1 Developmental Biology Program, Institute of Biotechnology, PO Box 56
(Viikinkaari 9), University of Helsinki, 00014, Finland
2 Department of Biochemistry, University of Lausanne, 1066 Epalinges,
Switzerland
Author for correspondence (e-mail:
marja.mikkola{at}helsinki.fi)
Accepted 23 July 2004
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SUMMARY |
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Key words: Ectodysplasin, Hair placode, Tooth placode, Mammary placode, Mouse
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Introduction |
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In most ectodermally derived organs, the mesenchyme seems to supply the
first signal required to initiate organogenesis, whereas the epithelium
responds according to its inherent capabilities. Classic tissue recombination
experiments using mouse and chick embryonic skin from different body parts
have revealed that an initial signal(s) arising in the dermis causes the
formation of placodes in epidermis from non-hair bearing regions
(Hardy, 1992). Similarly,
mesenchyme from mouse mammary region is able to induce functional mammary
epithelium when recombined with dorsal epidermis
(Veltmaat et al., 2003
). The
issue of whether tooth initiation is determined by neural crest derived
mesenchyme or by the ectoderm is controversial
(Mina and Kollar, 1987
;
Mitsiadis et al., 2003
).
An epithelial placode can be considered a basal unit of ectodermal
organogenesis. A characteristic feature of epithelial placodes is that many
signal molecules including Wnts, bone morphogenetic proteins (Bmps),
fibroblast growth factors (Fgfs), and sonic hedgehog (SHH) are expressed by
the placodes themselves, or by the underlying condensed mesenchyme
(Millar, 2002;
Thesleff and Mikkola, 2002a
;
Pispa and Thesleff, 2003
;
Veltmaat et al., 2003
). These
molecules are shared by many ectodermal organs, and recent studies have
revealed that they regulate the initial stages of morphogenesis in these
organs (Pispa and Thesleff,
2003
). Therefore, it is tempting to assume that they also play
identical roles, i.e. regulate similar cellular mechanisms in the initiation
of all appendage types.
Ectodysplasin (Eda), a recently characterised member of the tumour necrosis
factor (TNF) ligand superfamily, has also been implicated in the early
development of ectodermal appendages
(Barsh, 1999;
Thesleff and Mikkola, 2002b
;
Mikkola and Thesleff, 2003
).
Mice deficient for Eda (Tabby mice), its receptor Edar
(downless mice) or the intracellular adapter protein Edaradd that is
required for Edar signalling (crinkled mice) have identical
phenotypes characterised by defective hair development
(Falconer et al., 1951
;
Claxton, 1967
;
Headon and Overbeek, 1999
;
Headon et al., 2001
;
Laurikkala et al., 2002
;
Yan et al., 2002
). Other
abnormalities of these mice include missing or misshapen teeth, as well as
defects in a number of exocrine glands, such as sweat and salivary glands
(Grüneberg, 1965
;
Sofaer, 1969
;
Grüneberg, 1971
;
Blecher et al., 1983
;
Pispa et al., 1999
). Mutations
in the corresponding human genes result in a malformation syndrome called
hypohidrotic ectodermal dysplasia (HED)
(Kere et al., 1996
;
Monreal et al., 1999
;
Headon et al., 2001
). The
symptoms of HED are similar to those found in mice, including missing or
sparse hair, abnormal dentition and absent or reduced sweating
(Kere and Elomaa, 2002
).
Accumulating evidence suggests that Eda is an early and necessary signal
required for placode formation (for reviews, see
Mikkola and Thesleff, 2003;
Pispa and Thesleff, 2003
).
First, Tabby mice lack primary hair follicles that give rise to guard
hairs as well as the placodes for auchene and zigzag hairs
(Claxton, 1967
;
Laurikkala et al., 2002
). None
of the placode marker genes tested displays a patterned expression in
Tabby or downless mice at E14-E15, when the first hair
follicles are forming in wild-type mice
(Headon and Overbeek, 1999
;
Andl et al., 2002
;
Laurikkala et al., 2002
).
