ß 2006 Wiley-Liss, Inc.

American Journal of Medical Genetics Part A 140A:2521 – 2529 (2006)

Looking Different:
Understanding Diversity in Facial Form
S.A. Brugmann, J. Kim, and J.A. Helms*
Department of Plastic and Reconstructive Surgery, Stanford University, Stanford, California
Received 1 May 2006; Accepted 20 May 2006

The face is perhaps the most distinguishing feature of the
vertebrate body. Six billion human faces decorate the earth,
each of them unique and exceptional in their own way.
Likewise, facial variation is the cornerstone of species-
specific diversity within the animal kingdom. Yet despite
this multiplicity in form, the underlying architecture of the
vertebrate face is remarkably conserved. If early embryos of
different species first resemble one another, then how is this
facial diversity generated? Our primary goal is to elucidate
the molecular origins of species-specific craniofacial mor-
phogenesis. We examined one facial primordia, the fronto-
nasal prominence, of phylogenetically related (chick vs.
quail vs. duck) and distant (mouse vs. chick) embryos and
asked how such drastically different forms (e.g., beak, bill, or
muzzle) could be generated from a once-similar entity. We
examined the morphological ontogeny and a number of
molecular expression patterns in an attempt to shed light on
when species-specific variations occur and what molecules
(BMPs, FGFs, etc.) are implicated in its differential growth.
We hypothesize that subtle changes in the signaling of these
morphogens can reproducibly alter the morphology of the
frontonasal prominence. Taken together, these data facilitate
our fledgling understanding of the process by which facial
morphogenesis is regulated. ß 2006 Wiley-Liss, Inc.
Key words: craniofacial; frontonasal prominence; species
variations; BMP; FGF; mouse; chick; quail; duck

How to cite this article: Brugmann SA, Kim J, Helms JA. 2006. Looking different: Understanding diversity in
facial form. Am J Med Genet Part A 140A:2521–2529.

INTRODUCTION MATERIALS AND METHODS

‘‘Somethin’ tells me it’s all happening at the zoo’’
(Paul Simon).
Nature has nowhere created greater diversity, nor
illustrated a better sense of humor, than in her
elaborations of the vertebrate face. A quick perusal of
the residents of any zoo—as well as their visitors—
will surely illustrate the enormous facial variations
that exist throughout the animal kingdom. Elephants
are a glorious illustration of this point, as is the
spoonbill and the awe-inspiring anteater (Fig. 1).
Noting these facial features, an inquisitive craniofa-
cial biologist might begin to wonder how such
diversity actually arises, whereas a craniofacial
geneticist may observe comparable alterations in
human facial form. In the latter case, however, the
extreme variations represent pathological condi-
tions. Is the molecular machinery so handily
employed by mother nature to generate the unique
faces in the animal kingdom also responsible for
generating the diversity—from normal to abnor-
mal—of human faces? This was the question that
captured our interest and which we pursued, in the
context of how facial patterning is established.
Embryo Collection and Photography
All procedures followed protocols that were
approved by Stanford University. Mouse embryo
collections followed standard procedures, where
mice were mated in the evening and inspected in the
morning; upon detection of a vaginal plug, mice
embryos were designated as e0.5. Chick embryos
were staged according to Hamburger Hamilton
criteria [Hamburger and Hamilton, 1951].
To better visualize the morphology of the fronto-
nasal prominence, all embryos were soaked briefly
in phosphate buffered saline (PBS) containing
ethidium bromide. Embryos were photographed in
fluorescent light, and images were then changed to
grey scale.
Grant sponsor: NIH; Grant numbers: R01-DE12462, F32-DE017499.
*Correspondence to: J.A. Helms, Department of Plastic and Recon-
structive Surgery, 257 Campus Drive, Room GK207, Stanford University,
Stanford, CA 94305. E-mail: jhelms@stanford.edu
DOI 10.1002/ajmg.a.31361

