RESEARCH ARTICLE
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Development 136, 107-116 (2009) doi:10.1242/dev.026583
A SHH-responsive signaling center in the forebrain regulates
craniofacial morphogenesis via the facial ectoderm
Diane Hu and Ralph S. Marcucio*
Interactions among the forebrain, neural crest and facial ectoderm regulate development of the upper jaw. To examine these
interactions, we activated the Sonic hedgehog (SHH) pathway in the brain. Beginning 72 hours after activation of the SHH pathway,
growth within the avian frontonasal process (FNP) was exaggerated in lateral regions and impaired in medial regions. This growth
pattern is similar to that in mice and superimposed a mammalian-like morphology on the upper jaw. Jaw growth is controlled by
signals from the frontonasal ectodermal zone (FEZ), and the divergent morphologies that characterize birds and mammals are
accompanied by changes in the FEZ. In chicks there is a single FEZ spanning the FNP, but in mice both median nasal processes have a
FEZ. In treated chicks, the FEZ was split into right and left domains that resembled the pattern present in mice. Additionally, we
observed that, in the brain, fibroblast growth factor 8 (Fgf8) was downregulated, and signals in or near the nasal pit were altered.
Raldh2 expression was expanded, whereas Fgf8, Wnt4, Wnt6 and Zfhx1b were downregulated. However, Wnt9b, and activation of
the canonical WNT pathway, were unaltered in treated embryos. At later time points the upper beak was shortened owing to
hypoplasia of the skeleton, and this phenotype was reproduced when we blocked the FGF pathway. Thus, the brain establishes
multiple signaling centers within the developing upper jaw. Changes in organization of the brain that occur during evolution or as
a result of disease can alter these centers and thereby generate morphological variation.
INTRODUCTION
Previous research has examined the role of various epithelia during
development of the craniofacial complex. For example, signals from
endoderm and ectoderm are required for skeletogenesis in the face
(Brito et al., 2006; Couly et al., 2002; Graham et al., 2005; Ruhin et
al., 2003; Tyler and Hall, 1977). Our research focuses on mechanisms
regulating development of the upper jaw. The skeleton in this region
is formed from neural crest-derived cells generated along the dorsal
surface of the forebrain and midbrain (Couly et al., 1993; Evans and
Noden, 2006; Noden, 1978). These cells migrate adjacent to the
forebrain, reside in the frontonasal (FNP) and maxillary processes
(MXP), and are encased by ectodermal and neuroectodermal epithelia.
Subsequent interactions among these tissues control morphogenesis
of the skeleton (Hu et al., 2003; Marcucio et al., 2005; Schneider and
Helms, 2003; Schneider et al., 2001).
Patterning and growth of the upper jaw is controlled by discrete
signaling centers located in the ectoderm. The frontonasal ectodermal
zone (FEZ) regulates development of the distal tip of the upper jaw
(Hu et al., 2003). In chicks, the FEZ forms at approximately stage 20
[HH20 (Hamburger and Hamilton, 1951)] when Shh transcripts are
detected in ectodermal cells comprising the roof of the stomodeum
(Marcucio et al., 2005). At this time, Shh- and Fgf8-expressing cells
form a boundary that spans the mediolateral axis of the FNP. Shortly
after this boundary forms, Fgf8 is downregulated and becomes
restricted to epithelium near the nasal pit, whereas Shh expression is
maintained for a longer period of time. Retroviral mapping studies
revealed that the boundary presages the distal tip of the upper beak,
and our studies demonstrate that the FEZ regulates dorsoventral
patterning within the distal tip of the upper jaw (Hu et al., 2003). Mice
have a similar signaling center, but Shh transcripts do not span the
width of the upper jaw. Rather, distinct domains of Shh expression are
associated with the left and right median nasal processes (Hu and
Marcucio, 2008). In addition to Shh and Fgf8, multiple genes
encoding bone morphogenetic proteins (Bmp2, Bmp4 and Bmp7) are
expressed in the FEZ (Abzhanov and Tabin, 2004; Foppiano et al.,
2007; Hu et al., 2003; Marcucio et al., 2005), but the role of these
molecules is unknown. However, our observations indicate that the
FEZ functions, in part, by regulating expression of Bmps in neural
crest mesenchyme (Hu and Marcucio, 2008). In turn, BMP signaling
regulates growth of the upper jaw (Abzhanov et al., 2004; Wu et al.,
2006; Wu et al., 2004). Recently, the nasal pit has been shown to
pattern the jaw skeleton. FGF signaling in this region controls cell
proliferation and antagonizes Bmp4 expression (Szabo-Rogers et al.,
2008). Thus, interactions among the brain, ectoderm and neural crest
sculpt this region of the head.
SHH signaling within the forebrain is required for induction of
Shh in the FEZ (Marcucio et al., 2005), and differences in Shh
expression correlate with morphological variation in mice and
chicks. Therefore, we hypothesized that the forebrain may generate
morphological variation by regulating signals from the ectoderm
that control development of the upper jaw. To test this, we activated
SHH signaling within the forebrain after emigration of neural crest
cells was complete. Our results demonstrate that a SHH-responsive
center in the brain imprints information on the ectoderm that
controls morphogenesis of the upper jaw.
MATERIALS AND METHODS
Bead preparation
Department of Orthopedic Surgery, San Francisco General Hospital, University of
California at San Francisco, School of Medicine, San Francisco, CA 94110, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 17 October 2008
Affi-gel blue beads (50-100 mesh, 200-250 μm diameter; BioRad) were
soaked in recombinant SHH-N protein [800 μg/ml in PBS 0.1% bovine
serum albumin (BSA), Ontogeny] for 1 hour at 37°C; beads were placed into
the forebrain of HH11 embryos (see Fig. S1D in the supplementary
material). Alternatively, beads soaked in SU5402 (3 mg/ml) were implanted
in the FNP of ~HH17 embryos.