Second, Edar is one of the earliest markers of newly formed placodes. During
early development, Eda and Edar are colocalised in the simple ectodermal sheet
covering the embryo (Tucker et al.,
2000
; Laurikkala et al.,
2001
; Laurikkala et al.,
2002
). Upon initiation of tooth and hair development, Edar becomes
restricted to the forming placodes, whereas Eda shows complementary expression
in the flanking tissue. In transgenic mice that lack epithelial
ß-catenin, the development of hair follicles is inhibited
(Huelsken et al., 2001
). In
these mice Edar, but no other placode marker tested, retains its punctuate
placodal expression pattern suggesting that Edar is relatively high in the
hierarchy of genes regulating placodal fate. Edar is also expressed in the
newly formed mammary gland placodes (Pispa
et al., 2003
). However, the number of mammary glands is normal in
Tabby mice but the nipple morphology is altered
(Mustonen et al., 2003
). In
addition, HED is occasionally associated with absent or rudimentary nipples
(Clarke et al., 1987
).
Third, we have recently shown that transgenic mice overexpressing the
Eda-A1 isoform of ectodysplasin (ligand of Edar) under keratin 14 (K14)
promoter, which drives the expression to the developing ectoderm as early as
embryonic day 9 (E9), well before the development of any ectodermal appendage
is initiated, are featured by supernumerary ectodermal organs
(Mustonen et al., 2003). Most
notably, these mice have extra mammary glands and teeth. Furthermore, hair
follicles are produced continuously between E14 and birth in
K14-Eda-A1 mice, unlike in wild-type mice where they develop in three
separate waves giving rise to the different pelage hair types. In the current
study, we have analysed in more detail the dynamics of ectodermal placode
formation in K14-Eda-A1 transgenic, in Eda-deficient Tabby
and in wild-type mice, as well as in organ culture. Our results suggest that
Edar signalling regulates cell fate decisions by promoting placodal fate.
Epithelial placodes of K14-Eda-A1 transgenic embryos were enlarged,
and exogenous Eda-A1 stimulated the growth and fusion of placodes also in
vitro. This function of Eda-A1 appears to be downstream of the initial primary
inductive signal required for placode initiation during skin patterning and
appears to involve cellular mechanisms other than increased proliferation of
placodal cells.
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Materials and methods |
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Histology and in situ hybridisation
Tissues from mouse embryos were dissected and processed for in situ
hybridisation as previously described
(Mustonen et al., 2003).
Radioactive in situ hybridisation on paraffin sections was performed according
to Wilkinson and Green (Wilkinson and
Green, 1990
). Patched 1 (Kim
et al., 1998
), Bmp4
(Vainio et al., 1993
) and
Lef1 (Travis et al.,
1991
) probes were labelled with 35S-UTP. Whole-mount in
situ hybridisation analysis of embryonic day 11 (E11) to E14 whole embryos,
and E11 to E13 lower jaws was carried out as described earlier
(Kettunen and Thesleff, 1998
)
using the InsituPro robot (Intavis, Germany). The following plasmids were used
as templates: ß-catenin (Laurikkala
et al., 2002
), Edar
(Laurikkala et al., 2001
),
Lef1 (Travis et al., 1991
),
Pitx2 (Dassule and McMahon,
1998
) and sonic hedgehog
(Vaahtokari et al., 1996
). For
plastic histology, tissues were embedded in Historesin as specified by the
manufacturer (Leica) and stained using Haematoxylin and Eosin.
Scanning electron microscopy (SEM)
Embryonic mice were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer
pH 7.3 for at least 12 hours. Embryos were dehydrated in graded series of 50%,
70%, 94% ethanol for 1 hour each, and 100% ethanol for 12 hours, and subjected
to crucial point drying and platinum coating. The back skin of shoulder
regions was viewed with Zeiss DSM 962 scanning electron microscope (Zeiss,
Oberkochen, Germany).
Expression vectors
Expression construct for the ectodomain of murine Eda-A1 (amino acids 180
to 391) in pSecTag vector (Invitrogen) has been previously described
(Koppinen et al., 2001). The
tags of the vector were eliminated due to the usage of the authentic Eda-A1
stop codon; instead a 6x His tag was added at the N-terminal end of the
recombinant protein. Plasmid coding for a single amino acid mutation in Eda-A1
coding sequence (Y343C) which abolishes receptor binding without affecting
trimer formation (Schneider et al.,
2001
) was made by in vitro mutagenesis using the overlap extension
method (Ho et al., 1989
). Both
constructs were verified by sequencing.
