is to understand how tissues are
induced and patterned to generate dif-
ferent organs at the correct location and
time. Evidence suggests that signals
from the anterior visceral endoderm,
anterior neural ridge, and the meso-
derm are required for the development
of head structures (Spemann, 1938;
Shimamura and Rubenstein, 1997;
Beddington and Robertson, 1999; De-
pew et al., 2002a). Furthermore, the for-
mation of vertebrate “New Head” de-
pends upon the presence of CNC cells
and additional sensory placodes. Multi-
ple molecules have been identified as
critical regulators at these signaling
centers. However, the challenge re-
mains to determine how various signal-
2360 CHAI AND MAXSON
ing centers coordinate with each other
and build complicated structures that
make the head.
In the facial region, each branchial
arch contains a central blood vessel,
the aortic arch, which is surrounded
by cells of paraxial mesoderm. The
mesoderm core is enveloped by the
more peripherally located CNC cells.
The inner surface of the branchial
arch contains cells derived from the
pharyngeal endoderm, while the outer
surface is covered by the ectoderm
(Fig. 3). The spatial relationship of the
boundary of cells with different em-
bryonic origin is crucial for normal or-
ganogenesis and may reflect the regu-
latory interactions that control the
development of complex structures in
the craniofacial region.
Ectoderm
Craniofacial ectoderm plays a critical
role in regulating the fate of CNC cells
during craniofacial morphogenesis,
whereas the establishment of ecto-
derm identity is independent of CNC
cells. Later, the continuous and recip-
rocal interaction between the ecto-
derm and the CNC-derived ectomes-
enchyme controls the position, size,
and shape of craniofacial organs dur-
ing embryogenesis. It is imperative to
appreciate the inductive capability of
ectoderm and CNC cells in the context
of time. This concept is best illus-
trated by the seemingly contradictory
conclusions regarding the ability of
the oral ectoderm or the CNC-derived
ectomesenchyme to induce tooth mor-
phogenesis.
Tissue recombination experiments
show that the tooth inductive signal
first resides in the oral ectoderm and
then shifts into the underlying CNC-
derived ectomesenchyme at a later de-
velopmental stage. In mouse, dental
epithelium before embryonic day (E)
12 is capable of inducing tooth forma-
tion when combined with nondental
mesenchyme, whereas dental mesen-
chyme after E12 can induce tooth for-
mation when combined with nonden-
tal epithelium (such as the second
branchial arch epithelium; Mina and
Kollar, 1987).
Growth and transcription factors
are responsible for establishing the
patterning of craniofacial develop-
ment. In the first branchial arch oral
ectoderm, FGF8 and BMP4 are criti-
cal for setting up the proximal– distal
axis during development. In the upper
and middle face, where structures de-
rive from the frontonasal process,
Sonic hedgehog (Shh) and FGF signal-
ing appear to be critical for setting up
a boundary in the neural and surface
ectoderm (Helms et al., 2005). In
craniofacial patterning, definitive evi-
dence only supports the critical func-
tion of ectodermal FGF signaling, as
conditional inactivation of the Fgf8
signaling resulted in the disappear-
ance of the proximal portion of the
mandible (Trumpp et al., 1999). More
recently, studies have shown that sig-
naling through FGFR1 is critical for
the neural crest independent pattern-
ing of the pharyngeal ectoderm. Ecto-
derm FGF signaling patterns the pha-
ryngeal region to create a permissive
environment for the entry of CNC
cells (Trokovic et al., 2003, 2005).
Craniofacial ectoderm regulates the
expression of transcription factors to
specify the fate of CNC cells. For ex-
ample, FGF8 signaling controls the
expression of two Lim-homeobox do-
main genes, Lhx6 and Lhx7, in the
CNC-derived ectomesenchyme. Lhx6
and Lhx7 are mainly expressed in the
oral side of ectomesenchyme, while
Gsc is expressed in the aboral region
(Tucker et al., 1999). Endothelin-1 in
the mandibular epithelium controls the
expression of Gsc. Mutations in either
Endothelin-1 or Gsc result in mandibu-
lar development defects, demonstrating
the endothelin-mediated Gsc expres-
sion is critical for controlling the pat-
terning of mandible (Clouthier et al.,
1998). In addition, BMP signaling has
been shown to be a critical regulator for
Msx1 function, which is exclusively ex-
pressed in the CNC-derived ectomesen-
chyme (Chen et al., 1996; Tucker et al.,
1998a). Conditional inactivation of
Bmpr1a in the oral ectoderm results in
tooth agenesis (Andl et al., 2004). Inhi-
bition of BMP signaling by noggin
causes ectopic Barx-1 expression in the
distal, presumptive incisor ectomesen-
chyme and a transformation of tooth
identity from incisor to molar, thus
demonstrating the significance of BMP
signaling in patterning of craniofacial
structures (Tucker et al., 1998b). Taken
together, it is clear that ectoderm-
mediated signaling plays an important
role in determining the patterning of
craniofacial structures. At this point,
one cannot help but ask the question of
what is responsible for setting up the
signaling domain in the ectoderm. It
turns out that pharyngeal endoderm is
critical for this process.
Pharyngeal Endoderm
The pharyngeal endoderm makes a
limited contribution to craniofacial
development, but it serves as an in-
dispensable inducer during tissue–
tissue interactions, controlling
craniofacial development (Fig. 3).
The traditional view that the neural
crest plays the key role in patterning
the branchial arches must be recon-
sidered, because studies have found
that the early ablation of CNC cells
did not affect the development and
function of endoderm cells. These
crestless branchial arches were pat-
terned normally and had a sense of
individual identity, strongly sug-
gesting that the patterning of
endoderm is not dependent on CNC
cells (Veitch et al., 1999). Hox genes
are known to be critical for the iden-
tity of neural crest cells. Hoxa1 and
Hoxb1 double mutant mice had pat-
terning defects in the hindbrain;
specifically, rhombomere 4 lost its
ability to generate neural crest cells.
Consequently, the second branchial
arch crest population was not gener-
ated. Despite the neural crest defect,
the formation of the second branchial
arch endoderm and epithelial region-
alization was normal (Gavalas et al.,
2001).
Limited information is available re-
garding the patterning of pharyngeal
endoderm. Retinoic acid signaling
clearly plays a crucial role in regulating
pharyngeal endoderm development as
inactivation of retinaldehyde-specific
dehydrogenase type 2 (Raldh2), the reti-
noic acid synthetic enzyme, resulted in
the absence of all branchial arches cau-
dal to the first (Niederreither et al.,
1999). Inhibition of retinoid action by
pan-retinoic acid receptor (pan-RAR)
specifically perturbed the development
of the third and fourth branchial
arches, although the first and second
branchial arches formed normally
(Wendling et al., 2000). Recent studies
have shown that Raldh2 is expressed in
the lateral mesoderm flanking the
endoderm during early embryonic de-