DEVELOPMENT
KEY WORDS: Shh, Fgf, Wnt, Bmp, RA, Facial morphogenesis, Zfhx1b, Chick, Mouse
RESEARCH ARTICLE
Embryo processing
Mouse and chick embryos were fixed in 4% paraformaldehyde (4°C
overnight), transferred to PBS containing 0.01% ethidium bromide and
photographed using a Leica MFLZIII dissecting microscope with
epifluorescence. Embryos were dehydrated, embedded in paraffin and
sectioned (8 μm). Mouse embryos were collected at E10.5, E11 and E12.
The day we observed a plug was E0. No further staging was performed.
In situ hybridization
In situ hybridization was performed on sections or in whole mount as
described (Albrecht et al., 1997). Subclones of Shh, Nkx2.1, Fgf8, Bmp2,
Bmp4, Bmp7, Wnt4, Wnt6, Wnt9b, Wnt14, Raldh2 and Zfhxb1 were
linearized for transcription of 35S- or dig-labeled antisense riboprobes.
Sections were counterstained with bis-benzimide, and images are
superimpositions of fluorescent and pseudo-colored dark-field image.
Safranin O staining
Cartilage (Red) was visualized on sections with Safranin O/Fast Green
(SO/FG) staining (Lu et al., 2005). Alternatively, embryos were stained with
Alcian Blue and Alizarin Red (Wassersug, 1976).
BrdU labeling
Twenty minutes before sacrifice, 1 μl of BrdU (Zymed, South San
Francisco, CA) was injected into the vitelline vein. Embryos were processed
as above. BrdU incorporation was assessed by immunohistochemistry using
diaminobenzidine (DAB) followed by counterstaining with hematoxylin
(Zymed). The number of proliferating cells was determined on images
captured using Adobe Photoshop of the Olympus CAST system. Analysis
of Variance (ANOVA) was performed on medial and lateral sections
separately to determine statistical significance, and on treated and control
sides of embryos 24 hours after implantation of SU5402 beads.
TUNEL analysis
DNA fragmentation was examined using a TUNEL kit following the
manufacturer’s instructions (Apoptag Plus, Intergen).
Electroporation and X-Gal staining
To visualize activation of the Wnt pathway we injected the Top-Gal plasmid
DNA (200 nl; 2 μg/μl) into the space between the ectoderm and the forebrain
on the right side of embryos. Then, electroporation [five pulses (10 volts) 20
mseconds apart] was used to transfect cells. Twenty-four hours later,
embryos were fixed in 0.4% gluteraldehyde (15 minutes at room
temperature), washed and then standard X-Gal reaction was performed.
RESULTS
Neural crest cell emigration from the brain
Prior to experimentation we analyzed the timing of neural crest
emigration from the brain by immunohistochemical detection of the
neural crest marker HNK-1. At HH9–, emigration of neural crest
cells from the neural tube was under way (see Fig. S1A in the
supplementary material), and cells had moved laterally and rostrally
by HH9 (see Fig. S1B in the supplementary material). At HH10, the
HNK-1-positive cells reached the lateral and rostral edge of the
dorsal surface of the embryo (see Fig. S1C in the supplementary
material). We activated the SHH pathway by placing a bead soaked
in the N terminus of SHH into the lumen of the forebrain at HH11.
Thus, generation of neural crest cells destined for the upper jaw was
not affected, and molecular, cellular and morphological changes
resulted from alterations in the interactions among the forebrain,
neural crest cells and surface ectoderm.
Ectopic SHH signaling alters the telencephalon
SHH signaling specifies the dorsoventral axis of the brain (reviewed
by Lupo et al., 2006). Therefore, we assessed the extent to which the
forebrain was altered by examining expression of the ventral
markers Nkx2.1 and Shh, and the dorsal marker Pax6 near the
sagittal midline of embryos. Within 24 hours (~HH15), we observed
Development 136 (1)
an expansion of Nkx2.1 and Shh expression domains (Fig.
1A,B,D,E), and a downregulation of Pax6 (Fig. 1C,F). At 48 and 72
hours, we observed similar changes in gene expression in the brain
(not shown). Additionally, we observed changes in Fgf8 expression;
24 hours after bead placement, controls exhibited Fgf8 expression
in the ectoderm and in the brain (Fig. 1G). However, in the brain of
treated embryos Fgf8 expression was mosaic (Fig. 1J). At 60 hours
(~HH20/21), Fgf8 transcripts were detected in the forebrain, optic
recess and ectoderm of control embryos (Fig. 1H), but in treated
embryos expression in the optic recess and facial ectoderm was
absent (Fig. 1K). In control and treated embryos, Fgf8 expression
was downregulated in the facial ectoderm at 72 hours (Fig. 1I,L). At
each time point we never observed Fgf8 transcripts in the optic
recess of treated embryos, but we did observe a continuous domain
of Shh and Nkx2.1 expression in the floor of the brain, which is
normally disrupted by the optic recess. Thus, our treatment disrupted
the normal appearance of the optic recess.
We confirmed that these changes resulted from altered
specification of the brain rather than alterations in cell survival. We
only observed TUNEL-positive cells in basal regions of the brain in
control and treated embryos at 24, 48 and 60 hours after bead
implantation. No significant signs of cell death were detected in
dorsal regions of the brain, in the surface ectoderm or in the
mesenchyme of treated embryos (see Fig. S2 in the supplementary
material).
Ectopic SHH signaling in the forebrain disrupts
facial morphology
The changes in the brain agree with studies illustrating the role of
SHH in patterning neural tissues (reviewed by Lupo et al., 2006).
