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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02708
1 Institute of Biotechnology, Developmental Biology Program, University of
Helsinki, 00014 Helsinki, Finland.
2 Department of Biochemistry, University of Lausanne, 1066 Epalinges,
Switzerland.
* Authors for correspondence (e-mail: irma.thesleff{at}helsinki.fi; marja.mikkola{at}helsinki.fi)
Accepted 20 October 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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B (NF-B), but
the transcriptional targets of Edar have remained unknown. Using a
quantitative approach, we show in cultured embryonic skin that Eda induced the
expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective
tissue growth factor) and follistatin. Moreover, our data indicate that Shh is
a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh
was unable to rescue primary hair placode formation in Eda-deficient
skin explants.
Key words: Ccn2, Ectodermal dysplasia, Lateral inhibition, NF-B, Tabby, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Typically, placodes develop sequentially in specific patterns, such as teeth
at precise locations along the dental lamina or hairs and feathers at regular
intervals within the integument. Individual organ primordia need signals for
their initiation, expansion and termination. It is apparent that both positive
and negative regulators of placodal fate are involved in these processes, and
a reaction-diffusion model has been set forth to explain the establishment of
periodic patterning of hair and feather buds
(Oro and Scott, 1998;
Jung and Chuong, 1998;
Jiang et al., 1999). Many
signalling molecules (and their inhibitors), including Wnts, fibroblast growth
factors (Fgfs), transforming growth factor-ßs (Tgf-ßs) and sonic
hedgehog (Shh), are expressed in the placodes or by the underlying condensed
mesenchyme (Pispa and Thesleff,
2003; Schmidt-Ullrich and
Paus, 2005; Mikkola and
Millar, 2006). Wnts and Fgfs are wellknown promoters of placodal
cell fate (Gat et al., 1998;
Jung et al., 1998;
Noramly et al., 1999;
Huelsken et al., 2001;
Andl et al., 2002), whereas
bone morphogenetic proteins (Bmps) of the Tgf-ß superfamily are generally
regarded as placode inhibitors (Jung et
al., 1998; Noramly and Morgan,
1998; Botchkarev et al.,
1999).
Ectodysplasin-A (Eda), a member of the tumour necrosis factor (Tnf)
superfamily is an early and necessary signal required for placode formation
(for reviews, see Thesleff and Mikkola,
2002; Mikkola and Thesleff,
2003). Recent studies have indicated that the Eda pathway is
downstream of the primary inductive signal required for placode initiation,
yet lies high in the hierarchy of molecules positively regulating placodal
cell fate (Mustonen et al.,
2004). Although dental, mammary and secondary hair placodes form
normally in Eda-deficient mice (Tabby mice)
(Pispa et al., 1999;
Laurikkala et al., 2002;
Kangas et al., 2004) (M.P. and
M.L.M., unpublished), mice carrying mutations in any of the necessary
components of the Eda signalling pathway lack primary hair placodes giving
rise to guard hairs (Mikkola and Thesleff,
2003). In humans, mutations in the Eda pathway genes
cause hypohidrotic ectodermal dysplasia syndrome featured by missing or
malformed teeth, sparse hair and the absence of a number of exocrine
glands.
Many of the details in the Eda signalling pathway have been uncovered
during recent years. Studies with cultured cells transfected with wild-type or
mutant Edar, the receptor for the Eda-A1 isoform of ectodysplasin, have
suggested that activation of the transcription factor NF-B is crucial
for Eda signalling (Yan et al.,
2000; Koppinen et al.,
2001; Kumar et al.,
2001). In addition, phenotypic analyses of mice and humans with
compromised NF-B responses indicate that Edar signalling is mediated
for most part, if not totally, by the I-B kinase (Ikk)-dependent
canonical NF-B pathway in vivo
(Schmidt-Ullrich et al., 2001;
Puel et al., 2004). Recently,
NF-B reporter activity in primary hair placodes was shown to be
dependent on Eda (Schmidt-Ullrich et al.,
2006) in line with our own observations (M.L.M., unpublished).
Thus far, the direct downstream target genes regulated by Edar have not been
found.
Overexpression of Eda-A1 in developing epidermis results in supernumerary
tooth and mammary placodes, which develop into mature organs. Moreover, Eda-A1
transgenic embryos are characterized by increased placodal size, and treatment
of embryonic skin with recombinant Eda-A1 in vitro promotes placodal cell fate
in a dose-dependent manner (Mustonen et
al., 2004). The effects of Eda-A1 are highly similar to those
brought about by the best-known positive regulators of placode formation such
as noggin, a potent inhibitor of Bmps
(Noramly and Morgan, 1998;
Botchkarev et al., 1999).