Production of recombinant Eda-A1 and Eda-A1 (Y343C)
Cos7 cells were maintained in Dulbecco's minimum essential medium (DMEM)
supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin,
and 100 µg/ml streptomycin. For the production of recombinant proteins,
100,000 Cos7 cells were plated on 50 mm plates. The following day,
semiconfluent cells were transfected with empty pSecTag vector, Eda-A1 or
Eda-A1 (Y343C) expression vectors using the Fugene 6 reagent, according to the
instructions of the manufacturer (Roche Molecular Biochemicals). After 5
hours, a fresh culture medium was changed, and the cells were allowed to grow
for further 24 or 48 hours before the medium was collected and used for organ
culture experiments. A sample of the conditioned medium was taken and analysed
for the production of recombinant molecules by western blotting
(Koppinen et al., 2001
) using
anti-penta-his antibody as recommended (Qiagen) or antibody specific to the
COOH-terminal end of Eda (Elomaa et al.,
2001
). Production of Fc-Eda-A1 has been previously described
(Gaide and Schneider,
2003
).
Organ cultures
Back skin of E12-14 wild-type or Tabby embryos was dissected in
Dulbecco's PBS; pH 7.4 under a stereomicroscope. Skin explants were grown for
24-72 hours on nucleopore filters at 37°C in a Trowell type culture
containing DMEM supplemented with 10% fetal calf serum, glutamine and
penicillin-streptomycin. When indicated, conditioned medium from Eda-A1 or
Eda-A1 (Y343C), or vector transfected cells was added to maximum one-fifth of
the total volume. In some experiments, recombinant purified Fc-Eda-A1
(Gaide and Schneider, 2003)
was added to the culture medium to a final concentration of 0.05-5.00
µg/ml. When indicated, Affigel-Blue agarose beads soaked in three
concentrations (20, 75 or 500 µg/ml) of Bmp4 (R&D Systems) were placed
on top of the explant.
Analysis of tooth placode size
The lower jaws of Eda-A1 transgenic embryos and their wild-type litter
mates were processed for whole-mount in situ hybridisation using a probe
specific to Shh to visualise the tooth placodes. Specific care was
taken in determination of the age of individual embryos. After staining, the
lower jaws were photographed with a digital camera along with a 1 mm grid that
was used as a standard. The mesio-distal length and the linguobuccal width of
the molar placodes were measured using NIH Image software. The measurements
were visualised as box plots, the height of the box showing 50% and bars 90%
of the results, median is shown as a horizontal line inside the box.
Altogether, 23 molar placodes from transgenic embryos and 18 placodes of their
wild-type littermates derived from two litters were analysed. For statistical
analysis, the non-parametric Mann-Whitney U-test was chosen because the size
of the placodes did not seem to be normally distributed. The tests were
performed using JMP computer software.
In vivo cell proliferation assay
Two FVB/N mice mated with a K14-Eda-A1 transgenic male were
injected i.p. at E14 with 600 µl of 5'-bromo-2'-deoxyuridine (BrdU)
labelling reagent (Zymed). After 2 hours labelling, the mice were sacrificed
and the embryos collected and fixed in 4% paraformaldehyde overnight. Samples
were dehydrated in ethanol series and embedded in paraffin wax. Four wild-type
and four transgenic embryos were cut into 7 µm sections at the mid-back
skin area. BrdU incorporation was visualised by
antibody-streptavidin-biotin-peroxidase system (Zymed) and background tissue
was stained with Haematoxylin. BrdU-positive and negative cells were counted
under 200x magnification (Olympus Provis). Two-thousand five-hundred and
eighty-four transgenic interfollicular cells, 3441 wild type interfollicular
cells, 1937 transgenic placodal cells (40 placode sections) and 818 wild-type
placodal cells (26 placode sections) were counted. The mitotic index was the
proportion of BrdU-positive cells of all cells in a microscopic view or in the
section of an epithelial hair placode. The P values were obtained
using Student's t-test with the statistical program SPSS.
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Results |
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First, we analysed the first wave of follicles by several placodal marker
genes by in situ hybridisation at embryonic day 14 (E14), a time point when
the placodes have just been formed. It has recently been shown that mRNA of
the ubiquitously expressed ß-catenin is strongly upregulated in the
epithelium of the newly formed placodes and K14-Cre mediated deletion of the
ß-catenin gene causes failure of placode development
(Huelsken et al., 2001).
