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First published online 18 February 2009
doi: 10.1242/dev.032912
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1 Whitehead institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA
02102, USA.
2 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA
02139, USA.
* Author for correspondence (e-mail: sive{at}wi.mit.edu)
Accepted 27 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Primary mouth, Xenopus, Wnt, sFRP, Frzb-1, Crescent, Basement membrane, Laminin, Fibronectin
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In vertebrates, the primary mouth becomes the
opening to the pharynx as surrounding tissues form the jaws, teeth, tongue and
palate, which together constitute the secondary or adult mouth.
We previously defined morphological changes that lead to primary mouth
formation in Xenopus laevis during early tailbud and hatching stages
(Dickinson and Sive, 2006).
The earliest step identified is disappearance of the basement membrane between
the ectoderm and endoderm, which occurs at early tailbud stage. Later, during
tadpole stages, the presumptive primary mouth ectoderm undergoes invagination
to form the `stomodeum'. Subsequently, this invagination deepens, accompanied
by a burst of cell death in the ectodermal layer. Ectoderm and endodermal
layers intercalate, leading to thinning of the cell layers in the primary
mouth anlage. Finally, the thin covering (the `buccopharyngeal membrane')
perforates at swimming tadpole stage to open the primary mouth.
Three regions of the embryo are required to induce formation of the primary
mouth. These are the deep anterior endoderm, the anterior neural plate and the
lateral mesectoderm, including the neural crest
(Dickinson and Sive, 2006).
These regions are likely to secrete regulatory factors that govern primary
mouth development, but these signals and other genes involved are not known,
and their identification forms the basis of this paper.
Early during development, substantial data indicate that anterior
development in Xenopus and other vertebrates requires the inhibition
of β-catenin-mediated Wnt signaling
(Agathon et al., 2003;
De Robertis, 2006;
De Robertis et al., 2000;
Kemp et al., 2005;
Lewis et al., 2008;
Niehrs, 1999). Wnt signaling
can be inhibited by several secreted antagonists, which target the Wnt
co-receptors Frizzled and LRP6 (Semenov et
al., 2008; Yamamoto et al.,
2008). The secreted Frizzled Related Proteins (sFRPs) comprise
another class of Wnt antagonists, which contain a cysteine-rich domain with
homology to the extracellular domain of Frizzled receptors. sFRPs are believed
to bind Wnt ligands, thereby preventing their interaction with Frizzleds
(Jones and Jomary, 2002;
Kawano and Kypta, 2003). Some
sFRPs also inhibit other pathways, including BMP signaling
(Bovolenta et al., 2008;
Lee et al., 2006).
Wnt antagonists are required for anterior specification during primary axis
formation. For example, during gastrula stages of X. laevis, dkk-1
and the sFRPs frzb-1, crescent, sfrp-2 and sizzled are
expressed in the Spemann organizer and are important for formation of the head
(Glinka et al., 1998;
Niehrs et al., 2001;
De Robertis, 2006;
Jones and Jomary, 2002;
Kawano and Kypta, 2003).
Later, during Xenopus and zebrafish neurulation, Wnt antagonists are
expressed anteriorly and are required for formation of the forebrain and
placodes (Carmona-Fontaine et al.,
2007; Houart et al.,
2002). Although it is clear that inhibition of Wnt/β-catenin
signaling is important for early stages of anterior patterning, it is not
clear whether these antagonists function later during anterior organogenesis,
including formation of the primary mouth.
In order to define signaling pathways that regulate primary mouth formation, we used expression microarrays to identify genes with enriched expression in the primary mouth anlage. Through this screen, we isolated two Wnt antagonists, the sFRPs Frzb-1 and Crescent, as potential molecular regulators of primary mouth development. We show that sFRP function is crucial for primary mouth formation, and to locally promote dissolution of the basement membrane. These data are the first to connect Wnt signaling and basement membrane integrity during primary mouth development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Embryos were staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber,
1994).
Microarray analysis
Tissue was collected from three regions of the embryo at stage 25-26. (1)
The presumptive primary mouth (PMo), including endoderm and ectoderm, dorsal
to the cement gland, ventral to the telencephalon and central to the hatching
gland (Fig. 1A, PMo, red). (2)
The anterodorsal (neural) region (AD) (Fig.