However, our goal was to examine the relationship between
patterning the forebrain and development of the facial skeleton.
Therefore, we analyzed the ontogeny of the malformations in treated
embryos. At 48 hours (~HH19), mild defects were apparent (n=8).
The relationship between the nasal pits and the MXP were different
in control (bead soaked in PBS; n=7) and experimental embryos
(Fig. 2A,B). At this time, neural crest cells have arrived in the FNP
but growth has not begun yet. Similarly, in mice, neural crest cells
have begun populating the upper jaw anlagen, but there has not been
much growth by this time (Fig. 2C). However, at this time, the chick
and mouse faces appear distinct. The chick has well-developed nasal
pits whereas these are small in mice. Thus, unique attributes
characterize these regions of the face from the beginning of
development. By 72 hours (~HH23), treated embryos exhibited
severe signs of malformation. Normally, the FNP has begun to grow
at this time (Fig. 2D). In treated embryos the forebrain was small,
the eyes were small, the nasal pits were malformed, and the FNP and
LNP exhibited aberrant growth, but the MXPs appeared unaffected
(Fig. 2E) (n=14). Therefore, we focused on morphological changes
within the FNP. In treated embryos, growth was occurring in lateral
regions and converging toward the middle part of the FNP. These
embryos resembled E11.0 mouse embryos (Fig. 2F). At this time in
mice, growth was occurring in lateral regions near the nasal pit, and
was converging towards the middle of the upper jaw anlagen.
Hence, in mice, the midline of the upper jaw anlagen is filled in by
lateral to medial growth of the embryonic primordia. By 96 hours
(~HH28) after bead implantation, the phenotype had become more
severe. In controls, growth was centered in the middle of the FNP
(Fig. 2G). However, in treated embryos growth was concentrated in
lateral regions of the FNP, and these developing primordia had
converged to the midline to touch each other (Fig. 2H). In mice at
this time, the right and left median nasal processes had expanded and
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abutted each other in the midline where a furrow formed (Fig. 2I).
Interestingly, in severe cases (n=8/17 at 96 hours, and 1 at day 13),
some of the treated chicks had midfacial clefts (see Fig. S3 in the
supplementary material), a malformation observed in a number of
mouse mutants, including the Fgf8 hypomorph (D.H. and R.S.M.,
unpublished). Owing to changes in the growth characteristics within
the FNP, we assessed the extent to which the FEZ in the treated
chicks was altered.
Spatial organization of the FEZ correlates with growth patterns
observed in chicks and mice (Hu and Marcucio, 2008). In normal
chicks, Shh expression was evident along the tip of the growing FNP.
The Shh expression domain in these embryos was small and was
centered within the medial ectoderm of the expanding FNP (Fig. 2J).
At 72 (data not shown) and 96 hours after treatment, Shh expression
in the FEZ was downregulated in the midline (Fig. 2K). This
expression pattern resembles that observed in mice at E12. Shh
transcripts were not detected in the midline (Fig. 2L). At 72 hours
after implantation of beads, the total number of proliferating cells
was reduced in the midline, but unaltered in lateral regions, of the
FNP in treated embryos compared with controls (Fig. 3). Thus, the
FEZ controls growth in the medial region of the upper jaw.
Embryos were allowed to develop to later stages to examine
formation of the upper beak. Eleven (n=5; not shown), 12 (n=5) (Fig.
4A-D) and 13 (n=6) (Fig. 4E,F) days after bead implantation, the
eyes were small, the upper beak was shortened, the nasal capsule
was underdeveloped and bones comprising the proximal portion of
DEVELOPMENT
Fig. 1. Activation of the SHH signaling pathway in the prosencephalon expands the ventral forebrain. Sections near the midline of each
chick embryo were chosen for analysis based on the presence of or proximity to Rathke’s pouch (RP). (A) In control embryos 24 hours after bead
implantation (~HH15), Nkx2.1 transcripts (green) were present in the diencephalon and telencephalon (arrows). Cells located in the optic recess also
expressed Nkx2.1. (B) At this time, Shh (red) was expressed in the diencepahlon, but the expression domain did not extend beyond the optic recess
(arrow). (C) In the brain, Pax6 (pink) was expressed in dorsal regions of the telencephalon, but was absent from the basal diencephalon. (D-F) In
treated embryos, Nkx2.1 (D) and Shh (E) expression was expanded in the forebrain, while Pax6 (F) was repressed. Arrows indicate the dorsal
location of the Pax6 expression domain. (G) Fgf8 (yellow) was expressed in the ectoderm (arrowhead) and forebrain (arrow) of controls at 24 hours
after bead implantation. (H) At 60 hours, the ectoderm (arrowhead), brain (arrow) and optic recess (OR) expressed Fgf8. (I) By 72 hours, Fgf8 was
expressed in the brain (arrow) and OR but was absent from the ectoderm. (J) At 24 hours after bead implantation, Fgf8 expression in the brain was
mottled (arrow), and there was no expression in the optic recess. Expression of Fgf8 in the ectoderm appeared unaltered (arrowhead). (K) At 60
hours, Fgf8 was not expressed in the OR, and in the forebrain Fgf8 was downregulated. In the ectoderm of treated embryos, Fgf8 was not
expressed (arrowhead). (L) By 72 hours, Fgf8 expression in the brain was maintained, but the OR domain was not apparent. Fgf8 was not expressed
in the ectoderm at this time. Scale bar: 300 μm.
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Development 136 (1)
the upper jaw were small and malformed. The premaxillary bone
located in the distal part of the upper jaw appeared smaller, but was
not as severely affected as the more proximal skeletal elements. As
changes in the FEZ corresponded to the reductions in the
premaxillary bone, we focused on malformations in the proximal
region of the jaw and nasal capsule.