Interestingly, the consequences of the ablation of noggin and Eda are
converse to each other in terms of hair placode formation: primary hair
follicle formation is dependent on Eda, whereas secondary hair follicles
require noggin for initiation (Botchkarev
et al., 2002).
Eda signalling also influences later stages of ectodermal organ
development. Absence of Eda leads to an obvious molar cusp patterning
defect associated with a smaller enamel knot, an epithelial signalling centre
regulating tooth shape (Pispa et al.,
1999). Forced expression of Eda-A1 or Edar results in a lack of
enamel in incisors, which is associated with the absence of ameloblasts, the
epithelial cells producing the enamel matrix
(Mustonen et al., 2003;
Pispa et al., 2004;
Tucker et al., 2004). A
similar phenotype was recently reported in mice overexpressing follistatin or
noggin (Wang et al., 2004a;
Plikus et al., 2005). These
findings together with the similar effects of Eda-A1 and noggin on placode
formation prompted us to test whether Edar activity could counteract Bmp
signalling.
In this study, we provide evidence that recombinant Eda antagonizes the
activity of Bmp4 in developing incisors and provide evidence indicating that
suppression of Bmp activity is compromised in Eda-deficient skin. By
using a quantitative approach, we found that the expression of Ccn2/Ctgf (CCN
family protein 2/connective tissue growth factor), a multifunctional secreted
protein (Perbal, 2004) known
to antagonize Bmp4 activity (Abreu et al.,
2002) was strongly induced by Eda-A1 in cultured embryonic skin.
The expression pattern of Ccn2 correlated with that of Edar
in nascent hair and tooth placodes. In addition, follistatin was moderately
upregulated by Eda-A1. Finally, we show that Shh was strongly induced
by Eda-A1 in developing skin, but, unlike noggin, recombinant Shh did not
rescue hair placode formation in Eda-null skin.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Organ cultures
Wild-type E15 incisors were dissected in Dulbecco's PBS pH 7.4 under a
stereomicroscope. Embryonic tooth explants were grown on nuclepore filters at
37°C for 24 hours in a Trowell-type culture containing Dulbecco's minimum
essential medium (DMEM) supplemented with 10% fetal calf serum (FCS),
glutamine and penicillin-streptomycin. Affi-Gel agarose beads (BioRad) were
soaked in bovine serum albumin (BSA, 1 µg/µl, Sigma) or in recombinant,
purified Fc-Eda-A1 protein (250 ng/µl)
(Gaide and Schneider, 2003),
and heparin acrylic beads (Sigma) were soaked in Bmp4 protein (100 ng/µl,
R&D Systems) for 45 minutes at 37°C. The beads were placed on top of
the explants using fine forceps, and explants were cultured for 24 hours. When
indicated, Eda-A1 or BSA-releasing beads were introduced 6 hours before Bmp4
beads followed by a further 24 hours of culture.
In rescue experiments, back skin from carefully staged E13
Eda-null or wild-type embryos was dissected and cultured as
previously described (Laurikkala et al.,
2002). Recombinant noggin (R&D Systems) or sonic hedgehog
(R&D Systems) was administered to the culture medium as indicated in the
text.
In situ hybridization
Whole embryos, isolated mandibles or cultured explants were treated with
cold methanol for 2 minutes, fixed in 4% paraformaldehyde overnight, and
processed for whole-mount in situ hybridization as described earlier
(Mustonen et al., 2004) by
using the InSituPro robot (Intavis AG, Germany). The digoxigenin-labeled
probes were detected with BM Purple AP Substrate Precipitating Solution
(Boehringer Mannheim Gmbh, Germany). The following plasmids were used as
templates: ameloblastin (Wang et al.,
2004a); ß-catenin, Edar and Shh
(Laurikkala et al., 2002);
follistatin (Wang et al.,
2004b); and a 0.8 kb probe specific to the 3' end of
Ccn2 (Friedrichsen et al.,
2003). Noggin (McMahon et al.,
1998), gremlin (Khokha et al., 2001), Dan
(Dionne et al., 2001) and
bambi (Grotewold et al., 2001)
probes were labelled with 35S-UTP, and radioactive in situ
hybridization on paraffin sections was performed according to standard
procedures as described previously
(Laurikkala et al., 2002).
