1 Department of Developmental and Cell Biology, University of California,
Irvine, 5210 McGaugh Hall, Irvine, CA 92697-2300, USA
2 Centre for Regenerative Medicine, Developmental Biology Programme, School of
Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
* Author for correspondence (e-mail: tschilli{at}uci.edu)
Accepted 16 June 2005
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SUMMARY |
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Key words: Craniofacial, Cleft palate, Neural crest, Holoprosencephaly, Danio rerio
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Introduction |
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The anterior neurocranium (ANC) of larval zebrafish consists of paired
trabecular rods joined at the midline anteriorly to form the ethmoid plate
(Fig. 1A), and these form a
striking example of M-L polarity. Genetic studies in zebrafish and mice have
revealed that one member of the Hedgehog family of secreted proteins expressed
at the midline, Sonic hedgehog (Shh), is required for ANC formation. Hh
proteins act as polarizing factors in other contexts. Shh is
expressed in the midline prechordal plate mesoderm and floorplate of the CNS
adjacent to NC progenitors of the ANC
(Strahle et al., 2004).
Inactivation of zebrafish shh or other components of its signal
transduction pathway causes ML patterning defects in which trabecular
cartilages fuse at the midline, which is consistent with shh having a
role in polarity (Brand et al.,
1996
; Schilling,
1997
; Kimmel et al.,
2001
). Similarly, loss-of-function mutations in Shh in
the mouse cause severe defects in cranial growth and loss of virtually all of
the craniofacial bones (Chiang et al.,
1996
). Humans with mutant SHH exhibit holoprosencephaly
(HPE), often accompanied by midline craniofacial defects and palatal clefting
(Gorlin et al., 1990
;
Roessler et al., 1996
;
Muenke and Beachy, 2000
).
Recent studies in mice have also shown that NC cells require the essential Hh
receptor co-factor Smoothened (Smo) for craniofacial development, suggesting
that Shh acts directly on skeletal progenitors
(Jeong et al., 2004
). Given
its well-established role in regulating polarity in the neural tube and limb
bud in a concentration-dependent manner
(Ingham and McMahon, 2001
),
Shh is a good candidate for a signal in the NC environment that polarizes
skeletal fates along the ML axis. In zebrafish, shh may share this
role with its close relative tiggy-winkle hedgehog (twhh),
which is expressed in a similar pattern.
Shh is expressed in several craniofacial tissues, including the
early embryonic mesendoderm, ventral brain and oral ectoderm, and one or more
of these sources could mediate its roles in ANC patterning. Surgical removal
of oral ectoderm causes midfacial clefting in chicken embryos, whereas
exogenous SHH protein causes midfacial expansion and, in some cases,
duplications of midline structures of the ANC such as the nasal bone
(Helms et al., 1997;
Hu and Helms, 1999
;
Hu et al., 2003
). These and
other results have implicated Hh signaling from the facial ectoderm in ML
patterning of the face, and in the regulation of NC proliferation and/or
survival (Ahlgren et al.,
2002
). Endodermal expression of Shh is less important in
this process, as zebrafish mutants that eliminate endoderm, such as
casanova, lack viscerocranial cartilages but still form an ANC
(David et al., 2002
). Thus,
Shh from the oral ectoderm may play a crucial role in ML polarity
that is conserved in all vertebrates. However, the precise source of the Hh
that controls facial patterning remains unclear.
Here, we present a characterization of NC morphogenesis and skeletal patterning in the sonic you (syu) mutant zebrafish, which disrupts the shh gene, and in embryos treated with the Hh inhibitor cyclopamine (CyA), at different stages of NC development. By constructing a transgenic line in which NC cells fluoresce in the living embryo, we are able for the first time to follow their migratory pathways in detail. We show that two distinct groups of NC form the trabecular and medial ethmoid cartilages of the ANC, contrary to the classical notion that the ethmoid simply forms by trabecular fusion. Both sets of NC cells require Hh signaling, but we argue that they do so at different times and in different regions, and that this helps to explain the spectrum of ML polarity defects observed in fish, mice and humans that are mutant in the Hh pathway. We suggest that Hh signals act both early, to separate skeletogenic NC at the midline, and later, to promote chondrogenesis, and that multiple sources of Shh induce the final skeletal pattern.