Effect of ventralizing the brain on signals near
the nasal pit
We identified molecular changes in other regions of the face that may
generate the phenotype that we observed. A number of genes in, or
adjacent to, the nasal pit were altered within 72 hours of bead
placement. Wnt4 was normally observed in the nasal pit epithelium
(Fig. 5A), but in treated embryos Wnt4 transcripts were not detected
(Fig. 5E). Similarly, Wnt6 was normally expressed in two domains
in the nasal pit (Fig. 5B). In treated embryos, Wnt6 was reduced in
these areas (Fig. 5F). Wnt9b was normally expressed in the ectoderm
covering the lateral region of the FNP, the lateral nasal processes and
the MXPs, but was excluded from the epithelium of the nasal pit (Fig.
5C). In treated embryos, we observed malformations in this region
of the face that distorted expression of Wnt9b. The epithelium of the
nasal pit normally intersects the surface ectoderm at right angles, but
in treated embryos, the nasal pit was sloped. Therefore, when the face
of the embryos were viewed from the front, the nasal pit epithelium
was visible and this tissue did not express Wnt9b (Fig. 5G).
To examine the extent to which canonical WNT signaling was
altered in treated embryos, we electroporated the TOP-Gal plasmid
(DasGupta and Fuchs, 1999) into the face 48 hours after treatment.
Twenty-four hours later we observed strong expression of βgalactosidase throughout the FNP and MXPs in control embryos
(Fig. 5D). Similarly, we observed strong expression of βgalactosidase in treated embryos (Fig. 5E). Although we could not
quantify activation of the WNT pathway owing to variation in
electroporation efficiency among embryos, our results indicate that
the canonical WNT pathway was active in medial and lateral regions
of the FNP in all embryos.
DEVELOPMENT
Fig. 2. Altering the dorsoventral polarity of the brain perturbed facial development. (A) Control chick embryo 48 hours after bead
implantation illustrating the medial (m) and lateral (l) regions of the FNP and their relationship with the lateral nasal processes (LNP). mn,
mandibular process; mx, maxillary process. (B) In treated chick embryos, the nasal pits were not elongated, and the FNP was not well defined.
(C) Growth of the upper jaw anlagen has not begun in mouse embryos at E9.5. (D) Control chick embryo 72 hours after bead implantation
appeared normal. (E) Treated chick embryos had micro-opthalmia. In the FNP, growth occurred in lateral FNP (l; red arrows), whereas there was a
lack of growth in the midline (m; arrowhead). (F) Growth of the middle and upper jaw in E11.0 mouse embryos occurred in lateral regions and
began to form median nasal processes (arrows). Arrowhead indicates the median furrow present in the upper jaw of mouse embryos. (G) A control
chick embryo 96 (~HH28) hours post-implantation illustrates the normal morphology of the face. Arrow indicates growth in the medial portion of
the FNP. (H,I) In treated chick embryos (H), growth of the FNP occurred in lateral domains (l; arrows) reminiscent of (I) median nasal processes
(arrows) in mammals. Arrowheads indicate the median furrow in mouse embryos. (J) At HH28, Shh expression in the FEZ was restricted to a small
domain located at the tip of the expanding FNP in chicks (red arrow, n=4). (K) In treated chicks, the Shh domain was divided into two halves that
were larger than in normal chicks (red arrows; n=9/11). Black arrow indicates the lack of Shh expression in the midline. (L) In mice at E12 (n=6), Shh
expression was evident in two large, distinct domains (red arrows), whereas in the midline, no Shh transcripts were observed (black arrow). The
circled expression domain is in the basal forebrain. Scale bars: 1 mm in A,B,D,E,G,H,J,K; 500 μm in C,F,I,L.
Fig. 3. The FEZ establishes proliferative zones in the
mesenchyme of chick embryos. (A) Control embryo 72 hours after
bead implantation. (B) Boxed area in B is shown in C. (C) BrDU
incorporation illustrates the uniform distribution of proliferating cells
across the FNP. (D) Treated embryos were malformed at 72 hours after
bead implantation. (E) Boxed area is shown in F. (F) In treated embryos,
fewer proliferating cells were detected in the midline. (G) The number
of proliferating cells in midline (boxed areas) was reduced in treated
embryos (n=10, P<0.05). Scale bar: 2 mm in A,D; 500 μm in B,E;
100 μm in C,F.
Additionally, 72 hours after bead placement, Raldh2 was
expressed in mesenchyme of the maxillary and lateral nasal
process (Fig. 5I). After treatment, we observed expansion of
Raldh2 in the mesenchyme of the FNP (Fig. 5M). By contrast,
Fgf8 was normally expressed in medial and lateral epithelium of
the nasal pit (Fig. 5J). After activation of SHH signaling in the
brain, we observed that the nasal pit was malformed and Fgf8
expression was not apparent in the medial epithelium (Fig. 5N).
In the developing neural plate, Zfhx1b (Zeb-2/Sip1) is positively
regulated by FGF signaling (Sheng et al., 2003). Therefore, we
wanted to determine whether expression of Zfhx1b was altered in
treated embryos. Normally, Zfhx1b is expressed in the
mesenchyme of the FNP adjacent to the ectoderm (Fig. 5K), and
we observed downregulation of Zfhx1b in mesenchyme of the
FNP (Fig. 5O). However, whether this change was a direct result
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Fig. 4. Morphology of the upper jaw skeleton. (A) Gross
morphology of a control chick embryo at day 12 illustrates the
relationship between the jaws. (B) In treated chick embryos, the upper
jaw was shorter than in controls. Small eyes were also evident in these
embryos. (C) A sagittal section through the distal tip of the upper jaw
and stained with Milligan’s modified Trichrome revealed the highly
turbinated nasal capsule (arrow), the prenasal process of the
premaxillary bone (arrowhead), the prenasal cartilage (pnc) and the egg
tooth (asterisk). (D) In treated embryos, the nasal capsule (arrow) was
rudimentary, but the egg tooth (*) was present. The prenasal process of
the premaxillary bone was smaller and does not appear in this section.