1A, dark gray), comprising the central telencephalon, excluding
the eyes. (3) The ventral region including the cement gland (V+CG)
(Fig. 1A, light gray). One
hundred dissections were performed for each of three biological replicates and
stored in Trizol (Invitrogen) at -80°C. Total RNA was isolated using
Trizol extraction followed by a lithium chloride solution (Ambion)
precipitation. Total RNA (100 ng) was used to prepare biotinylated cRNA using
the Two Cycle cDNA Synthesis Kit (Affymetrix), according to the manufacturer's
protocol. Briefly, SuperScript II-directed reverse transcription used a
T7-Oligo(dT) Promoter Primer to create first strand cDNA. RNase H-mediated
second strand cDNA synthesis was followed by MEGAscript T7 (Ambion) directed
in vitro transcription, which generated unmodified cRNA. cRNA was used as a
template for a second round of cDNA synthesis, followed by a second in vitro
transcription reaction, which incorporated a biotinylated nucleotide analog
during cRNA amplification. Samples were prepared for hybridization using 15
µg biotinylated cRNA in a 1xhybridization cocktail. Additional
hybridization cocktail components were provided in the Affymetrix GeneChip
Hybridization, Wash and Stain Kit. GeneChip arrays (Xenopus) were
hybridized in a GeneChip Hybridization Oven at 45°C for 16 hours at 60
rotations per minute. Washing was done using a GeneChip Fluidics Station 450
according to the manufacturer's instructions, using the buffers provided in
the Affymetrix GeneChip Hybridization, Wash and Stain Kit. Arrays were scanned
on a GeneChip Scanner 3000 and images were extracted and analyzed using
GeneChip Operating Software v1.4. The generated CHP and CEL files have been
deposited in the Gene Expression Omnibus (NCBI, GSE13377). Expression level
differences and statistical significance were calculated using Excel, and both
were considered in identifying candidate genes.
qRT-PCR
cDNA was prepared using the Sensiscript Kit (Qiagen). qRT-PCR was performed
using ABI Prism 7000 or 7900 (ABI). Fluorescence detection chemistry used the
SYBR green dye master mix (Roche). Primers sequences are available on request.
The relative amount of product was calculated using 
CT and products
normalized to ef1-alpha.
In situ hybridization
cDNAs used to transcribe in situ hybridization probes were frzb-1
[GA# U68059 (Wang et al.,
1997a; Wang et al.,
1997b)], crescent (also called frzb-2)
(Bradley et al., 2000),
XCG (Sive et al.,
1989) and nrp-1 [GA#, BC084198
(Richter et al., 1990)]. In
situ hybridization was performed as described by Sive et al.
(Sive et al., 2000), omitting
the proteinase K treatment. Double-staining analysis was performed as
described previously (Wiellette and Sive,
2003).
Morpholinos and RNA injections
Antisense morpholinos were purchased from Gene Tools. To design
frzb-1 morpholinos, we sequenced the start site of frzb-1 in
X. laevis (primer sequences are available on request). Morpholinos
included two frzb-1 start site morpholinos (morpholino sequences are
available on request), a previously published splice blocking crescent
morpholino (Shibata et al.,
2005) and a standard control morpholino. For rescue assays, 10
mutations were introduced into the start site of the frzb-1 cDNA,
using a site-directed mutagenesis kit (Stratagene). One nanogram per embryo of
the mutated frzb-1 mRNA produced an overexpression phenotype
comparable to the wild-type construct, as described
(Wang et al., 1997a).
F0 transgenics and heat shock
For heat-shock inducible transgenics, we used a construct [pSGH2;
ISceI-GFP-HSE plasmid (Bajoghli et al.,
2004)] with a multimerized heat-shock element (HSE) promoter, a
multiple cloning site and I-SceI sites. The genes dkk-1 [GA# AF030434
(Glinka et al., 1998)],
wnt-8b (GA# U22173 (Christian et
al., 1991)], chordin [GA#, L35764
(Sasai et al., 1994)],
frzb-1 and crescent (see above) were inserted into the
multiple cloning site (GFP::HSE::gene of interest). The meganuclease method
was used to create F0 transgenics as previously described
(Pan et al., 2006). Heat shock
was achieved by moving embryos from 15°C to 35-37°C at the appropriate
stage, and maintaining embryos at this temperature for 2 hours. The construct
minus inserted genes served as a control.