2522
FIG. 1. Nature’s variation. The most striking variations in differential growth of the frontonasal prominence are seen at any local zoo. Compare the long pliable
frontonasal prominence of an elephant (A), the flat frontonasal prominence of a spoon bill (B), and the elongated, bony frontonasal prominence of an anteater (C).
In Situ Hybridization
Embryos were embedded in paraffin and sectioned
at a 7-mm thickness. The relevant mRNAs for in situ
hybridization were prepared using sequence-speci-
fic primers and polymerase chain reaction. Tissue
sections or whole mount embryos were incubated in
hybridization buffer (Ambion Corporation, Austin,
TX) containing digoxigenin-labeled riboprobe at an
approximate concentration of 0.2–0.3 mg/ml probe
per kilobase of probe complexity. Non-specifically
bound probe was hydrolyzed with RNase A, and
final washes were carried out at high stringency
(0.2
SSC, 658C). For color detection, embryos or
slides were blocked with 10% sheep serum and
levamisole, and developed using nitro blue tetra-
zolium chloride (NBT) and 5-bromo-4-chloro-3-
indolyl phosphate (BCIP; Roche, Indianapolis, IN).
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
BRUGMANN ET AL.
After developing, the slides were cover-slipped with
aqueous mounting medium.
RCAS-Sprouty and RCAS-Noggin Infection
The response of cells to FGF signaling (in the case
of Sprouty 2) or to BMP signaling (in the case
of Noggin) was prevented by the injection of a
replication competent retrovirus (RCAS) encoding
either the FGF antagonist Sprouty 2, or the BMP
antagonist Noggin (RCAS-Noggin) or, as a control,
alkaline phosphatase (RCAS-AP). Injections were
carried out between Hamilton Hamburger (HH)
stages 8 and 10. In both cases this approach led to
widespread protein production by HH stage 15.
Analyses of the resulting phenotypes were carried
out at HH stage 25.
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

UNDERSTANDING DIVERSITY IN FACIAL FORM 2523

In Ovo Delivery of Sorafenib
A concentrated solution of Sorafenib was prepared
(2.8
109 mg/ml), and an aliquot of 1.0 ml was
injected via a micro-injector near the head region of
HH stage 5 chick embryos. Control embryos were
injected with PBS; all embryos were collected after
72 hr, and processed as described above for
photography.
RESULTS
Class Differences: When Facial Form
Diverges in Mammals and Aves
We began with a relatively straightforward ques-
tion; namely, when are species-specific craniofacial
features first evident? Centuries ago, embryologists
recognized that late-staged embryos already exhibit
the craniofacial features characteristic of their post-
natal phenotypes. Therefore, we began by analyzing
two readily available vertebrate embryos, mammals
and birds, for the first evidence of their species-
specific characteristics.
The mouse muzzle shares features of other
mammalian faces in that the tip of the muzzle
projects some distance from the sides of the face
(Fig. 2A). The tip of the muzzle is derived from the
frontonasal prominence, and in mice is characterized
by the closely positioned nasal pits that are separated
by an infranasal depression (IND, Fig. 2B). The IND
is sometimes erroneously referred to as a cleft.
Histological analyses reveal, however, that this
‘‘cleft’’ is actually the epithelial seam-lined frontona-
sal prominence which appears compressed between

FIG. 2. Time course of murine frontonasal prominence development. Embryos were collected at various stages and stained with ethidium bromide to illustrate the
origins of the characteristic shape of the mouse muzzle (dotted black box, A; high magnification, B) and the IND (arrow, B and C). The origin of this structure becomes
evident upon a review of early embryonic stages (D–I). The frontonasal prominence is divided into medial (yellow overlay) and lateral (blue overlay) regions. The
medial aspect has a deep furrow that becomes exaggerated with the growth of the maxillary prominences (MX). Still earlier, the ‘‘waveform’’ of the frontonasal
prominence (dotted white line, G and H) is evident, with the medial region retruding, relative to the protruding lateral aspects of the frontonasal prominence. Less than
24 hr earlier in development this indentation is undetectable and the frontonasal prominence appears almost sphere-like with the only obvious features being the nasal
pits (dotted black lines, I).
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2524 BRUGMANN ET AL.

