? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
Gas1 is a modifier for holoprosencephaly
and genetically interacts with sonic hedgehog
Maisa Seppala,1 Michael J. Depew,1 David C. Martinelli,2 Chen-Ming Fan,2
Paul T. Sharpe,1 and Martyn T. Cobourne1,3
1Department of Craniofacial Development, Dental Institute, King’s College London, London, United Kingdom. 2Department of Embryology, Carnegie Institution,
Baltimore, Maryland, USA. 3Department of Orthodontics, Dental Institute, King’s College London, London, United Kingdom.
Holoprosencephaly (HPE) is a clinically heterogeneous develop-
mental field defect of the CNS, in which the embryonic forebrain
or prosencephalon fails to divide into distinct halves (1, 2). The
underlying brain malformation can have a profound affect upon
midline development of the face. In the most severe form of HPE,
the forebrain remains as a single undivided vesicle and there is
cyclopia, with a single midline eye situated below a rudimentary
nose or proboscis and midline clefting of the lip and palate. How-
ever, the severity varies in both the brain malformation and the
craniofacial features, even among members of the same pedigree.
In microform HPE, milder craniofacial features such as ocular
hypotelorism, premaxillary agenesis, and solitary median maxillary
central incisor (SMMCI) can occur in the absence of defects within
the CNS (3). The etiology of HPE is complex, with both environ-
mental and genetic factors being implicated (4). Maternal diabetes,
alcohol or drug ingestion, and defects in cholesterol metabolism
have all been associated with HPE (5, 6), while a number of candi-
date genes have also been identified in humans, including sonic
hedgehog (SHH) (7, 8).
Shh signaling is essential for normal development of the early
forebrain, as transcripts are expressed in mesendoderm of the
prechordal plate (9, 10) and are required for formation of ventral
midline structures. Shh–/– mice have otocephaly, with only a single
forebrain vesicle and cyclopia (11). Moreover, mutations in human
SHH that have been identified suggest a role in both familial and
sporadic HPE (7, 8) and represent a substantial proportion of cases
demonstrating autosomal-dominant inheritance of this condition
(12). Interestingly, the spectrum of phenotypic severity character-
istic of HPE can be seen in association with identical mutations in
SHH, even among members of the same pedigree (8). SHH muta-
tion can also lead to SMMCI in the primary or secondary denti-
tion, occurring as an isolated syndrome (12, 13) or as a manifesta-
tion of HPE (14).
Shh is a member of the hedgehog family of vertebrate signal-
ing molecules and is essential for normal development of many
regions within the embryo (15). The versatility of Shh is reflected
in a complex signaling pathway, with unique mechanisms of gen-
eration, distribution, reception, and intracellular transduction of
the signal (16). Reception at target cells is mediated via direct phys-
ical interaction with the Patched1 (Ptc1) transmembrane domain
protein (17); in the resting state, Ptc1 inhibits transduction,
which is only relieved by the binding of ligand (18). This inhibi-
tory activity is indirect, via Ptc1-mediated membrane localization
and phosphorylation of Smoothened (Smo), a G protein–coupled
receptor whose activity is essential for signal transduction (19).
Because Ptc1 is also a direct transcriptional target of signaling,
pathway activity is therefore buffered by the relative availability
of bound and unbound receptor at the cell surface (20, 21). In
vertebrates, signaling is mediated and interpreted principally via
Gli transcription factors (Gli1–Gli3) through a large complex of
intracellular mediators. Gli2 and Gli3 function as both activators
and repressors and are the principal transducers of Shh signal-
ing activity (22–24). Gli1 is a sole activator and specific target of
Shh transduction, but plays a secondary role in signaling (23, 25).
The precise events that mediate reception of Shh on the surface of
receiving cells are not fully understood. However, several plasma
membrane–associated proteins have been identified that can bind
Shh and regulate pathway activity in vertebrates. These include
Megalin, a low-density lipoprotein receptor (26), the Ig/fibronec-
tin type III repeat transmembrane domain proteins Cdo and Boc
(27, 28), and the membrane glycoproteins hedgehog-interacting
protein (29, 30) and growth arrest–specific 1 (Gas1; ref. 31).
Nonstandard?abbreviations?used: Gas1, growth arrest–specific 1; HPE, holoprosen-
cephaly; Ptc1, Patched1; Shh, sonic hedgehog.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 117:1575–1584 (2007). doi:10.1172/JCI32032.