Laminin immunohistochemistry and auto-fluorescent rendering
Specimens were embedded in 4% low-melt agarose (SeaPlaque GTG, Cambrex) and
sectioned with a 1000 Series Vibratome at 100 µm. Immunohistochemistry was
performed as described (Dickinson and
Sive, 2006) using a polyclonal anti-laminin antibody (Sigma,
L-9393) diluted 1:150, a goat anti-rabbit Alexa Fluor-conjugated antibody
(Molecular Probes) diluted 1:500 and a 0.1% propidium iodide (Sigma)
counterstain. Embryos were prepared for optical auto-fluorescent sections as
described (Dickinson and Sive,
2006).
Extirpations and transplants
Extirpations and transplants were performed in 1.0xMBS (Modified
Barth's Solution) in plasticine-lined Petri dishes, using 1-mm diameter
capillary tubes pulled to a fine point. Glass bridges were used to hold tissue
together while healing. Two types of transplants were performed. (1) `Face
transplants', which involve dissecting the ectodermal and endodermal layers of
the cement gland and the region just dorsal, which fate maps to the
presumptive primary mouth, and transplanting this entire region to a donor
embryo in which the same tissue has been removed. (2) `Ectoderm transplants',
which involve removing the ectoderm, both superficial and deep, from the
presumptive primary mouth and transplanting to a donor embryo in which the
same tissue has been removed.
JNK inhibition
Embryos were bathed in a 20 µM solution of SP600125 (Sigma) with 1% DMSO
in 0.1% MBS from stage 17 to stage 40 in culture dishes at room
temperature.
β-Catenin protein levels and activity
β-Catenin protein levels were analyzed by western blotting. Tissue
lysates were prepared from pooled samples of 10 embyros. Primary antibodies
were anti-β-catenin (Zymed, 71-2700) and anti-β-actin (Sigma,
A5441), both diluted 1:1000. Secondary antibodies were HRP-conjugated
anti-mouse or anti-rabbit IgG (Cell Signaling), and detection was by
chemiluminescence using LumniGLO Reagent and Peroxide (Cell Signaling). The
bands were quantified by densitometry using Photoshop (Adobe).
β-catenin activity was measured using the TOPFLASH system. Briefly,
embryos were injected with 10 pg of the TOPFLASH construct
(Clevers and van de Wetering,
1997), 10 pg pRL-SV40/TK as a reference plasmid, and 1 ng of
frzb-1 RNA. Luciferase assays were performed using the
Dual-Luciferase Assay Kit (Promega). The primary mouth and surrounding area
were dissected at stage 20-22, suspended in 50 µl of 1xPassive Lysis
Buffer and stored at -80°C. Luciferase was detected on a luminometer
(Molecular Devices). All values were expressed as relative luciferase units
(Firefly luciferase activity/Renilla luciferase activity) and scaled and
plotted with Microsoft Excel.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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In situ hybridization showed that frzb-1 was expressed in the prechordal plate, presumptive anterior pituitary and primary mouth, as well as in the endoderm lying beneath the cement gland, at neurula stages (stage 17-20; Fig. 2A,A'). At early tailbud stages (stage 24 and 26), expression appeared strong in the deep ectoderm of the future anterior pituitary and primary mouth, while fainter expression persisted in underlying endoderm (Fig. 2B-C'). At stage 28, frzb-1 mRNA was present in the developing anterior pituitary (Fig. 2D,D'). By tadpole, stage 32, expression appeared to be absent in the head (data not shown). Increased probe concentrations and longer incubation times revealed a low level of frzb-1 expression in the brain (data not shown), consistent with expression values from the microarray analysis.
Frzb-1 belongs to a large class of Wnt antagonists, the secreted frizzled related proteins (sFRPs). Because our microarray screen consisted of only one time point, and primary mouth formation takes place over many hours, we examined the expression of other sFRPs, including sFRP1, sFRP2, sFRP5 and crescent (see Fig. S2A-L in the supplementary material), to detect redundantly expressed genes. Only crescent was expressed in or near the future primary mouth (Fig. 2E-H'). At stage 17, crescent was expressed in a broader domain than was frzb-1, including the prechordal plate, the presumptive anterior pituitary and primary mouth, and in tissue lying beneath the cement gland (Fig. 2E,E'). At stages 20-24, crescent expression was primarily confined to the prechordal plate, posterior and anterior to the presumptive primary mouth and the anterior pituitary (Fig. 2F,F'). By stage 26, crescent mRNA was no longer detected in the head region (Fig. 2E-H'), although microarray analysis showed very low levels of crescent expression in the future primary mouth at stage 26 (data not shown).