the two maxillary prominences (MX, Fig. 2D). The
origins of this arrangement became more obvious
when we examined earlier staged embryos: at e13.5
we found that the frontonasal prominence itself is
divided into a medial (midline, yellow) and lateral
region (blue) and the medial aspect has a deep
furrow (arrow, Fig. 2C). These lateral aspects of the
frontonasal prominence are initially separated from
one another by the medial region. With the ensuing
expansion of the lateral regions relative to the medial
region, along with growth of the maxillary pro-
minences, the lateral aspects of the frontonasal
prominence approximate and give the impression
of a midline cleft (Fig. 2F–D).
We examined younger embryos to gain an
appreciation for how this differential growth began.
At this developmental age we observed that the
medial region of the frontonasal prominence is
retruded relative to the protruding lateral regions of
the frontonasal prominence, resulting in the indicat-
ed ‘‘waveform’’ (dotted white line, Fig. 2G). Twelve
hours earlier, at e9.5, we detected only a subtle
indentation in the medial region of the frontonasal
prominence (Fig. 2H), and less than 24 hr earlier than
that, at e9.0, this indentation is undetectable (Fig. 2I).
At this point in development the murine frontonasal
prominence appeared almost sphere-like, delimi-
tated from the MXs by the nasal pits (black dotted
lines Fig. 2I).
We next compared the morphology of the mouse
muzzle with that of a bird beak, as both structures are
primarily derived from the frontonasal prominence.
At HH stage 40, chicken embryos exhibit a pointed
beak reminiscent of its post-hatching phenotype
(Fig. 3A). At HH stage 30, the origins of the pointed
upper beak become obvious: the midline of the
frontonasal prominence in avians has grown out
past the lateral aspects, creating a waveform that is
inverted relative to that of the mouse frontonasal
prominence (dotted line, Fig. 3B). Still earlier, at
stage 27, this arrangement, where the medial region
of the frontonasal prominence protruded relative
to the lateral regions, is just becoming established
(Fig. 3C). At HH stage 25, the medial and lateral
regions protrude the same amount, giving the
impression of an almost flattened frontonasal pro-
minence (dotted line, Fig. 3D).
At HH stage 20 the frontonasal prominence,
which encompasses the region between the nasal
pits, is nearly spherical (Fig. 3E). This arrange-
ment is nearly unchanged at earlier time points
(HH stage 17, Fig. 3F). From these comparisons
between a mammal and a bird we drew two
conclusions. First, we confirmed that unique, spe-
cies-specific features within the frontonasal pro-
minence are established during the embryonic
period. Second, despite their ultimate dissimi-
larities, the frontonasal-derived features of early

FIG. 3. Time course of chicken frontonasal prominence development. Embryos were collected and stained with ethidium bromide to illustrate the origins of the
characteristic shape of the chicken beak. Stage 40 chicken embryos exhibit a pointed beak reminiscent of its post-hatching phenotype (A). At an earlier stage, the origins
of the pointed upper beak are obvious: the midline of the frontonasal prominence has grown out past the lateral aspects in an inverse ‘‘waveform’’ (dotted white line, B).
An asterisk marks the nasal pit (*). At earlier stages, the medial region (yellow overlay) of the frontonasal prominence still protrudes relative to lateral regions (blue
overlay) (C and D). At stage 20 the frontonasal prominence appears more flattened (dotted white line, E). At stage 17, the frontonasal prominence is nearly spherical and
defined from the adjacent MX by the nasal pits (dotted white lines, F).
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
UNDERSTANDING DIVERSITY IN FACIAL FORM
embryos converge until different species closely
resemble one another.
All in the Family: Species-Specific Differences
in Chick, Quail, and Duck Faces
Our next analyses centered on determining when
this morphological diversity in the frontonasal
prominence was first detectable. We hypothesized
that more distantly related animals (i.e., those from
different classes, such as mammals and aves) would
diverge earlier than those that shared the same class
(Fig. 4). Similarly, we reasoned that animals that were
from the same class (aves) but from different orders
(i.e., Galliformes vs. Anseriformes) would exhibit
species-specific features sooner during the embryo-
nic period than those that shared the same family
(i.e., Phasianidae) but arose from different Genera
(i.e., Coturnix vs. Gallus; Fig. 4). To directly test this
hypothesis we compared embryos from chicken
(Gallus gallus), quail (Coturnix japonica), and duck
(Genus Anas) for evidence of facial similarities, and
facial distinctions.
We began our analyses using avian embryos from
the same family (Phasianidae), namely the quail and
chicken. Both birds have short, sharp beaks that are
ideal for cracking small seeds that make up the bulk
of their diets [Grant and Grant, 2002]. During the
latter period of embryonic development, embryonic
chick and quail beaks are discernible predominantly
because of size differences (Fig. 5A,D). Their general
morphologies, however, are quite alike. In fact, by
HH stage 25, the frontonasal prominence of a chick
and a quail are nearly indistinguishable (Fig. 5B,E),
and at earlier stages than that (HH stage 17) the
entire head region of the embryos is identical
(Fig. 5C,F). Thus, as we had hypothesized, animals
that share the same family share nearly identical
facial features for a greater period of their embryonic
development.
Next we compared the quail and chick embryos to
duck embryos. Since Galliformes (quails, chicks) and
Anseriformes (ducks) arise from different orders we
predicted that their facial features would diverge
FIG. 4. A cladogram of murine and avian species. This cladogram illustrates
phylogenetic relationships and shows points at which various species have
diverged from common ancestral forms.
2525
earlier than the faces of animals that shared the same
order. We found that by HH stage 25, the shape of
the frontonasal prominence was notably broader in
the duck, and the morphology of the midline distinct
from that of the stage matched quail and chick
embryos (compare Fig. 5B,E,H), whereas the fronto-
nasal prominence in quails and chicks looked nearly
identical until HH stage 27 (data not shown;
[Schneider and Helms, 2003]. At earlier embryonic
stages, however, the chick, quail, and duck embryos
were impossible to differentiate (compare Fig. 5C,F,I).
Thus, when considered together, these morphologi-
cal assessments indicate that more distantly related
embryos exhibited species-specific craniofacial char-
acteristics earlier during the embryonic period.
Conversely, more closely related animals had more
similar facial features for a longer period of their
embryonic development. In addition, we found that
during the earliest stages of craniofacial develop-
ment, animals from different classes looked remark-
ably similar. Therefore, the fundamental structural
characteristics, or ‘‘bauplan,’’ of the face appear to be
conserved. How, then, do animals develop species-
specific craniofacial features?
Molecular Pathways Involved in
Species-Specific Craniofacial Form
Shortly after the neural tube fuses and the cephalic
flexure position the head in an anterior location,
embryos from a wide variety of classes and families
look remarkably similar, yet shortly afterwards,
around the time of organogenesis, species-specific
facial features are achieved. Therefore, we reasoned
that there must be a window of time during which the
growth and patterning of the frontonasal promi-
nence is differentially regulated. We undertook a
series of in situ hybridization analyses in an attempt
to identify genes whose differential expression might
account for some of these species-specific changes in
frontonasal growth.
We began our analyses comparing the expres-
sion patterns of three morphogens that have
well-documented roles in craniofacial development
[Helms et al., 2005]. Our first analyses were carried
out on HH stage 22 quail and duck embryos because
at this early stage the shape of the frontonasal
prominence is morphologically similar. Any gene
that played a causal role in species-specific shape
changes, we reasoned, would show a difference in
its expression pattern prior to any shape change. At
HH stage 25 the Sonic hedgehog (Shh) expression
domain in ventral facial (stomodeal) ectoderm was
indistinguishable between chick (data not shown),
quail, and duck embryos (compare Fig. 6A,A0 , red
arrow; also see [Cordero et al., 2004]). Therefore, it
was unlikely that differences in the facial expression
of Shh were responsible for differences in frontona-
sal form between these avian species.
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2526 BRUGMANN ET AL.