1576? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
Gas1 encodes a glycosylphosphatidyl-inositol–linked membrane
glycoprotein that is repressed in response to Shh and has previ-
ously been demonstrated in vitro to have an antagonistic effect on
Shh signaling in the somites (31). The human homolog of mouse
Gas1 maps to chromosome 9q21.3–q22 (32), a locus previously
associated with a number of human disorders that involve defects
within the craniofacial region, including Chiari type I malforma-
tion (OMIM 118420; ref. 33), autosomal-dominant and -reces-
sive neurosensory deafness (OMIM 606705 and OMIM 600974,
respectively; refs. 34, 35), and nonsyndromic cleft lip with or
without cleft palate (36). Moreover, mosaic trisomy 9 has been
associated with both HPE (37) and defects of the facial midline in
the absence of gross brain malformation (38). Therefore, we ana-
lyzed the craniofacial phenotype of mice generated with targeted
mutation of Gas1 and found that they demonstrated features
associated with microform HPE. There was lack of development
in the maxillary region, fusion of the premaxillary incisors, cleft
secondary palate, and pituitary anomalies; however, the telence-
phalic ventricles remained intact. Surprisingly, these defects were
associated with a lack of Shh transduction in cell populations at
a distance from the source of transcription, suggesting that Gas1
function can also potentiate signaling in the early face. Indeed,
the loss of a single Shh allele in a Gas1 mutant background signifi-
cantly worsened the midline craniofacial phenotype, which sug-
gests that Shh and Gas1 genetically interact.
Gas1 mutants have microform HPE associated with multiple craniofa-
cial defects. Newborn Gas1–/– mice exhibit microphthalmia and a
lack of retinal pigmentation; despite also being smaller, the gross
morphology of Gas1–/– heads appears normal (39). In a mixed
CD1-129Sv background these mice are viable until adulthood,
Craniofacial defects associated with the perinatal
Gas1–/– skull. (A) Comparison of WT and Gas1–/– skulls
differentially stained for bone (red) and cartilage (blue)
demonstrated the reduced size of the Gas1–/– skull. (B)
Norma basalis of WT and Gas1–/– skulls. Blue arrows
indicate the absence of optic pillars in the Gas1–/–
mouse; yellow arrows highlight the Gas1–/– cleft palate;
black arrow indicates fenestration of the Gas1–/– neuro-
cranial base; green arrows indicate dysmorphic ecto-
tympanic rings; and white arrows denote absence of
the hypoglossal foramen. (C and D) Magnified norma
basalis views of the maxillary and premaxillary palate
of WT (C) and 2 Gas1–/– (D) neonates without fully cleft
palates. Red arrows highlight the morphologic range
of fused incisors in Gas1–/– mice; green arrows indi-
cate the range of midline hypoplasia in premaxillary
palatal shelves and paraseptal cartilages; and yellow
arrows show the developing maxillary palatal shelves.
(E–L) Comparison of WT (E) and Gas1–/– (F–L) middle
ear skeletal elements demonstrating the range of dys-
morphology evident in Gas1–/– mice. Outlined are the
styloid process (green), stapes (orange), incus (yel-
low), and malleus (purple) in WT mice. Green arrows
indicate tympanohyal segments; pink arrows indicate
stylohyal segments; purple arrows indicate the mallei;
yellow arrows indicate the incuses; blue arrows high-
light the gonials; orange arrows highlight the stapes;
red arrows indicate the ectotympanics; black arrows
indicates ectopic cartilage between the stapes, styloid
process, and ectotympanic; and white arrows indicate
hypoglossal foreman. Exemplified in K is the fusion of
the stapes to the otic capsule. Original magnification,
×1.8 (C and D); ×2.3 (E and F); ×1.3 (G–L).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
while in a mixed 129Sv-C57BL/6 background the majority die
within 3 days of birth. At birth, gross morphology of the brain
has been reported to be normal in both mutant lines compared
with Gas1+/– WT mice (39, 40).
Because of the putative link between Gas1 function and Shh sig-
naling (31) as well as the pattern of expression (41) and association
of the human GAS1 locus with craniofacial anomalies, we analyzed
perinatal ontogeny of the craniofacial skeleton in 129Sv-C57BL/6
Gas1–/– mice. Chondrocranial (including both neurocranial and
splanchnocranial) and dermatocranial defects were evident, particu-
larly along the neurocranial midline and in the middle and inner ear,
but including all 3 primary sensory capsules (nasal, optic, and otic;
Figure 1, A and B). These midline defects varied in severity, ranging
from bilateral cleft of the secondary palate to absence of elaborated
premaxillary palatal processes or failure of the maxillary and pala-
tine palatal processes to fuse in the midline. The premaxillae were
hypoplastic, and variable synostosis occurred across the midline,
accompanied by the presence of a single premaxillary incisor crown
within the premaxillae. While the external nares were patent, the
nasal capsular cartilages also exhibited midline defects, such as the
absence of elaborated paraseptal cartilages (Figure 1, C and D).
Further defects were identified within the cranial base. The basi-
sphenoid had a large midline basicranial hypophyseal fenestration,
usually indicative of abnormal early development at the junction
of Rathke’s pouch and the anterorostral-most notochord. Later-
ally, the ala temporali were reduced in size. Overall, varying combi-
nations of midline defects were present in 90% of mice examined at
various stages of development (Table 1). Together, these anomalies
all present as features within the clinical spectrum of HPE and in
the absence of gross defects within the CNS, suggestive of micro-
form HPE in Gas1–/– mice.
