RESEARCH ARTICLE 2681
Development 138, 2681-2691 (2011) doi:10.1242/dev.057711
© 2011. Published by The Company of Biologists Ltd
Ectodysplasin and Wnt pathways are required for salivary
gland branching morphogenesis
Otso Häärä1, Sayumi Fujimori2,*, Ruth Schmidt-Ullrich3, Christine Hartmann2, Irma Thesleff1,†
and Marja L. Mikkola1,†
SUMMARY
The developing submandibular salivary gland (SMG) is a well-studied model for tissue interactions and branching morphogenesis.
Its development shares similar features with other ectodermal appendages such as hair and tooth. The ectodysplasin (Eda)
pathway is essential for the formation and function of several ectodermal organs. Mutations in the signaling components of the
Eda pathway lead to a human syndrome known as hypohidrotic ectodermal dysplasia (HED), which is characterized by missing
and malformed teeth, sparse hair and reduced sweating. Individuals with HED suffer also from dry mouth because of reduced
saliva flow. In order to understand the underlying mechanism, we analyzed salivary gland development in mouse models with
altered Eda pathway activities. We have found that Eda regulates growth and branching of the SMG via transcription factor NFkB in the epithelium, and that the hedgehog pathway is an important mediator of Eda/NF-kB. We also sought to determine
whether a similar reciprocal interplay between the Eda and Wnt/b-catenin pathways, which are known to operate in other skin
appendages, functions in developing SMG. Surprisingly and unlike in developing hair follicles and teeth, canonical Wnt signaling
activity did not colocalize with Edar/NF-kB in salivary gland epithelium. Instead, we observed high mesenchymal Wnt activity and
show that ablation of mesenchymal Wnt signaling either in vitro or in vivo compromised branching morphogenesis. We also
provide evidence suggesting that the effects of mesenchymal Wnt/b-catenin signaling are mediated, at least in part, through
regulation of Eda expression.
INTRODUCTION
Ectodysplasin belongs to the tumor necrosis factor (TNF) family
of signaling molecules and its function has been shown to be
vital for the formation of the ectodermal organs in vertebrates
from teleost fish to mammals (Kondo et al., 2001; Laurikkala et
al., 2001; Laurikkala et al., 2002). The ectodysplasin pathway
includes the ligand ectodysplasin A1 (Eda-A1, hereafter Eda),
the receptor Edar and the adapter molecule Edaradd; mutations
in any of the genes encoding these proteins lead to a human
disease called hypohidrotic ectodermal dysplasia (HED; MIM
305100 and 224900), with the characteristic features being
missing or mis-shapen teeth, sparse hair, and absent or reduced
sweating (Kere et al., 1996; Mikkola, 2009). The mouse mutants
for the same components of the ectodysplasin pathway are called
Tabby (Eda), Downless (Edar) and Crinkled (Edaradd)
(Srivastava et al., 1997; Headon and Overbeek, 1999; Headon et
al., 2001). These mice phenocopy individuals with HED: they
have hair abnormalities, defective dentition and lack sweat
glands. Numerous studies have shown that Eda operates
throughout the development of skin appendages, from initiation
to late morphogenesis and differentiation (for a review, see
Mikkola, 2009).
1
Developmental Biology Program, Institute of Biotechnology, University of Helsinki,
P.O.B. 56, 00014 Helsinki, Finland. 2Research Institute of Molecular Pathology, Dr
Bohrgasse 7, 1030 Vienna, Austria. 3Max-Delbrück-Center for Molecular Medicine,
Robert-Rössle-Str. 10, 13125 Berlin, Germany.
*Present address: Laboratory of Biological Imaging, Immunology Frontier Research
Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
†
Authors for correspondence ([email protected]; [email protected])
Accepted 14 April 2011
Individuals with HED suffer from dry mouth caused by
dysfunctional salivary glands and heterozygous female carriers
are best identified by reduced saliva flow and altered saliva
protein composition (Lexner et al., 2007). Despite the obvious
connection to human disease, very little is known about the
defects and the pathogenic mechanisms of the salivary gland
defects in the mouse models. The submandibular salivary glands
(SMG) of the Eda deficient mouse model (Tabby) have reduced
weight and less granular convoluted tubules in the adult,
indicating permanent dysfunction in saliva production (Blecher
et al., 1983). Eda-null glands are reported to have less ductal
structures, whereas Eda-overexpressing K14-Eda mice show
larger lumens (Nordgarden et al., 2004) and adult mice with
enhanced Edar signaling activity were reported to have more
elaborately branched salivary glands (Chang et al., 2009).
Another study reported that Eda-deficient SMGs are hypoplastic,
whereas Edar deficient SMGs are severely dysplastic and
suggested involvement of the Eda pathway in lumen formation
(Jaskoll et al., 2003). Addition of recombinant Eda protein to
salivary glands in culture has been shown to increase epithelial
branching (Jaskoll et al., 2003). These few reports indicate that
Eda influences SMG morphogenesis. Interestingly, Eda is
expressed in the mesenchyme of the developing mouse
submandibular salivary glands, whereas in all other ectodermal
organs studied so far, Eda expression is confined to epithelium
(Pispa et al., 2003). The receptor Edar is expressed in the
epithelium of the submandibular glands as in other ectodermal
appendages. Currently, information about the mechanism of Eda
regulation of SMG development, as well as a detailed analysis
of the branching phenotypes of Eda loss- and gain-of-function
mice, is lacking.
DEVELOPMENT
KEY WORDS: Tabby, Ectodysplasin, Edar, Salivary gland, HED, Branching morphogenesis, NF-kB, Wnt, Sonic hedgehog
2682 RESEARCH ARTICLE
MATERIALS AND METHODS
Animals
The following mouse strains and their genotyping have been described
earlier: K14-Eda (Mustonen et al., 2003), Eda deficient (Tabby) (Pispa et
al., 1999), IkBaDN (Schmidt-Ullrich et al., 2001) and NF-kB REP mice
(Bhakar et al., 2002). Tabby mice were of CBAB6 background, K14-Eda
mice were of FVB background and NF-kB REP of C57Bl/6 background.