FIG. 5. Comparison of different avian embryos including a chick (A, B, C), quail (D, E, F), and duck (G, H, I). Despite their unique facial features upon hatching (A, D,
G) embryos with in the same family (chick and quail, family: Phasianidae) resemble each other. By HH stage 25, the frontonasal prominence of a chick and a quail are
nearly indistinguishable (compare B and E), and at early stages than that (HH stage 17) the entire head region of the embryos are identical (compare C and F). Embryos
from different orders (Galliformes; chicks and quails and Anseriformes; ducks) diverge earlier than the faces of animals that shared the same order. By HH stage 25, the
shape of the frontonasal prominence was notably broader in the duck (H) and the morphology of the midline distinct from that of the stage matched quail and chick
embryos (B, E, H). At stage 17, however, the chick, quail, and duck embryo were impossible to differentiate (compare C, F, I).

We next evaluated Fibroblast growth factor 8
(FGF8) expression and found that the maxillary
domain was identical between the duck and quail
embryos (Fig. 6). The domain surrounding the nasal
pit, however, was different between the two species:
while the FGF8 domain circumscribed the nasal pits
of quail embryos, it was restricted to the medial
aspect of the nasal pits in duck embryos (compare
Fig. 6B,B0 , red arrows).
Among the genes we analyzed the most striking
difference in an expression pattern was seen in our
analyses of BMP4. In HH stage 24 duck embryos,
BMP4 was expressed in two broad strips in the
midline of the duck frontonasal prominence (com-
pare Fig. 6C, red arrow, and C0 ). In addition, BMP4
was more broadly expressed in the region around
the nasal pits, and its expression extended into
the underlying mesenchyme of the MXs (compare
Fig. 6C,C0 , black arrows). In quail embryos, the BMP4
domain was much more laterally restricted (Fig. 6C0 ,
black arrows), and was all but undetectable in
the midline of the frontonasal prominence
(Fig. 6C0 , red arrow).
We also examined younger quail and duck
embryos (around HH stage 17) and found that the
BMP4 expression domain was nearly identical at the
earlier stage (compare Fig. 6D,D0 ), indicating that
any species-specific differences in expression likely
occurred in a 24 hr period, between HH stages
20 and 25.
Taken together, these data indicate that molecular
changes, ranging from very subtle (e.g., FGF8) to
much more distinct (e.g., BMP4), precede the
morphological variations that characterize a particu-
lar avian species. Since these molecular pathways are
likely essential for sculpting the face of a wide range
of vertebrates, we speculated that disruptions in
these same pathways would necessarily lead to
morphological variations that include pathological
alterations in facial form. To test this hypothesis
we undertook a series of experiments where we
inhibited FGF and BMP signaling and examined
the morphological consequences of these two
treatments.
Disrupting FGF Signaling in the Face Produces
a Range of Dysmorphologies
We made an initial foray into understanding the
role of two of these pathways, mediated by FGFs and
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