These data indicate that during early neurula stages, crescent and frzb-1 have overlapping expression patterns in the presumptive primary mouth, consistent with a role in the development of this organ.
Frzb-1 and Crescent are required for primary mouth formation
We investigated whether Frzb-1/Crescent function was necessary for primary
mouth formation, and whether the function of these sFRPs was redundant, using
antisense morpholinos against frzb-1 and/or crescent, as
detailed in the Materials and methods. Injection of frzb-1
morpholinos alone resulted in embryos with a small stomodeum (invagination,
dotted yellow) but no primary mouth opening, whereas the same amount of
control morpholino had no effect on the primary mouth opening (dotted black;
Fig. 3A, parts a,b). The
injection of crescent morpholinos resulted in a smaller primary mouth
opening relative to controls (Fig.
3A, part c). However, co-injection of frzb-1 and
crescent morpholinos resulted in morphants with neither stomodeum nor
primary mouth opening (Fig. 3A,
part d). The specificity of the phenotype was confirmed by rescue with low
levels (200 pg) of mutated frzb-1 mRNA that does not hybridize to the
morpholino (see Materials and methods). When injected into control embryos,
this amount of frzb-1 mRNA did not alter primary mouth morphology
(3A, parts e,f). Ninety-three percent of embryos injected with frzb-1
and crescent morpholinos together with the control GFP mRNA had
neither a stomodeum nor a primary mouth opening
(Fig. 3A, part g). This
phenotype was rescued in 86% of morphant embryos injected with frzb-1
mRNA (Fig. 3A, part h).
Although these embryos were not completely normal, possibly because of the
effects of crescent loss of function
(Shibata et al., 2005), the
primary mouth opening was relatively normal in size. crescent mRNA
alone was not able to rescue the primary mouth defect (data not shown), and
gave a cyclopic phenotype that resulted from effects on prechordal plate
migration (Pera and De Robertis,
2000).
We did not observe abnormal levels of cell death or proliferation in the
primary mouth region of morphants at stage 23-24 (see Fig. S3B, parts a-d in
the supplementary material). Importantly, despite the absence of the primary
mouth at stage 40, this region was correctly specified at stage 23-24, as
indicated by the expression of two markers specifically expressed in the
primary mouth anlage, pitx3 and vgl-2
(Fig. 3B, parts a-d).
Furthermore, the morphology of the morphants appeared to be relatively normal
at stage 23-24, with structures around the future primary mouth present,
including the eyes and cement gland (see Fig. S3A, part a in the supplementary
material). At later times, frzb-1/crescent morphants show a phenotype
consistent with a function in primary axis formation
(Bradley et al., 2000;
Pera and De Robertis, 2000)
(see Fig. S3A, parts b-h in the supplementary material). In agreement with a
requirement for Frzb-1/Crescent, removal of the frzb-1 expression
domain at early tailbud by extirpation resulted in neither a stomodeum nor a
primary mouth (Fig. S4 in the supplementary material).
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A specific requirement for Frzb-1 and Crescent in the primary mouth region during tailbud stages
Because frzb-1/crescent morphants showed a whole embryo phenotype,
we investigated whether gene function is required in the forming primary
mouth, or whether the primary mouth phenotype is secondary to earlier defects.
To answer this, we performed face transplants to localize morphant tissue
specifically to the future primary mouth during early tailbud stages (stage
23-24; see Fig. 3B, part a, and
3C, part a; see also Materials and methods). In the first transplant, donor
tissue originated from morpholino-injected embryos, while recipients were
uninjected sibling embryos (Fig.
3B, part a). Using morphant donor tissue, 83% of embryos did not
form a stomodeum or a primary mouth opening and 17% had a smaller stomodeum
and no opening; controls all had normal primary mouth morphology
(Fig. 3B, parts b-c'). We
note that localized loss of Frzb-1/Crescent results in a smaller surrounding
face, suggesting that this region may organize other aspects of face
development.
In the second transplant, donor tissue originated from uninjected embryos and the recipients were embryos injected with frzb-1 and crescent or control morpholinos (Fig. 3C, part d). All of the controls and 80% of the morphant recipients had a primary mouth opening, albeit of variable size and shape (Fig. 3B, parts e-f').
These results indicate that Frzb-1 and Crescent are necessary locally in tissue that will form the primary mouth, from the time this organ begins to develop.