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Materials and methods |
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Morpholinos
Morpholinos targeting zebrafish shh (shh-MO) and
tiggy-winkle hedgehog (twhh-MO) were obtained (GeneTools) as
reported previously (Nasevicius and Ekker,
2000). These were microinjected at the one- to two-cell stage,
either alone or in combination. Defects depended on the amount of MO injected;
for most experiments, we injected 2.0 ng shh-MO and 0.55 ng
twhh-MO (Fig. 1).
Phenotypic analysis
For skeletal analysis, larvae were fixed at 4-5 dpf and stained for
cartilage with Alcian Blue, after which they were dissected and flat-mounted
as described (Javidan and Schilling,
2004). In situ hybridization was performed as described previously
(Thisse et al., 1993
). Probes
used in this study were foxd3
(Odenthal and Nusslein-Volhard,
1998
), shh (Krauss et
al., 1993
), ptc1
(Concordet et al., 1996
),
sox9a (Yan et al.,
2002
) and sox10
(Dutton et al., 2001
).
Confocal imaging
For live imaging of NC, heterozygous
sox10:egfp+/ embryos (homozygous embryos showed
variable defects in NC survival), were dechorionated at 12 hpf and mounted in
0.75% agarose on bridged coverslips. To prevent drying, petroleum jelly
(Vaseline) was used to form a chamber of embryo medium containing tricaine
anaesthetic, and this was sealed with a second coverslip. Time-lapsed imaging
was performed using a Zeiss LSM 510 laser confocal microscope at 10 minute
intervals. At each time point, approximately 100-µm z-stacks at 5
µm intervals were captured. Movies of projected z-stacks, as well
as individual z-sections, were configured and individual cells
followed manually. To analyze the later distribution of egfp+ cells,
embryos were fixed with 4% paraformaldehyde (PFA) for 8 hours at 4°C,
washed several times with PBS, and embedded in 1.5% agar after manual yolk
removal. Images were captured at 2 µm intervals.
Cell labeling and fate mapping
Premigratory and postmigratory NC in sox10:egfp fish were labeled
with either the lipophilic fluorescent dye, PKH26 (Sigma), or by intracellular
microinjection of TRITC-dextran (Molecular Probes, #1817) into single cells.
The distribution of their descendants was determined in the ANC 2-3 days
later. Anesthetized embryos were mounted in 3% methylcellulose and a small
volume of PKH-solution (20% in dilution solution) was injected through a small
cut in the surface ectoderm made with a tungsten needle. On average, this
labeled five to ten cells. Injection sites were recorded and the embryos
subsequently raised until 80-85 hpf, when they were fixed in PFA for 8-12
hours, dissected and flat-mounted for observation of PKH+ cells.
Intracellular, single-cell labeling with tetramethylrhodamine-dextran
(TRITC-dextran; 10,000 MW) was performed by iontophoresis, as previously
reported (Schilling and Kimmel,
1994). Briefly, dechorionated embryos were mounted in 1.5% agar,
agar was removed adjacent to the injection site and TRITC (3% in 0.2 M KCl)
was injected iontophoretically; fluorescence was monitored continuously on a
Zeiss Axioplan2 fluorescence microscope. Injection sites were recorded within
1 hour after injection, and the embryos were incubated for another 2-3 days to
analyze cartilage.
Cyclopamine treatments
A 10 mM stock solution of cyclopamine (CyA), dissolved in 95% methanol, was
diluted in embryo medium to different concentrations between 5-100 µM.