UNDERSTANDING DIVERSITY IN FACIAL FORM 2527

FIG. 6. Species-specific morphological differences within the frontonasal prominence are preceded by differential gene expression patterns. Sonic hedgehog (Shh)
expression domain in ventral facial (stomodeal) ectoderm was indistinguishable between quail and duck embryos (compare A and A0 , red arrow). Fibroblast growth
factor 8 (FGF8) expression within the maxillary domain was identical between the duck and quail embryos (B and B0 ) however, the FGF8 domain circumscribed the
nasal pits of quail embryos whereas it was restricted to the medial aspect of the nasal pits in duck embryos (compare B, B0 , red arrows). BMP4 was expressed in two
broad strips in the midline of the duck frontonasal prominence but was undetectable in the midline of the quail frontonasal prominence (compare C and C0 , red arrows).
Further, BMP4 was more broadly expressed in the region around the ducks nasal pits (compare C and C0 , black arrows). BMP4 expression in younger quail and duck
embryos (HH stage 17) was nearly identical (compare D and D0 ).

BMPs, in shaping the frontonasal prominence by
disrupting the pathways during earlier stages of
development. For example, we used three different
methods to inhibit FGF signaling: overexpression of
a dominant negative form of FGF receptor 1 (FGFR1),
the addition of a compound that inhibits FGF sig-
naling [Amaya et al., 1991; Hannon et al., 1996; Kudla
et al., 1998], and overexpression of a molecular
antagonist of FGF signaling, Sprouty 2 [Chambers
and Mason, 2000; Nutt et al., 2001]. Each one of these
approaches has some drawbacks; for example, over-
expression of a truncated form of FGFR1 reduces, but
does not eliminate, FGF signaling within a tissue
[Flanagan-Steet et al., 2000]. Likewise, the compound
used to inhibit FGF signaling (Sorafenib; Bayer
Pharmaceuticals Corporation, West Haven, CT) also
disrupts EGF signaling [Schoffski et al., 2006]. And
while Sprouty 2 has known antagonistic activities to
FGF signaling [Hacohen et al., 1998; Kramer et al.,
1999; Nutt et al., 2001] there are also data demon-
strating that it can act as an agonist of the same
pathway [reviewed in Christofori, 2003]. Lastly,
there are no known direct targets of FGF signaling
in the face at these stages, which complicates anal-
yses since we cannot demonstrate target gene
downregulation.
Despite these caveats, all three approaches pro-
duced a remarkably similar craniofacial phenotype
in chick embryos treated at HH stage 5 (Fig. 7). In
each case the frontonasal prominence was dramati-
cally reduced in both its mediolateral and proximo-
distal growth (Fig. 7A–C). This truncation resulted in
the near-approximation of the MXs to one another
(Fig. 7A–C, dotted white lines). The reduction in
mediolateral growth resulted in the coalescence of
the bilateral nasal pits into a single nasal pit or

FIG. 7. Perturbing FGFR1 signaling results in inhibition of the outgrowth of the frontonasal prominence. Using three different mechanisms to block signaling through
FGFR1 resulted in the reduction in proximal/distal growth of the frontonasal prominence, the near-approximation of the MXs (dotted white lines, A–C) and the
coalescence of the bilateral nasal pits into a single pit or proboscis (red arrows, A–C). In addition, the collapse of the frontonasal prominence also produced a fused eye
field (yellow arrow, C).
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2528 BRUGMANN ET AL.