Hanging drop cultures and quantitative RT-PCR
To analyse the induction of putative target genes by Eda-A1, tissues were
grown submerged in hanging drops. E14 wild-type or Eda-/-
back skin was dissected in Dulbecco's PBS pH 7.4 and cut in two halves along
the midline: one half was used as the control and the other one was exposed to
Eda-A1. A minimum of triplicate samples was assayed each time. Skin-halves
were placed in culture medium and allowed to recover in a cell culture
incubator for about 30 minutes. When grown in the absence of serum, MEM was
supplemented with glutamine, 0.2% bovine serum albumin and 20 mmol/l Hepes, pH
7.2. Each skin half was cultured individually in one drop of 40 µl
pre-warmed medium supplemented with Eda-A1, or equivalent proportion of
protein dissolvent, placed under the lid of a 35 mm diameter plastic Petri
dish containing medium or PBS to prevent evaporation
(James et al., 2006).
Tissues from hanging drops (or freshly isolated E14 wild-type or K14-Eda-A1
skin) were placed straight into 350 µl lysis buffer of the RNeasy mini kit
(Qiagen) containing 1% ß-mercaptoethanol (Sigma). Total RNA was isolated
as specified by the manufacturer and quantified using UV spectroscopy. One
hundred to 700 ng of total RNA was reverse transcribed using 500 ng of random
hexamers (Promega) and 100 units of Superscript II (Invitrogen) according to
the manufacturer's instructions. Quantitative PCR (qPCR) was carried out using
the 2x SYBR-green PCR master mix (Applied Biosystems) and Applied
Biosystems' default PCR conditions for the ABI 7000 as described
(James et al., 2006). Primer
sequences are available upon request. PCR products were run on a 2% agarose
gel to verify their correct size and the absence of non-specific reaction
products and primer dimers. Gene expression was quantified by comparing the
sample data against a dilution series of PCR products (amplicons) of the gene
of interest. Data were analysed using Applied Biosystems' Prism SDS software
and normalized against Ranbp1.
Promoter analysis
The mouse and human promoter sequences of Ccn2, follistatin and
Shh genes were aligned with LALIGN, and analysed for the presence of
putative NF-B binding sites by Match and P-Match programs that are
freely available on the Internet.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Wang et al., 2004a;
Plikus et al., 2005). We
showed recently that the K14-follistatin phenotype results from suppression of
Bmp activity by ectopic follistatin expressed in the labial side of the
incisor, thereby leading to the inhibition of ameloblast differentiation. In
cultured incisor explants, recombinant follistatin antagonized Bmp4-induced
expression of the ameloblastspecific gene ameloblastin
(Wang et al., 2004a). Due to
the obvious similarity of the three transgenic phenotypes, we considered the
incisor culture as a useful system to monitor Bmp activity and to test whether
Eda-A1 can suppress it. In accordance with our previous results,
Bmp4-releasing beads induced intense ameloblastin expression (5/5 explants)
compared with control explants, which displayed the typical expression pattern
on the labial side of the incisor epithelium
(Fig. 1A,B). However, unlike
follistatin (Wang et al.,
2004a), Eda-A1-releasing beads placed next to a Bmp4 bead did not
affect the expression of ameloblastin in the majority of the explants (5/7
explants) (Fig. 1D), although
occasionally a prominent reduction was detected (2/7 explants; data not
shown), which we never observed with BSA-soaked beads
(Fig. 1C). However, it is
unlikely that the Tnf family protein Eda could interfere with Bmp activity by
directly binding to Bmps and/or binding to Bmp receptors. Its effects, if any,
would more probably be indirect and might require induced expression of a Bmp
antagonist. In order to test this possibility, we introduced beads soaked with
Eda, or control beads releasing BSA, 6 hours before a Bmp4 bead. Remarkably,
we noticed a near total absence of ameloblastin expression in explants
pretreated with Eda (24/27 explants), whereas BSA had no effect (21/23
explants) (Fig. 1E,F). Eda also
appeared to reduce the endogenous level of ameloblastin mRNA to a certain
extent (compare Fig. 1G with
1A). These results demonstrate that activation of the Edar pathway
is able to counteract Bmp4 signalling and suggest a plausible explanation for
the observed enamel phenotype of K14-Eda and K14-Edar mice
(Mustonen et al., 2003;
Pispa et al., 2004;
Tucker et al., 2004).
Search for the physiological targets of Edar
A putative physiological target of Edar should have an overlapping
expression pattern in developing teeth and/or hair follicles (see also below).
During tooth development, Edar becomes restricted to dental placodes
as they form (E12) (Fig. 2A)
(Tucker et al., 2000;
Laurikkala et al., 2001). At
the bud stage (E13), expression of Edar was intense at the tip of the
tooth bud, and at the cap stage (E14) it was confined to the enamel knot, an
epithelial signalling centre regulating tooth shape
(Fig. 2B). Similarly, during
hair development Edar is first detected throughout the epithelium,
becomes localized to nascent placodes, and is later most intense at the tip of
the growing hair follicle (Headon and
Overbeek, 1999; Laurikkala et
al., 2002).