velopment and may pattern the pha-
ryngeal endoderm development
(Niederreither et al., 2003; Graham
et al., 2005). Tbx-1 is another impor-
tant gene for pharyngeal endoderm
development. Tbx-1 mutant mice
show failure to form caudal pouches,
whereas the first pharyngeal pouch
develops normally (Jerome and Pa-
paioannou, 2001; Lindsay et al.,
2001). Of interest, Tbx-1 is also ex-
pressed in the lateral mesoderm
flanking the endoderm before the
patterning of the pharyngeal
endoderm (Brown et al., 2004).
The pharyngeal endoderm exerts its
regulatory function through tissue–tis-
sue interactions. For example, the pha-
ryngeal pouches generate several spe-
cialized epithelial structures, such as
the thyroid, parathyroid, and thymus
(Graham et al., 2005). The development
of these organs involves the interaction
between pharyngeal endoderm and its
flanking cranial neural crest cells. Com-
promised retinoic acid signaling from
the pharyngeal mesoderm affects the
development of pharyngeal endoderm,
which in turn causes defects in CNC
migration and development of pharyn-
geal pouch-derived organs, such as the
thymus and parathyroid glands (Nied-
erreither et al., 2003). Clearly, the pha-
ryngeal endoderm plays an important
role in regulating the morphogenesis of
pharyngeal arch derivatives.
Mesoderm
The cranial paraxial mesoderm has an
organization different than the trunk
paraxial mesoderm. The cranial parax-
ial mesoderm can be roughly divided
into (1) preotic head mesoderm, which
lacks any overt sign of segmentation
and never forms somites; and (2) occip-
ital somites, which are caudal to the otic
vesicle and give rise to epaxial and hy-
paxial muscles of the neck, the pharyn-
geal and laryngeal muscles that develop
in the caudal branchial arches, and the
tongue muscle (Fig. 3B; Noden, 1983;
Couly et al., 1992; Huang et al., 1999;
Mootoosamy and Dietrich, 2002).
There are profound morphological
and molecular differences between the
cranial and trunk paraxial mesoderm.
In comparison to the clearly seg-
mented somite development within
the trunk mesoderm, studies have
suggested that head mesoderm has
RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2361
become arrested evolutionarily as
somitomeres (Jacobson, 1963; Pack-
ard and Meier, 1983). Despite several
gene expression analyses suggesting
some degree of regionalization in the
cranial paraxial mesoderm (such as
Paraxis, Tbx1, and Hoxb-1), there is
no evidence supporting the existence
of metameric patterns. The impor-
tance of somitomeric head mesoderm
is yet to be determined (Noden and
Trainor, 2005). At the molecular level,
genes that drive mesoderm segmenta-
tion in the trunk are missing from the
pre-otic mesoderm in the head. When
cranial paraxial mesoderm was
grafted to the trunk region, activation
of the myogenic program in this ec-
topic position was inhibited, suggest-
ing that there are distinct regulatory
cascades acting in the development of
trunk and head muscles, possibly re-
flecting their distinct function and
evolution (Mootoosamy and Dietrich,
2002). Of interest, however, hetero-
topic grafting of paraxial mesoderm
cells to different regions of the cranial
mesoderm in the mouse showed no re-
striction in cell potency in the cranio-
caudal axis, revealing considerable
plasticity in the fate of the cranial me-
soderm (Trainor et al., 1994). Signals
from surface ectoderm and endoderm
as well as CNC may influence the fate
of mesoderm cells (Trokovic et al.,
2003).
In terms of patterning capability,
cranial paraxial mesoderm provides a
permissive substratum for the migrat-
ing CNC cells to populate the
branchial arch. Studies using chick
embryos suggest that cranial paraxial
mesoderm is able to direct CNC cell
movement independently of their epi-
thelial or mesenchymal organization
(Noden, 1986; Ferguson and Graham,
2004). Of interest, recent cell fate
mapping analysis has suggested that
myoblast precursors contain posi-
tional identity inherited from their so-
matic mesenchymal stem cell precur-
sors and can help to determine
skeletal homologies that are based on
muscle attachments (Matsuoka et al.,
2005). Conversely, CNC cells, which
provide most of the connective tissues
and tendons in the head, may pattern
and shape the individual cranial mus-
cle (Noden, 1986; Kontges and Lums-
den, 1996). The intimate relationship
between cranial paraxial mesoderm
and the CNC is clearly established
from the moment that CNC cells enter
the branchial arch and is maintained
throughout craniofacial development.
In parallel, studies have also demon-
strated that cranial mesoderm is ca-
pable of providing crucial signals for
endoderm development.
To date, there is not a clear demon-
stration of the patterning potential of
cranial paraxial mesoderm to regulate
craniofacial development in mam-
mals. This finding is largely because
of (1) lack of information on molecular
signals involved in the interaction be-
tween cranial paraxial mesoderm and
adjacent tissue and (2) our inability to
generate tissue-specific gene inactiva-
tions. Recently, using the Cre/loxp re-
combination approach, we have learned
that Myf5-Cre- or Mesp1-Cre-mediated
recombination can be used to generate a
targeted gene inactivation in the cra-
nial paraxial mesoderm-derived first
branchial arch myogenic core (our un-
published data). In Myf5-Cre;R26R
mouse embryos at E9.5, the first
branchial arch myoblasts are lacZ-
positive, accurately reflecting their ori-
gin from the paraxial mesoderm (Fig.
4). Targeted gene inactivation in the
cranial paraxial mesoderm-derived
mesenchyme will reveal important in-
formation on its patterning potential in
regulating craniofacial development.
Collectively, it is clear that cranio-
facial development requires contin-
ued interaction and contribution by
cell populations derived from the ec-
toderm, the neural crest, the parax-
ial mesoderm, and the endoderm.
These interactions occur en route
and at the terminal site of tissue and
organ genesis. There is no example
of any craniofacial developmental
event that can occur in a totally cell
autonomous or completely depen-
dent manner. Such a dichotomous
view can only impede our progress
toward a better understanding of the
regulatory mechanism of craniofa-
cial development. Instead, we need
to focus our efforts to unveil the mo-
lecular signals involved in tissue–
tissue interactions at each critical
time point and to gain a better under-
standing of tissue boundary establish-
ment, because a disturbed tissue
boundary during early embryonic de-
velopment can lead to craniofacial mal-
formations.
2362 CHAI AND MAXSON
SIGNALING INTERACTIONS
IN THE PATTERNING AND
MORPHOGENESIS OF
CRANIOFACIAL ORGANS
Recent studies have uncovered spe-
cific signaling cascades that play cru-
cial roles in regulating the patterning
and morphogenesis of craniofacial or-
gans. One of the best-studied models
of craniofacial organogenesis is tooth
development, which involves contin-
uous tissue–tissue interactions be-
tween the ectoderm-derived enamel
organ epithelium and the cranial
neural crest-derived ectomesen-
chyme (Slavkin et al., 1968; Kollar,
1972; Lumsden, 1988; Jernvall and
Thesleff, 2000; Tucker and Sharpe,
2004, and references therein). Multi-
ple growth and transcription factors
belonging to several signaling fami-
lies have been identified as critical
regulators at the initiation and
throughout all stages of tooth devel-
opment (http://bite-it.helsinki.fi).
Furthermore, most of the signaling
networks are used reiteratively
throughout tooth development and
are common to the regulatory sys-
tems critical for governing the devel-
opment of other organs (such as the
development of feather, hair, mam-
mary gland, salivary gland, and pan-
creas; see reviews by Nuckolls et al.,
1999; Shum et al., 2000). The grow-
ing scientific evidence suggests a
highly conserved biological mecha-
nism to regulate the patterning and
morphogenesis of craniofacial or-
gans, but studies are now beginning
to reveal the unique features of the
signaling network in regulating
tooth morphogenesis. This topic will
be the focus of our discussion here.
Patterning of the
Mammalian Dentition
The patterning of dentition depends
on the proper development of the oral
cavity, where maxillary and mandib-
ular teeth are housed and involves the
determination of location, shape, num-
ber, and size of tooth development. Mul-
tiple developmental decisions are
made in the patterning and develop-
ment of mammalian dentition. The
development of the oral cavity be-
gins with the establishment of the
frontonasal prominence and the first
branchial arch. The migrating CNC
cells are the major driving forces for
branchial arch development. As the
first branchial arch extends ventral–
medially, it gives rise to both man-
dibular and maxillary prominences
(although recent study suggests that
the maxillary prominence has a sep-
arate origin from the mandibular
prominence in chicken; Lee et al.,
2004). The primitive oral cavity
forms as a consequence of the fusion
among intermaxillary segments of
the frontonasal prominence and the
paired maxillary and mandibular
prominences. The oral epithelium, in
contrast to the aboral (suboral) epi-
thelium, becomes thickened to form
the dental lamina, marking the time
and location for tooth development
to begin.
Before the initiation of tooth devel-
opment, the mandibular ectoderm can
be roughly divided into the proximal
domain, which expresses FGF8 and
gives rise to molars, and the distal
domain, which expresses BMP4 and
gives rise to incisors (Fig. 5). Signifi-
cantly, FGF and BMP act antagonis-
tically to restrict Barx1 and Dlx2 ex-
pression to the proximal domain of the
first arch ectomesenchyme and Msx1
and Msx2 expression to the distal do-
main, respectively. The biological sig-
nificance of such a regional molecular
specification has been demonstrated
elegantly with inhibition of BMP sig-
naling resulting in ectopic Barx-1 ex-
pression in the distal, presumptive in-
cisor mesenchyme and producing
transformation of tooth form from in-
cisor-form to molar-form (Tucker et
al., 1998b). Recently, study has shown
that Barx1 played a key role in the
development of the dentition and di-
gestive system, which was vital for the
evolution of mammals (Miletich et al.,
2005). Overall, FGF and BMP growth
factor gradients are critical determi-
nants for specifying the initiation
sites of tooth formations as well as for
specifying the CNC-derived dental
mesenchyme to become incisor-form
vs. molar-form tooth organs.
Besides the proximal and distal do-
mains within the oral ectoderm, the
mandibular arch mesenchyme can
also be divided into an oral and aboral
(rostral– caudal) axis (Fig. 5). Signals
from the oral ectoderm appear to be
responsible for coordinating this divi-
sion. Of interest, the postmigratory
CNC cells have an intimate associa-
tion with the oral ectoderm (Chai et
al., 2000). This CNC distribution pat-
tern significantly overlaps with the
expression of two specific Lim-ho-
meobox domain genes, Lhx6 and
Lhx7, within the CNC-derived ecto-
mesenchyme, suggesting that they
may have critical functions in direct-
ing CNC cells to reach their destina-
tion (Grigoriou et al., 1998). In the
aboral region, the homeobox gene
Goosecoid (Gsc) is expressed where
Lhx6 and Lhx7 are excluded (Tucker
et al., 1999). Apparently, FGF8 is re-
sponsible for regulating the expres-
sion of both Lhx and Gsc genes. The
mandibular arch mesenchyme can be
roughly divided into the Lhx-positive
rostral (oral) domain and the Gsc-pos-
itive caudal (aboral) domain (Fig. 5).
Clearly, patterning of the mamma-
lian dentition is a three-dimensional
process. One aspect that is beginning
to be explored is the determination of
lingual– buccal (medial–lateral) den-
tal cusp patterning (Fig. 5). Lingual
and buccal cusps of the molar are dif-
ferent (in number and shape), and left
and right first molars, for example,
have a mirror image in patterning. It
will be fascinating to learn how sig-
nals are set up to achieve this har-
mony during tooth development.
The development of each dental
cusp is under the control of a transient
signaling center known as the enamel
knot, which is a dense population of
epithelial cells without any prolifera-
tive activity (Fig. 6). The enamel knot
contains multiple signaling molecules
(such as Shh, BMP, and FGF) and
controls the size and shape of cusp
formation. Thereafter, it is removed
by programmed cell death (Jernvall
and Thesleff, 2000; Tucker and
Sharpe, 2004). Unlike the incisor
tooth organ, molar development relies
on additional enamel knots (second-
ary and tertiary) to form multicusp
patterning.
BMP signaling is involved in regu-
lating the distance between adjacent
secondary enamel knots to control the
positioning of cusps. Two recently
published studies have provided im-
portant information regarding the
contribution of BMP signaling inhibi-
tion to the regulation of cusp pattern-
ing. Ectodin is a secreted BMP inhib-
itor with an inverse expression
 RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2363