Wnt overexpression and loss of Frzb-1/Crescent give similar phenotypes
Previous studies have shown that both frzb-1 and crescent
can antagonize Wnt-8 (Bradley et al.,
2000; Leyns et al.,
1997; Schneider and Mercola,
2001; Wang et al.,
1997a). However, sFRPs can also interact with other signaling
pathways, such as BMP (reviewed by
Bovolenta et al., 2008). In
order to determine whether the Wnt signaling pathway is targeted during
primary mouth formation, we investigated whether modulators of this pathway
could phenocopy the effects of changing Frzb-1/Crescent expression.
First, we tested whether an increase in Wnt signaling would phenocopy the effects of frzb-1/crescent morpholinos. This would be predicted if these sFRPs targeted Wnt signaling (Fig. 4A). Because overexpression of wnt-8 during early development has profound effects on axial patterning, we restricted wnt-8 expression to neurula and tailbud stages by driving the expression of this gene in transgenic embryos under the control of a heat-shock element (HSE) promoter (GFP::HSE::wnt-8). Two periods of heat shock were administered to determine whether the effects of Wnt-8 overexpression correlated with the expression of frzb-1 and crescent in the primary mouth anlage. Maximal overexpression was expected at the end of the 2-hour heat shock. Overexpression of wnt-8, during the time of frzb-1 and crescent expression (stage 17-24), resulted in embryos with a reduced head, and neither a stomodeum nor a primary mouth opening (Fig. 4B,C). This phenotype resembled that observed with loss of Frzb-1/Crescent function in the whole embryo (see Fig. 3A-D). When wnt-8 was overexpressed later (stage 25-28), after frzb-1 and crescent expression is downregulated in the primary mouth anlage, a normal or slightly smaller primary mouth opening formed (Fig. 4D).
Because Wnt-8 has many functions in the whole embryo, we examined the effect of overexpression exclusively in the presumptive primary mouth by performing `ectoderm transplants'. These transplants involved a smaller region than the face transplants described (see Materials and methods) to limit the cells exposed to Wnt-8 (Fig. 4E). Expression was controlled temporally using the HSE promoter. Donor embryos were heat shocked at stage 17-24 or stage 25-28, and were transplanted into host embryos at stage 24 or stage 28, respectively. In recipient embryos, where heat shock was administered at stage 17, and Wnt-8 expressing tissue was transplanted at stage 24, neither a stomodeum nor a primary mouth opening formed (Fig. 4F,G). The timing of this experiment correlates with the time of frzb-1/crescent expression in the presumptive primary mouth. When heat shock was administered later (stage 24) and wnt-8 expressing tissue was transplanted at stage 28 (when frzb-1 and crescent are no longer expressed in the future primary mouth) a normal opening formed (Fig. 4H).
These data show that temporally and spatially restricted Wnt-8
overexpression phenocopies loss of Frzb-1/Crescent function, suggesting that
these sFRPs target the Wnt pathway. Because Wnt-8 predominantly activates
β-catenin-mediated signaling (Darken
and Wilson, 2001), these data suggest a role for Frzb-1 and
Crescent in modulating the Wnt/β-catenin pathway.
Frzb-1 and Crescent may inhibit the function of one or several Wnt proteins during primary mouth formation. Data gathered in our microarray screen indicate that the expression of wnt-8, wnt-8b, wnt-3a, wnt-2 and wnt-4 is lower in the primary mouth than in the surrounding regions (see Fig. S1 in the supplementary material). This suggests that other mechanisms may downregulate Wnt gene expression at the RNA level.
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), phenocopies the effects of Frzb-1 or Crescent
overexpression. Using a heat-shock element (HSE) promoter, we overexpressed
frzb-1 (GFP::HSE::frzb-1) during late neurula and early tailbud
stages (Fig. 5A, part a) in
transgenic embryos. This resulted in a very large stomodeum
(Fig. 5A, part b-d'),
indicating that Frzb-1 is sufficient to expand the size of the primary mouth,
consistent with the mRNA overexpression effects
(Pera and De Robertis, 2000).