Embryos were soaked in CyA solutions at different stages between 0-48 hpf,
washed several times in embryo medium, and either fixed at 24-36 hpf for in
situ hybridization for ptc1 or raised for another 2-3 days for Alcian
staining.
mRNA injections and bead implantation
Shh was overexpressed by injecting 0.3 ng of zebrafish shh mRNA
into embryos at the 1-2 cell stage. For misexpression experiments at later
stages, beads coated in recombinant Shh protein were implanted. CM-AffiGel
Blue beads (70-100 µm diameter, Bio-Rad) were incubated in Shh protein
(mouse recombinant, 10 µg/ml, R&D Systems) at 4°C for 1 hour. Using
a tungsten needle, a small slit was made posterior to the left eye. A
protein-coated bead was inserted, and positioned between eye and brain (see
Fig. 7C inset). Control beads
were coated in 0.1% BSA-PBS. Treated embryos were incubated and fixed for
cartilage analysis.
Cell transplantation
Wild-type donors were injected at the one- to two-cell stage with a mixture
of 3% TRITC-dextran and 3% biotinylated-dextran, and cells were transplanted
into syutq252 mutants (or
shh-MO/twhh-MO-injected embryos). Transplants were either
placed near the animal pole to target surface ectoderm, or further vegetally
to target the ventral brain in these experiments. Donors for ventral brain
transplants were injected with small amounts of Taram-A (Tar*; 0.5 ng/embryo),
which biases them toward dorsal fates, including floorplate. At 24 hpf, the
positions of fluorescent donor cells were recorded and then embryos were
raised to 4 dpf for skeletal analysis. Biotin-labeled donor cells were
detected with a Vectastain Elite ABC kit (Vectastain) and DAB as the
substrate.
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Results |
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To determine the stage at which cranial cartilage formation is disrupted in Hh-deficient embryos, we first analyzed chondrogenesis with Alcian staining. In wild types, trabecular cartilage is first detected between 45-48 hpf (Schilling and Kimmel, 1997). Two parallel, single-cell rows of chondrocytes form between the eyes (Fig. 2A), and these fuse in the midline at their anterior ends to form the ethmoidal plate by 56 hpf (Fig. 2B,C). At the same time, the trabeculae elongate rapidly and their posterior ends fuse to the basal plate of the skull (Fig. 2B-D). To examine pre-chondrogenic cells of the ANC in wild type, we analyzed expression of the Sox transcription factor sox9a during NC migration and condensation beneath the brain (Fig. 2I-L). Expression of sox9a mRNA is first detected at 24 hpf (data not shown), and by 30 hpf marks bilateral clusters of cells between the diencephalon and the eyes (Fig. 2I). These elongate by 36 hpf to prefigure the trabeculae (Fig. 2J). Expression of sox9a in ethmoid progenitors is first detected at 42 hpf, and these bridge the anterior ends of the trabecular condensations by 45 hpf (Fig. 2K).
|
Following skeletal precursors in NC with sox10:egfp
Cartilage fusions at the midline in syu mutants could be due to
abnormal NC migration or later ectopic fusions of NC streams after their
migration beneath the brain. To distinguish between these possibilities, we
followed cranial NC morphogenesis in wild-type and Hh-deficient embryos using
a newly generated transgenic line in which 4.9 kb of the sox10
promoter drives egfp expression in NC. A detailed description of the
expression patterns of this transgene in other body regions will be presented
elsewhere. Expression in the head begins at 11 hpf and, for the first time,
has allowed us to follow whole populations of cranial NC in the living embryo,
from their premigratory origins at 14 hpf
(Fig. 3A,B), and along their
migratory pathways up to 20 hpf (Fig.
3C). Expression also persists in cartilage at 48 hpf
(Fig. 3I). Time-lapsed,
confocal movies of sox10:egfp expression between 14-20 hpf revealed
that NC at midbrain levels migrates anteriorly, between the eyes, into the
position of the future ANC (Fig.