NF-kB REP mice were bred into Eda–/– and K14-Eda backgrounds to
monitor NF-kB activity. IkBaDN mice were in a C57Bl/6 background, and
were crossed with NF-kB REP mice for the analysis of NF-kB reporter
expression. BATgal mice (Maretto et al., 2003) were kindly provided by
Stefano Piccolo (University of Padua, Italy) and maintained in C57Bl/6;
TOPgal mice (DasGupta and Fuchs, 1999) and B6;129SGt(ROSA)26Sor/J
(Rosa 26) mice were from Jackson Laboratories; Axin2/ConductinLacZ/LacZ
(Axin2LacZ/LacZ) mice (Lustig et al., 2002) were kindly provided by Walter
Birchmeier (MDC, Berlin, Germany). Heterozygous Shh-CreGFP mice
(Harfe et al., 2004) expressing green fluorescent protein under Shh
promoter, were maintained in NMRI background. To obtain DermoCre+;
b-cat–/fl mice, male mice of the genotype DermoCre (neo-); b-catlacZ/+
(Huelsken et al., 2000) were crossed with female mice of the genotype bcatfl/fl (Huelsken et al., 2001). The FRT-neo cassette of the DermoCre mice
(Yu et al., 2003) had been removed using the FLPeR mice (Farley et al.,
2000). The appearance of a vaginal plug was taken as embryonic day (E)
0, and embryos were carefully staged according to limb morphogenesis.
Organ cultures
Submandibular/sublingual salivary gland rudiments (referred to as SMGs)
were cultivated in Trowell-type culture as described previously (Fliniaux
et al., 2008; Häärä et al., 2009). In short, the explants were cultured in airliquid interface on filters (Whatman Nuclepore, 0.1 mM) in Dulbecco’s
minimum essential medium (DMEM) supplemented with 10% fetal calf
serum (PAA laboratories, Pasching, Austria), 2 mM glutamine, 100 U/ml
penicillin and 100 mg/ml streptomycin. For cultures longer than 2 days,
DMEM and F12 medium (1:1) was used and supplemented with 10% fetal
calf serum, ascorbic acid (0.075 g/l), glutamine and penicillinstreptomycin. The following proteins or inhibitors were used at
concentrations indicated in the text: Fc-Eda-A1 (Gaide and Schneider,
2003), sonic hedgehog (R&D Systems), CKI-7 (Sigma-Aldrich),
cyclopamine (Sigma-Aldrich) and XAV939 (Stemgent). For microscopy
and morphometric analyses, the samples were imaged under Olympus
SZX9 stereomicroscope or under Olympus AX70 microscope. Data were
analyzed by freely available ImageJ software (NCBI). All statistical
comparisons were made between mutants and their littermate controls,
except for Eda–/– mice, which were kept as a homozygous colony. For
number of explants analyzed, see figure legends and Fig. S1 in the
supplementary material.
Histology
The tissues were fixed in 4% paraformaldehyde and taken through ethanol
series and xylene into paraffin and sectioned at 7 mm for histology. The size
of the salivary gland epithelium of DermoCre+; b-cat–/fl mice and their
littermate controls was measured from serial tissue sections: the area of the
middle (largest) cross-section (maxA) of the epithelium in each gland was
measured (in mm2) using ImageJ software and the diameter (d) of the
glands was calculated from the number of sections times the thickness of
one section (7 mm). The size of the gland was calculated from the equation:
size=maxA ⫻ d. The mean number of end buds in DermoCre+; b-cat–/fl
SMG was counted from the same tissues sections.
In situ hybridization
The embryonic tissues were fixed in 4% paraformaldehyde (PFA) for 24
hours in +4°C and dehydrated through methanol series (25, 50, 75, 100%).
Whole-mount in situ hybridization was performed with InSituPro robot
(Intavis AG, Germany) and section in situ hybridization as previously
described (Fliniaux et al., 2008) using probes specific to Wnt4, Wnt6 and
Wnt11 (Sarkar and Sharpe, 1999). Digoxigenin-labeled probes were
detected with BM Purple AP Substrate Precipitating Solution (Boehringer
Mannheim Gmbh, Germany). Radioactive in situ hybridization on paraffin
sections was carried out according to standard protocols using 35S-UTP
labeling (Amersham) and probes specific to Shh, Dhh, Ptch1, Eda and
Edar (Bitgood and McMahon, 1995; Laurikkala et al., 2002). The
quantification of Eda expression was carried out using ImageJ from darkfield images (threshold=200), measuring the mean luminosity in the
mesenchyme in each gland.
DEVELOPMENT
The development of the submandibular gland in the mouse
embryo begins at ~E12, when the epithelium invaginates into the
underlying mesenchyme and forms a bud (Patel et al., 2006;
Tucker, 2007). The first signs of epithelial branching
morphogenesis are detected at E13, when clefts are formed in the
growing epithelial bud, finally giving rise to multiple separate buds.
These buds thereafter branch continuously throughout the prenatal
development and early postnatal development, but the most intense
branching is detected between E13 and E15. The differentiation of
the ducts starts at E14.5-E15, when the first columnar epithelial
cells appear in the proximal end of the main duct. Thereafter, the
epithelium differentiates into saliva-producing terminal buds (acini)
and transporting ducts (Borghese, 1950).
The factors regulating SMG morphogenesis have been studied
intensively during past 10 years (Patel et al., 2006; Tucker, 2007).
The roles of the fibroblast growth factor (Fgf) family members,
including Fgf1, Fgf3, Fgf7, Fg8 and Fgf10 and their receptors, as
well the function of the epidermal growth factor (Egf) pathway
have been well characterized in submandibular salivary gland
development (Kashimata and Gresik, 1997; Hoffman et al., 2002;
Koyama et al., 2008; Häärä et al., 2009). Fgf proteins and Egf
family ligands act in concert with extracellular matrix and
basement membrane components, such as fibronectin, collagen IV,
matrix metalloproteinases and laminins (Sakai et al., 2003; Larsen
et al., 2006; Tucker, 2007).