A similar, yet less profound, phenotype is observed when Crescent is
overexpressed at neurula and tailbud stages (data not shown). Overexpressing
Dkk-1 under a heat-shock element promoter (GFP::HSE::dkk-1) in transgenic
embryos, during neurula and tailbud stages, to prevent an early patterning
phenotype (Glinka et al.,
1998) mimics Frzb-1 overexpression
(Fig. 5A, part c). These
results further suggest that Frzb-1/Crescent function to inhibit Wnt
signaling, similar to Dkk-1. Another prediction is that Dkk-1 overexpression would rescue the frzb-1/crescent morphant phenotype. We tested this by injecting dkk-1 or GFP mRNA together with frzb-1/crescent morpholinos and performing face transplants (Fig. 5B, part a) to localize the control or morphant tissue. Ninety percent of embryos receiving donor tissue containing the morpholinos and control GFP mRNA form neither a stomodeum nor a primary mouth, and 10% form a small stomodeum and no opening (Fig. 5B, parts b,b'). Using donor tissue containing the morpholinos and dkk-1 mRNA, 66% of embryos form a primary mouth opening, while 33% of the embryos have a large stomodeum, but no opening (Fig. 5B, parts c,c'). Other controls, including donor tissue containing dkk-1 mRNA plus control morpholinos, or GFP mRNA plus control morpholinos, almost always formed a primary mouth opening (75% and 100%, respectively; data not shown). These results show that overexpression of dkk-1 mRNA can restore primary mouth formation to frzb-1/crescent morphants.
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),
although recent evidence suggests that this protein can also regulate
non-canonical Wnt signaling (Cha et al.,
2008).
Frzb-1/Crescent do not target the JNK or BMP signaling pathways
sFRPs can antagonize Wnt signaling mediated by both β-catenin and
c-Jun NH2-terminal kinase [JNK; planar cell polarity (PCP)], as
well as the BMP pathway (reviewed by
Bovolenta et al., 2008). We
therefore investigated whether perturbation of the Wnt/PCP and BMP pathways
gave a similar primary mouth phenotype to that caused by Frzb-1
overexpression.
Both Frzb-1 and Crescent can inhibit Wnt/PCP signaling
(Qian et al., 2007;
Shibata et al., 2005) by
activation of the JNK pathway. If Frzb-1 functions to inhibit Wnt/PCP
signaling in the primary mouth, inhibition of JNK would phenocopy the Frzb-1
overexpression phenotype (Fig.
6A). However, treatment of late neurula stage embryos (stage 17)
with a JNK chemical inhibitor, SP600125
(Han et al., 2001), led to an
absence of a stomodeum and a primary mouth opening, which was opposite to the
Frzb-1 gain-of-function phenotype (Fig.
6B,C). Thus, although the Wnt/PCP pathway seems to be important
for primary mouth formation, Frzb-1 probably does not regulate this pathway.
In addition to acting through the JNK pathway, non-canonical Wnt signaling can
also use other signaling pathways, including those involving calcium and Src
(van Amerongen et al., 2008).
It is possible that Frzb-1 and Crescent can target these other pathways during
primary mouth formation.
The sFRP Sizzled inhibits the Xolloid-like protease, which is essential for
BMP regulation (Lee et al.,
2006). If Frzb-1 acts similarly and inhibits Xolloid-like in the
primary mouth anlage, then overexpression of the BMP inhibitor Chordin should
phenocopy the Frzb-1 overexpression phenotype
(Fig. 6D). However, contrary to
this prediction, overexpression of Chordin under the HSE promoter
(GFP::HSE::chrd) in transgenic embryos led to a very small stomodeum and no
opening (Fig. 6E,F). A similar
phenotype was observed by overexpression of a dominant-negative BMP receptor
during neurula stages (data not shown). Therefore, although the BMP signaling
pathway is likely to be important for primary mouth development, it does not
appear to be a target of Frzb-1 and Crescent.
Frzb-1 is likely to be the major sFRP regulating primary mouth formation owing to the higher level and longer period of expression of the mRNA in the presumptive primary mouth. Therefore, we tested whether increased frzb-1 mRNA could decrease Wnt signaling in the primary mouth region by using β-catenin protein and activity as a readout. We found that overexpression of Frzb-1 significantly decreased β-catenin protein and activity levels in the presumptive primary mouth and flanking tissues (see Fig. S5 in the supplementary material). Furthermore, the microarray screen reveals that β-catenin, and Wnt ligands associated with the Wnt/β-catenin pathway, are expressed at lower levels in the presumptive primary mouth than in surrounding regions (see Fig. S1C in the supplementary material).
Together, these data suggest that Frzb-1 and Crescent primarily act to inhibit β-catenin-mediated Wnt signaling, rather than the Wnt/PCP or BMP pathways, during primary mouth formation.