(E) Alcian Blue and Alizarin Red staining of a control head (day 13)
illustrates the nasal bone (ns), jugal bar (Ju), maxillary bone (mx) and
premaxillary bone (pmx). (F) In treated embryos, an ectopic cartilage
nodule was apparent (arrow). The nasal bone, the maxillary bone and
the jugal were greatly reduced in size, and the premaxillary bone
appeared smaller. Scale bars: 2 mm in A,B; 100 μm in C,D; 1 mm in E,F.
of downregulation of FGF signaling is unknown. Finally, FGF
signaling represses expression of Bmp4 in lateral regions of the
FNP (Szabo-Rogers et al., 2008; Wu et al., 2006), and we
observed the expected upregulation of Bmp4 expression in lateral
regions of the ectoderm covering FNP in treated embryos (Fig.
5L,P). However, we did not detect changes in mesenchymal
expression of Bmp4, Bmp2 and Noggin (see Fig. S4 in the
supplementary material). Hence, multiple signaling pathways
were altered after activation of SHH signaling within the brain.
Effect of blocking FGF signaling in the face
Activation of SHH signaling in the brain created defects in the
proximal region of the upper jaw and nasal capsule (Fig. 4), and
these alterations were accompanied by changes in expression of
Fgf8 (Figs 1, 5). To further explore these changes, we performed
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Development 136 (1)
whole-mount in situ hybridization to compare expression of Fgf8 in
control and treated embryos. Within 48 and 72 hours of treatment,
the nasal pits were malformed and Fgf8 was downregulated near the
nasal pit (Fig. 5Q-T).
Next, we blocked FGF signaling by implanting beads soaked in the
FGF receptor inhibitor SU5402 (Eblaghie et al., 2003; Szabo-Rogers
et al., 2008) into the mesenchyme comprising the right side of the FNP
at HH17. We examined the skeletons at day 13, and we observed a
similar phenotype in SHH- and SU5402-treated embryos (Figs 4, 6).
The nasal capsule was reduced and proximal skeletal elements were
small or absent in treated embryos (Fig. 6C-F). At 24 hours after bead
placement, we did not observe evidence of increased cell death (see
Fig. S5 in the supplementary material), but we detected reduced cell
proliferation in mesenchyme adjacent to the bead (Fig. 7). Thus,
blockade of FGF signaling generates a phenotype that resembles that
produced when we activated the SHH pathway in the brain. However,
we are unable to determine the extent to which blockade of signaling
by specific Fgf ligands produce the phenotype.
DEVELOPMENT
Fig. 5. Changes in signaling near the nasal pit in chick embryos. (A,B) Seventy-two hours after bead placement, Wnt4 (A, pink, arrow) and
Wnt6 (B, green, arrow) were expressed in the epithelium of the nasal pit. (C) At this time, whole-mount in situ hybridization reveal Wnt9b
expression throughout the epithelium covering the developing upper jaw (arrows), as well as in the lateral nasal (lnp) and maxillary (mxp) processes.
However, Wnt9b was not expressed in the nasal epithelium (arrowhead). (D) X-gal reaction revealed activation of the canonical Wnt pathway
throughout the developing FNP, lnp and mxp. (E,F) Wnt4 (E) and Wnt6 (F) were absent from the nasal epithelium in treated embryos 72 hours after
implantation. (G) In treated embryos, Wnt9b transcripts spanned the mediolateral axis of the FNP (arrow). In these embryos, the nasal epithelium
was exposed on the surface of the face and Wnt9b transcripts were absent from this tissue (arrowhead). (H) X-gal staining revealed that Wnt
signaling still occurred in treated embryos. (I) Raldh2 was expressed (red) in the mesenchyme of the lnp and mxp 72 hours after bead implantation.
(J) Fgf8 was expressed in the nasal epithelium of control embryos at this time. (K) Zfhx1b transcripts were present in the mesenchyme of the FNP in
control embryos at this time. (L) Bmp4 transcripts were evident in the lateral regions of the FNP of control embryos 72 hours after bead
implantation. (M) At this time Raldh2 was expressed (red) ectopically in mesenchyme of the FNP (arrows) and mxp. Expression in the lnp was not
evident. (N) Fgf8 was absent form the medial nasal epithelium (arrow). (O) Zfhx1b was downregulated in the mesenchyme of treated embryos.
(P) Bmp4 expression was expanded (red arrows) to surround the nasal pit in treated embryos at 72 hours. (Q) Fgf8 was expressed near the nasal pit
in controls at 48 hours, but (R) in treated embryos Fgf8 was downregulated. (S) At 72 hours after bead placement, Fgf8 expression was strong
surrounding the nasal pit, but (T) in treated embryos Fgf8 was downregulated. Scale bars: 250 μm in A,B,E,F,I-K,M-O; 1 mm in C,D,G,H,L,P,Q-T.
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Fig. 7. Blockade of FGF signaling reduces cell proliferation in
chick embryos. (A) Low magnification of the FNP after staining for
BrdU. Boxes indicate locations of sections in B and C. (B,C) High
magnification of cell proliferation data in regions near the bead (B) and
on the contralateral side (C). (D) Graph illustrating proliferation in
treated embryos (n=9, control 35.3%, s.d.=4.8%, treated 29.8%,
s.d.=3.4%, P<0.05). Scale bars: 500 μm in A; 200 μm in B,C.