3B,C; see Movie 1 in the supplementary material). Tracking of
individual cells in these movies revealed a general tendency for NC cells
originating in more anterior positions to migrate with more anterior
trajectories (Fig. 3D). NC
cells that originate at anterior midbrain levels, tend to move along the
dorsal edge of the optic vesicle to the anterior tip of the embryo
(n=4). By contrast, NC cells that originate further posteriorly, near
the midbrain-hindbrain boundary, follow a more ventral trajectory posterior to
the eyes by 20 hpf (n=8). Through these movements, NC eventually
forms a continuous band of cells that surround the posterior and medial
portions of the eyes, the future locations of cartilages of the ANC.
|
Next, we investigated NC migration in Hh-deficient embryos using sox10:egfp. This revealed little difference from controls at early stages (n=3, data not shown). At 24 hpf, the head is slightly reduced but the pattern of egfp+ NC cells is similar to controls when viewed laterally (Fig. 3J). A ventral view, however, reveals a dramatic difference. NC cells lying anterior to the mandibular arch fuse across the ventral midline, between the eyes (Fig. 3K,L). Fusion is more pronounced at 36 hpf (Fig. 3M), and leads directly to a midline condensation that forms a single rod of cartilage (Fig. 3N). This midline structure is most likely a fusion of trabeculae, based on the relative positions of the adjacent palatoquadrate and the anterior end of the notochord in Hh-deficient embryos. These defects in Hh-deficient embryos do not appear to result from elevated NC cell death, as determined by acridine orange staining (data not shown). Taken together, these results suggest that Hh signaling is required for the morphogenetic movements that separate NC streams at the ventral midline.
Separate origins for trabecular and medial ethmoid cartilages in the NC
To confirm cartilage identities in syu mutants, we labeled
individual premigratory NC cells with fluorescent lineage tracers and tracked
their fates in the ANC at 80-85 hpf. In the initial experiments, the
lipophilic fluorescent dye, PKH26, was injected extracellularly at different
positions in the NC of sox10:egfp transgenics at 13 hpf and followed
until they formed cartilage (Fig.
4). This technique typically labeled 5-10 cells. When NC were
labeled at the anterior midbrain, just dorsal to the eye
(Fig. 4A,K; 200-300 µm from
the anterior end), cells were later found in the medial ethmoid, confined to a
triangular group of chondrocytes wedged between the anterior ends of the
trabeculae (Fig. 4B;
n=15). By contrast, cells labeled further posteriorly, by the
midbrain-hindbrain boundary (Fig.
4E,K; 300-400 µm) generated progeny confined to the trabecular
rods and lateral ethmoid, but not in the medial ethmoid
(Fig. 4F; n=15). We
confirmed these results by labeling single NC cells intracellularly with
iontophoresis of fluorescent dextrans in sox10:egfp transgenics (see
Fig. S1 in the supplementary material). Our fate map for the ANC roughly
coincides with the anterior limits of sox10 and foxd3
expression in NC (Fig. 4I,J).
These domains are changing rapidly, as one hour earlier (12 hpf) we could not
distinguish the positions of ethmoid and trabecular precursors.
|
To confirm the identity of the single midline cartilage in Hh-deficient animals, we performed similar fate mapping studies (Fig. 5A,C). Although posterior cells that normally form the trabeculae and lateral ethmoid contributed to the midline cartilage rod in Hh-deficient animals (Fig. 5D; n=5), more anterior NC cells that normally form the medial ethmoid did not form cartilage (Fig. 5B; n=5). Some labeled, undifferentiated cells were found in the tissue surrounding the cartilage, suggesting that medial ethmoid progenitors were eliminated in these embryos. Thus, the midline cartilage in Hh-deficient animals arises from the same NC population as trabecular precursors in wild type do.