proboscis (Fig. 7A–C, red arrows). In addition, the
collapse of the frontonasal prominence also pro-
duced a single eye field (e.g., synophthalmia) in
some embryos (Fig. 7C, yellow arrows). Collectively,
these data indicate that disruptions in FGF signaling
at an early stage of avian development produces a
range of craniofacial midline malformations.
Perturbing BMP Signaling Alters
Frontonasal Morphology
We extended our study into the regulation of
frontonasal development by exploring the role that
BMP signaling plays in its morphogenesis. Our
previous analyses indicated that a number of BMPs
are expressed in the craniofacial primordia (data
not shown; see [Ashique et al., 2002]). Therefore,
we used the overexpression of a BMP antagonist,
Noggin [Lamb et al., 1993; Brunet et al., 1998] to block
most endogenous BMP signaling in the frontonasal
prominence. Infecting embryos with RCAS-Noggin
at HH stage 15 (Fig. 8A,B) produced widespread
expression of Noggin by HH stage 20 (Fig. 8A,B). The
consequences of this mis-expression included the
downregulation of Shh in the ventral facial ectoderm
(compare Fig. 8C,D) and the expansion of FGF8
(compare Fig. 8E,F). The expression pattern of other
genes, such as BarX1, however, did not appear to
change (Fig. 8G,H). Morphologically, the attenua-
tion of BMP signaling resulted in a reduction in the
proximal/distal outgrowth of both the frontonasal
and MX prominences (compare Fig. 8I,J). As a result,
the nasal pits remain proximal and were unable to
merge with the lateral nasal and maxillary processes
(Fig. 8I,J; np, dotted black line).
DISCUSSION
The human face shows remarkable variability and
because of this it is oftentimes the singular feature
used to distinguish and discriminate among indivi-
duals. Despite this exclusivity, the structural edifice
of the face is so highly conserved that its underlying
pattern is shared by nearly all vertebrates [reviewed
in Brugmann et al., 2006]. One might then wonder,
what forces act to establish the craniofacial bauplan?
And what are the driving influences behind the
divergence in craniofacial form? As Darwin and many
other scientists speculated, the answer to both ques-
tions lies in natural selection. Just which molecular
pathways establish the global organization of the
face, however, remain unknown. Likewise, how

FIG. 8. Perturbing BMP signaling results in the reduction in the proximal/distal outgrowth of the frontonasal and MXs. Molecularly, the overexpression of the BMP
antagonist, Noggin (compare A and B) results in the reduction of Shh (compare C and D) and the expansion of FGF8 (compare E and F) in the facial ectoderm of the
frontonasal prominence. Other genes expressed in these areas, such as BarX1, however, did not appear to change (compare G and H). Morphologically, perturbing
BMP signaling resulted in a reduction in the proximal/distal outgrowth of the frontonasal and MXs (compare I and J). As a result, the nasal pits (np, dotted black line)
remain proximal and were unable to merge with the lateral nasal and maxillary processes (I and J).
 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
UNDERSTANDING DIVERSITY IN FACIAL FORM
much genetic change has to occur in order to
generate such craniofacial diversity is still a mystery.
New studies, however, suggest that even slight
variations in gene or protein expression can lead to
huge morphological alterations [Terai et al., 2002;
Albertson et al., 2003a,b]. How these morphological
alterations are brought about by the gentle tweaking
of a particular molecular pathway is still being
determined, but at least one avenue of study that
has proved fruitful is the identification of genes
responsible for human craniofacial anomalies.
Malformations involving the craniofacial region
comprise approximately one-third of all birth
defects, a remarkable statistic that underscores our
need to determine the genetic and environmental
etiologies that cause craniofacial anomalies. Even
when genes have been identified, a hefty challenge
remains: just how do specific gene mutations actually
cause the morphological changes that result in
craniofacial defects? In the end, a detailed under-
standing of the molecular, cellular, and tissue
interactions that are interfered with, as a conse-
quence of an initial genetic disruption, will undoubt-
edly shed more light on the developmental steps that
underlie species-specific craniofacial diversity, as
well as the pathologies associated with a range of
craniofacial birth defects.
ACKNOWLEDGMENTS
This manuscript is dedicated to Dr. Robert Gorlin,
whose commitment to understanding craniofacial
anomalies laid the foundation for our studies. His
allegiance to the field of craniofacial biology has
been, and will continue to be, an inspiration. We
thank Henry Goodnough for careful reading of this
manuscript. This work was supported by NIH R01-
DE12462 (J.A.H.) and F32-DE017499 (S.A.B.).
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