These were not the only craniofacial defects. Consistent with the
earlier reported ophthalmic deficits (39), the ala hypochiasmatica
and associated optic pillars of the optic capsules were lacking in the
perinatal Gas1–/– skull. The external auditory meatus was hypoplas-
tic or nearly absent in the Gas1–/– mouse, and the tympanic rings
varied in both size and shape, although they were usually much
smaller in diameter and thicker. Gonial bones, when present, were
hypoplastic. Defects were also identified in the 3 ear ossicles. In gen-
eral, the malleus lacked a normal neck, manubrium, and processus
brevis and was sometimes synchondrotic with the remnant of the
incus. Moreover, a process extended from
either the incal or the malleal portion of
the fused elements toward the stapes. This
process was often synchondrotically fused
with the stapes. While the stapes was less
affected, it was not completely normal
and was occasionally synchondrotic with
the otic capsule. The styloid process was
always, although variably, affected, typi-
cally being markedly hypoplastic and sepa-
rated into tympanohyal and stylohyal por-
tions. The otic capsule was dysmorphic, as
were the middle ear–associated tegmen
tympani and retroarticular processes of
the squamosals (Figure 1, E–L).
Gas1 and Ptc1 are coexpressed in peripheral
regions of hedgehog transduction in the early
craniofacial region. Shh signaling is known
to be important for normal development
of the facial midline (7, 8, 42). The findings that lack of Gas1
function is associated with abnormalities in development of the
facial midline led us to examine and compare expression domains
of Gas1 and members of the Shh signaling pathway during early
development of the facial region.
Shh transcripts demonstrated a very specific temporospatial
expression domain in the early craniofacial region. At E10.5 and
E11.5, Shh was expressed within the CNS, in the floorplate of the
mesencephalon and neuroectoderm of the diencephalon, and in
the pharyngeal endoderm and facial ectoderm of the frontonasal
process (Figure 2, A, B, F, and G) (42). As targets of Shh signaling,
Ptc1 and Gli1 were strongly expressed in these regions, showing
a graded distribution of transcriptional activity across fields of
responding cells (Figure 2, C, D, H, and I). In contrast, Gas1 was
expressed at a distance from the source of Shh transcription, in a
domain largely reciprocal to that of Ptc1 and Gli1 (Figure 2, E and J).
However, examination of serial sections revealed that regions
of coexpression existed among Ptc1, Gli1, and Gas1 along the
peripheral margins of their respective expression domains. These
overlapping regions of transcription were clearly identifiable at
known sites of Shh signaling activity in the craniofacial tissues,
particularly the neural tube, first branchial arch, and frontonasal
region (Figure 2, C–E and H–J). Given that Shh is able to signal
over a distance equal to many cell diameters from the source of
transcription (43, 44), these findings suggest that Gas1 may inter-
act with Shh in peripheral regions of the signaling domain within
the craniofacial region.
Shh signal transduction is reduced in the frontonasal region of Gas1–/–
mice. We next compared expression of Shh pathway genes in the
craniofacial and midfacial regions of WT and Gas1–/– mice at E10.5
and E11.5. The domains of Shh transcription were normal at both
developmental stages within these regions in Gas1–/– embryos (Fig-
ure 2, K, L, O, and P) compared with WT embryos (Figure 2, A, B, F,
and G). Ptc1 and Gli1 were expressed in response to this signaling
throughout the frontonasal region of WT embryos (Figure 2, C, D,
H, and I, arrows); however, in Gas1–/– mice there was a clear reduc-
tion in the extent of this expression. Ptc1 and Gli1 failed to extend
throughout the length of the frontonasal process in Gas1–/– mice at
both stages (Figure 2, M, N, Q, and R, arrows). This reduction was
in the peripheral regions of Ptc1 and Gli1 expression, at a distance
from the source of Shh transcription and in regions coincident
Frequency of midline-centered craniofacial deficiencies in Gas1–/–, Gas1–/–Shh+/+,
and Gas1–/–Shh+/– mice
Fused premaxillary incisor
Complete secondary cleft palate
Partial secondary cleft palate
Premaxillary incisor agenesis
Fused mandibular incisor
Clefting of the basisphenoid
Ectopic proximal dentaryA
ANot considered a midline-centered deficiency.
1578? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
with those expressing Gas1. Gas1 transcripts were only detected at
background levels in the Gas1–/– mice as a result of the targeting
strategy (data not shown and refs. 39, 40). Therefore, a reduction
in Shh signaling activity, as assayed by Shh-responsive genes, was
identified in the frontonasal region of Gas1–/– embryos, suggesting
that in regions of coexpression, Gas1 might act as an agonist of
Shh signal transduction.