Loss of Frzb-1 and Crescent, or increased Wnt-8 expression, results in a persistent basement membrane
One of the earliest morphological changes during primary mouth formation is
loss of the basement membrane (Dickinson
and Sive, 2006) between ectoderm and endoderm when frzb-1
and crescent are expressed. We therefore hypothesized that
Frzb-1/Crescent regulate basement membrane dissolution. Consistent with this,
in frzb-1/crescent morphants, expression of Laminin, a
basement membrane protein, persisted in the presumptive primary mouth, whereas
control embryos lacked Laminin in this region
(Fig. 7A, parts a-b').
This phenotype was specific, as co-injecting a small amount of frzb-1
mRNA restored the loss of Laminin staining to 70% of the morphants
(Fig. 7A, parts d,d').
This level of frzb-1 mRNA did not alter primary mouth morphology (not
shown), nor normal basement membrane loss
(Fig. 7A, parts
c,c').
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We further investigated whether Wnt signaling can regulate basement membrane breakdown by performing ectoderm transplants (Fig. 7C, part a; see also Materials and methods), where a small piece of presumptive primary mouth ectoderm was transplanted from donor embryos transgenic for wnt-8::HSE::GFP to uninjected recipient embryos (Fig. 8C, part a). With donor tissue overexpressing Wnt-8, Laminin immunoreactivity persisted in the primary mouth region compared with controls (Fig. 7C, parts b-c'). Thus, dissolution of the basement membrane in the presumptive primary mouth region requires the inhibition of Wnt signaling.
We next tested whether Wnt signaling modulates basement membrane proteins
at the level of RNA expression (Fig.
7D). Specifically, we expressed frzb-1 mRNA or a
wnt-8 construct under the control of the HSE promoter and isolated
the presumptive primary mouth region at early tailbud (stage 20-22), prior to
basement membrane dissolution. Increased frzb-1 mRNA resulted in
fibronectin and laminin-
1 mRNA levels that
were approximately 50% of the control levels, with little effect on
integrin-β1 mRNA levels
(Fig. 7D, part a). Conversely,
overexpression of Wnt-8 from mid-neurula (stage 17) to harvest, resulted in
fibronectin mRNA levels that were 232% of the control level, and
laminin mRNA levels that were 170% of the control level, while having
little effect on integrin-β1 mRNA levels
(Fig. 7D, part b). These
results indicate that Frzb-1 can regulate basement membrane dissolution by
downregulating laminin and fibronectin mRNA expression.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The present study identifies a fourth domain, which
expresses the sFRPs frzb-1 and, to a lesser extent,
crescent. Removal of this domain ablates primary mouth formation, and
antisense assays show that this is due to loss of Frzb-1 and Crescent
expression, rather than because the domain contains the future primary mouth.
In Xenopus, this domain expresses both frzb-1 and other
secreted Wnt inhibitors, including dkk-1 and wnt inhibitory
factor-1 (wif-1), suggesting that there is a requirement for low
Wnt function in this region (Glinka et
al., 1998; Hsieh et al.,
1999) (see Fig. S1 in the supplementary material). Interestingly,
when frzb-1/crescent expression was inhibited locally in the future
primary mouth region, the entire face was smaller than controls, suggesting
that the frzb-1/crescent domain may organize other aspects of the
face.
|
;
De Robertis, 2006;
De Robertis et al., 2000;
Kemp et al., 2005;
Lewis et al., 2008;
Niehrs, 1999). Our data
indicate that local inhibition of Wnt signaling is also crucial to primary
mouth formation at the extreme anterior of the embryo. Gene expression
analyses indicate that a primary anteroposterior axis is in place by the end
of gastrulation (Gamse and Sive,
2001; Zaraisky et al.,
1995). Primary mouth specification begins many hours later, by
late neurula/early tailbud, when a discrete region can be fate mapped as the
future primary mouth, when region-specific gene expression is activated, and
when the basement membrane between the ectoderm and endoderm breaks down
locally (Dickinson and Sive,
2006). Our data demonstrate that inhibition of Wnt signaling is
required for anterior organogenesis after the primary anteroposterior pattern
is formed. It is not clear whether this represents a sustained requirement for
inhibition of Wnt signaling to maintain `anteriorness', or a new period that
involves more local inhibition of this signaling pathway.