Whole-mount in situ hybridisation with ß-catenin probe revealed the
normal hexagonal pattern of forming follicles on wild-type skin at E14
(Fig. 1A,E). Conversely, in
Eda-A1-overexpressing embryos, the follicles appeared enlarged, their
shapes were more irregular and fusions between placodes were observed
(Fig. 1B,F). Edar mRNA
encoding the receptor specific to Eda-A1, retained its normal placodal
expression in transgenic skin but again the placodes had irregular appearance
(Fig. 1C,D,G,H). The mRNA for
transcription factor Lef1, which is crucial for hair follicle
induction (van Genderen et al.,
1994
), is upregulated both in the epithelium and the condensed
mesenchyme of the placodes (Kratochwil et
al., 1996
). Like ß-catenin, Lef1 mRNA was expressed
within the enlarged placodes, as evidenced by both sectional
(Fig. 1I,J) and whole-mount
(data not shown) in situ hybridisation. Sonic hedgehog (Shh) is
expressed in the epithelium and patched 1 is expressed both in the epithelium
and in the underlying condensed mesenchyme; in Shh-deficient mice,
hair follicles fail to develop beyond the plug stage (stage 2)
(Chiang et al., 1999
).
Expression of patched 1 is positively regulated by Shh and thus, reflects the
signalling activity of the epithelium
(StJacques et al., 1998
;
Chiang et al., 1999
). Sectional
in situ hybridisation with a probe specific for patched 1 revealed that the
domain of the condensed mesenchyme beneath the transgenic placodes was also
enlarged (Fig. 1K,L). The
expression of Bmp4, a well-characterised inhibitor of hair and
feather placodes, was also detected in transgenic embryos
(Fig. 1M,N). Thus, all placode
markers tested revealed the enlarged placodes of K14-Eda-A1 embryos,
indicating that more epidermal cells had adopted follicular fate.
|
Eda-A1 promotes enlargement of placodes in a dose-dependent manner
The enlarged guard hair placodes of Eda-A1 transgenic embryos and the
absence of primary hair placodes in Eda- and Edar-deficient
mice (Headon and Overbeek,
1999; Laurikkala et al.,
2002
) suggest that Edar regulates their initiation and/or growth.
To gain further insight into the role of Eda-Edar signalling on placode
formation, we performed a series of in vitro experiments with cultured skin
explants in the absence or presence of exogenous recombinant Eda-A1. Under
culture conditions hair follicles are formed essentially as in vivo, i.e.
primary placodes are detected at E14, and secondary placodes giving rise to
awl hairs at E16-E16.5.
Endogenous Eda is initially produced as a trimeric membrane-bound protein
but a soluble signalling molecule, consisting of a collagenous domain followed
by the TNF domain, is released by furin cleavage
(Chen et al., 2001;
Elomaa et al., 2001
;
Schneider et al., 2001
)
(Fig. 2A). We applied
conditioned media from transfected Cos7 cells, producing the recombinant
ectodomain of Eda-A1 that mimicked the processed soluble endogenous ligand, to
skin explants (Fig. 2B). The
recombinant Eda protein runs as several bands on an SDS-PAGE gel, unlike the
full-length Eda (which produces a doublet band)
(Mikkola et al., 1999
). It
appears that these multiple bands are mainly due to differential
N-glycosylation (data not shown), which appears not to affect the activity of
the molecule [as we have previously shown that this recombinant molecule
activates Edar in a biochemical assay
(Koppinen et al., 2001
)]. As a
control, we used medium either from cells transfected with Eda-A1 carrying a
point mutation (Y343C) that has previously been shown to abolish receptor
binding without affecting trimer formation
(Schneider et al., 2001
), or
from cells transfected with an empty vector.
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Next, we tested whether we could advance the appearance of hair placodes by application of Fc-Eda-A1. Wild-type E12 skin explants were dissected and cultured with 2 µg/ml of purified Eda-A1, and analysed for placode formation after 1 or 2 days of culture. As expected, a clear effect on placode size was detected after 2 days of culture (equivalent to E14) (data not shown). However, in line with our in vivo analysis of K14-Eda-A1 mice, no evidence of placode formation was detected after 24 hours exposure to Eda-A1 (equivalent to E13) (Fig. 4J,K). Hence, stimulated Eda-Edar signalling did not accelerate the initiation of the first hair follicles.