Premaxillary hypoplasia and incisor fusion in Gas1–/– mice. At E12.5,
a clear restriction in development of the facial midline was identi-
fied in Gas1–/– embryos. The frontonasal region was narrower, and
the early incisor tooth buds were approximated together (Figure
3, A and B, double arrows). During early tooth development, Shh
expression was restricted to these early thickenings of dental epi-
thelium, and in the frontonasal process of WT embryos, these early
dental placodes were separated by a region of non–Shh-expressing
epithelium (Figure 3, C and E). However, analysis of Shh in the
Gas1–/– mice demonstrated a continuous zone of expression, repre-
senting fusion of the incisor placodes (Figure 3, D and F). Further-
more, the response of the frontonasal tissues to Shh signaling was
reduced in Gas1–/– mice: Ptc1 expression, while normally detectable
in the epithelium and mesenchyme of the frontonasal process up
to the boundary of the nasal capsule, was not seen in the mutant
(Figure 3, G and H). Consistent with these findings, both histo-
logical analysis and differential staining of skeletal preparations
derived from perinatal Gas1–/– mice demonstrated fusion between
the premaxillary incisors (see Figure 1D and Figure 3J). Instead
of 2 separate tooth germs situated adjacent to the nasal cavity,
the Gas1–/– mouse had a fused incisor crown positioned below the
nasal septum in the midline (Figure 3, I and J). Thus, the single
premaxillary incisor crown was a direct result of a lack of develop-
ment in the facial midline of the Gas1–/– embryo.
Reduced Shh signal transduction and cell proliferation in the develop-
ing palate of Gas1–/– mice. A complete or partial cleft of the second-
ary palate was present in 60% of Gas1–/– mice analyzed (Figure 1
and Figure 4, A and B). Standard histological analysis suggested
that the initially vertical palatal shelves elevated above the tongue
and oriented themselves into a horizontal position; however, in
affected mice these shelves were unable to contact each other in
the midline (Figure 4, C–F). By E15.5 in WT mice, the shelves
began the process of fusion along the anteroposterior axis, while
in Gas1–/– mice these shelves failed to meet their counterparts in
the midline, resulting in cleft secondary palate.
The Shh signaling pathway is known to play an important role
in mediating growth of the palatal shelves (45, 46). At E13.5, Shh
was strongly expressed in epithelium of WT palatal processes, and
Ptc1 exhibited a gradient of activity within the mesenchyme in
response to signaling (Figure 5, A, B, E, and F). In addition, Ptc1
was also expressed within bilateral regions of mesenchyme in the
bend of the palatal vault (Figure 5F, arrow). We examined Shh sig-
naling activity in palatal shelves of Gas1–/– mice at E13.5 and found
a noticeable reduction in the range of Ptc1 transcriptional activity
within the mesenchyme (n = 3 out of 5 mice analyzed); however, Shh
was expressed in the palatal epithelium of these mutants (Figure 5,
C, D, G, and H). Ptc1 expression was present in close proximity to
the palatal shelf epithelium in affected Gas1–/– mice, but the extent
of this expression within the mesenchyme was less than that seen
in the WT embryos. In addition, Ptc1 expression was absent from
palatal bend mesenchyme in Gas1–/– mice (Figure 5H, arrow).
Shh pathway gene expression in
the craniofacial region of WT and
Gas1–/– mice. (A, B, F, and G) At
E10.5 and E11.5, sagittal sections
demarcated Shh in the pharyngeal
endoderm, diencephalon, and facial
ectoderm of WT embryos. (C, D, H,
and I) Ptc1 and Gli1 demonstrated
a gradient of transcriptional activity
across these regions in response to
signaling. (E and J) In contrast, Gas1
was expressed at a distance from the
source of Shh transcription, in a par-
tially overlapping domain with Ptc1
and Gli1, at their peripheral margins.
(K–R) In Gas1–/– embryos at both
E10.5 and E11.5, there was a marked
reduction in the domain of both Ptc1
(M and Q) and Gli1 (N and R) expres-
sion within the frontonasal (black
arrows) and mandibular processes
(blue arrows) compared with that of
WT embryos (compare with respec-
tive arrows in C, D, H, and I), while
Shh expression was normal in Gas1–/–
compared with WT embryos (B and
G) at both stages. di, diencephalon;
fe, facial ectoderm; fnp, frontonasal
process; md, mandibular process;
ne, neural ectoderm; pe, pharyngeal
endoderm; rp, Rathke’s pouch; t, tel-
encephalon. Scale bar: 200 μm.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
We further addressed the cleft phenotype of Gas1–/– mice by ana-
lyzing cell proliferation in epithelium and mesenchyme of the pal-
atal shelves at E12.5 and E13.5 (Figure 6, A–F). No statistically sig-
nificant difference between WT and Gas1–/– shelves was detectable
at E12.5 (data not shown). At E13.5, the shelves were positioned
bilaterally adjacent to the tongue just prior to their elevation above
the dorsum. There was no significant difference in epithelial and
mesenchymal proliferation within the anterior palate between WT
and Gas1–/– embryos, and while some reduction was seen in epithe-
lium of the apex in the middle and posterior regions, this was not
identifiable in the bend region. However, there was a significant
reduction in the percentage of mesenchymal cells proliferating
within the middle and posterior regions of the palatal shelves at
the apex (42% for both; P < 0.001) and bend (40% and 32%, respec-
tively; P < 0.001) in Gas1–/– compared with WT embryos (Figure 6,
G and H). Therefore, in the absence of Gas1 function, a reduction
in Ptc1 transcription in mesenchyme of both the palatal apex and
bend was associated with a statistically significant reduction in cell
proliferation within these regions.