Much less is known about the roles of the other conserved signal
families that regulate the development of other ectodermal organs.
In vitro studies have suggested that sonic hedgehog (Shh) signaling
regulates epithelial duct formation in salivary glands (Hashizume
and Hieda, 2006) and also indicated involvement of Shh in
branching morphogenesis (Jaskoll et al., 2004a). One of the key
players in ectodermal organ formation is the Wnt/b-catenin
(hereafter Wnt/b-cat) pathway. The canonical Wnt pathway was
recently implicated in postnatal salivary gland development and
regeneration (Hai et al., 2010), but practically nothing is known
about its role during embryonic salivary gland development. The
role of Eda has been addressed in few studies that have indicated
the stimulatory effect of Eda signaling on salivary gland
development (see above). However, the pathogenesis behind the
salivary gland defects associated with HED, and the intracellular
mediators and downstream targets of Eda, have not been
characterized in detail.
To address the molecular mechanisms that lead to impaired
function of salivary glands in individuals with HED, we analyzed
submandibular salivary gland development in Eda mutant mice,
both loss- and gain-of-function, followed by in vitro
morphometrical and functional studies. We report that branching is
highly dependent on Eda activity and that NF-kB is an essential
mediator of Eda signaling. We provide evidence to indicate that
Shh is a crucial downstream component of Eda/Edar/NF-kB
pathway in the salivary gland and is required to mediate Edainduced branching morphogenesis. Our data also reveal that
suppression of mesenchymal Wnt/b-cat signaling leads to
decreased SMG branching morphogenesis accompanied by
downregulation of Eda expression.
Development 138 (13)
Ectodysplasin and Wnt in salivary gland
RESEARCH ARTICLE 2683
X-gal staining
Embryos were fixed for 30 minutes in 2% paraformaldehyde, 0.2%
glutaraldehyde, washed three times for 10 minutes in Dulbecco’s PBS (pH
7.5) containing 2 mM MgCl2 and 0.02% Nonidet P-40. Samples were
stained overnight at room temperature with X-gal staining solution [1
mg/ml X-gal, 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2 and 10%
Nonidet P-40 in PBS (pH 7.5)]. Whole-mount stained tissues were postfixed in 4% PFA overnight at +4°C, embedded in paraffin and sectioned at
7 mm.
Quantitative PCR
Dissected salivary glands were placed into 350 ml lysis buffer of the
RNeasy mini kit (Qiagen) containing 1% b-mercaptoethanol (Sigma). Total
RNA was isolated as specified according to the manufacturer’s instructions
and quantified using a nanodrop spectrophotometer. A minimum of eight
samples were analyzed in each experiment. 100 ng of total RNA was
reverse transcribed using 500 ng of random hexamers (Promega) and 100
units of Superscript II (Invitrogen) following the manufacturer’s
instructions. Lightcycler DNA Master SYBR Green I (Roche) was used
with a Lightcycler 480, and the software provided by the manufacturer was
used for analysis. The data were normalized against Ranbp gene (Ranbp1
– Mouse Genome Informatics) (Fliniaux et al., 2008). Primer sequences
are available upon request. Gene expression was quantified by comparing
the sample data against a dilution series of PCR products of the gene of
interest.
RESULTS
Branching depends on, and correlates with, Eda
activity
To study the effect of Eda signaling on branching morphogenesis
of the salivary glands, we focused on the early stages of branching
at E13-E15, and used a well-established ex vivo culture system for
submandibular salivary glands (SMG), which is known to
recapitulate the branching pattern that occurs in vivo and allows an
accurate quantification of forming branches (Borghese, 1950; Patel
et al., 2006). SMG development was compared between Eda lossof-function (Eda deficient, Tabby), wild-type and Eda gain-offunction (K14-Eda) mice. The SMG rudiments were dissected at
E13 and cultured for 2 days. The glands of Eda–/– mice were
similar to wild type in appearance at the onset of branching, at
E12.5-E13. However, after 2 days of culture, the epithelium was
less branched and smaller, with significantly fewer (~65%) end
buds compared with wild type (Fig. 1A,B,D). Overexpression of
Eda under the K14 promoter led to a substantial increase in
branching and end bud formation (Fig. 1C,D).
Shh is a crucial target of Eda in the salivary gland
Shh has been shown to be involved in SMG development (Jaskoll
et al., 2004a), and there is evidence that Shh may be a direct
downstream target of Eda in hair follicles (Cui et al., 2006;
Schmidt-Ullrich et al., 2006; Pummila et al., 2007). Therefore, we
analyzed the connection between Eda and Shh in the developing
salivary gland. Quantification of Shh transcripts in Eda null, control
and K14-Eda transgenic SMGs at E14 revealed that the amount of
Shh correlated with the Eda status (Fig. 2A). However, Shh
expression was not completely absent in Eda–/– glands, indicating
that other factors besides Eda are likely to regulate Shh expression.
Supplementation of Shh protein to cultured E13 and E14 SMG was
previously shown to increase branching morphogenesis, whereas
hedgehog pathway antagonist cyclopamine suppressed branching
(Jaskoll et al., 2004a). To test whether the effect of Shh signaling
depends on the level of Eda expression, we treated E13 Eda–/–,
control and K14-Eda salivary glands with recombinant Shh protein
(500 ng/ml). We observed a prominent increase in branching in
Eda-null explants, a significantly weaker response in control and
no apparent effect in K14-Eda SMGs (Fig. 2B). Next, we
compared the ability of Shh and recombinant Eda protein (Fc-Eda
500 ng/ml) (Gaide and Schneider, 2003) to restore the Eda–/–
phenotype. Both Fc-Eda and Shh induced a similar, about 1.4-fold,
increase in the number of end buds (Fig. 2C-F). The rescue was not
complete though, as the average number of end buds after 2 days
culture was less than in wild-type glands (see Fig. S1 in the
supplementary material). By contrast, treatment of E13 Shh+/–
salivary glands, which were less branched than wild type, for 2
days with Eda protein, had no significant effect, yet Shh protein
rescued branching as expected (see Fig. S2 in the supplementary
material).