pattern to p21, a hallmark cell cycle
regulator expressed in the enamel
knot during tooth development (Kas-
sai et al., 2005). Loss of ectodin results
in enlargement of the enamel knot,
highly altered cusp patterns, fusion of
molars, and extra teeth. Interestingly,
the most dramatic changes in cusp
patterning occurs along the buccal
side of the cusps, suggesting that ec-
todin functions in the evolution of lat-
eral bias in teeth. Noggin is another
widely distributed BMP inhibitor (Chen
et al., 2004). Overexpression of Noggin
in the dental epithelium blocked the de-
velopment of all mandibular and max-
illary third molars at the bud stage and
severely altered the patterning of max-
illary molars (Plikus et al., 2005). The
differential defects between mandibu-
lar and maxillary molars suggest a re-
quirement for varying thresholds of
BMP signaling.

Fig. 4. Two component genetic system for in-

delibly marking the fate of cranial neural crest-
(CNC), or mesoderm-, or ectoderm-derived
cells. A: Contribution of CNC cells during early
craniofacial development. CNC-derived cells
(blue) populate the frontonasal process (FN)
and the first branchial arch (arrow). B: Cross-
section of an embryonic day (E) 9.5 mouse em-
bryo (Wnt1-Cre/R26R) shows CNC-derived
cells (blue) in the first branchial arch. The arrow
indicates the paraxial mesoderm-derived myo-
Fig. 4. genic core in the first arch. The arrowhead in-
dicates ectoderm. C: Contribution of paraxial
mesoderm-derived cells during early craniofa-
cial development. The arrow indicates the
paraxial mesoderm-derived myogenic core
(blue) in the first branchial arch of the Myf5-Cre/
R26R sample. D: Cross-section of the first and
second branchial arch contains paraxial meso-
derm-derived myogenic cells (blue; arrow). No-
tice these cells are pink in B. E: Contribution of
ectodermal cells during craniofacial develop-
ment. Ectoderm-derived cells (blue) cover the
surface of the K14-Cre/R26R embryo. The ar-
row indicates the first branchial arch surface
ectoderm. F: Cross-section of the first branchial
arch shows ectoderm-derived cells (arrow) on
the surface.

Fig. 5. Patterning of the first branchial arch.