Eda-A1 cannot override the inhibitory effect of Bmp4
Our results indicate that Eda-A1 promotes placodal cell fate. Exogenous
Bmp4, however, has been shown to inhibit both hair and feather placode
formation (Jung et al., 1998;
Noramly and Morgan, 1998
;
Botchkarev et al., 1999
). Next,
we wanted to test whether Bmp4 can counteract the placode promoting effect of
Eda-A1. Wild-type E13 skin was cultured for 24 hours in the presence of Eda-A1
(2 µg/µl) simultaneously with agarose beads releasing Bmp4 placed on top
of the explants. As the effect of Bmp4 may be concentration dependent
(Vainio et al., 1993
), we used
beads soaked in three different concentrations. The activity of Bmp4 beads was
verified by their ability to induce the expression of Msx2
(Laurikkala et al., 2002
)
(data not shown). At all concentrations tested, placode formation was
inhibited around the Bmp4 bead (Fig.
5). With highest concentrations used, sometimes most of the
explant was devoid of placodes (Fig.
5C). Whether Bmp4 specifically inhibits Eda-A1 signalling or
placode inititation at an even earlier step is currently unclear.
|
|
|
The increased hair placode size of K14-Eda-A1 embryos is not due to proliferation
Basically, the growth of an epithelial thickening or placode could result
from local cell proliferation, cell migration, altered cell adhesion or all of
them. To learn more about the mechanistic role of Edar signalling in placode
formation, we examined cell proliferation by counting mitotic indexes of newly
formed hair placodes at E14. Pregnant wild-type mice mated with
K14-Eda-A1 males were injected with BrdU and sacrificed 2 hours
later. Epithelial cells of skin sections from equivalent back-skin regions
were analysed for BrdU incorporation by immunohistochemical staining
(Fig. 8). First, we counted
mitotic indexes (percentage of BrdU-positive cells) from wild-type hair
placode and interplacode regions (Table
1). Proliferation was detected in both regions. However, the
interplacodal area showed much higher mitotic index (38.6%) than the placode
area (24.4%) (P=0.004). Similar figures were obtained from analysis
of transgenic embryos: 42.9% of interplacode cells were BrdU positive compared
with 24.2% of placode cells (P<0.001). No differences in mitotic
figures were observed between wild-type and transgenic animals in placode area
(24.4% versus 24.2%, P=0.964), or in interplacode region (42.9%
versus 38.6%, P=0.338). This suggests that although increased Edar
signalling in the transgenics results in larger placodes, this phenomenon was
not due to propagated proliferation.
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Discussion |
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Our results from the analysis of Eda-A1-overexpressing mouse embryos
indicate that increased signalling via Eda-A1 receptor Edar promotes placodal
fate in developing mammary glands and teeth, as well as in the first wave of
hair follicles that give rise to guard hairs. In particular, the placodes of
molar teeth as well as guard hairs were increased in size. The more thorough
analysis of the effects Eda-A1 recombinant protein on hair placodes in organ
cultures indicated that the stimulation of placodal fate took place at the
expense of interplacode region, as evidenced by the expression patterns of
placodal marker genes as well as histology. Clearly, lateral inhibition
regulating the size as well as the distance of placodes from one another was
relieved. In principle, this could be achieved either by inhibiting the action
of molecules that mediate lateral inhibition or by promoting the action of
placodal activators. This may also account for the other phenomena typical to
K14-Eda-A1 mice we have reported earlier, i.e. the continuous
development of new hair follicles abnormally close to the pre-existing ones,
and the development of extra teeth and mammary glands
(Mustonen et al., 2003).
Recently, several other reports on transgenic mice overexpressing Eda-A1 have
been published (Srivastava et al.,
2001
; Cui et al., 2003; Zhang
et al., 2003
; Newton et al.,
2004
). None of these reported any changes in mammary glands or
teeth, whereas defects in distinct hair types were frequent. Cui et al. used a
full-length Eda-A1 and a Tet-inducible promoter, whereas Newton et al.
expressed the ectodomain of Eda-A1 under K5 or muscle specific myosin
light-chain 2 promoter. The two groups reported opposite results on the effect
of Eda-A1 on hair follicle density in adult mice. This, in principle, could be
taken as an indication of the role of Eda-A1 in the regulation of follicle
fate. Unfortunately, no embryonic analyses were made. Zhang et al. used the
involucrin promoter to drive the expression from E16.5 onwards, and thus no
effects on primary hair placodes could be expected. At later stages, they
found, at periodic locations, multiple hair follicles side by side, as well as
branched follicles, similar to previous reports
(Mustonen et al., 2003
). In
line with our results, occasionally hair buds with increased width were
detected, suggesting that Eda-A1 also affects the expansion of the secondary
placodes.