Separate early and late requirements for Hh signaling
To determine the precise temporal requirements for Hh signaling in the ANC,
we blocked Hh signaling using the plant-derived steroidal alkaloid cyclopamine
(CyA). CyA directly antagonizes the Hh signal-activation component Smoothened
(Cooper et al., 1998;
Chen et al., 2002
). Embryos
were bathed in CyA at different stages between 0-48 hpf and stained for
cartilage. Siblings treated in parallel were stained by in situ hybridization
for patched 1 (ptc1) at 24-36 hpf as a control for loss of
Hh activity in NC (data not shown). Embryos treated with low concentrations (5
µM) for a 4-hour period during gastrulation between 4-8 hpf had trabecular
fusions similar to syu mutants
(Fig. 6B,C; n=30;
50%), and, at these concentrations, ptc1 expression was reduced but
not eliminated in the NC (data not shown). Higher concentrations, 50-100
µM, applied over a similar time period, effectively blocked all
ptc1 expression and prevented all cartilage formation anterior to the
basicapsular commissure (Fig.
6D). We were surprised at first to find that treatments with these
high concentrations for any 4-hour period between 0-24 hpf caused a complete
loss of the ANC (Fig. 6E),
indicating an absolute requirement for Hh signaling in ANC development during
NC migration. However, even early treatments suppress ptc1 expression
up to at least 36 hpf, indicating that suppression of Hh signaling by CyA
persists long after its removal.
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Because syu mutants lack a functional Shh protein, we also tested whether Shh-coated beads could rescue syu or shh/twhh-MO injected embryos at different stages of ANC development. Implantation of a BSA-soaked control bead had no effect at any stage (Fig. 7D). By contrast, beads soaked in 10 µg/ml Shh implanted as late as 22 hpf partially restored both head size and ANC patterning in syu (Fig. 7E; n=15). Surprisingly, despite bead placement between the eye and midbrain on one side of the head, rescue often occured bilaterally to nearly a wild-type configuration. Others showed cartilage distortions or ectopic chondrocytes along the medial trabeculae (data not shown; n=10). To determine whether the postmigratory NC distribution is restored by these late Shh applications, we analyzed ANC precursors in MO-injected sox10:egfp embryos after bead implantation. With BSA-beads, egfp+ cells were observed in the midline at 36 hpf (Fig. 7G), showing that the beads themselves did not physically disrupt NC distribution. By contrast, when a Shh-coated bead was implanted at 22 hpf, separation was restored between groups of sox10:egfp+ cells on either side of the midline in syu mutants, although a few cells remained at the midline in some cases (Fig. 7H). Overall head size was not rescued in these experiments (compare Fig. 7G and H with I), suggesting that the role of Hh signaling in midline separation of NC is not simply in growth. These results show that exogenous Hh can rescue ANC pattern in Hh-deficient zebrafish embryos by restoring the cranial NC distribution. They also suggest that the pre-chondrogenic pattern in the ANC is not specified at 22 hpf. By 30 hpf, Shh-coated beads can only partially rescue the syu phenotype (Fig. 7F), and a few hours later they have no effect.
Distinct Shh sources in the floorplate and oral ectoderm induce the ANC
Shh is a secreted protein expressed in several different cranial tissues in
the embryo, including the ventral brain, facial ectoderm and pharyngeal
endoderm. To address which is required for ANC development, we used mosaic
analysis to test the capacity of wild-type cells to rescue ANC development
when transplanted into Hh-deficient hosts. Surface ectodermal cells that form
epidermis arise from ectoderm near the animal pole of the early gastrula at 6
hpf (Kimmel et al., 1990). Cells were grafted from this location in a
wild-type donor embryo co-injected with fluorescent lineage tracers, into the
same region in syu mutants and raised to 96 hpf for cartilage
staining. These ectodermal grafts expressed shh mRNA (data not shown)
and partially rescued trabecular and ethmoid chondrogenesis
(Fig. 8A-C; n=3).
Rescue only occurred when transplanted cells were located in dorsal oral
ectoderm, just above the mouth, that comes to lie beneath the ethmoid
(Fig. 8B). To target cells to
the ventral forebrain, similar transplants were performed with ectoderm from
the dorsal side of the early gastrula near the margin at 6 hpf, where these
cells share a common lineage with floorplate cells at posterior hindbrain and
spinal levels (Hatta et al., 1991). Dye-labeled cells were grafted from this
location in wild type into the same region of syu or
shh/twhh-MO injected hosts, and stained for cartilage
(Fig. 8D-I). In some cases,
these transplants also rescued bilateral trabecular separation
(Fig. 8E; n=5).