A: The frontal view of a scanning electronic
microscopic image of an embryonic day (E) 9.5
mouse embryo (25–29 somites) shows the fron-
tonasal process (FN), the maxillary (max), and
the mandibular (mand) process. B: Schematic
drawing of A. C: The first branchial arch can be
divided into proximal/distal, oral/aboral, and
buccal/lingual (see D) domains. In the distal
domain, bone morphogenetic protein (BMP)
signaling in the ectoderm regulates the expres-
sion of Msx1 and Msx2. It also provides inhibi-
tion for Barx1 expression in the cranial neural
crest (CNC) -derived mesenchyme in the prox-
imal domain. In the proximal domain, fibroblast
growth factor (FGF) signaling regulates the ex-
pression of multiple transcription factors (mod-
ified from Tucker and Sharpe, 2004). D: Molar
teeth (m1, m2, m3) show different cusp patterns
Fig. 5. along buccal and lingual aspects of the jaw.
2364 CHAI AND MAXSON
Control of Signaling Level
and Tooth Development
Recent studies show that local feed-
back provides a tightly controlled FGF
and BMP gradient to ensure proper
tooth development. For example, Is-
let1 positively regulates the expres-
sion of Bmp4, whereas a range of
Pitx2 levels can differentially regulate
the expression of Fgf8 and Bmp4 dur-
ing initial tooth development (Lu et
al., 1999; Mitsiadis et al., 2003; Liu et
al., 2003). Pitx2 is restricted to the
dental epithelium throughout tooth
morphogenesis (Fig. 6). Mutation of
the human PITX2 gene results in
Rieger’s syndrome, an autosomal
dominant disorder, that leads to the
absence of certain teeth and defects of
the eye (Rieger, 1935; Semina et al.,
1996). Loss of Pitx2 results in re-
tarded tooth development at the initi-
ation/early bud stage, clearly demon-
strating the important function of
Pitx2 gene in regulating tooth devel-
opment (Lu et al., 1999). Significantly,
loss of one copy of the Pitx2 gene can
also affect tooth development in mice
(Gage et al., 1999). Therefore, Pitx2
acts in a dosage-specific manner in hu-
mans and mice. The expression of
Pitx2 gene is sensitive to the level of
BMP4 or FGF8 signaling, indicating a
feedback loop among these signaling
molecules (St. Amand et al., 2000).
Shh, another important epithelial
signaling molecule, controls the prolif-
eration of enamel organ epithelial
cells during initiation of tooth devel-
opment (Fig. 6; Bitgood and McMa-
hon, 1995; Dassule and McMahon,
1998; Hardcastle et al., 1998; Co-
bourne et al., 2001). The Wnt signal-
ing pathway interacts with Shh signal-
ing to establish boundaries during tooth
development. Specifically, Wnt-7b acts
to repress the expression of Shh in non-
dental oral ectoderm, whereas Shh ex-
pression is restricted to the dental ecto-
derm and can instruct, permit, or
induce tooth bud formations (Sarkar et
al., 2000).
Determination of Tooth
Number
Humans have a dental formula of
3.2.1.2/2.1.2.3 in the permanent den-
tition [two incisors, one canine, two
premolars (bicuspids) and three mo-
lars, with “/” marking the front mid-
line]. Some rodents, such as mice,
have a dental formula of 3.0.0.1/
1.0.0.3. The initiation of each tooth
germ is marked by the formation of
dental lamina. Ectodysplasin (EDA)
signaling cascade molecules, which
belong to the tumor necrosis factor
family of ligands, are critical regula-
tors for the determination of tooth
number during embryonic develop-
ment (Fig. 6). Mice with a compro-
mised EDA signaling cascade, such
as Tabby (EDA), Downless (EDAR,
EDA receptor), and Crinkled (EDAR-
ADD, EDA intracellular adaptor
protein) mutations, show abnormal
tooth numbers and defects in the de-
velopment of other ectodermal or-
gans (hair follicles and exocrine
glands; Sofaer, 1969, 1977; Headon
et al., 2001; Pispa and Thesleff,
2003; Tucker et al., 2004). On the
other hand, overexpression of EDA
signaling molecules results in an ex-
pansion of the molar tooth field and
the formation of a supernumerary
tooth distal to the first molar (Mus-
tonen et al., 2003; Tucker et al.,
2004). Of interest, the supernumer-
ary tooth has the cusp patterning of
a premolar, suggesting that dental
cusp patterning is highly dependent
on the exact position in the oral cav-
ity where tooth development occurs.
BMP signaling is another morpho-
regulator for the number, size, and
shape of tooth development. Loss of
BMP receptor 1a (Bmpr1a) caused
retarded tooth development (Andl et
al., 2004). Overexpression of BMP
inhibitor Noggin in the oral ecto-
derm resulted in miniaturization of
maxillary first and second molars,
and retarded maxillary third and
mandibular molar development at
the lamina stage (Plikus et al.,
2005). The selective loss of molars in
Noggin-overexpressing mice, along
with the abnormal buccal cusp pat-
terning in ectodin null mutant mice,
strongly suggest that various tooth
germs and dental cusps have differ-
ential requirements for the level of
BMP signaling. Modulation of BMP
signaling may account for one of the
mechanisms responsible for changes
in tooth number, size, and shape
through evolution.
Continued
Epithelial–Mesenchymal
Interactions and the Success
of Tooth Morphogenesis
After the initiation of tooth develop-
ment, there is continued interaction
between the dental epithelium and
the CNC-derived ectomesenchyme
throughout tooth morphogenesis. The
initial tooth generating potential re-
sides within the epithelium and is ca-
pable of inducing nontooth forming ec-
tomesenchyme to develop teeth (Mina
and Kollar, 1987; Jernvall and Thesleff,
2000). Soon after development, this
tooth-forming potential shifts to the
dental mesenchyme. This shift of
tooth-forming potential coincides
with a shift in BMP signaling from
the epithelium to the mesenchyme.
Morphologically, at this time, tooth
development advances into the bud
stage (Fig. 6). Multiple signaling
molecules within the enamel organ
epithelium have been implicated in
the regulation of transcription fac-
tors in the surrounding ectomesen-
chyme during the bud to the cap
stage transition (Fig. 6; Jernvall and
Thesleff, 2000; Tucker and Sharpe,
2004). Interestingly, individual null
mutations of Msx1, Lef1, or Pax9 all
result in retarded tooth development
at the bud stage, indicating the crit-
ical roles of these transcription fac-
tors during the transition from the
bud to cap stage tooth development
(Satokata and Maas, 1994; Kratoch-
wil et al., 1996; Peters et al., 1998;
Sasaki et al., 2005). Ectopic overex-
pression and tissue recombination
experiments have shown that these
transcription factors are regulated
by epithelial signals, can control the
expression of growth factors and
other transcription factors in the ec-
tomesenchyme and provide instruc-
tive feedback to the enamel organ
epithelium. Thus, they are essential
determining factors during the cap
stage tooth development.
To date, no null mutation experi-
ments have resulted in failure of ini-
tiation of tooth development (thicken-
ing of the dental lamina). Some of the
compound null mutations, however,
have resulted in retardation of tooth
development at an early bud stage,
such as Msx1⫺/⫺/Msx2⫺/⫺, Dlx1⫺/⫺/
Dlx2⫺/⫺, and Gli2⫺/⫺/Gli3⫺/⫺.