In the present study, we demonstrated that the placodes for the extra teeth
and mammary glands developed in close proximity to the pre-existing placodes
on the dental lamina and mammary line. During feather and hair development,
Bmps of the transforming growth factor ß (TGFß) family are the best
characterised candidates for placode inhibitors and mediators of lateral
inhibition, whereas during tooth and mammary gland development lateral
inhibition has been less studied. Ectopic expression of Bmp2 or Bmp4, or beads
releasing Bmp4 inhibit hair and feather placode formation
(Jung et al., 1998;
Noramly and Morgan, 1998
;
Botchkarev et al., 1999
), and
our data showed that the placode promoting effect of Eda-A1 was inhibited by
Bmp4. Forced expression of the Bmp inhibitor noggin during early feather
development results in fused and enlarged placodes
(Jung et al., 1998
;
Noramly and Morgan, 1998
).
Noggin also promotes murine hair placode formation
(Botchkarev et al., 1999
).
Hence, these phenotypes are very similar to the effects of increased Eda-A1
signalling we observed both in vivo and in vitro.
Molecules that promote placode fate include members of the Wnt and Fgf
pathway (Pispa and Thesleff,
2003). Exogenous Fgfs induce ectopic placodes in wild-type chick
embryos (Song et al., 1996
;
Jung et al., 1998
). In mice,
expression of a soluble dominant-negative form of Fgfr2 IIIb results in total
absence of hair follicles in the most severely affected embryos
(Celli et al., 1998
). Fgf
signalling is also indispensable for tooth and mammary gland development
(Trumpp et al., 1999
;
Mandler and Neubüser,
2001
; Mailleux et al.,
2002
). Canonical Wnt activity is mediated by the co-operation of
ß-catenin with transcription factors of the Lef1/TCF family.
Interestingly, forced stabilisation and activation of ß-catenin in
developing chick skin can override lateral inhibition and elicit a placodal
response in interfollicular ectoderm
(Noramly et al., 1999
). This
very much resembles the response of mouse skin to excess of Eda-A1.
Constitutively active ß-catenin can also induce ectopic hairs
(Gat et al., 1998
), whereas
lack of ß-catenin in the developing ectoderm inhibits hair follicle
initiation (Huelsken et al.,
2001
). In conclusion, the effect of Eda-A1 is highly similar to
the best characterised proteins promoting hair follicle fate.
What is then the position of Edar signalling in the hierarchy of molecules
regulating placodal cell fate? It is well established that the primary
inductive signal at least during hair and mammary gland development is derived
from the mesenchyme, whereas both Eda and Edar are expressed by the ectoderm
(Laurikkala et al., 2001;
Laurikkala et al., 2002
). We
were not able to accelerate the initiation of hair follicles either in vivo or
in vitro by exogenous Eda-A1, suggesting that Eda-A1 cannot override the
normal requirement of the first dermal signal. Accordingly, the initial
development of the mammary line and five mammary primodia were normal in
K14-Eda-A1 mice. In addition, the dental lamina, the incisor and
molar placodes were normally defined in time and space. Ectopic mammary and
tooth placodes were detected slightly later and only next to already formed
placodes within the already specified morphogenetic fields, mammary line and
dental lamina, respectively. All these data combined with earlier results (see
Introduction) suggest that Eda-A1 does not regulate the primary induction of
epithelial placodes, but instead plays a pivotal role in the next phase, i.e.
in the expansion of the placodes. In this process, Eda-A1 signalling
apparently is very high in hierarchy. The fact that in the absence of Eda
activity only some placodes are totally missing (primary and some of the
secondary hair placodes but not tooth and mammary gland primordia) may merely
reflect redundancy with another TNF family member or with a parallel
signalling route required for the same process. Intriguingly, TNFRSF19, an
orphan TNF receptor with sequence similarity to Edar shows close co-expression
with Edar in tooth, hair and mammary placodes
(Pispa et al., 2003
).