DISCUSSION
The brain participates in regulating morphogenesis of the face. Our
current work revealed that a SHH-responsive signaling center
communicates patterning information to the ectoderm that covers the
developing upper jaw. The brain regulates expression of signaling
molecules in the ectoderm that then control morphogenesis of the
upper jaw. Morphogenesis of the distal region of the upper jaw is
regulated by the FEZ (Hu et al., 2003), whereas the proximal part of
the upper jaw is controlled by signals near the nasal pit (Szabo-Rogers
et al., 2008). Our results indicate that development of these modules
is integrated by signals coming from the brain. Previously, we
demonstrated that the forebrain regulates the onset of Shh expression
in the FEZ (Marcucio et al., 2005). Now, we demonstrate that the brain
regulates the spatial expression domain of Shh in the FEZ and
expression of other signaling molecules near the nasal pit. Thus, the
brain directs development of the middle and upper jaw by establishing
discrete signaling centers in the ectoderm that covers the developing
upper jaw. These signaling centers are then responsible for
coordinating growth and patterning of the upper jaw anlagen.
Interactions between the brain, ectoderm and neural crest occur
as part of a set of signaling interactions that pattern the facial
skeleton. During early stages of facial development, tissue
interactions establish regional identity within ingressing neural crest
cells. Epithelial-mesenchymal interactions mediated by BMP4 and
FGF8 define the maxillary and mandibular regions of the first
pharyngeal arch (Shigetani et al., 2000). These signaling interactions
may act to establish unique patterns of gene expression in the
adjacent mesenchyme that control regional characteristics within the
skeleton. In part, nested expression of Dlx genes in mesenchymal
cells comprising the upper jaw anlagen pattern the developing
skeleton (Depew et al., 2002). Expression of Dlx genes is regulated
by signals from the epithelium covering the pharyngeal arches, and
homeotic transformations occur in the jaws of animals with
mutations in various Dlx genes (reviewed by Depew et al., 2005).
Finally, signals from the pharyngeal endoderm appear necessary for
development of the cartilages that comprise the ventromedial part of
the upper jaw, but the nature and timing of these interactions are
unknown (Benouaiche et al., 2008). At later stages, the ectoderm
controls proximodistal extension and dorsoventral polarity of the
upper jaw, but does not contain information that specifies upper or
lower jaw structures. For example, when the FEZ was transplanted
to the mandible, the lower jaw was duplicated rather than
transformed into an FNP-like structure (Hu et al., 2003). Hence, a
series of interactions occur among tissues that comprise the facial
primordia. In our work, we have focused on identifying the role of
the brain in these signaling interactions.
Ectopic SHH signaling ‘ventralizes’ the
telencephalon
Signaling by SHH controls dorsoventral patterning of the forebrain
(reviewed by (Bertrand and Dahmane, 2006). Therefore, we
examined dorsoventral polarity in treated and control embryos. We
observed expansion of Shh and Nkx2.1 and a repression of Pax6
expression. These changes are consistent with other reports and
indicate a shift in dorsoventral patterning of the forebrain (Goodrich
et al., 1997; Manuel and Price, 2005).
DEVELOPMENT
Fig. 6. Blockade of FGF signaling may underlie skeletal changes
after activation of SHH in the brain of chick embryos. (A) A
control chick embryo illustrates morphology at day 13. (B) In treated
embryos, the upper jaw was shorter than in controls and ‘clefts’ were
apparent (arrows). (C) Dorsal view of the head of a control stained with
Alcian Blue and Alizarin Red at day 13 of development illustrates the
nasal bone (nb), jugal bar (Ju), maxillary bone (mx) and premaxillary
bone (pmx). The nasal capsule is indicated with an asterisk. The lower
jaw is labeled mn. (D) The nasal bone (black nb), premaxillary bone
(black pmx) and jugal bar (not shown) on the untreated side appeared
normal. On the treated side, the premaxillary bone (red pmx) and nasal
bone (red nb) appeared hypoplastic, and in this embryo the maxillary
bone was absent. The nasal capsule (*) was greatly reduced on the
treated side of embryos. The jugal bar is not in view. (E) Lateral view of
control embryo illustrating the bones of the upper jaw. (F) Hypoplasia of
the upper jaw skeleton was apparent in treated embryos. Scale bars:
2.5 mm in A,B; 2 mm in C-F.
RESEARCH ARTICLE
The regulatory mechanisms controlling Shh expression in the
neural tube are complex and involve multiple enhancers (Epstein
et al., 2000; Epstein et al., 1999; Jeong et al., 2006; Jeong and
Epstein, 2003) and signals (reviewed by Takahashi and Liu,
2006). However, published data indicate that a cis-regulatory
element controlling Shh expression in the telencephalon contains
an Nkx2.1-binding site (Jeong et al., 2006), and Shh expression is
absent from the telencephalon in the Nkx2.1–/– mouse (Sussel et
al., 1999). In the telencephalon, Nkx2.1 expression appears to be
regulated by a combination of signals, including FGF8 and SHH.
The allelic series of Fgf8 mutant mice (Meyers et al., 1998) have
a progressive loss of Nkx2.1 and Shh expression in the
telencephalon that corresponds to decreasing gene dose (Storm et
al., 2006), and our unpublished observations using SU5402 to
block FGF signaling in the forebrain produce identical results
(data not shown). However, we also observed similar changes in
the telencephalon after blocking SHH signaling within the brain
(Marcucio et al., 2005). Collectively, these data indicate that
FGF8 and SHH signaling regulate expression of Shh via NKX2.1
in the telencephalon. In our current experiments, increased SHH
signaling reorganized expression of Nkx2.1 and Shh and shifted
the anterior boundary of the ventral telencephalon.