Clearly, there is functional redun-
dancy among different members of the
same transcription factor family in
regulating the advancement of tooth
morphogenesis. Of interest, some of
the null mutations reveal regional
specificity of certain signaling mole-
cules in regulating tooth morphogene-
sis. For example, only maxillary molar
tooth organs were affected in Dlx1⫺/
⫺/⫺
⫺/Dlx2 double mutants. Further-
more, loss of Dlx1 and Dlx2 genes re-
sulted in a change of cell fate in the
dental mesenchyme region from odon-
togenic to chondrogenic (Thomas et
al., 1997; Tucker and Sharpe, 1999).
This finding also raises the possibility
that a subpopulation of CNC cells car-
rying Dlx1/Dlx2 genes migrates into
the maxillary molar region and, upon
receiving the proper instruction from
the dental epithelium, can contribute
to the formation of tooth organ. By
using the two-component genetic sys-
tem (Wnt1-Cre/R26R) for indelibly
marking the progenies of CNC cells
(Fig. 4), it may be feasible to test the
hypothesis that a subpopulation of
CNC cells designated for the maxil-
lary molar region is affected in Dlx1/
Dlx2 double-mutant mice (Chai et al.,
2000; Han et al., 2003). The outcome
of such experiments may address the
speculation that a predetermined sub-
population of CNC cells possesses in-
structive functions in the induction of
tooth development.
The activin ␤A null mutation re-
veals another example of region-spe-
cific signaling in regulating tooth de-
velopment. All teeth, except the
maxillary molars, are arrested at the
bud stage in activin ␤A⫺/⫺ mice, a
reverse phenotype compared with the
Dlx1/Dlx2 double mutant (Ferguson
et al., 1998). Because activin ␤A is
expressed in the CNC-derived ecto-
mesenchyme, it may help to specify
the fate of CNC cells during tooth de-
velopment.
Overall, continuous and reciprocal
epithelial–mesenchymal interaction is
the key for successful tooth organ mor-
phogenesis. From initiation to odonto-
blast/ameloblast differentiation to ma-
trix formation, this interaction provides
precise communication between two ad-
jacent tissue types and governs the
number, size, and shape of tooth forma-
tion. Our understanding of these biolog-
ical processes may serve as a founda-
RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2365
tion for the future design and
fabrication of tooth regeneration.
SKULL DEVELOPMENT
AND TISSUE BOUNDARY
The vertebral skull represents an ex-
cellent model for the investigation of
development and evolution. In mam-
mals, both CNC- and mesoderm-de-
rived cells contribute to the develop-
ment of the skull (Fig. 3D). Different
elements of the skull are established
by the formation of tissue boundaries,
which reflect an evolutionary contri-
bution and are critical for the pre- and
postnatal dynamic development of the
head and face. Recent studies are now
beginning to address the molecular
regulation of patterning and size de-
termination of different elements of
the skull. Multiple excellent reviews
have addressed the regulation of skull
vault development (Wilkie and Mor-
riss-Kay, 2001; Santagati and Rijli,
2003; Morriss-Kay and Wilkie, 2005),
but there are very few reviews that
address the molecular regulatory
mechanism of facial bone develop-
ment. Here, using mandible and pal-
ate development as examples, we
summarize recent advancements to-
ward the understanding of the regula-
tory mechanism of craniofacial bone
development.
The skull consists of the neurocra-
nium and viscerocranium. The neuro-
cranium (skull vault and base) sur-
rounds and protects the brain. In
humans, eight bones compose the neu-
rocranium: the paired temporal and
parietal bones and the singular fron-
tal, sphenoid, ethmoid, and occipital
bones. Fourteen bones compose the
viscerocranium (the jaws and other
pharyngeal arch derivatives): the
paired nasal bones, maxillae, palatine
bones, lacrimal bones, zygoma and in-
ferior nasal conchae, along with the
singular vomer and mandible. The
viscerocranium derives from the neu-
ral crest, forms the face, and supports
the functions of feeding and breath-
ing.
Until recently, the tissue origin of
the skull vault has been controversial
because of conflicting reports using
quail– chick grafting. Noden (1978,
1988) reported that the CNC only con-
tributes to the rostral portion of the
frontal bones and that the remainder
of the skull vault is of mesoderm ori-
gin. In contrast, Couly and coworkers
(1993) concluded that the skull vault
is entirely neural crest derived. In the
mouse model, it has been demon-
strated elegantly that frontal bones
are neural crest-derived and parietal
bones are of mesoderm origin (Jiang et
al., 2002). The posterior part of the
skull vault (supraoccipital and exoc-
cipital bones) derives from the occipi-
tal somites (Fig. 3D). The dura mater
that underlies the frontal and parietal
bones is also neural crest derived
(Jiang et al., 2002; Ito et al., 2003;
Sasaki et al., 2006). The coronal and
sagittal sutures are of mesoderm ori-
gin (Morriss-Kay and Wilkie, 2005).
Clearly, the skull vault elements are
developed at the boundary between
CNC- and mesoderm-derived tissue.
This boundary is of paramount impor-
tance in mediating tissue–tissue in-
teraction that control skull vault de-
velopment. When there is mixing of
the two cell populations as the result
of a gene mutation (such as in the
ephrin-B1 or Twist mutant), the
boundary between CNC and the me-
soderm is lost, resulting in the prema-
ture fusion of cranial sutures known
as craniosynostosis (Twigg et al.,
2004; Merrill et al., 2006).
MANDIBULAR
MORPHOGENESIS
Patterning of the branchial arches re-
quires the establishment of both inter-
branchial arch and intrabranchial
arch identities (Depew et al., 2002a).
It is well established that Hox, Pbx,
and Otx homeobox genes are critical
for the normal patterning of inter-
branchial arch identities (Gendron-
Maguire et al., 1993; Rijli et al., 1993;
Matsuo et al., 1995; Selleri et al.,
2001). For example, there clearly is a
Hox gene code that controls branchial
arch development. To give rise to the
derivatives of the first branchial arch,
the structure needs to be Hox gene
negative. Until recently, however, less
information has been available about
the genetic control of the establish-
ment of the intrabranchial arch iden-
tities. This is an important area in
craniofacial development, because hu-
man mandibular dysmorphogenesis
appears to be a common malformation
and appears in multiple congenital
2366 CHAI AND MAXSON
birth defect syndromes, ranging from
agnathia (agenesis of the jaw) to mi-
crognathia to patterning malforma-
tions.
Based on the mouse model, we know
now that the first branchial arch
(mandibular arch) becomes apparent
at E8.0 –E8.5 (6 – 8 somites) as small
swellings on the side of the developing
head. As CNC cells migrate into and
proliferate within the mandibular
arch, it develops rapidly toward the
ventral midline. The CNC-derived
cells are localized immediately subja-
cent to the covering epithelium (Chai
et al., 2000). Mandibular development
depends upon the interaction between
the oral ectoderm and the CNC-de-
rived mesenchyme within the first
branchial arch. Within the oral ecto-
derm, signaling molecules, such as
BMP, TGF-␤, and FGF, are expressed
in a region-specific manner (Chai et
al., 1994, 1997; Trumpp et al., 1999;
Ito et al., 2002; Liu et al., 2005). They
may regulate homeobox-containing
genes (such as Dlx, Lhx, and Gsc)
within the CNC-derived mesenchyme
to generate early polarity and are re-
sponsible for patterning of the first
branchial arch.
Ectodermal FGF signaling is criti-
cal for CNC cell survival in the man-
dibular arch as conditional inactiva-
tion of Fgf8 in the ectoderm caused
increased apoptotic activity and a dra-
matic loss of all first branchial arch
skeletal structures (except the most dis-
tal portion of the mandible; Trumpp et
al., 1999). During early embryonic de-
velopment, however, FGF signaling is
not required for cell proliferation and
survival, but instead is required for cell
migration. Therefore, FGF signaling
has differential functional specificity in
different developmental contexts (Sun
et al., 1999).
In the proximal domain of the first
branchial arch, FGF signaling directly
or indirectly regulates the expression
of homeobox genes in the mesenchyme
to control the development of mandi-
ble (see previous discussion). In the
distal domain of the first branchial
arch, BMP signaling appears to be a
crucial regulator for mandibular mor-
phogenesis. BMP4 is expressed within
the distal ectoderm that covers the
first branchial arch mesenchyme at
E9.5. The postmigratory CNC cells
are exposed to the instructive signal-
ing (such as BMP) from the ectoderm
and become committed to give rise to
structures associated with the distal
portion of the first arch. As embryonic
development progresses, there is a
shift of the instructive capability for
patterning the mandible from the ec-
toderm to the CNC-derived mesen-
chyme. The patterning of the proxi-
mal– distal domain of the first
branchial arch is achieved through
the antagonistic interaction between
BMP and FGF, which controls the
mesenchymal expression of signaling
molecules, such as Msx1 and Barx1,
respectively (Tucker et al., 1998b;
Tucker and Sharpe, 2004). BMP sig-
naling clearly has an important role in
regulating mandibular morphogene-
sis. Specifically, loss of BMP signaling
in the oral ectoderm and the pharyn-
geal endoderm results in extreme phe-
notypes, ranging from an almost com-
pletely missing mandible to severe
defects in the distal region (Liu et al.,
2005). BMP target genes Msx1 and
Msx2 have differential dose require-
ments for BMP4 signaling, and they
act in a functionally redundant man-
ner. Both Msx1 and Msx2 are ex-
pressed in the midline region, and loss
of BMP4 signaling results in compro-
mised Msx gene expression. Mutation
of Msx1 and Msx2 genes results in
midline cleft of the first arch and se-
vere defects in mandibular morpho-
genesis (Satokata et al., 2000; Ishii et
al., 2005; our unpublished data).
Mandibular development requires
proper modulation of BMP and FGF
signaling in the ectoderm. This modu-
lation is achieved through the antag-
onistic interaction between BMP and
FGF signaling, the inhibition of BMP
signaling by Noggin or Chordin and by
the feedback from the underlying CNC-
derived mesenchyme (Stottmann et al.,
2001; Wilson and Tucker, 2004; Liu et
al., 2005). For example, BMP signaling
is required for maintaining FGF signal-
ing in the proximal domain of the man-
dibular arch, but it represses FGF sig-
naling in the distal domain (Liu et al.,
2005). Furthermore, BMP signaling is
subject to multiple points of regulation,
at the ligand, receptor, Smad, and tran-
scription complex level (Massague et
al., 2005). Clearly, tightly controlled
BMP and FGF signaling is of para-
mount importance for mandibular mor-
phogenesis.
Similar to the proximal– distal do-
mains within the ectoderm of the first
branchial arch, there are discrete pop-
ulations within the underlying CNC-
derived mesenchyme. Dlx genes are
known to play important roles in reg-
ulating the patterning of the develop-
ing jaw. Six Dlx genes (Dlx1, Dlx2,
Dlx3, Dlx5, Dlx6, and Dlx7) have been
described in mice (Dolle et al., 1992;
Robinson and Mahon, 1994; Simeone
et al., 1994; Qiu et al., 1995, 1997;
Stock et al., 1996). During the devel-
opment of the branchial arches, Dlx
genes show nested expression patterns
and play important roles in establishing
the identity along the proximodistal
axis within each branchial arch (Depew
et al., 2002a, 2005). Specifically, Dlx1/2
are expressed in the CNC-derived ecto-
mesenchyme both in the proximal and
distal region of the branchial arch,
whereas Dlx5/6 and Dlx3/7 show pro-
gressively restricted expression do-
mains toward the distal region of the
branchial arch.
Recent studies have shown that a
combinatorial Dlx code regulates the
establishment of the distinct skeletal
elements within a given branchial
arch unit (Qiu et al., 1995, 1997; De-
pew et al., 2002a,b; Cobourne and
Sharpe, 2003). Loss of Dlx5 and Dlx6
results in a homeotic transformation
of the lower jaw into an upper jaw,
supporting a model of patterning
within the branchial arch that relies
on a nested pattern of Dlx gene ex-
pression (Depew et al., 2002b). In ad-
dition, jaw development is sensitive to
the dosage of Dlx genes, as haploinsuf-
ficiency of single or multiple Dlx genes
has a gradient effect on mandibular
development (Depew et al., 2005).
This observation clearly suggests that
the expression of Dlx genes must be
tightly regulated. To date, little infor-
mation is available about the direct
regulation on Dlx gene expression. Al-
though FGF8-soaked beads placed in
the first arch epithelium are able to
induce Dlx2 and Dlx5 expression in
the mandibular mesenchyme, loss of
Fgf8 in the ectoderm shows unaltered
Dlx2 and Dlx5 expression in the first
branchial arch (Trumpp et al., 1999).
Similarly, BMP-soaked beads are able
to induce Dlx gene expression, but the
endogenous BMP and Dlx expression
patterns do not suggest a direct regu-
latory relationship. Overall, Dlx genes
 RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2367