Extracellular high-affinity antagonists of Bmps include, among others,
noggin, gremlin (gremlin 1, Grem1 - Mouse Genome Informatics), Dan
(Neuroblastoma, Nbl1 - Mouse Genome Informatics), chordin,
follistatin, ectodin (scelerostin domain containing 1, Sostdc1 -
Mouse Genome Informatics), and Ccn2/Ctgf (also known as Fisp12)
(Balemans and Van Hul, 2002).
The expression profiles during tooth and/or hair development of only some of
these have been previously described. Therefore we analysed the expression of
a number of Bmp inhibitors between E12 and 16. Noggin is expressed in the
mesenchymal condensate under the epithelial hair placode
(Botchkarev et al., 1999), but
we found no expression in the developing dental placode, although intense
expression was observed in Meckel's cartilage
(Fig. 2C). Only faint
expression of noggin was detected at later developmental stages
(Fig. 2D, and data not shown).
Expression of gremlin and Dan, two Bmp inhibitors with similar
structural motifs and inductive activities
(Balemans and Van Hul, 2002),
was limited to the mesenchyme of the developing tooth at E12
(Fig. 2E,G), and in subsequent
developmental stages (Fig.
2F,H, and data not shown). Expression of gremlin is confined to
the interplacodal mesenchyme during feather development
(Ohyama et al., 2001).
Interestingly, follistatin is expressed in the tooth, hair and feather
placodes (Ferguson et al.,
1998; Patel et al.,
1999; Nakamura et al.,
2003), and is localized to the enamel knot of E14 molar teeth
(Wang et al., 2004b). Also
Ccn2, a modular multifunctional protein with known ability to inhibit
Bmp signalling (Abreu et al.,
2002), is expressed in the epithelium of developing teeth, and is
localized to the enamel knot of the cap stage tooth and is found later in
preameloblasts (Shimo et al.,
2002; Friedrichsen et al.,
2003; Yamaai et al.,
2005). Although ectodin, a modulator of Bmp and Wnt pathways, is
mainly epithelial, its expression domain does not significantly overlap with
that of Edar (Laurikkala et al.,
2003).
|
).
At the placode and bud stage it was expressed in the condensed mesenchyme
(Fig. 2I, and data not shown),
while at the cap stage prominent expression was also evident in the epithelium
overlapping the enamel knot region (Fig.
2J). Smad6 and Smad7 are inhibitory Smads, which antagonize the
Tgf-ß pathway, binding either to the type I receptor or Smad4
(Derynck and Zhang, 2003).
Interestingly, Smad7 transcripts have been detected in the epithelium
of developing teeth (Luukko et al.,
2001), and the expression of this gene can be regulated by
NF-B (Derynck and Zhang,
2003). In conclusion, based on the published data and our own
expression analyses, we considered follistatin, Ccn2, bambi and Smad7 as best
candidates mediating the Bmp-inhibitory action of Eda-A1.
Noggin is able to partially restore hair placode formation in Eda-null embryos
Eda-deficient mice lack primary hair follicles and the localized
expression of a battery of placode markers at E14
(Laurikkala et al., 2002).
Bmps have a well-established role as placode inhibitors during feather and
secondary hair placode formation (for reviews, see
Millar, 2002;
Schmidt-Ullrich and Paus,
2005), and at least Bmp4 and Bmp7 are expressed
in developing murine skin, and their uniform expression is retained in E13 and
14 Eda-deficient skin (data not shown). Therefore we reasoned that
one crucial outcome of Eda signalling during hair placode formation could be
suppression of Bmp activity. If this was the case it might be possible to
restore primary hair placode in Eda-deficient mice by an exogenous
Bmp inhibitor. Low doses of recombinant Eda-A1 induce normally sized and
spaced placodes in E13 Eda-/- skin explants cultured for
24 hours, whereas high doses of Eda-A1 cause enlargement and fusion of
placodes (Mustonen et al.,
2004) (Fig. 3D,E).
Treatment of E13 Eda-/- skin explants with 0.5 µg/ml of
recombinant noggin induced the formation of some placodes
(Fig. 3A,B), whereas 2 µg/ml
of noggin led to the development of multiple placodes seen as a more prominent
punctuate expression of placode-specific genes throughout the explant
(Fig. 3C). In wild-type skin,
noggin slightly increased the size and number of hair placodes
(Fig. 3F,G), in line with
previous reports (Noramly and Morgan,
1998; Botchkarev et al.,
1999). Interestingly, the spacing of noggin-rescued follicles of
Eda-deficient skin was not as regular as that seen in untreated wild-type skin
or in explants rescued by low doses of Eda-A1
(Fig. 3D,F), and we never
noticed as prominent enlargement of placodes as seen with superfluous Eda-A1
(Fig. 3E)
(Mustonen et al., 2004). These
results strongly suggest that lack of primary hair placode formation in
Eda-null mice is at least partly due to insufficient inhibition of
Bmp activity, but that additional Eda targets are likely to be involved.