To test whether changes caused by differences in Eda expression
levels were affected by suppression of hedgehog signaling,
cyclopamine (5 mM) was added to salivary glands dissected from
E13 Eda–/–, control and K14-Eda embryos. Cyclopamine reversed
excessive branching in K14-Eda glands significantly and reduced
branching clearly in control glands, but had no effect on Eda–/–
glands (Fig. 2G-M). Thus, inhibition of hedgehog signaling by
cyclopamine in wild-type and K14-Eda salivary glands
phenocopied Eda–/– salivary glands. Taken together, the effect of
both enhancing and inhibiting hedgehog pathway was strictly
dependent on Eda signaling activity.
If Shh was a downstream target of Eda in the developing salivary
gland, Shh transcripts should be localized in the epithelium where
Edar expression and signaling activity is confined (Pispa et al.,
2003) (see also below). Radioactive in situ hybridization with a
Shh-specific probe revealed low level of Shh expression in the
epithelium of wild-type salivary glands, which was augmented in
K14-Eda glands, but was similar to negative control in Eda–/–
glands (Fig. 3A-C). In addition, Ptch1, a transcriptional target of
Shh, was expressed at low levels in developing SMGs (see Fig. S2
in the supplementary material). To address whether other hedgehog
DEVELOPMENT
Fig. 1. Eda regulates SMG branching morphogenesis. E13 SMGs were cultured for 2 days. (A-C) Severe reduction in epithelial branching was
seen in Eda–/– salivary glands compared with wild type, whereas overexpression of Eda under the keratin 14 promoter in K14-Eda mice led to a
significant increase in branch number. (D) Mean number of end buds+s.e.m. for data shown in A-C. **P<0.01, ***P<0.001.
2684 RESEARCH ARTICLE
Development 138 (13)
Fig. 2. Shh mediates the effects of Eda in developing SMG. (A) The
in vivo steady-state expression level of Shh was quantified by qRT-PCR.
The amount of Shh mRNA was reduced in E14 Eda–/– salivary glands and
increased in K14-Eda glands compared with wild type. (B-M) E13 SMGs
were cultured for 2 days. (B) Comparison of the effect of Shh protein
(fold-increase in the number of end buds) on Eda–/–, wild-type and K14Eda SMGs. Shh protein induced a nearly 1.5-fold increase in the number
of end buds in Eda–/– SMGs (n=14), had a slight effect (1.2-fold increase)
on wild-type glands (n=11) and no effect on K14-Eda salivary glands
(n=7). (C) Mean number of end buds+s.e.m. for data shown in D-F. Eda–/–
SMGs were cultured in the control medium, or in the presence of Shh or
Eda protein. (D-F) Shh (n=13) and Eda (n=12) proteins increased
branching of Eda–/– SMGs to the same extent. (G-L) Effect of the
hedgehog pathway inhibitor cyclopamine (5 mM) on Eda–/– (n=10), wildtype (n=6) and K14-Eda (n=6) SMGs. Branching was strongly reduced in
wild-type and K14-Eda glands, while Eda–/– SMGs did not respond to
cyclopamine. (M) Number of end buds±s.e.m. for data shown in G-L.
*P<0.05, **P<0.01.
family members, desert (Dhh) and Indian hedgehog (Ihh), could be
involved in SMG morphogenesis, we analyzed their expression by
qRT-PCR at E13 and E14. Ihh expression levels were very low at
both stages, whereas the amount of Dhh transcripts exceeded that
of Shh (Fig. 3D). However, expression of Dhh was not localized to
the epithelium (Fig. 3E) and did not differ significantly between
Eda-null, wild-type and K14-Eda salivary glands (Fig. 3F). Taken
together, our results (Figs 2, 3) indicate that Shh is an important
downstream mediator of Eda in SMG branching morphogenesis.
These observations, however, do not exclude the possible role of
other hedgehog family members in salivary gland morphogenesis.
Eda signaling in SMG is mediated by NF-kB
In developing teeth and hair follicles, the effects of Eda are
mediated largely by transcription factor nuclear factor-kB (NF-kB)
(Schmidt-Ullrich et al., 2001; Pispa et al., 2008). Inhibition of
NF-kB in vitro by cell-permeable peptide SN50 was reported to
inhibit SMG branching morphogenesis (Melnick et al., 2001a).
However, more recent studies by the same group (Melnick et al.,
2009) suggested that NF-kB activity is not essential for Eda
signaling in the salivary gland. To study this obvious discrepancy,
we first analyzed NF-kB activity with a NF-kB lacZ reporter
mouse (Bhakar et al., 2002; Pispa et al., 2008) in the developing
salivary gland. At all stages studied (E12-E17), we observed strong
NF-kB signaling activity in the epithelium (Fig. 4A-E; see Fig. S3
in the supplementary material), where also Edar was expressed
(Fig. 4F,G) (Pispa et al., 2003). However, NF-kB activity was not
distributed equally throughout the epithelium, but was more intense
in the outermost cell layer (Fig. 4B-E) adjacent to the mesenchymal
source of Eda (Pispa et al., 2003).
Next, we analyzed the NF-kB reporter activity in the Eda–/–
background. We did not detect NF-kB activity in Eda-null salivary
glands at any stage studied (E12-E17) (Fig. 4H,I; and data not
shown), indicating that NF-kB signaling in SMG is entirely
dependent on Eda. Furthermore, increased NF-kB reporter activity
was observed in K14-Eda transgenic embryos compared with their
control littermates (Fig. 4J; and data not shown). To study whether
Eda can induce NF-kB activity in vitro, we treated Eda–/–; NF-kB
reporter glands with different concentrations of Eda protein and
followed the expression of the reporter. After 1 day of treatment, NFkB activity appeared dose dependently in Eda–/– salivary glands (Fig.