are clearly important for intrabranchial

arch patterning, and further studies in
this area will advance our understand-
ing of their role and the regulatory
mechanism in this process.

PALATOGENESIS AND THE

MOLECULAR MECHANISM
OF CLEFT PALATE
The mammalian palate develops from
two primordia: the primary palate and
the secondary palate. The primary
palate represents only a small part of
the adult hard palate. The secondary
palate is the primordium of the hard
and soft parts of the palate. Palate
development is a multistep process that

Fig. 6.

Fig. 6. Schematic drawing of reciprocal and

reiterative signaling in regulating epithelial–
mesenchymal interactions throughout sequen-
tial stages of tooth morphogenesis. A precise
spatial and temporal orchestration of multiple
growth and transcription factors (as listed
within each box) is critical for the initiation as
well as patterning of tooth germ development.
These signaling molecules tightly regulate both
the enamel organ epithelia and the cranial neu-
ral crest-derived dental mesenchyme. Specifi-
cally, when tooth development is initiated with
formation of dental lamina, its underlying mes-
enchyme is almost entirely populated with cra-
nial neural crest (CNC) -derived cells (dark
blue). As tooth germ develops from the bud to
the cap stage, CNC-derived cells are concen-
trated (dark blue) at the interface with the
enamel organ epithelium, whereas the periph-
eral portion of the dental sac is populated with
both CNC- and non–CNC-derived cells (light
blue). Molecular signaling residing within the
enamel organ epithelium and the CNC-derived
dental mesenchyme are engaged in a constant
and reciprocal dialogue to mediate tooth mor-
phogenesis. The dark red dot represents
enamel knot-a signaling center within the
enamel organ epithelia, whereas light pink rep-
resents enamel organ epithelium (modified from
Jernvall and Thesleff, 2000; Chai et al., 2000).