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B
activation (see Introduction). To correlate the kinetics of Ccn2
expression with that of a validated NF-B target gene, we also analysed
the expression of I-B
in the same samples
(Scott et al., 1993;
Hoffmann et al., 2003).
I-B is an inhibitor of NF-B, and its expression is
induced by a number of Tnf receptors, thereby participating in a feedback loop
of NF-B activity (Hayden and Ghosh,
2004). Expression of I-B
correlates with that of Edar and NF-B activation in developing
molars (Laurikkala et al.,
2001; Ohazama et al.,
2004). Consistently, its localized expression in primary hair
placodes is dependent on Edar
(Schmidt-Ullrich et al.,
2006). Expression of I-B was
induced to about 2.5-fold after 1 hour exposure to Eda-A1, was highest (about
5-fold) at 3-4 hours and slowly declined thereafter
(Fig. 4A).
Next, we performed a similar series of experiments in the absence or
presence of serum (Fig. 4B) in
order to eliminate the contribution of serum-derived factors in our
experimental set-up, as Ccn2 has been identified as one of the
immediate-early genes induced by serum growth factors
(Rachwal and Brigstock, 2005).
After 3 hours of exposure to Eda-A1, Ccn2 expression was highly
induced (about 10-fold) under both conditions. At 6 hours, a 15-fold induction
of Ccn2 was detected in the absence of serum, whereas again a
decrease to about 2.5-fold was seen in the presence of serum. When the actual
numbers of Ccn2 transcripts induced by 6 hour treatment of Eda-A1
were compared, the difference between the two samples was less prominent (data
not shown). The reason for this is that in control explants (no Eda-A1 added)
there was more Ccn2 in the presence of serum, and therefore the fold
of induction was lower (data not shown). However, about twice as many
Ccn2 transcripts were induced by Eda-A1 at 6 hours in the absence of
serum, suggesting that a serum component may inhibit Eda-A1 activity. After 22
hours of culture, a sustained level of Ccn2 expression (about 5-fold)
was detected in both culture conditions
(Fig. 4B).
Finally, we tested the ability of Eda-A1 to induce Ccn2, follistatin and Smad7 in wild-type E14 skin explants (Fig. 4C). Like in Eda-deficient skin, we noticed no effect in the expression of Smad7 upon Eda-A1 treatment, whereas a 4-fold and 10-fold augmentation in Ccn2 levels was observed at 3 hours and 5 hours, respectively. The fold of induction was slightly lower than in Eda-deficient skin (Fig. 4B), mainly due to the fact that the initial amount of Ccn2 transcripts was higher in wild-type skin (data not shown). qPCR analysis of epithelia separated after 3 hours exposure to Eda-A1 confirmed that Ccn2 was specifically induced in the epithelium (data not shown). A 3-fold induction in the expression levels of follistatin was evident at 5 hours of culture with Eda-A1 (Fig. 4C). A modest increase of Ccn2 transcripts was also observed in K14-Eda-A1 skin at E14 compared with non-transgenic littermates (data not shown).
Expression of Ccn2 correlates with that of Edar during early stages of pelage hair and tooth development
Currently, only limited knowledge on the expression profile of
Ccn2 during ectodermal organ development is available
(Shimo et al., 2002;
Friedrichsen et al., 2003;
Yaamai et al., 2005). Therefore, we analysed the expression of Ccn2
by whole-mount in situ hybridization during early hair and tooth development
(Fig. 5). At E14, Ccn2
was expressed in nascent primary hair placodes of wild-type embryos
(Fig. 5A,B), whereas the
epithelium of the developing mystacial vibrissae of the snout was devoid of
Ccn2 (Fig. 5A)
(Friedrichsen et al., 2003).
Interestingly, closer examination of pelage hair follicles revealed that
Ccn2 was sometimes concentrated at the periphery of placodes
(Fig. 5C,C').
Ccn2 expression was detected also in the cartilage of the digits as
previously described (Fig. 5A)
(Friedrichsen et al., 2003).
No localized expression of Ccn2 was detected in the skin ectoderm of
Eda-null embryos at E14 (Fig.
5D), although expression in the digits was unaltered (data not
shown). However, treatment of Eda-/- skin explants with
recombinant Eda-A1 induced localized upregulation of Ccn2 in placodes
(Fig. 5E-G and data not shown).