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Discussion |
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We also show that the AP position of a premigratory NC cell beside the
midbrain predicts its fate in the skull
(Fig. 4K). By following clones
of single cells or small groups labeled with fluorescent lineage tracer dyes,
we confirmed that cells migrating along the more anterior pathway above the
eye exclusively form median ethmoid cartilage, whereas cells migrating along
the more posterior path behind the eye form the trabeculae. Our results
corroborate previous fish, amphibian and avian fate maps, showing that the
trabeculae cranii derive from mesencephalic NC
(Landacre, 1921;
Stone, 1929
;
Langille and Hall, 1988
) but
reveal an additional, more anterior, ethmoid progenitor population. Thus,
rather than simply arising as a medial growth and fusion of the trabeculae
(trabeculae communis; Fig. 9A,
top panels), as previously assumed (De
Beer, 1937
; Bertmar,
1959
), the medial ethmoid and perhaps other midline skeletal
elements have a distinct cellular origin
(Fig. 9A, bottom panels),
possibly under distinct genetic control. In support of this hypothesis, in
schmalspur (sur) mutant zebrafish, trabecular defects occur
without corresponding changes in the ethmoid plate
(Brand et al., 1996
), whereas
other mutants show defects restricted to the ethmoid
(Neuhauss et al., 1996
;
Piotrowski et al., 1996
). More
long-term fate mapping studies are required to determine whether nasal and
palatal progenitors, which differentiate between 1 and 3 weeks
postfertilization in zebrafish (Cubbage
and Mabee, 1996
), also arise from this anterior NC. These results
also have important implications for the interpretation of gene expression
patterns in the premigratory NC, which in some cases do not include the most
anterior, ethmoid domain.
We also labeled NC cells later, during migration, and followed the
contributions of cells along each pathway to cartilage in the ANC. Consistent
with our fate maps at earlier stages, we found that only cells at the anterior
end near the midline contributed to the medial ethmoid plate. The trabeculae
are formed by NC cells that lie more posteriorly, many developing in close
association with the mandibular arch. Several recent papers have highlighted
similar cells in amphibian and avian embryos, and have argued that they are a
part of the mandibular arch, blurring the distinction between maxillary and
mandibular (Cerny et al.,
2004). Our results indicate that a majority of these cells lie
anterior to the stomodeum in zebrafish, just outside of the mandibular arch.
In other respects, our fate map is similar to those for the facial prominences
in other species (McGonnell et al.,
1998
).
Hh signaling at the midline is required for both NC morphogenesis and chondrogenesis
The variety of skeletal defects caused by Hh-deficiencies remains
unexplained. For example, within the spectrum of human patients with
Hh-associated HPE, some show midline collapse of facial structures, while
others show clefting (Roessler and Muenke,
2001; Roessler and Muenke,
2003
). Our studies suggest that such phenotypic differences
reflect stage-dependent requirements for Hh signaling; and separate roles in
midline separation and chondrogenesis. We have shown that early NC migration
is unaffected in Hh-deficient embryos and that Hh acts later to prevent NC
cells from crossing the midline. Shh may induce midline tissue expansion that
acts as a physical barrier to movement
(Fig. 9B, Step 1). Shh has
anti-apoptotic activity (Charrier et al.,
2001
), and also promotes cell survival and growth in the ventral
brain (Britto et al., 2002
).
Our analysis of sox10:egfp suggests that this is not simply an affect
on growth, although we have not formally ruled out this possibility. We did
not observe elevated NC apoptosis in Hh-deficient embryos (data not shown),
which is consistent with previous reports
(Cordero et al., 2004
).