Gas1 and Shh genetically interact. These data suggest that Gas1 is
able to positively regulate Shh signal transduction and that this
activity is relevant for signaling at a distance from the source of
transcription. It is clear that the absence of Gas1 function had
important consequences for normal midline development of the
craniofacial region. In order to determine whether Gas1 and Shh
interact genetically, we analyzed the craniofacial phenotype of
mice lacking a single Shh allele in a Gas1–/– background.
Individually, both Gas1+/– and Shh+/– mice were phenotypically nor-
mal in the craniofacial region; however, the loss of a Shh allele in a
Gas1–/– background caused a progressively more severe HPE pheno-
type. Gas1–/–Shh+/– embryos survived to birth but only had a single
external nostril, while Gas1–/–Shh–/– mice had severe craniofacial
defects and only survived to around E9.5 (data not shown). Indica-
tive of a significant genetic interaction, Gas1–/–Shh+/– perinatal skulls
exhibited exacerbated neurocranial, splanchnocranial, and dermato-
cranial defects relative to the Gas1–/– single mutants, as well as signifi-
cant and novel defects of patterning and development (Figure 7).
Skull size was further reduced in compound Gas1–/–Shh+/– perina-
tal mice, and patent sutures between the frontal and parietal ossi-
fications were not evident. Development of the neurocranial base
was severely disrupted: the trabecular basal plate, which runs from
the rostral basisphenoid, presphenoid, and ethmoid through the
nasal septum, was discontinuous and cleft. The nasal capsules and
associated dermal ossifications were drastically reduced, without
proper midline manifestations. The premaxillaries were hypoplas-
tic, synostotic, and lacking teeth. Lateral extensions from the neu-
rocranial base were all disrupted, the ala temporali were hypoplas-
tic, the lamina obturans failed to properly invest the cartilage of
the ala, and ectopic preotic cartilaginous pillars extended toward
the crista parotica of the otic capsule (Figure 7, A–D).
The frontonasal region is reduced in Gas1–/– embryos. (A and B) At
E12.5, histological analysis revealed a lack of transverse development
in the maxillary incisor region in Gas1–/– compared with WT embryos
(double arrows). In addition, the vomeronasal organ surrounded by the
early paraseptal cartilage (A, arrow) failed to develop in Gas1–/– mice.
(C–F) This reduced development caused fusion of the early incisor
placodes and continuous expression of Shh across the frontonasal
region of Gas1–/– mice. (C and D) Radioactive section in situ hybrid-
ization. (E and F) Whole-mount in situ hybridization. (G and H) Ptc1
expression was reduced in the frontonasal region of Gas1–/– compared
with WT embryos. (I and J) In perinatal WT embryos, the bilateral
maxillary incisor teeth formed in their normal positions adjacent to the
nasal capsule, while in Gas1–/– mice, a single enlarged maxillary inci-
sor crown was present, representing fusion between 2 separate tooth
germs. The paraseptal cartilage, which failed to form in Gas1–/– mice, is
denoted by an arrow. i, premaxillary incisor. Scale bar: 250 μm (A–D,
G and H); 500 μm (E and F); 750 μm (I and J).
1580? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
Meckel’s cartilage was truncated and lacked a malleal end.
Instead, the proximal end bifurcated slightly, apparently being
shared by the dysmorphic proximal dentary and an ectopic ossifi-
cation that appeared to be a mirror-image duplication of the prox-
imal dentary, which included a secondary cartilage-containing
condylar process and a molar alveolus. The distal dentary lacked a
symphysis, as it was synostotic across the midline, and contained
a single lower incisor (Figure 7, C and E). Middle ear–associated
splanchnocranial elements either failed to develop (malleus and
incus) or were represented by a cartilaginous remnant (stapes). The
associated dermatocranial elements, the ectotympanic and gonial
bones, also failed to form. The squamosal bones were repatterned
and separated into distinct retrotympanic and squamosal portions
(Figure 7F). Together, these data demonstrate a marked worsening
of the midline craniofacial phenotype with the loss of a single Shh
allele in a Gas1–/– background.
HPE, cleft palate, and congenital deafness are 3 of the most com-
mon craniofacial anomalies that affect human populations. Both
cleft palate and congenital deafness have a birth prevalence of
around 1 in 1,000 (47, 48), while HPE is seen in 1 in 10,000–20,000
live births and in 1 in 250 during embryogenesis, making it the
most common cause of brain abnormality in humans (1, 49). In
many cases the etiology of these conditions remains unknown;
however, the 9q21.3–q22 locus has previously been associated
with both neurosensory deafness (34, 35) and nonsyndromic cleft
palate (36), while mosaic trisomy 9 has been implicated in HPE
(37) and isolated defects of the facial midline (38). Given that the
human GAS1 gene resides at this locus, we examined the conse-
quences of a loss in Gas1 function using a mouse model.