Vibratome sections of the whole mounts of wild-type embryos confirmed the
colocalization of Ccn2 and Edar in nascent hair placodes
(Fig. 5H,I).
|
; Laurikkala et
al., 2001). Likewise, expression of Ccn2 was detected in
both molar and incisor placodes in addition to a prominent signal in Meckel's
cartilage (Fig. 5L). In
Eda-deficient embryos, Edar transcripts were fairly normally
distributed (Fig. 5M), as
demonstrated previously during a more advanced stage of molar development
(Tucker et al., 2000).
Interestingly, and in contrast to what was seen in skin, there was no gross
difference in the expression of Ccn2 between wild-type and
Eda-null E12 mandibles (Fig.
5N).
|
). It is possible that this was due to improper amount,
location or timing in the application of exogenous noggin, but it may also
suggest that suppression of Bmp activity is not sufficient to mimic all of the
effects of Eda-A1. We have previously shown that Shh transcripts are
detected upon rescue of Eda-null skin with overnight treatment of
recombinant Eda (Mustonen et al.,
2004). Moreover, Shh is coexpressed with Edar in
developing hair follicles, and in embryonic teeth from bud stage onwards
(Bitgood and McMahon, 1995;
Laurikkala et al., 2001;
Laurikkala et al., 2002).
These data prompted us to analyse whether Shh is a target of Edar.
Strikingly, a 2.5-fold induction of Shh was evident already after 1
hour treatment of E14 skin-halves with Eda-A1, and by 3 hours it had increased
to 15-fold (Fig. 6A). The
overall Shh induction displayed kinetics highly similar to those of
Ccn2 and I-B
(Fig. 4A). The amount of
Shh in K14-Eda-A1 whole skin samples was increased 2.5-fold compared
with control littermates (Fig.
6B). In mice lacking Shh, hair follicle formation is
initiated and Shh is thought to regulate proliferation and downgrowth of the
follicular epithelium (St-Jacques et al.,
1998; Chiang et al.,
1999). In line with the proposed later role of Shh in follicular
morphogenesis, we found no indication of placode formation in
Eda-/- skin explants treated with recombinant Shh protein
(Fig. 6C-F). |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
Shh and to a lesser extent follistatin, was rapidly induced in
embryonic skin explants upon Eda-A1 treatment. Noggin partially restored
primary hair placode formation in Eda-/- embryos,
indicating that suppression of Bmp activity was compromised in embryonic skin
in the absence of Eda. Moreover, recombinant Eda antagonized the activity of
Bmp4 in incisor explants. These actions are likely to be mediated by Ccn2,
possibly together with follistatin. We do not exclude the possibility that Eda
may also regulate Bmp activity by other means, e.g. via Tak1 (Tgf-ß
activated kinase 1; Map3k7 - Mouse Genome Informatics), which is thought to
mediate Ikk activation by Edar (Morlon et
al., 2005). Although in some cell types Tak1 acts synergistically
with Bmps, it may also dramatically antagonize Bmp signalling, possibly
through its effects on subcellular localization of R-Smads
(Hoffmann et al., 2005).
While this paper was under review, the Headon group reported sporadic
rescue of hair follicle formation in noggin-treated Eda-/-
skin (Mou et al., 2006). The
more noticeable placode formation obtained by us
(Fig. 2) might be due to the
earlier onset of the rescue experiment (E13 versus E14) or the higher noggin
concentration used (2 versus 1 µg/ml). In agreement with our results, Mou
et al. (Mou et al., 2006)
noticed upregulation of Ccn2 expression by recombinant Eda-A1, as
well as suppression of Bmp4-dependent Smad1/5/8 phosphorylation in Eda-A1
treated Eda-/- skin.
To our knowledge, Ccn2 and Shh are the first likely bona
fide transcriptional targets of the Eda signalling pathway discovered. The
maximal fold of induction of Ccn2 and Shh after pertinent
Eda treatment was about 11- and 15-fold in cultured skin explants,
respectively, but due to the presence of the mesenchyme (which lacks Edar and
is therefore unresponsive to Eda-A1), these figures are likely to be
underestimates. The rapid induction of the two genes evident already after 1
hour's exposure to Eda and the similar kinetics of I-B
expression strongly suggest that they are direct transcriptional targets of
Edar mediated via NF-B. Comparison of mouse and human Ccn2
promoters revealed a conserved putative binding site of NF-B (see Table
1 in the supplementary material) (Blom et
al., 2002), which is practically identical with the validated
binding sites in I-B and M-CSF promoters [see Hoffmann et al.