4K-M). However, even at the highest tested concentration of Fc-Eda,
the NF-kB activity was lower than in wild-type glands cultured
under the same conditions (Fig. 4N), and was limited to the margins
of the epithelium more strictly than in control glands. This suggests
that the recombinant protein had a limited ability to penetrate through
the salivary tissue ex vivo. This finding may also explains the
incomplete rescue of Eda–/– salivary glands by the Eda protein (Fig.
2C; see Fig. S1 in the supplementary material).
DEVELOPMENT
Fig. 3. Expression of hedgehog family members in developing
SMGs. (A-C) Expression of Shh was analyzed by radioactive in situ
hybridization at E14. Expression of Shh in SMG epithelium (arrows)
correlated with the expression level of Eda, and pronounced
upregulation of Shh was detected in K14-Eda SMGs. (D) Expression of
Shh, Dhh and Ihh at E13 and E14 was determined by qRT-PCR. Data are
mean+s.e.m. (E) Radioactive in situ hybridization revealed that Dhh
expression did not localize to the epithelium. (F) Quantification of Dhh
transcripts by qRT-PCR in Eda–/–, wild-type and K14-Eda SMGs showed
that expression of Dhh did not correlate with the Eda pathway activity
at E14. Data are mean+s.e.m.
Ectodysplasin and Wnt in salivary gland
RESEARCH ARTICLE 2685
In order to study the importance of NF-kB signaling in salivary
gland branching morphogenesis, we analyzed IkBaDN mice, which
lack NF-kB activity (Schmidt-Ullrich et al., 2001). Suppression of
NF-kB signaling in these mice is achieved by ubiquitous
expression of the transdominant super-repressor IkBaDN. Analysis
of NF-kB reporter expression confirmed that NF-kB activity was
completely inhibited in SMGs of IkBaDN embryos (Fig. 5A-C).
Importantly, loss of NF-kB activity led to a similar branching
defect as seen in Eda-null mice (Fig. 5D,E,H; compare to Fig. 1).
To test whether overexpression of Eda can induce branching
independently of NF-kB activity, we crossed IkBaDN mice with
K14-Eda mice and analyzed the branching pattern of cultured
SMGs. IkBaDN and compound IkBaDN; K14-Eda mutants
showed a similar branching defect, with reduced branching
compared with wild-type and K14-Eda littermates (Fig. 5D-H).
These findings show that Eda-induced branching is completely
dependent on NF-kB in developing salivary glands.
Wnt activity is restricted to the mesenchyme
during early stages of salivary gland development
In many ectodermal appendages, canonical Wnt activity
colocalizes with Edar/NF-kB activity in the epithelium and studies
on hair follicles have shown that Wnt and Eda pathways regulate
partially the same epithelial target genes (Fliniaux et al., 2008;
Zhang et al., 2009). Moreover, in hair follicles Wnt/b-cat pathway
has been placed upstream of Edar, which in turn is thought to
regulate expression of Wnt ligands (Zhang et al., 2009). To address
whether a similar interplay between the two pathways operates also
in developing salivary glands, we analyzed the localization of Wnt
activity using the Axin2-lacZ reporter mice (Lustig et al., 2002).
At E13-E14, Axin2-lacZ expression was entirely confined to the
mesenchyme surrounding the branching epithelium and was
strikingly different from the NF-kB lacZ reporter expression (Fig.
6A,A⬘,A⬙,B,B⬘,B⬙; compare to Fig. 4A-E). Only at E15, when the
main salivary duct starts to differentiate and forms columnar
epithelium, was Axin2-lacZ detected in the ductal epithelium in
addition to mesenchyme (Fig. 6C,C⬘,C⬙). From E16 onwards, Wnt
activity was exclusively epithelial and localized to the developing
ducts (Fig. 6D,D⬘,D⬙). Side-by-side comparison of Axin2 and NFkB reporter tissues confirmed mirror-image Wnt and NF-kB
activities (Fig. 6E,F), and even when Wnt activity was present in
the epithelium, it was strictly limited to the ducts, whereas NF-kB
activity was localized to the branching buds and alveoli (Fig.
6G,H). Analysis of two other Wnt/b-catenin reporters, TOP-gal and
BAT-gal (DasGupta and Fuchs, 1999; Maretto et al., 2003), did not
reveal any canonical Wnt activity in the branching epithelium at
DEVELOPMENT
Fig. 4. NF-kB activity is dependent on
Eda signaling. NF-kB reporter activity
was analyzed by b-gal whole-mount
staining in developing SMGs. (A) b-Gal
activity (blue) was localized to the
epithelium throughout prenatal salivary
gland development. (B-E) Sections of
whole-mount stained tissues show that
NF-kB reporter expression was highest in
the epithelial cell layer closest to the
mesenchyme. C and E are higher
magnifications of B and D, respectively.
(F,G) Expression of Edar was localized to
the epithelium by radioactive in situ
hybridization. (H-J) In vivo, NF-kB reporter
expression was absent in Eda–/– SMGs and
increased in K14-Eda transgenic embryos.
(K-N) E13 Eda–/–;NF-kB REP or wild-type
NF-kB REP salivary glands were cultured
for 1 day. (K) No NF-kB activity was
observed in Eda–/– salivary glands (arrow).
(L,M) Application of recombinant Eda to
Eda–/– explants induced the reporter
expression in a dose-dependent manner.
Arrows indicate NF-kB reporter
expression. (N) NF-kB reporter expression
was higher in cultured wild-type SMGs
than in Eda-treated Eda–/– explants.
Compare N with L and M. Arrows indicate
NF-kB reporter expression. Scale bars:
500 mm for A,B,D,F,H-N; 100 mm for
C,E,G.
2686 RESEARCH ARTICLE
Development 138 (13)
E13 to E15 either (see Fig. S4 in the supplementary material).