Fig. 7. Anatomy of palatogenesis and five cat-

egories of palatal shelf defects that result in
cleft palate. A: Mouse palatogenesis starts at
embryonic day (E) 11.5. By E13.5, palatal
shelves (P) are on both sides of the tongue (T).
Each palatal shelf can be divided into the me-
dial (m) and lateral (l) aspects. t, tooth germ.
B,C: Between E13.5 and E14.0, palatal shelves
turn horizontally above the tongue and face
each other along the midline. D: At E14.5, pal-
atal fusion begins to take place. The arrow in-
dicates the midline epithelial seam. n, nasal
epithelium; o, oral epithelium. E,F: From E15.5
to E16.5, palatal fusion is completed through-
out the entire palate. Five different categories of
palatal shelf defects that result in cleft palate
Fig. 7. (also see Zhang et al., 2002). P, palatal shelf.
2368 CHAI AND MAXSON
involves palatal shelf growth, elevation,
midline fusion of palatal shelves, and
the disappearance of midline epithelial
seam (Fig. 7). The palatal structures
are composed of the CNC-derived ecto-
mesenchyme and pharyngeal ectoderm
(Ferguson, 1988; Shuler, 1995). The ep-
ithelia that cover the palatal shelves
are regionally divided into oral, nasal,
and medial edge epithelium (Fig. 7D).
The nasal and oral epithelia differenti-
ate into pseudostratified and squamous
epithelia, respectively, whereas the me-
dial edge epithelium (MEE) is removed
from the fusion line by means of pro-
grammed cell death and cell migration
(Martinez-Alvarez et al., 2000; Vaziri
Sani et al., 2005).
Mouse palatogenesis initiates at
E11.5 as marked by the formation of
palatal shelves extending from the in-
ternal aspects of maxillae. After initi-
ation, the palatal shelves project
downward on each side of the tongue
between E12.5 and E13.5 (Fig. 7A,B).
As the jaws develop, the tongue de-
scends, thus providing space to accom-
modate the horizontal apposition of
palatal shelves above the tongue (Fig.
7C). The fusion of palatal shelves oc-
curs at E14.5, resulting in the forma-
tion of continuous palate (Fig. 7D).
Both the elevation and fusion of the
palatal shelves occur in an anterior to
posterior sequence. The complete fu-
sion of palatal shelves results in the
separation of the oral cavity from the
nasal cavity (Fig. 7E,F).
Fate of Medial Edge
Epithelial Cells and the
Contribution of Cranial
Neural Crest Cells During
Palatogenesis
There has been a tremendous interest
in the fate of medial edge epithelial
cells during palatal fusion. Apoptosis
is clearly one of the important mecha-
nisms for eliminating MEE cells dur-
ing palatal fusion (Farbman, 1968;
Saunders, 1966; Dudas and Kaarti-
nen, 2005). At the molecular level,
TGF-␤ and RA are critical inducers for
apoptosis of the MEE. The dosage of
these signaling molecules may differ-
entially control the progression of the
cell cycle in the MEE (Cuervo et al.,
2002; Martinez-Alvarez et al., 2000;
Cuervo and Covarrubias, 2004). An al-
ternative fate for the MEE is migra-
tion along the midline toward the na-
sal and oral epithelia, resulting in the
loss of MEE (Carette and Ferguson,
1992, Hilliard et al., 2005). Studies
from multiple laboratories have sug-
gested that epithelial–mesenchymal
transformation (EMT) is an important
cellular mechanism for the disappear-
ance of MEE (Fitchett and Hay, 1989;
Griffith and Hay, 1992; Shuler et al.,
1992). These studies were largely
based on cell lineage tracing using
membrane-intercalating dye and epi-
thelial and mesenchymal cellular
markers. Recently, however, studies
using genetic cellular markers have
challenged the theory of EMT during
palatal fusion. Both in vivo and in
vitro studies show that EMT does not
occur during palatal fusion (Cuervo et
al., 2002; Vaziri Sani et al., 2005; Xu
et al., 2006). Apoptosis and cell migra-
tion may be sufficient to account for
the cellular mechanism for the disap-
pearance of MEE cells.
CNC cells are critical for palatogen-
esis. Until recently, however, little
has been known about the fate of the
CNC-derived palatal mesenchyme or
the molecular mechanisms that regu-
late the epithelial–mesenchymal in-
teractions during palate development.
The lack of information is largely
due to the difficulty in CNC cell la-
beling and fate analysis in the palate.
Significantly, using the Wnt1-Cre/
R26R model, we have systematically
followed the migration, proliferation,
and differentiation of CNC cells
throughout embryogenesis. We show
that the CNC-derived ectomesen-
chyme contribute significantly to the
palatal mesenchyme and that there is
a dynamic distribution of these CNC
cells during palatogenesis (Chai et al.,
2000; Ito et al., 2003). This two-
component genetic system provides
the opportunity to integrate analysis
of the fate and function of the mam-
malian neural crest with mouse mo-
lecular genetics in both normal and
abnormal embryonic development.
Mouse Models for
Investigating the Molecular
Regulatory Mechanism of
Palatogenesis
Multiple genetically mutated mouse
models have made significant contri-
butions to our understanding of the
gene pathways involved in palate de-
velopment and the nature of signaling
molecules that act in a tissue-specific
manner at critical stages of embryonic
development. Interestingly, however,
most of genetic mutations cause mul-
tiple structural and functional defects
in the developing embryo. Conse-
quently, assessment of the role of a
particular gene in regulating palato-
genesis has been a challenge. To fur-
ther complicate the issue, cleft lip
and/or palate is a complex trait caused
by multiple genetic and environmen-
tal factors (Murray, 2002). Neverthe-
less, these animal models have ad-
vanced our understanding of some
important gene functions in human
palate development.
One of the important discoveries
has been the existence of genetic het-
erogeneity along the anterior–poste-
rior and medial–lateral axes of the de-
veloping palate (for review, see
Hilliard et al., 2005). This heterogene-
ity may provide differential regula-
tory mechanisms for the fusion of the
anterior vs. posterior region of the pal-
ate. For example, MEE cells begin to
undergo apoptosis at different times
during palatal fusion, depending on
their location. It has been shown that
apoptosis of MEE cells is triggered by
palatal shelf contact in the anterior
region, whereas it is initiated before
any contact between the opposing
shelves in the posterior region (Cu-
ervo et al., 2002). This may be the
result of differential molecular signals
in the palatal mesenchyme along the
anteroposterior (A-P) axis that in-
struct different fates to the palatal ep-
ithelium (Ferguson and Honig, 1984).
More recent studies have demon-
strated that constant and reciprocal
interactions between the palatal epi-
thelium and the CNC-derived mesen-
chyme are responsible for setting up
this genetic heterogeneity along the
A-P axis and are crucial for normal
palatal development and fusion
(Zhang et al., 2002; Murray and
Schutte, 2004; Rice et al., 2004).
Multiple genes have been found to
be critical for the development of the
anterior region of the palate. For ex-
ample, Msx1, Bmp4, Bmp2, Fgf10,
and Shox2 show restricted expression
patterns in the anterior region of the
palate (Rice et al., 2004; Hilliard et al.,