Gas1 was originally identified as a gene highly expressed in a
fibroblast cell line maintained under conditions of growth arrest
(50); it encodes a membrane glycoprotein that in vitro has been
implicated as an inhibitor of cell cycle progression (51, 52), a par-
ticipant in excitotoxic neuronal cell death (53), and a negative reg-
ulator of Shh (31, 43).However, Gas1–/– mice do not demonstrate
generalized growth excess or tumor progression: while Gas1 does
negatively regulate retinal proliferation (39), it is actually required
for normal postnatal proliferation in regions of the cerebellum
(40). Similarly, the phenotype of these animals is not indicative of
excess Shh signal transduction. The limbs develop with small auto-
Palatogenesis in WT and Gas1–/– mice. (A and B) Scanning electron
microscopy demonstrating that palate development was complete
in WT mice at P0 (A), while there was a full-penetrance cleft of the
secondary palate in affected Gas1–/– mice at the same time point (B).
(C and D) Histological analysis showed comparable morphology of
the palatal shelves at E13.5 in WT and Gas1–/– embryos. (E and F)
At E15.5, while the shelves rose above the tongue and fused in the
midline in the WT embryos, fusion failed to occur in affected Gas1–/–
embryos. Scale bar: 1.25 mm (A and B); 500 μm (C–F).
Shh pathway gene expression in the palatal shelves of WT and Gas1–/–
mice. (A–D) At E13.5, Shh was expressed in the epithelium of WT
and Gas1–/– palatal shelves. (E and F) Ptc1 was expressed throughout
the epithelium and mesenchyme of WT shelves, in addition to iso-
lated regions in the palatal bend mesenchyme (F, arrow). (G and H) In
contrast, Gas1–/– palatal shelves had reduced Ptc1 expression, which
failed to extend along their length and was lost in the palatal bend
mesenchyme (H, arrow). Scale bar: 250 μm.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
pods, missing phalanges, and anterior digit syndactyly, but this is
secondary to defective proliferation in the apical ectodermal ridge
(54). Gas1 also exhibits structural homology to the GFR-α receptor
for the GDNF family of ligands (GFL), capable of independently
binding the receptor tyrosine kinase ret and interfering with signal-
ing (55). Therefore, Gas1 would appear to have multiple functions,
influencing the cell cycle in a cell context–dependent manner and
potentially interacting with several signaling pathways.
Our findings suggest that Gas1 can also act cooperatively dur-
ing Shh signaling in both the early facial processes and develop-
ing palate. Gas1 demonstrates coexpression with Ptc1 in peripheral
domains of Shh-mediated signal activity; it is within these regions
that signaling is reduced in Gas1–/– mice. This is consistent with pre-
vious reports that the active signaling form of Shh can bind Gas1
(31). This interaction is known to occur through a common inter-
face with that for Ptc1, inviting speculation that Gas1 might act as
a coreceptor, presenting Shh to Ptc protein in regions of coexpres-
sion (56). However, our present data are inconsistent with previous
reports that Gas1 activity can attenuate the mitogenic response to
Shh signaling in the somite and downregulate Ptc1 induction in
isolated diastema mesenchyme of the jaw (31, 43). However, these
contrary observations might be explained within the context of
endogenous Ptc1 and Gas1 protein levels, which would be expected
to strongly influence signal output. From our analysis, it is clear
that Gas1 positively influences hedgehog transduction at the mar-
gins of signal activity, where membrane quantities of Gas1 and Ptc1
are at minimal levels. In regions where either Gas1 or Ptc1 are in
gross excess, these effects would appear to be negated.
Gas1–/– mice demonstrated a number of features associated
with HPE, each with varying levels of penetrance.The evidence of
reduced hedgehog target transcriptional activity in the craniofa-
cial region of these mice is consistent with Gas1 mediating its
effects through the Shh pathway. HPE is a common congenital
anomaly, which in the most severe form is characterized by a com-
plete failure of forebrain division and cyclopia (57). However, the
phenotypic spectrum associated with this condition is extensive,
and a gradation of severity exists in the malformations that can
affect both the brain and the face (58). The basis of this clinical
heterogeneity is not fully understood, but it has been suggested
that HPE represents a multifactorial condition (even in cases
demonstrating autosomal-dominant inheritance patterns), as
penetrance of the phenotype reflects the varying contributions of
multiple genetic and environmental influences within the indi-
vidual (3). Indeed, the association between hedgehog signaling
and HPE provides clear evidence of this complexity. The early
craniofacial region is sensitive to perturbations in Shh signaling
and this changes with time; disruption of the pathway during
discrete periods of embryonic development can recapitulate the
phenotypic spectrum of HPE, inducing hypotelorism, midfacial
hypoplasia, facial clefting, or frank cyclopia, depending on the
timing and magnitude of signal loss (42). In addition, modifying
loci have also been identified that act in concert with SHH in the
induction of HPE in human populations; these include TGIF and
ZIC2 (12). Here, we provide evidence to suggest that GAS1 might
represent a further locus for HPE in humans.