Thus, the complementary activities of NF-kB and Axin2 reporters
in epithelium and mesenchyme, respectively, suggest that the
situation in salivary glands may differ from other ectodermal
appendages.
Inhibition of mesenchymal Wnt/b-catenin
signaling impairs SMG branching morphogenesis
To assess the significance of mesenchymal Wnt signaling in the
early branching morphogenesis of SMG, we analyzed the effect of
Wnt inhibition in vitro and in vivo. First, we treated E13 wild-type
salivary glands for 2 days with the Wnt signaling inhibitors
XAV939 (Huang et al., 2009) or CKI-7 (Peters et al., 1999;
Fliniaux et al., 2008) (Fig. 7A-D). As the reporter studies indicated
that there was no canonical Wnt signaling in the epithelium at these
stages, these inhibitors were expected to affect mesenchymal Wnt
activity only. Twenty-four hour treatment with XAV939 (10 mM or
100 mM) did not have any obvious effect, yet 2 day treatment led
to a significant reduction in epithelial branching and growth (Fig.
7E), which resembled the Eda-null and IkBaDN mutant
phenotypes. Under these conditions, XAV939 inhibited Wnt
signaling, visualized by reduced lacZ expression in Axin2-lacZ
SMGs (see Fig. S5 in the supplementary material). In addition,
treatment with 100 mM CKI-7 caused a decrease in the number of
end buds (Fig. 7F). To validate these in vitro findings, we analyzed
conditional b-catenin-null mice, DermoCre+; b-cat–/fl mice, in
which b-catenin is deleted using Cre expressed in the mesenchyme.
Analysis of lacZ expression in DermoCre mice crossed with the
Rosa26 reporter mice confirmed widespread mesenchymal Cre
activity in SMG at E14 (data not shown). SMGs of DermoCre+; bcat–/fl were analyzed at E13 to E15, and showed a significantly
smaller size and reduced branching as compared with control
littermates (Fig. 7G-I). Collectively, these results imply that
mesenchymal Wnt signaling is required for the growth and
branching of the submandibular salivary gland. The identity of the
Wnt ligand(s) regulating SMG morphogenesis is currently unclear,
but we detected Wnt4 and Wnt6 initially in the mesenchyme (E13),
and 1 day later in the epithelium. In addition, Wnt11 was expressed
in the mesenchyme (see Fig. S4 in the supplementary material).
Lef1, a transcription factor mediating Wnt/b-cat signaling, has
been suggested to regulate the expression of Eda, and a
conserved Lef1-binding site is found in the promoter region of
Eda (Durmowicz et al., 2002; Laurikkala et al., 2001; Srivastava
et al., 1997); therefore, we tested whether mesenchymal
expression of Eda in developing SMG was affected by inhibition
of mesenchymal Wnt signaling. Twenty-four hour treatment of
E13 SMGs with 100 mM XAV939 led to a notable, but
statistically non-significant, reduction in the amount of Eda
mRNA (data not shown). Importantly, in situ hybridization
revealed that expression of Eda was downregulated in vivo in
salivary glands of DermoCre+; b-cat–/fl embryos compared with
their control littermates (Fig. 7J-L). These findings led us to
hypothesize that Eda could be an important downstream
mediator of the Wnt/b-cat pathway in developing SMGs. If this
was the case, exogenous Eda protein should overcome the
branching defect caused by suppressed Wnt/b-cat activity. To
test this, we cultured control and XAV939-treated E13 salivary
glands for 2 days in the presence of recombinant Eda protein
(Fig. 7M-P). We observed that Fc-Eda had a significant restoring
effect on branching of XAV939-treated SMG explants, the
number of end buds being normalized close that of control
explants (Fig. 7O-Q). Moreover, the ability of Eda to stimulate
branching was substantially higher in XAV939-treated glands
compared with control glands (Fig. 7R).
DEVELOPMENT
Fig. 5. NF-kB activity is required for Eda-mediated
branching morphogenesis. (A-C) Expression of the NF-kB
reporter was lost in the IkBaDN background. (D-G) E13
SMGs were cultured for 2 days. SMGs of IkBaDN embryos
(n=14) displayed reduced branching compared with control
littermates (n=6), while branching was increased in K14-Eda
littermates (n=10). Eda-induced branching was completely
suppressed in compound I-kBaDN; K14-Eda mutants (n=9).
Scale bars: 500 mm. (H) Mean number of end buds+s.e.m.
for data shown in D-G. ***P<0.001.
Ectodysplasin and Wnt in salivary gland
RESEARCH ARTICLE 2687
DISCUSSION
Owing to the apparent phenotypic similarity of Eda mutant (Tabby)
mice and humans carrying inactivating mutations in Eda, the mouse
models have turned out to be highly useful tools with which to
unravel the molecular pathogenesis of HED. Thus far, the glandular
aspects of HED have not been studied intensively, although over 20
different exocrine glands are known to be affected in Eda mutant
mice and/or individuals with HED (Clarke et al., 1987; Grüneberg,
1971). In general, the glands are either missing or hypoplastic. The
first case is exemplified by sweat glands, tracheal submucosal glands
and minor salivary glands where a lack of the initial outgrowth leads
to organ agenesis (Grüneberg, 1971; Rawlins and Hogan, 2005;
Kunisada et al., 2009; Wells et al., 2011). For most glandular organs,
however, the Eda pathway appears dispensable for initiation, but is
required either for epithelial growth, branching and/or
cytodifferentiation (Grüneberg, 1971). Here, we report a
comprehensive morphological and molecular analysis of salivary
gland development in mice mutant for the Eda/NF-kB pathway using
the ex vivo SMG explant culture system. We show that loss of Eda
compromised SMG growth and branching, whereas excess of Eda
led to highly accelerated branching morphogenesis. We propose that
the diminished branching of embryonic Eda-null glands eventually
leads to smaller salivary glands with reduced epithelial surface area
resulting in decreased saliva production seen in individuals with
HED. In addition, the saliva composition is altered in individuals
with HED (Lexner et al., 2007), but it is currently unclear whether
this reflects a functional defect in mature salivary glands or whether
the development of distinct salivary glands (sublingual,
submandibular and parotid), which are known to produce unequal
types of saliva (Tucker, 2007), is differentially affected by the lack
of Eda.