Relationship between brain and face
Our objective was to examine the effect of the brain on
development of the face. We used a loss-of-function approach to
demonstrate the requirement for SHH signaling within the brain
for the onset of FEZ function (Marcucio et al., 2005). Now, we
complemented our previous research by performing a gain-offunction experiment. Investigators have reported that activation
of SHH in the brain alters the dorsoventral polarity of the
forebrain, inhibits neural crest generation and produces defects
resembling holoprosencephaly (HPE) (Nasrallah and Golden,
2001). Therefore, we initiated our experiments after neural crest
cells left the neural tube. We determined that activation of SHH
signaling in the brain altered expression of signaling molecules in
the ectoderm covering the upper jaw, and these changes created
defects in the face.
In this work, we observed changes in skeletal elements that
comprise the proximal part of the upper jaw and the nasal capsule.
These elements are derived from lateral nasal and MXPs (Lee et al.,
2004), and the malformations hinted at the presence of a signaling
center located proximally in the jaw. Signals from the nasal pit, and in
particular FGFs, have been shown to control development of proximal
regions of the upper beak (Szabo-Rogers et al., 2008). We discovered
that a number of genes were misexpressed in this region of the face
after activating SHH signaling in the brain. We focused on the changes
that we observed in the BMP, WNT and FGF signaling pathways
owing to their importance for facial development (Ashique et al.,
2002; Brugmann et al., 2007; Foppiano et al., 2007; Lan et al., 2006;
MacDonald et al., 2004; Richman et al., 1997; Song et al., 2004;
Storm et al., 2006; Szabo-Rogers et al., 2008).
We examined several candidate genes in and around the nasal pit
to determine the extent to which the brain regulates expression of
genes in this area. We observed upregulation of Bmp4 in the
ectoderm but not mesenchyme near the nasal pit. A previous report
demonstrated that increased expression of Bmp4 in the
mesenchyme, but not in the ectoderm, causes expansion of the upper
beak (Abzhanov et al., 2004). Hence, the reduction in the upper beak
in conjunction with expanded Bmp4 expression in the ectoderm is
consistent with this earlier report. In addition to the expansion of
Bmp4, we observed other misregulated genes in treated embryos.
Development 136 (1)
One of the genes that was misexpressed in treated embryos was
the transcriptional co-factor Zfhx1b. This molecule was nearly
undetectable in the FNP of treated embryos. Interestingly, Zfhx1b
is thought to act primarily as a transcriptional repressor of
Tgfβ/Bmp signaling (Postigo, 2003; Postigo et al., 2003), and
coordinated Bmp signaling is required for development of this
region of the face (e.g. Wu et al., 2006). Mutations in Zfhx1b cause
a variety of developmental disorders, including Hirschsprungs
disease and Mowat-Wilson Syndrome in humans (Mowat et al.,
2003; Yamada et al., 2001; Zweier et al., 2002). Mice that lack exon
7 of Zfhx1b have deficiencies in neural crest generation and
migration that may explain the malformations observed in humans.
However, the role of this molecule during later stages of
development is unknown.
The TOP-Gal plasmid has been used to identify sites of WNT
signal activation in response to stabilized to β-catenin and TCF/Lef
(DasGupta and Fuchs, 1999), and we used this plasmid to visualize
Wnt activity in treated and control embryos. Our results revealed
strong activation of the Wnt pathway in all embryos. These results
lead us to conclude that activation of the SHH pathway in the
forebrain did not impair canonical Wnt signaling in the developing
upper jaw. The Wnt ligands that were downregulated, Wnt4 and
Wnt6, are members of the non-canonical family of Wnt ligands that
function through the planar cell polarity pathway rather than the βcatenin pathway (Chang et al., 2007; Schmidt et al., 2007).
Therefore, effects of changes in these molecules would not be
detected by the TOP-Gal assay.
Additionally, we detected modest expansion of Raldh2 in the
mesenchyme near the nasal pit. Mutations that destabilize and
reduce protein levels of retinol dehydrogenase 10 create defects in
the nasal region of mouse embryos, including clefting and
malformations of the nasal septum (Sandell et al., 2007).
Interestingly, increased signaling by retinoic acid has been show to
downregulate Fgf8 expression in the epithelium of the first
pharyngeal arch (Vieux-Rochas et al., 2007), and we observed a
similar downregulation of Fgf8. Whether the changes in Raldh2 and
Fgf8 are related remains to be determined.
The downregulation of Fgf8 in the nasal pit was one of the most
notable changes in gene expression that we observed after
ventralizing the brain. In our final experiment, we blocked the
FGF pathway by inhibiting the ability of FGF ligands to activate
Fgf receptors, and we observed a phenotype that was similar to
that observed when the SHH pathway was activated in the brain.
Hence, we conclude that alterations in FGF signaling from
regions near the nasal pit appear to contribute to the
morphological defects in the upper jaw. However, the extent to
which the changes can be attributed to the absence of signaling by
a specific ligand, such as FGF8, is unknown. Mice that express
reduced levels of FGF8 have median clefts of the face that
resemble the most severe cases that we observed (see Fig. S4 in
the supplementary material), but a variety of FGF ligands are
expressed throughout the face (Francis-West et al., 1998) and may
have changed in our treated embryos. Although we are not able to
attribute our results to the loss of signaling by specific FGF
ligands, we (Hu et al., 2003), and others (Song et al., 2004; SzaboRogers et al., 2008; Wu et al., 2006), have demonstrated a
requirement for FGF signaling throughout development of the
FNP. The FGF signaling pathway establishes proliferative zones
in the mesenchyme by positioning the expression domains of
Bmp4 in the FNP (Szabo-Rogers et al., 2008; Wu et al., 2006), and
these authors suggest that this may contribute to species-specific
widening of the upper beak in birds.