Frzb-1/Crescent regulate β-catenin-mediated Wnt signaling
Several assays indicate that Frzb-1 and Crescent antagonize Wnt signaling
in the primary mouth anlage. For example, overexpression of Dkk-1, a Wnt
inhibitor, gives a similar phenotype to overexpression of Frzb-1/Crescent, and
can substitute for these sFRPs in regulating primary mouth formation. Our data
suggest that these sFRPs target β-catenin-mediated Wnt signaling. Thus,
overexpression of Wnt-8, a known Wnt/β-catenin ligand, mimics the
phenotype seen after loss of Frzb-1/Crescent, whereas Frzb-1 overexpression
decreases β-catenin levels in the presumptive primary mouth (see Fig. S5
in the supplementary material). Further indication that Frzb-1/Crescent
targets Wnt/β-catenin signaling in the primary mouth region comes from
the examination of putative promoters for the basement membrane genes
laminin and fibronectin, whose expression is inhibited by
Frzb-1/Crescent. In the X. tropicalis genome, one
TCF/LEF/β-catenin-binding site lies within 5 kb upstream of each of the
fibronectin and the laminin start sites (data obtained using
Transfac software; not shown). These data suggest that Wnt signaling regulates
the transcription of basement membrane genes, via the modulation of
β-catenin.
Frzb-1/Crescent do not appear to target either the Wnt/PCP pathway, or the BMP pathway, as neither BMP nor JNK inhibition could mimic the Frzb-1 overexpression phenotype. However, the inhibition of BMP and JNK results in a very small or absent primary mouth, suggesting that both pathways regulate other aspects of primary mouth development.
Basement membrane dynamics and Wnt signaling
Basement membrane dissolution is important in many developmental contexts
and may be necessary for invagination, cell death and intercalation
(Davidson et al., 2004;
Ingber, 2006;
Svoboda and O'Shea, 1987). For
example, during cavitation of embryoid bodies, loss of contact with the
basement membrane initiates cell death
(Murray and Edgar, 2000).
Basement membrane breakdown is required for changes in cell polarity and
movement during chick gastrulation (Nakaya
et al., 2008). Basement membrane dissolution may promote
subsequent steps in primary mouth formation; however, this possibility has not
yet been addressed.
This study provides the first connection between Wnt signaling and basement
membrane modulation during primary mouth development in any species. This
connection has not been made extensively in any embryonic context. In organ
culture of mouse lung, addition of Dkk-1 protein resulted in depressed levels
of Fibronectin protein and aberrant lung branching
(De Langhe et al., 2005). In
addition to in Xenopus, sFRPs and other Wnt inhibitors are expressed
in anterior domains at a time that could influence primary mouth development
in zebrafish, chick and mouse (Chapman et
al., 2004; Duprez et al.,
1999; Hoang et al.,
1998; Houart et al.,
2002; Tylzanowski et al.,
2004).
In cancer, basement membrane breakdown is pivotal to metastasis
(Spaderna et al., 2006), and
connections have been made between Wnt signaling and the expression of
basement membrane components. For example, β-catenin controls the
expression of laminin during tumor progression
(Hlubek et al., 2001), and
increased frzb-1 (sFRP3) expression correlates with
decreased Fibronectin protein (Guo et al.,
2008).
It is not clear whether inhibition of Wnt signaling is the sole mediator of
basement membrane dissolution in the primary mouth region. Other mechanisms
downstream of Frzb1/Crescent, or in an independent pathway, may contribute.
These could include proteolytic degradation of extracellular matrix components
by metalloproteases (Page-McCaw et al.,
2007), and we are presently investigating this possibility.
Model: local repression of Wnt signaling leads to basement membrane dissolution in the primary mouth anlage
Previous data have shown that loss of the basement membrane in the primary
mouth region is a very early step in the formation of this organ
(Dickinson and Sive, 2006).
Our data identify a molecular mechanism that locally regulates basement
membrane dissolution (Fig. 8).
Specifically, the Wnt antagonists Frzb-1 and Crescent act redundantly to
inhibit β-catenin activity in cells adjacent to the basement membrane in
the primary mouth anlage. We suggest that the transcription of pivotal
basement membrane genes, including laminin and fibronectin,
is dependent on β-catenin, and therefore decreases after Wnt inhibition.
After synthesis of basement membrane proteins ceases, the basement membrane
breaks down. Without Wnt inhibition, the basement membrane is maintained. Loss
of the basement membrane may be required for subsequent steps in primary mouth
development, including invagination, cell death, intercalation and
perforation. However, these putative connections have not been explored. In
addition to the local inhibition of Wnt signaling, other mechanisms may
regulate the fine spatial control of basement membrane dissolution. We suggest
that the modulation of Wnt signaling is a widespread regulator of basement
membrane remodeling during development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1071/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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