Our analysis of mice with suppressed NF-kB signaling activity,
singly and in combination with mice overexpressing Eda,
conclusively demonstrated the necessity of Eda/Edar/NF-kB for
SMG morphogenesis. Furthermore, the ability of Eda to regulate
salivary gland development relied on intact NF-kB activity.
Analysis of NF-kB reporter mice revealed that activation of NFkB is entirely dependent on Eda during embryonic SMG
development and not, for example, on TNFa, as recently suggested
(Melnick et al., 2009). Previous studies have shown that
supplementation of TNFa substantially increases epithelial cell
proliferation and branching of cultured SMGs (Melnick et al.,
2001b), and in light of our findings it is possible that as a powerful
inducer of NF-kB, exogenous TNFa may have mimicked the effect
of Eda, as previously shown in embryonic Eda–/– skin explants
where TNFa rescued primary hair placode formation (SchmidtUllrich et al., 2006). Contrary to previous speculations (Jaskoll et
al., 2003), analysis of the NF-kB reporter mice indicated that the
Eda/NF-kB pathway is active already at the earliest developmental
stages, although our data show that it is dispensable for initiation
DEVELOPMENT
Fig. 6. Wnt reporter activity does not
colocalize with NF-kB activity at any
stage. (A-D⬙) Wnt/b-cat activity in
developing SMGs was assessed using
Axin2LacZ/+ embryos. (A-B⬙) b-Gal activity
localized to the mesenchyme at E13-E14;
compare with NF-kB reporter expression in
Fig. 4A-E. (C-C⬙) At E15, Axin2-lacZ
expression was detected both in the
mesenchyme and the main epithelial duct.
(D-D⬙) At E16 Axin2-lacZ was solely
observed in developing ducts. Black arrows
highlight Axin2-lacZ expression, white
arrows its absence. (E-H) Comparison of
Axin2-lacZ and the NF-kB reporter activities
in wholemounts revealed non-overlapping
expression at E15 and E16. At E16, the
ducts (broken line in H) were positive for
Wnt but not for NF-kB reporter, whereas
the developing alveoli were positive for NFkB but not for Wnt activity. Scale bars: 500
mm for A-D and A⬘-D⬘; 100 mm for A⬙-D⬙.
2688 RESEARCH ARTICLE
Development 138 (13)
of SMG morphogenesis. The similarity of the SMG phenotypes of
Eda-null and IkBaDN mutant mice and the absence of any residual
NF-kB signaling activity in both mutants demonstrate that lack of
an early phenotype is not due to redundancy with another pathway
activating NF-kB.
Shh is an important mediator of Eda signaling in
the developing salivary gland
Downstream target genes of the Edar/NF-kB pathway have been
under intensive study during recent years. Shh, Wnt10a and
Wnt10b, Wnt pathway inhibitor Dkk4, and BMP inhibitors
Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor)
and follistatin have been shown to be downstream of the Eda
pathway in hair and/or tooth development (Cui et al., 2006;
Schmidt-Ullrich et al., 2006; Pummila et al., 2007; Fliniaux et al.,
2008; Zhang et al., 2009). Of the confirmed or suspected
transcriptional targets of Eda, Shh is so far the only one studied in
the context of salivary gland development (Jaskoll et al., 2004a).
Here, we report that the response of cultured SMGs to the
manipulation of the hedgehog pathway, either by application of
Shh protein or by inhibition with cyclopamine, is crucially
dependent on Eda. These data combined with the fact that the
expression level of Shh positively correlated with Eda signaling
activity in vivo, implicates Shh as a key mediator downstream of
Eda in developing salivary glands. While our manuscript was under
review, Wells et al. (Wells et al., 2010) reported the rescue of an
Edar-deficient SMG branching defect (which was similar to that
seen in Eda-null embryos) produced by recombinant Shh protein,
further confirming the importance of Shh downstream of Eda/Edar.
Although the function of Shh in SMG morphogenesis is not
entirely clear, Fgf8, a potent mitogen that is essential for the growth
of SMG (Jaskoll et al., 2004b), has been proposed to act downstream
of Shh, as exogenous Fgf8 was shown to rescue the growth defect of
cyclopamine-treated SMGs (Jaskoll et al., 2004a). In line with this,
qRT-PCR analysis of Eda-null SMGs at later stages (E16 to
newborn) indicated that both Shh and Fgf8 were downregulated
(Melnick et al., 2009). Wells et al. (Wells et al., 2010) reported that
treatment with Fgf8 protein did not rescue the branching defect of
Edar mutant SMGs, but did increase bud surface area. It is likely that
many signaling molecules, acting either in the same or in parallel
pathways, function as proliferative cues to promote epithelial growth
and branching of SMG (Patel et al., 2006).
DEVELOPMENT
Fig. 7. Suppression of mesenchymal Wnt/b-cat activity
reduces branching and size of the SMG. (A-D) E13 SMGs
were cultured for 2 days in the presence of tankyrase inhibitor
XAV939 or casein kinase inhibitor CKI-7. XAV939 [10 mM
(n=16) and 100 mM (n=6)] reduced branching by 40%
compared with controls; 100 mM CKI-7 also had a similar
effect. (E) Mean number of end buds±s.e.m. of SMGs cultured
for 1 or 2 days in the presence of XAV939. (F) Mean number
of end buds+s.e.m. of SMGs cultured for 2 days in the
presence of CKI-7. (G,H) Histology of the salivary glands of
DermoCre+; b-cat–/fl embryos at E14. Deletion of
mesenchymal b-catenin led to an overall smaller size and
fewer branches compared with control littermates. Scale bars:
500 mm. (I) Quantification of the epithelial size and the
number of end buds from serial sections in DermoCre+; bcat–/fl embryos at E13 to E15 (n=3 for E13; n=6 for E14, and
n=3 for E15) in comparison with control littermates. Values for
control SMGs were set at 1 (broken line). Data are
mean+s.e.m. (J,K) Radioactive in situ hybridization showed
reduced expression of Eda in DermoCre+; b-cat–/fl salivary
glands compared with control littermates at E14. (L) In situ
hybridization signal of mesenchymal Eda expression was
quantified in E14 DermoCre+; b-cat–/fl (n=4) and control
salivary glands (n=4), four sections per sample were analyzed.