The cellular mechanism regulating epithelial placode formation has been a
neglected area of research, despite being a matter of dispute for decades
(Magerl et al., 2001;
Pispa and Thesleff, 2003
;
Veltmaat et al., 2003
). Our
results have two main implications. First, hair placode formation seems not to
result from a locally enhanced proliferation of placodal cells. Almost twice
as much proliferation was detected in the interplacode area as compared with
the placode area. These results are in line with data from Wessells and
Roessner (Wessells and Roessner, 1965) who studied 3H-thymidine
incorporation and mitotic figures of hair follicle cells. They found that
primary hair and vibrissae placodes were almost devoid of label, and the
incorporation of label continued only after the placode had invaginated in the
mesenchyme. Similar results were obtained during feather development
(Wessells, 1965). However, the contrary has been suggested based on the fact
the hair placodes in newborn mice (giving rise to auchene and zigzag hairs)
are positive for the proliferation marker Ki67
(Magerl et al., 2001
). During
mammary gland development, the hypothesis of cell migration being the driving
force underlying the formation of mammary placodes has got more support
(Veltmaat et al., 2003
). As in
hair and feather placodes, the mammary region has lower mitotic index than the
adjacent ectoderm (Balinsky,
1950
). In addition, the shape of the epithelial cells of the
mammary line (Propper, 1978
)
as well as the dynamic expression profiles of mammary placode marker genes
(Mailleux et al., 2002
) have
been interpreted as strong evidence to support cell migration as the primary
mechanism of placode formation.
The second implication of our BrdU incorporation data is that the specific
role of Edar signalling during the growth of the placode appears not to
involve cell proliferation. The mitotic indices of wild-type and
K14-Eda-A1 epidermis were highly similar, yet the placodes of
transgenic animals were larger, indicating that Eda-A1 regulates their growth
via another mechanism. In addition, we have not been able to detect enhanced
proliferation in skin explants using Eda-A1-releasing beads (T.M. and M.L.M.,
unpublished). Moreover, Eda promoted not only hair but also tooth and mammary
placode fate, suggesting that it has a similar role during the early stages of
the development of most ectodermal appendages. The next task is now to reveal
what cellular processes Eda-A1-Edar signalling controls. We find it unlikely
that Eda-A1 suppresses apoptosis, as the epidermis of Tabby embryos
does not show increased cell death (J.L. and I.T., unpublished) nor does the
enamel knot, the site of Edar expression of the developing molar
(Koppinen et al., 2001).
Regulation of cell-cell contacts is intimately involved in hair follicle
formation and downregulation of the intercellular adhesion molecule E-cadherin
appears to be essential for hair placode formation
(Jamora et al., 2003
). This is
achieved through the concerted action of a Bmp inhibition, which is required
for the expression of Lef1, and of a Wnt molecule, which is required for the
stabilisation of ß-catenin to make a functional Lef1/ß-catenin
complex that directly binds to E-cadherin promoter. Eda-A1 could be involved
in the control of cell adhesion either directly or indirectly. However, it
could promote the migration of epithelial cells towards the placode and by
this means regulate the growth of the placode. Of course, the effects of
Eda-A1 need not be direct and could be mediated via other known
placode-promoting signals. The downstream responses of Edar are probably
mediated by transcription factor NF-
B as Edar signalling activates the
NF-
B pathway in cultured cells (Yan et al., 2000;
Koppinen et al., 2001
;
Kumar et al., 2001
), and
mutations in molecules required for the NF-
B activation result in HED
in humans (Zonana et al.,
2000
; Smahi et al.,
2002
; Courtois et al.,
2003
). However, the target genes of Edar signalling are still
unknown.
In conclusion, we have shown that Eda-A1-Edar signalling stimulates the
formation of ectodermal placodes in hair, teeth and mammary glands. However,
Eda-A1 did not affect the initiation of the first forming placodes in any of
the organ systems, indicating that it apparently does not influence the
initial patterning of the organs. It is plausible that the defects in the
patterning and morphogenesis of the ectodermal organs in humans and mice with
loss of function of Eda-Edar signalling as well as in the transgenic mice
overexpressing Eda-A1 result from early effects on ectodermal placode
formation. The significance of an early function of Eda-A1 is supported by
recent experiments where the Tabby phenotype was rescued by providing
recombinant Eda-A1 into the uteri of pregnant mice
(Gaide and Schneider, 2003).
Finally, our observations on the effects of increased Eda-A1 function both in
vivo and in vitro indicate that it stimulates placode enlargement by a
mechanism not involving cell proliferation. It apparently affects the balance
of activators and inhibitors, and impairs the lateral inhibition mechanism
responsible for both the expansion of the placodes and initiation of
successive placodes.
![]() |
ACKNOWLEDGMENTS |
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Footnotes |
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Present address: University Medical Center, 1 Rue Michel Servet, 1211
Geneva, Switzerland
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