(Hoffmann et al., 2003) and
references therein]. Furthermore, Ccn2 colocalized with Edar
in nascent hair placodes and in tooth germs.
Many of the actions of Ccn2 are thought to be mediated via Bmps and
Tgf-ß, such that it inhibits Bmp and enhances Tgf-ß signalling
(Abreu et al., 2002). It is
likely that during hair placode formation Ccn2 has a role as a Bmp antagonist.
As Ccn2 was originally isolated as a chemotactic factor for fibroblasts
(Rachwal and Brigstock, 2005),
it is tempting to speculate that it might also be involved in condensation of
dermal cells during placode formation, a process likely to result from cell
migration, rather than proliferation, as suggested by studies in chick
(Wessells, 1965;
Desbiens et al., 1991).
Targeted gene disruption has revealed the importance of Ccn2 for
chondrogenesis (Ivkovic et al.,
2003). The ectodermal phenotype of Ccn2-null mice, which
die shortly after birth, has not been described.
|
B recognition
sequences, in which the essential nucleotides were conserved between mouse and
human (see Table 1 in the supplementary material). In addition, sites not
conserved but with 100% match to the consensus sequence were identified in
both species (data not shown).
Our qPCR data revealed a low, yet reproducible, induction of follistatin
upon Eda treatment, suggesting that besides activin
(Ferguson et al., 1998;
Wang et al., 2004a), Edar may
also regulate follistatin expression in vivo. We identified a number of
putative NF-B-binding sites in mouse and human follistatin promoters,
and a conserved one (see Table 1 in the supplementary material) was located in
the middle of a 
60 nucleotide region of 100% identity in mouse and human.
Studies in chicken have suggested that follistatin locally antagonizes the
action of the Bmps, thereby permitting feather placode formation and
regulating the size of the bud (Patel et
al., 1999). Hair follicle morphogenesis is retarded in follistatin
knockout mice (Nakamura et al.,
2003), yet primary hair follicle formation appears to be
unaffected (M. Suomalainen and I.T., unpublished).
A key question is to what extent these novel functions of the Eda pathway
can explain the observed phenotypes of Eda/Edar-deficient and
Eda-overexpressing mice. As the molecular mechanisms involved in the
early stages of development of distinct ectodermal organs are shared to a
great extent (Pispa and Thesleff,
2003; Mikkola and Millar,
2006), it is possible that the same target genes are induced by
Eda in different epithelial appendages. However, genes with similar functions
may be induced by other signalling pathways in teeth and mammary glands but
not in primary hair placodes, thereby explaining the appendage-specific
phenotypes of Eda-null mice. The aberrant development of
Eda-/- molars is evident from bud stage onwards and
results in few shallow cusps associated with an overall smaller size of teeth
(Mikkola and Thesleff, 2003;
Kangas et al., 2004).
Intriguingly, ablation of Shh
(Dassule et al., 2000) and
follistatin (Wang et al.,
2004b) leads to small teeth with fewer cusps.
The molecular mechanism causing the supernumerary teeth and mammary glands
of K14-Eda mice has remained enigmatic. The role of lateral
inhibition, and its underlying molecular mechanism, in regulating the spacing
and number of teeth and mammary glands is poorly understood, and it is not
known whether Bmp signalling is involved. Instead, Shh signalling is required
for tooth development already at an early stage
(Hardcastle et al., 1998). The
development of the ectopic molar of K14-Eda mice is marked by a
Shh-expressing placode, and in wild-type embryos a weak and transient
upregulation of Shh is occasionally detected in the same location
(Kangas et al., 2004). We
propose that the extra Shh signal produced upon increased Edar activity is
crucial for promoting the development of this rudimentary dental placode into
a fully erupted tooth in K14-Eda-A1 mice. Intriguingly, in
Tg737orpk mice carrying a hypomorphic mutation in
polaris (Ift88 - Mouse Genome Informatics), a regulator of
Shh pathway (Haycraft et al.,
2005), a supernumerary tooth develops at the same position
(Zhang et al., 2003). Further
studies will be required to reveal other crucial downstream components of Eda
signalling in tooth and mammary development.
Previous studies have established a role for the Eda pathway as an
important activator of primary hair placode fate, downstream of the yet
unknown `first dermal signal' (Mustonen et
al., 2004; Houghton et al.,
2005; Schmidt-Ullrich et al.,
2006). During feather and hair follicle development, Bmp activity
is thought to mediate lateral inhibition, such that Bmps expressed in the
nascent placode prevent surrounding cells from adopting a follicular fate.
Simultaneously, the action of Bmps needs to be counteracted within the placode
itself (Oro and Scott, 1998).