DEVELOPMENT
114
Spatial organization of the FEZ regulates
morphological variation in the developing upper
jaw
In addition to the malformations evident in the proximal region of
the upper jaw, we also observed changes in the FNP of treated
embryos. These changes reflected a reorganization of gene
expression patterns in the FEZ that altered growth of the FNP. As
mentioned above, the establishment of species-specific growth
zones has been studied recently. Signaling by BMPs is thought to
underlie phenotypic variation in the developing upper jaw in a
variety of birds (Abzhanov et al., 2004; Wu et al., 2006; Wu et al.,
2004). We determined that the FEZ positively regulates expression
of Bmps in the neural crest mesenchyme of the upper jaw (Hu and
Marcucio, 2008). In this research, we observed an expansion of
Bmp4 expression in lateral ectoderm of the FNP. When taken
together, these studies indicate that signals from the FEZ induce or
maintain expression of Bmps in the mesenchyme whereas
antagonistic activity of FGF signaling restricts the expression
domain of Bmp4 in the ectoderm (Hu and Marcucio, 2008) (SzaboRogers et al., 2008; Wu et al., 2006). Thus, an intricate signaling
network establishes regionalized areas of gene expression and
growth in the facial primordia.
Given the role of the FEZ in regulating expression of patterning
genes in the FNP, we predicted that changes in gene expression
patterns in the FEZ would alter growth of the FNP. After activating
SHH signaling within the brain, chick embryos exhibited
morphological alterations. The eyes were smaller and the FNP was
divided into right and left ‘median nasal processes’. Similarly, mice
have small eyes and they exhibit well-defined median nasal
processes. In blind cavefish, increased SHH signaling is responsible
for atrophy of the optic vesicles (Yamamoto et al., 2004),
suggesting that the reduced eye size we observed was due to
atrophy rather than to a transformation to a mammalian
morphology. The reduced size of the eyes in treated chicks was not
likely to alter the growth zones within the FNP. We have observed
similar changes in the eyes during previous experiments, but
changes in the FNP did not resemble those observed here (Marcucio
et al., 2005). Furthermore, the morphological transformation of the
FNP into left and right median nasal processes was directly related
to the pattern of Shh expression in the FEZ. In chicks, a single
domain of Shh spans the mediolateral axis of the FNP, whereas in
mice two lateral domains of Shh are apparent (Hu and Marcucio,
2008). After activation of SHH signaling within the forebrain, Shh
expression domains in the FEZ resembled the murine pattern.
These similarities, together with our observations on the role of the
FEZ in regulating Bmp expression in the mesenchyme, lead us to
conclude that spatial organization of the FEZ establishes growth
zones that produce divergent morphologies distinguishing the early
stages of development of the upper jaws of avian and mammalian
embryos.
A recent report suggested that differential signaling by the
canonical WNT pathway distinguishes the unique growth
characteristics observed in mammals and birds. WNT
responsiveness was reported to be exclusively in the midline of the
developing avian FNP, whereas, in mice, WNT signaling occurred
in lateral regions (Brugmann et al., 2007). However, our current
work demonstrate that WNT signaling is active throughout the avian
FNP, lateral nasal processes and MXPs. These results are reinforced
by expression of Wnt ligands in ectoderm covering upper jaw in
birds. The reason for the discrepancies observed in these studies is
unknown but could reflect differences resulting from the mode of
delivery of the WNT-reporter constructs. We used electroporation to
RESEARCH ARTICLE
115
deliver the TOP-Gal or control plasmids. We achieved widespread
transfection and observed a large area of activation of the reporter
construct. Hence, our data suggest that WNT signaling participates
in regulating growth throughout the developing upper jaw anlagen
in both chicks and mice.
Together, these data indicate that a highly orchestrated set of tissue
interactions converge to regulate morphogenesis of the upper jaw.
Signals from the brain establish gene expression patterns within the
ectoderm. The ectoderm then signals to adjacent mesenchymal cells
to establish areas of cell proliferation and create growth zones within
the developing upper jaw. Interestingly, we determined that some
genes, like Wnt9b, are regulated independently of forebrain signals,
suggesting that intrinsic ectodermal properties or interactions between
other tissues participate in patterning ectodermal signaling centers that
control morphogenesis of the upper jaw. For example, Fgf8 expression
is evident in the developing facial ectoderm prior to emigration of
neural crest cells from the neural tube (Marcucio et al., 2005), and
interactions between the neural crest cells and ectoderm have been
shown to regulate expression of genes in the ectoderm (Eberhart et al.,
2006; Marcucio et al., 2005; Schneider and Helms, 2003).
Furthermore, signals from the pharyngeal endoderm appear to be
required for development of FNP derivatives in chick embryos, but
the exact nature of these interactions remain to be deduced
(Benouaiche et al., 2008). By understanding the intrinsic molecular
properties of the ectoderm, and the interactions that occur among the
endoderm, forebrain, neural crest and ectoderm, we will define the
signaling network that coordinates patterned growth of the upper jaw.
This will allow us to determine the extent to which morphogenesis of
these structures co-vary during development and will lead to an
understanding of mechanisms that generate normal variation, disease
phenotypes and evolutionary divergence in this region of the skull.
We thank Drs Richard Schneider, Benedikt Hallgrimsson, Melanie McCollum,
Silvia Foppiano, Nathan Young, Ophir Klein and Chuanyong Lu for discussions
of the manuscript. We thank Dr Elaine Fuchs for providing TOP-Gal plasmid, Dr
Claudio Stern for providing Zfhxb1, Dr Elena Frolova for Wnt9b and Stan ‘the
Eggman’ Keena of Petaluma Farms for farm fresh eggs. This work was funded
by the UCSF REAC and NIH (NIDCR: R01-DE018234) to R.S.M. Deposited in
PMC for release after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/107/DC1
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