We also analyzed the palatal phenotype of Gas1 mutants and pro-
vided further evidence that Shh signaling plays an important role
in mediating secondary palatogenesis, particularly in mesenchymal
proliferation within the palatal shelves (46). Gas1–/– mice demon-
strated a reduction in the range of Shh signal response across
mesenchyme of the palatal shelf. In these mutants, Ptc1 expression
remained localized to superficial regions of palatal mesenchyme
adjacent to the epithelium and failed to form a normal gradient of
activity across the shelves. Given that the cleft phenotype was not
completely penetrant, this implies that mesenchymal proliferation
must reach a certain threshold to ensure that the shelves elevate
above the tongue and meet their counterparts in the midline. The
incomplete penetrance associated with the palatal phenotype, in
Cell proliferation in the developing palate of WT and Gas1–/– mice. (A–F) BrdU analysis of E13.5 WT and Gas1–/– palatal shelves. In B, the upper
and lower boxes identify the area of mesenchymal cells counted in the palatal bend and apex, respectively; upper and lower dotted lines repre-
sent the regions of epithelial cells counted at the bend and apex, respectively. Scale bar: 125 μm. (G and H) Percentage of BrdU incorporation for
each area assayed. Proliferation rates were significantly reduced in epithelium of the middle and posterior apex and mesenchyme of the middle
and posterior apex and bend. a, anterior palate; m, middle palate; p, posterior palate. Data are mean ± SD. n = 4 mice per group analyzed for
each embryonic stage (total of 45 WT and 55 Gas1–/– sections counted). *P < 0.05 versus WT.
1582? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
addition to the finding that some Gas1–/– mice demonstrating
microform HPE were unaffected by cleft palate, indicates that this
phenotype can occur independently of HPE and shows the impor-
tance of modifying factors in the phenotypic manifestation of
this condition. Shh signaling also plays an important role during
development of the inner ear: morphogenesis of this region was
disrupted in Shh–/– mice, causing a lack of organization into dis-
tinct vestibular and cochlear portions and ventral otic derivatives,
including the failure of the cochlear duct and cochleovestibular
ganglia to develop (59, 60). Gas1–/– mice exhibited severe disrup-
tion in both the middle and inner ear, and it is possible that this is
secondary to defective hedgehog signaling.
Importantly, the loss of a single Shh allele in a Gas1–/– background
significantly worsened the midline craniofacial phenotype and
auditory defects of these animals. In particular, the neurocranial
base became severely disrupted with a cleft and discontinuous tra-
becular basal plate, while the premaxillary region lacked incisor
teeth. Notably, Gas1–/–Shh+/– perinates exhibited what appeared to
be a duplicated proximal dentary that included a condylar process
and an alveolus containing an ectopic molar. This form of pattern-
ing defect has not previously been described to our knowledge and
suggests an important interaction between Shh and Gas1 that helps
to establish mandibular and hyoid identity during early develop-
ment of the branchial arches. Our observations, along with the
previously documented association between 9q21.3–q22 and con-
genital malformations of the craniofacial region (34–36), provide
strong evidence that the GAS1 locus might act as a modifier for
some of these devastating human craniofacial anomalies.
Demonstration of a genetic interac-
tion between Gas1 and Shh. (A–D)
Comparison of Gas1+/–Shh+/–, Gas1–/–
Shh+/+, and 2 Gas1–/–Shh+/– perina-
tal skulls. The left 3 mice were litter-
mates. (A) Norma basalis. The black
line aligns the skulls, which were pho-
tographed at the same magnification.
(B) Norma lateralis. (C) Dentaries
of Gas1+/–Shh+/–, Gas1–/–Shh+/+, and
Gas1–/–Shh+/– perinates. Notably, the
Gas1–/–Shh+/– perinates, in addition
to midline fusion and a single incisor,
exhibited what appears to be a dupli-
cated proximal dentary that includes
a secondary cartilage-containing con-
dylar process (black arrows) and an
alveolus containing an ectopic molar
(green arrow). White arrowhead indi-
cates truncation and proximal bifur-
cation of Meckel’s cartilage. Original
magnification, ×4. (D) Calvaria of
Gas1+/–Shh+/–, Gas1–/–Shh+/+, and
Gas1–/–Shh+/– skulls. Black arrow-
heads highlight the loss of a patent
coronal suture in the calvarium of the
Gas1–/–Shh+/– skull. (E) Histological
sections of the developing ectopic
molar (red arrow) and dentary. MC,
Meckel’s cartilage; oc, otic capsule.
(F) Comparison of WT and Gas1–/–
Shh+/– middle ears. Outlined in the
WT image are styloid process (green),
stapes (orange), incus (yellow), and
malleus (purple). Yellow arrows high-
light the vestigial stapes; green arrows
indicate the ectopic preotic pillar run-
ning from the neurocranial base to the
otic capsule; red arrows indicate per-
sistent cartilage of the ala temporalis;
blue arrows indicate changes at the
squamosal; black arrows indicate the
ectopic condyles; dotted yellow line
denotes axis of symmetry. Original
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 6 June 2007
Generation of Gas1 and Shh mutant mice. Gas1–/– and Shh–/– mice were gener-
ated and genotyped as previously described (11, 39) and maintained in a
129Sv-C57BL/6 mixed background. Timed matings were set up such that
noon of the day on which vaginal plugs were detected was considered E0.5.