Data are mean+s.e.m. (M-P) E13 salivary glands were cultured
for 2 days in the presence or absence of 500 ng/ml of
recombinant Eda and 10 mM XAV939. Eda protein restored the
XAV939-induced branching defect (n≥6 in each group).
(Q) Mean number of end buds+s.e.m. of data shown in M-P.
(R) Response to Eda (fold increase in number of end buds) was
significantly higher in XAV939-treated (n=15) salivary glands
compared with untreated (n=6) ones. Data are mean+s.e.m.
***P<0.001, **P<0.01, *P<0.05.
Fig. 8. Regulation of salivary gland morphogenesis by Eda
signaling and its integration with Wnt and Shh pathways. Eda
expression is restricted to the SMG mesenchyme and is downstream of
Wnt/b-cat signaling. Eda is synthesized as an integral membrane
protein and its cleavage is required for biological activity. Eda
translocates to the epithelial compartment and binds to its receptor
Edar, which ultimately leads to the activation of the transcription factor
NF-kB. The degradation of the repressor IkBa promotes the transport of
NF-kB to the nucleus where it induces the expression of Shh and other
target genes essential for epithelial growth and branching.
It is conceivable that Shh is not the only important target gene
of the Eda pathway in the salivary gland. Besides Shh, exogenous
Eda protein induces the expression of follistatin in cultured
embryonic skin explants (Pummila et al., 2007). Although the
function of endogenous follistatin in salivary gland development is
unknown, it may have a role as an antagonist of activin, which
causes a severe disruption of SMG morphogenesis when applied
ex vivo (Ritvos et al., 1995). With increasing knowledge of the
downstream events of the Eda pathway (Mikkola, 2009), novel
target genes and their functional relevance are likely to be
uncovered and will be tested in the future.
Mesenchymal Wnt signaling is required for Eda
expression and SMG branching morphogenesis
Zhang et al. (Zhang et al., 2009) recently showed a sequential
interdependency between the Wnt and Eda pathways in developing
hair follicles: Wnt/b-cat signaling is essential for NF-kB activation
whereas Edar/NF-kB is thereafter required to strengthen and
maintain Wnt/b-cat activity. The link between the two pathways is
further supported by the conspicuous similarities of individuals
carrying mutations in Eda/Edar/Edaradd or in Wnt10a (Adaimy et
al., 2007; Cluzeau et al., 2011) (OMIM 257980), which is one of
the proposed targets of Edar/NF-kB (Zhang et al., 2009).
Moreover, the transcriptional targets of the two pathways are
partially overlapping (Fliniaux et al., 2008; Zhang et al., 2009), and
in developing hair, tooth and mammary buds, high Wnt/b-cat and
NF-kB signaling activities colocalize in the epithelium (Chu et al.,
2004; Liu et al., 2008; Pispa et al., 2008; Zhang et al., 2009).
Surprisingly, we did not find a similar correlation between Wnt and
NF-kB reporter expression patterns in developing salivary glands,
suggesting that unlike in hair follicles, Wnt/ b-cat and Eda/NF-kB
may not collaborate in regulation of target gene expression in
SMG.
RESEARCH ARTICLE 2689
The role of Wnt/b-cat signaling in branching morphogenesis has
not been studied in detail in cutaneous glands, with the exception
of mammary glands (for a review, see Boras-Granic and
Wysolmerski, 2008). High epithelial Wnt reporter activity is seen
during onset of embryonic branching morphogenesis (Chu et al.,
2004), and suppression of canonical Wnt activity via deletion of
the co-receptor Lrp6 compromises branching development
(Lindvall et al., 2009). In developing salivary glands, we did not
detect Wnt pathway activity in branching buds or alveoli with any
of the three reporters analyzed. The reporter studies seem to
suggest that epithelial Wnt signaling activity does not regulate
branching morphogenesis in the SMGs, but may instead be
involved in ductal differentiation and lumen formation.
Numerous studies have revealed the importance of epithelialmesenchymal interactions in salivary gland development, and
classic tissue recombination experiments suggest that the branching
pattern is controlled largely by signals from the mesenchyme (Patel
et al., 2006; Tucker, 2007). Our results indicate that mesenchymal
Wnt/b-cat activity regulates SMG morphogenesis. Inhibition of
Wnt signaling either in vivo or in vitro produced smaller glands
with fewer end buds. This function may be transient though, as
mesenchymal expression of Axin2-lacZ reporter vanished by E16.
The mechanism for how mesenchymal Wnt/b-cat signaling
regulates epithelial growth and branching has yet to be clarified,
but the Wnt target genes may include paracrine factors such as
some of the many Fgfs (Hoffman et al., 2002; Patel et al., 2006;
Steinberg et al., 2005). Our data suggest that transcriptional
regulation of Eda may be one important function of mesenchymal
Wnt/b-cat signaling, and that Eda in turn acts as a mesenchymal
cue to regulate epithelial branching via NF-kB and Shh (Fig. 8).
Acknowledgements
We thank Dr Walter Birchmeier and Dr Stefano Piccolo for mice; Dr Pascal
Schneider for Fc-Eda-A1; and Merja Mäkinen, Riikka Santalahti and Raija
Savolainen for excellent technical assistance. The work was financially
supported by the Academy of Finland, Sigrid Jusélius Foundation and Viikki
Graduate School in Molecular Biosciences.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.057711/-/DC1
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Ectodysplasin and Wnt in salivary gland