2005). Loss of Msx1 results in a cell
proliferation defect in the CNC-de-
rived palatal mesenchyme in the an-
terior region of secondary palate.
BMP4 functions downstream of Msx1
and controls the expression of Shh in
the palatal epithelium. Shh in turn
regulates the expression of Bmp2 in
the mesenchyme to promote cell prolif-
eration (Zhang et al., 2002). Meanwhile,
FGF10 is expressed in the anterior pal-
atal mesenchyme and functions in a
paracrine manner through its receptor
FGFR2 in the palatal epithelium to me-
diate Shh expression, which in turn reg-
ulates Bmp2 expression in the mesen-
chyme to promote cell proliferation. So
the BMP and FGF signaling pathways
converge on Shh signaling in the epithe-
lium to control the growth of the ante-
rior region of the palatal shelf. The
Shox2 gene is exclusively expressed in
the anterior palatal mesenchyme. Loss
of Shox2 results in an incomplete cleft
of the anterior hard palate, whereas fu-
sion of the posterior palate is normal
(Yu et al., 2005). Significantly, this
study clearly demonstrates that there
are different regulatory mechanisms
that control the development and fusion
of the anterior and posterior parts of the
palate and that a successful fusion of
the posterior part of the palate can oc-
cur, despite that there is a failure of
anterior palate fusion.
We know less about the specific
gene expression patterns in the poste-
rior region of the palate. Fgfr2 is ex-
pressed in the epithelium and the
CNC-derived mesenchyme in the mid-
dle and posterior palate. FGF8 signal-
ing selectively induces the expression
of Pax9 in the posterior region of pal-
atal mesenchyme. Loss of Pax9 results
in a palatal shelf development defect
and cleft palate (Peters et al., 1998;
Hilliard et al., 2005). To date, there is
little known about the regulation of
Pax9 expression in the developing pal-
ate. Therefore, the biological signifi-
cance of FGF8-mediated Pax9 expres-
sion during palatogenesis remains to
be determined. In addition to the dif-
ferential gene expression patterns
along the A-P axis of the developing
palate, there is also mesenchymal het-
erogeneity between the medial and
lateral regions of the palatal shelf
(Fig. 7A). For example, the odd
skipped-related genes Osr1 and Osr2
are expressed in a medial–lateral gra-
RECENT ADVANCES IN CRANIOFACIAL MORPHOGENESIS 2369
dient in the palatal shelf. Signifi-
cantly, mutation of the Osr2 gene re-
sults in the compromised development
of the medial aspect of palatal shelf
development and retards palatal shelf
elevation (Lan et al., 2004). The ex-
pression of Fgfr2 is focused on the me-
dial aspect of the developing palatal
shelf, suggesting a possible functional
significance in regulating the develop-
ment and elevation of palatal shelf.
In analyzing different mutant ani-
mal models with cleft palate, we pro-
pose to divide the palatal shelf devel-
opment defects into the following five
categories. (1) Failure of palatal shelf
formation (Fig. 7). Although this is a
severe type of palatal shelf develop-
ment defect, it has a rare occurrence.
Mutation of activin-␤A causes a se-
vere facial primordia development de-
fect, which is likely responsible for the
retardation of palatal shelf develop-
ment and complete cleft palate (Mat-
zuk et al., 1995). The Fgfr2 mutation
also affects the initial development of
the palatal shelf and results in com-
plete cleft palate (Rice et al., 2004). (2)
Fusion of the palatal shelf with the
tongue or mandible (Fig. 7). For exam-
ple, loss of function mutation of Fgf10
results in anterior palatal shelf fusion
with the tongue, whereas the middle
and posterior part of the palatal shelf
adheres to the mandible, thus pre-
venting the elevation of the palatal
shelf (Alappat et al., 2005). In hu-
mans, mutations in TBX22 have been
reported in families with X-linked
cleft palate and ankyloglossia (Bray-
brook et al., 2001). Similarly, Tbx22 is
expressed in the developing palate
and tongue in mice, suggesting an im-
portant role of Tbx22 in regulating
palate and tongue development (Bush
et al., 2002). (3) Failure of palatal
shelf elevation (Fig. 7). Studies have
shown that mutations of Pax9, Pitx1,
or Osr2 can lead to failed palatal shelf
elevation and cleft palate defect (Pe-
ters et al., 1998; Szeto et al., 1999; Lan
et al., 2004). The cellular defect is
mainly associated with the CNC-de-
rived palatal mesenchyme, suggesting
important functions of these tran-
scription factors in regulating the fate
of CNC cells during palatogenesis. (4)
Failure of palatal shelves to meet af-
ter elevation (Fig. 7). By far, this is the
most common type of cleft palate de-
fect documented in animal studies.
For example, mutations in Msx1 and
Lhx8 and conditional inactivation of
Tgfbr2 in CNC cells or Shh in the ep-
ithelium all result in retarded palatal
shelf development (Satokata and
Maas, 1994; Zhao et al., 1999; Ito et
al., 2003; Rice et al., 2004). (5) Persis-
tence of medial edge epithelium (Fig.
7). In Tgfb3 or Egfr mutant mice,
there is an alteration of the fate of
MEE cells (Kaartinen et al., 1995;
Proetzel et al., 1995; Miettinen et al.,
1999). In Tgfb3 null mutant mice,
MEE cells fail to undergo apoptosis
and persist along the midline to pre-
vent normal fusion. In addition to its
function in regulating the fate of
MEE, TGF-␤3 is also critical for
proper proliferation of the CNC-de-
rived palatal mesenchyme (our un-
published data). Furthermore, muta-
tions in TGF-␤3 have been associated
with cleft palate in humans, under-
scoring the crucial function of TGF-␤
signaling in regulating palatogenesis
(Lidral et al., 1998).
PROSPECTUS
Each year, approximately 250,000 in-
fants born in the United States have
some mental or physical defects.
Three fourths of all malformations
seen at birth involve craniofacial dys-
morphogenesis, affecting the develop-
ment of head, face, or neck. These
malformations are particularly devas-
tating, as our faces are our identity—
they are how we see ourselves and
how others see us. In recent years,
research has progressed so that we
know the precise genetic error that
leads to many craniofacial birth de-
fects.
At the conclusion of a recent Gordon
Research Conference on Craniofacial
Morphogenesis and Tissue Regenera-
tion (Ventura, CA), it was clear that
there has been tremendous interest
and development in recent years to-
ward a better understanding of the
molecular regulatory mechanism of
craniofacial development. Develop-
mental and evolutionary biologists as
well as tissue engineers are working
together to investigate and compare
the tissue origin, patterning, and
growth of various craniofacial organs
in an effort to reproduce and/or repair
defective tissue in the craniofacial re-
gion.
2370 CHAI AND MAXSON
To date, only approximately one
third of mouse mutations associated
with craniofacial malformations have
been linked to mutations of the or-
thologous human genes with similar
dysmorphogenesis (Wilkie and Mor-
riss-Kay, 2001). It is not known what
defects might arise from mutations of
the other two thirds as some of these
mutations in mice result in early em-
bryonic lethality. This lethality dem-
onstrates the important function of
these genes in early embryonic devel-
opment but makes it impossible to in-
vestigate the functional significance of
that particular gene in regulating
craniofacial development. It is con-
ceivable that the equivalent muta-
tions in human will also result in a
lethal phenotype. Nevertheless, an
important conclusion from studies us-
ing various animal models suggests
that there are no “craniofacial genes.”
The same morphogen (BMP, TGF-␤,
FGF, Shh, and so on) that controls
limb development, for example, also
plays a crucial role in regulating
craniofacial morphogenesis. Clearly,
the functional specificity and the de-
velopmental outcome of a signaling
molecule is determined by the cell
upon which the morphogen acts, as
different cell types are exposed to a
different combination of growth and
transcription factors and may respond
differently to the same growth factor
signaling (“Ask not what TGF-␤ can
do for the cell, ask what the cell can do
with TGF-␤,” Joan Massague, per-
sonal communication). Another im-
portant lesson from animal studies is
the available dosage of an important
regulatory gene, as haploinsufficiency
or gain of function of a critical gene is
often associated with congenital mal-
formations in humans. Overall, stud-
ies using mutant mice have signifi-
cantly improved our understanding of
human embryogenesis in general and
craniofacial development in particu-
lar. It is of paramount importance
that there are constant interactions
among researchers involved in both
human and animal studies as this will
help to ensure the rapid progress to-
ward a better understanding, treat-
ment, and prevention of human con-
genital malformations.
Tissue regeneration is another area
with many exciting new developments
based on research advancements in
developmental and evolutionary biol-
ogy (Chai and Slavkin, 2003). The re-
cent convergence of the human ge-
nome project and scientific advances
toward understanding the molecular
regulation of craniofacial morphogen-
esis, stem cell biology, and biotechnol-
ogy offer unprecedented opportunities
to realize craniofacial tissue regener-
ation. One of the next critical steps
will be to apply our knowledge of mo-
lecular regulation of tooth morpho-
genesis, for example, to manipulate
adult stem cells toward an odonto-
genic phenotype.
Significant progress has been made
in stem cell biological research, which
has advanced our understanding in
the area of hematopoiesis, tissue engi-
neering, and biomaterials (Lovell-
Badge, 2001). Recently, studies have
shown that adult stem cells have a
much higher degree of developmental
potential than previously thought,
and this has prompted considerations
to explore the potential of stem cell
mediated muscle, bone, cartilage, and
dentin regeneration (Gronthos et al.,
2000; Bianco et al., 2001; Bianco and
Robey, 2001). Stem cells are truly re-
markable. They have the potential to
grow into an array of specialized cells
and hold great promise for treating
medical and dental conditions. Sys-
temically injected mouse bone marrow
derived cells have given rise to mus-
cle, cartilage, bone, liver, heart, brain,
lung, alveolar epithelium, intestine,
and, of course, hematocytes (Ferrari et
al., 1998; Lagasse et al., 2000; Wood-
bury et al., 2000; Kotton et al., 2001).
Although most of these animal studies
serve now as precursors for future hu-
man clinical trials in treating certain
medical and dental conditions, the ba-
sic scientific principles learned from
these current analyses certainly have
advanced our understanding of the bi-
ological regulation of tissue engineer-
ing. The 21st century provides us with
tremendous opportunities to enable
biological solutions to biological prob-
lems.
ACKNOWLEDGMENTS
We thank Pablo Bringas, Jr., Jun
Han, Ryoichi Hosokawa, Julie Mayo,
Kyoko Oka, and Xun Xu for their as-
sistance with this manuscript. We
apologize to those colleagues whose
publications were not cited due to
space limitations. Both Dr. Chai’s lab
and Dr. Maxson’s lab were supported
by the NIH.
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