All animal procedures were reviewed, approved, and carried out under
license issued by the Animals and Scientific Procedures Division of the
Home Office, Her Majesty’s Government, London, United Kingdom.
Histological and skeletal analysis. For histological analysis, embryos were
fixed in 4% paraformaldehyde at 4°C, dehydrated through a graded etha-
nol series, embedded in paraffin wax, sectioned at 7 μm, and stained with
H&E. For differential staining of bone and cartilage, E18.5 and newborn
(i.e., P0) mice were fixed overnight in 95% ethanol, skinned, and eviscerated.
Cartilage staining was carried out by soaking in a solution of 76% ethanol,
20% glacial acetic acid, and 0.015% Alcian blue 8GX (Sigma-Aldrich) for 24
hours, differentiating for 7 days in 95% ethanol, macerating in 1% KOH
for 24 hours, and washing overnight under running tap water. Bone stain-
ing was carried out by transferring the heads to a freshly made 0.1% aque-
ous solution of alizarin red S (Sigma-Aldrich), with the addition of several
drops of 1% KOH to enhance the darkness of the red color. The samples
were then washed for 30 minutes under running tap water, decolorized in
20% glycerol in 1% KOH for 1–2 weeks, and prepared for storage in increas-
ing concentrations of glycerol in 70% ethanol to a final concentration of
100% glycerol. Skeletal preparations were photographed in light-field,
using a Leica stereomicroscope, while submerged in 100% glycerol.
In situ hybridization. Whole-mount and radioactive section in situ hybridiza-
tion was carried out as previously described (61). Whole-mount embryos were
photographed on an agarose background suspended in PBS using a Leica ste-
reomicroscope. Light- and dark-field images of sections were photographed
using a Zeiss Axioscop microscope and merged in Photoshop CS1 (Adobe).
Proliferation assay. Assays for cell proliferation were carried out using
a Zymed BrdU Labeling and Detection Kit (Invitrogen). Mouse embryos
were labeled with BrdU via intraperitoneal injection into pregnant females
(5 mg/100 g body wt) 2 hours prior to sacrifice. Embryos were fixed in
Carnoy’s fixative at 4°C overnight, dehydrated in methanol, embedded in
paraffin wax, and sectioned at 7 μm. The percentage of BrdU-positive cells
was calculated after counting by 1 researcher blinded to experimental group
on 2 separate occasions 1 week apart, and the mean value was calculated.
A total of 4 WT and 4 Gas1–/– mice were analyzed for each embryonic stage,
which included a total of 45 WT and 55 Gas1–/– sections. BrdU-positive cells
were counted in the epithelium and mesenchyme of the anterior and pos-
terior palate using an ocular scale grid orientated at the apex of the palatal
projection and in the bend region of the palatal shelf (46). Specifically, at
E12.5 this covered an area of mesenchyme that was 0.02 mm2 at the palatal
apex and 0.012 mm2 at the bend; these regions were bounded by epithelium
of lengths of 132 μm and 88 μm, respectively. At E13.5, the areas of mesen-
chyme counted were 0.03 mm2 and 0.015 mm2 and bounded by 263-μm and
165-μm lengths of epithelium at the palatal apex and bend, respectively.
Scanning electron microscopy. For scanning electron microscopy, tissues
were fixed and stored in 2.5% glutaraldehyde in 0.1 M sodium cacodyl-
ate buffer, rinsed in 0.1 M cacodylate buffer, and postfixed in 1% osmium
tetroxide in water for 90 minutes, followed by dehydration through a
graded series of acetone in water, critical point drying in liquid CO2, and
sputter coating with gold. Tissues were examined and recorded in a Phillips
SEM501B scanning electron microscope fitted with a Deben Pixie digital
scan generator and recorder.
Statistics. Student’s t test was used to analyze the significance of the differ-
ences in BrdU incorporation rates. A P value less than 0.05 was considered
We thank Andrew McMahon for Shh cDNA, Matthew Scott for
Ptc1 cDNA, and Alex Joiner for Gli1 cDNA as well as Tony Brain for
scanning electron microscopy. Maisa Seppala is a European Union
Marie Curie Early Stage Fellow (grant no. MEST-CT-2004-504025).
P.T. Sharpe is supported by the Wellcome Trust. M.J. Depew was
supported by the Royal Society.
Received for publication March 6, 2007, and accepted in revised
form April 10, 2007.
Address correspondence to: Martyn T. Cobourne, Floor 27, Guy’s
Hospital, London SE19RT, United Kingdom. Phone: 44-2071884432;
Fax: 44-2071884415; E-mail: email@example.com.
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