Apparently, in Eda-deficient embryos, no Bmp inhibition takes place
and Bmps repress the follicular fate to such an extent that primary hair
placodes are not discernible, whereas with increasing Edar signalling (such as
that seen in K14-Eda-A1 mice or in skin explants treated with
excessive Eda) rising amounts of Bmp inhibitors are expressed, thereby
allowing expansion of placodes. Our findings reveal the need for suppression
of Bmp activity within nascent primary hair placodes and thereby highlight the
mechanistic similarities in the induction of all pelage hair types. The fact
that Eda induces Bmp antagonists other than noggin also explains why primary
hair placodes are unaffected in noggin-/- embryos
(Botchkarev et al., 2002).
Taken together, our results provide a model for the crucial role of Edar
activity during primary hair placode initiation
(Fig. 7). First, Eda restricts
Bmp signalling in the placode by local upregulation of Bmp inhibitors. Second,
Eda may regulate proliferation and ingrowth of the hair follicle through
Shh expression, an action that is, however, required only after
placode initiation (St-Jacques et al.,
1998; Chiang et al.,
1999). During secondary hair initiation
(Fig. 7), noggin antagonizes
local Bmps, whereas Wnt/ß-catenin signalling is the best candidate as
inducer of Shh (Schmidt-Ullrich
and Paus, 2005; Mikkola and
Millar, 2006). During early stages of tooth development,
Shh induced by Eda together with other still unknown signals promotes
the growth of the dental bud (Fig.
7). The next goal is to investigate to what extent the two
signalling outcomes of ectodysplasin reported here can explain the other
ectodermal defects resulting from altered Eda signalling and which other
pathways are directly influenced by Edar activity.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02708/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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 G. D. Richardson, H. Bazzi, K. A. Fantauzzo, J. M. Waters, H. Crawford, P. Hynd, A. M. Christiano, and C. A. B. Jahoda KGF and EGF signalling block hair follicle induction and promote interfollicular epidermal fate in developing mouse skin Development, July 1, 2009; 136(13): 2153 - 2164. [Abstract] [Full Text] [PDF]
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 M. Kunisada, C.-Y. Cui, Y. Piao, M. S.H. Ko, and D. Schlessinger Requirement for Shh and Fox family genes at different stages in sweat gland development Hum. Mol. Genet., May 15, 2009; 18(10): 1769 - 1778. [Abstract] [Full Text] [PDF]
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 F. Clauss, M.-C. Maniere, F. Obry, E. Waltmann, S. Hadj-Rabia, C. Bodemer, Y. Alembik, H. Lesot, and M. Schmittbuhl Dento-Craniofacial Phenotypes and underlying Molecular Mechanisms in Hypohidrotic Ectodermal Dysplasia (HED): a Review Journal of Dental Research, December 1, 2008; 87(12): 1089 - 1099. [Abstract] [Full Text] [PDF]
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 E. Fuchs and J.A. Nowak Building Epithelial Tissues from Skin Stem Cells Cold Spring Harb Symp Quant Biol, November 6, 2008; (2008) sqb.2008.73.032v1. [Abstract] [PDF]
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J. Pispa, M. Pummila, P. A. Barker, I. Thesleff, and M. L. Mikkola Edar and Troy signalling pathways act redundantly to regulate initiation of hair follicle development Hum. Mol. Genet., November 1, 2008; 17(21): 3380 - 3391. [Abstract] [Full Text] [PDF]
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J. Hwang, T. Mehrani, S. E. Millar, and M. I. Morasso Dlx3 is a crucial regulator of hair follicle differentiation and cycling Development, September 15, 2008; 135(18): 3149 - 3159. [Abstract] [Full Text] [PDF]
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F. Michon, L. Forest, E. Collomb, J. Demongeot, and D. Dhouailly BMP2 and BMP7 play antagonistic roles in feather induction Development, August 15, 2008; 135(16): 2797 - 2805. [Abstract] [Full Text] [PDF]
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K. Narhi, E. Jarvinen, W. Birchmeier, M. M. Taketo, M. L. Mikkola, and I. Thesleff Sustained epithelial {beta}-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation Development, March 15, 2008; 135(6): 1019 - 1028. [Abstract] [Full Text] [PDF]
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 E. Fuchs Skin stem cells: rising to the surface J. Cell Biol., January 28, 2008; 180(2): 273 - 284. [Abstract] [Full Text] [PDF]
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 K. Kobielak, N. Stokes, J. de la Cruz, L. Polak, and E. Fuchs Loss of a quiescent niche but not follicle stem cells in the absence of bone morphogenetic protein signaling PNAS, June 12, 2007; 104(24): 10063 - 10068. [Abstract] [Full Text] [PDF]
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