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First published online 26 November 2008
doi: 10.1242/dev.026583
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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: ralph.marcucio{at}ucsf.edu)
Accepted 17 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Shh, Fgf, Wnt, Bmp, RA, Facial morphogenesis, Zfhx1b, Chick, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
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.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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HH17 embryos.
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.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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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 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 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.
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). 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 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.
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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 β-galactosidase throughout the FNP and MXPs in
control embryos (Fig. 5D).
Similarly, we observed strong expression of β-galactosidase 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.
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 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.
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; 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. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), 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.
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). 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).
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-of-function
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.
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
β-catenin 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;
Szabo-Rogers 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.
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) (Szabo-Rogers
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 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.
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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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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