Alternatively, Shh may directly alter NC cell movements in a
concentration-dependent manner, as it has been shown to modify adhesion and NC
migration in vitro (Testaz et al.,
2001
), or may act indirectly to induce a second signal from
midline tissue(s), that acts on NC. At least some of these requirements for Hh
signaling are direct, as NC cells transplanted from smo mutants into
wild-type hosts never form cartilage (see Fig. S3 in the supplementary
material), similar to recent findings in mice with a tissue-specific loss of
Smo function in NC (Jeong et al.,
2004
). Future studies are needed to dissect how these early
patterning influences of Hh on NC morphogenesis are coupled with effects on
growth.
Two lines of evidence point to the fact that there are separate early and
late roles for Hh-signaling in skull development
(Fig. 9B, Step 2 and Step 3).
First, CyA treatment between 24-36 hpf disrupts ANC chondrogenesis, but no
longer affects NC morphogenesis at the midline. Similarly, a lack of
Hh-signaling in Shh- or Smo-mutant mice causes severe
cranial bone loss, and in some cases palatal clefting, presumably when
ethmoidal chondrogenesis is disrupted. Late CyA treatment causes similar
clefts in chick, suggesting that this late requirement is conserved
(Cordero et al., 2004). Our
results are also consistent with numerous studies in mammals implicating Hh
proteins in skeletal differentiation (reviewed by
Karsenty and Wagner, 2002
;
Kronenberg, 2003
;
Zelzer and Olsen, 2003
). In
our model, if there is sufficient signaling to separate NC at the midline, but
not for ethmoid induction, clefting may result.
Multiple Hh sources induce neurocranial chondrogenesis
Not all forms of HPE in humans are accompanied by midline facial defects,
suggesting that skeletal defects are not simply secondary consequences of
forebrain defects. Our mosaic analyses help to explain this, and suggest that
ectodermal Hh sources are also crucial for ANC formation. Gastrulation is the
only period when CyA treatment in zebrafish causes midfacial fusions, and at
these stages our data suggest that the important Hh source is the neural tube.
Wild-type ventral neural tube cells locally rescue ANC development when
grafted into the forebrain in Hh-deficient embryos, and separate the
trabeculae at the midline. However, oral ectodermal grafts can also rescue
chondrogenesis in the ANC (Fig.
8). These results are consistent with studies in chick suggesting
that Shh from the facial ectoderm promotes maxillary and frontonasal outgrowth
(Hu and Helms, 1999;
Hu et al., 2003
;
MacDonald et al., 2004
).
Ectodermal sources may act together with Shh from the ventral diencephalon and
presumptive hypothalamus at these later stages to regulate chondrogenesis.
Taken together, our results suggest that the mechanisms by which Shh
controls the coordinated growth and fusion of facial primordia are highly
conserved among vertebrates. The ventral neural tube and oral ectoderm at the
midline appear to form an organizing center controlling skeletal growth and
morphogenesis. These tissues both express shh, and we argue that
skeletogenic NC cells must interpret their positions relative to these midline
sources of shh and differentiate accordingly. Interestingly,
mutations in mice and humans that disrupt squamous epithelia also often cause
cleft lip and palate (EEC syndrome), presumably reflecting defects in these
epithelial signaling centers (Celli et al.,
1999). An interesting direction for the future would be to explore
the roles of other growth factors in the cranial ectoderm, and their
interactions with Shh in skeletogenesis. As a starting point, mutagenesis
screens in zebrafish have uncovered large phenotypic classes of mutants that
disrupt the midline, many of which disrupt signaling by the TGFß family
member Nodal (Kimmel et al.,
2001
). Zebrafish Nodal-related proteins were recently shown to
directly regulate shh expression in the zebrafish midline
(Muller et al., 2000
) and
recent evidence in humans has implicated defects in Nodal signaling in HPE
(Gripp et al., 2000
). With the
large mutant collection in zebrafish, we can now begin to study of how such
pathways interact during palatal development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/17/3977/DC1
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