ectodysplasin and wnt pathways are required for salivary ... · the developing submandibular...

2681RESEARCH ARTICLE

INTRODUCTIONEctodysplasin belongs to the tumor necrosis factor (TNF) familyof signaling molecules and its function has been shown to bevital for the formation of the ectodermal organs in vertebratesfrom teleost fish to mammals (Kondo et al., 2001; Laurikkala etal., 2001; Laurikkala et al., 2002). The ectodysplasin pathwayincludes the ligand ectodysplasin A1 (Eda-A1, hereafter Eda),the receptor Edar and the adapter molecule Edaradd; mutationsin any of the genes encoding these proteins lead to a humandisease called hypohidrotic ectodermal dysplasia (HED; MIM305100 and 224900), with the characteristic features beingmissing or mis-shapen teeth, sparse hair, and absent or reducedsweating (Kere et al., 1996; Mikkola, 2009). The mouse mutantsfor the same components of the ectodysplasin pathway are calledTabby (Eda), Downless (Edar) and Crinkled (Edaradd)(Srivastava et al., 1997; Headon and Overbeek, 1999; Headon etal., 2001). These mice phenocopy individuals with HED: theyhave hair abnormalities, defective dentition and lack sweatglands. Numerous studies have shown that Eda operatesthroughout the development of skin appendages, from initiationto late morphogenesis and differentiation (for a review, seeMikkola, 2009).

Individuals with HED suffer from dry mouth caused bydysfunctional salivary glands and heterozygous female carriersare best identified by reduced saliva flow and altered salivaprotein composition (Lexner et al., 2007). Despite the obviousconnection to human disease, very little is known about thedefects and the pathogenic mechanisms of the salivary glanddefects in the mouse models. The submandibular salivary glands(SMG) of the Eda deficient mouse model (Tabby) have reducedweight and less granular convoluted tubules in the adult,indicating permanent dysfunction in saliva production (Blecheret al., 1983). Eda-null glands are reported to have less ductalstructures, whereas Eda-overexpressing K14-Eda mice showlarger lumens (Nordgarden et al., 2004) and adult mice withenhanced Edar signaling activity were reported to have moreelaborately branched salivary glands (Chang et al., 2009).Another study reported that Eda-deficient SMGs are hypoplastic,whereas Edar deficient SMGs are severely dysplastic andsuggested involvement of the Eda pathway in lumen formation(Jaskoll et al., 2003). Addition of recombinant Eda protein tosalivary glands in culture has been shown to increase epithelialbranching (Jaskoll et al., 2003). These few reports indicate thatEda influences SMG morphogenesis. Interestingly, Eda isexpressed in the mesenchyme of the developing mousesubmandibular salivary glands, whereas in all other ectodermalorgans studied so far, Eda expression is confined to epithelium(Pispa et al., 2003). The receptor Edar is expressed in theepithelium of the submandibular glands as in other ectodermalappendages. Currently, information about the mechanism of Edaregulation of SMG development, as well as a detailed analysisof the branching phenotypes of Eda loss- and gain-of-functionmice, is lacking.

Development 138, 2681-2691 (2011) doi:10.1242/dev.057711© 2011. Published by The Company of Biologists Ltd

1Developmental Biology Program, Institute of Biotechnology, University of Helsinki,P.O.B. 56, 00014 Helsinki, Finland. 2Research Institute of Molecular Pathology, DrBohrgasse 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 ResearchCenter, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan†Authors for correspondence ([email protected]; [email protected])

Accepted 14 April 2011

SUMMARYThe 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 theEda pathway lead to a human syndrome known as hypohidrotic ectodermal dysplasia (HED), which is characterized by missingand malformed teeth, sparse hair and reduced sweating. Individuals with HED suffer also from dry mouth because of reducedsaliva flow. In order to understand the underlying mechanism, we analyzed salivary gland development in mouse models withaltered Eda pathway activities. We have found that Eda regulates growth and branching of the SMG via transcription factor NF-kB in the epithelium, and that the hedgehog pathway is an important mediator of Eda/NF-kB. We also sought to determinewhether a similar reciprocal interplay between the Eda and Wnt/b-catenin pathways, which are known to operate in other skinappendages, functions in developing SMG. Surprisingly and unlike in developing hair follicles and teeth, canonical Wnt signalingactivity did not colocalize with Edar/NF-kB in salivary gland epithelium. Instead, we observed high mesenchymal Wnt activity andshow that ablation of mesenchymal Wnt signaling either in vitro or in vivo compromised branching morphogenesis. We alsoprovide evidence suggesting that the effects of mesenchymal Wnt/b-catenin signaling are mediated, at least in part, throughregulation of Eda expression.

KEY WORDS: Tabby, Ectodysplasin, Edar, Salivary gland, HED, Branching morphogenesis, NF-kB, Wnt, Sonic hedgehog

Ectodysplasin and Wnt pathways are required for salivarygland branching morphogenesisOtso Häärä1, Sayumi Fujimori2,*, Ruth Schmidt-Ullrich3, Christine Hartmann2, Irma Thesleff1,†

and Marja L. Mikkola1,†

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The development of the submandibular gland in the mouseembryo begins at ~E12, when the epithelium invaginates into theunderlying mesenchyme and forms a bud (Patel et al., 2006;Tucker, 2007). The first signs of epithelial branchingmorphogenesis are detected at E13, when clefts are formed in thegrowing epithelial bud, finally giving rise to multiple separate buds.These buds thereafter branch continuously throughout the prenataldevelopment and early postnatal development, but the most intensebranching is detected between E13 and E15. The differentiation ofthe ducts starts at E14.5-E15, when the first columnar epithelialcells appear in the proximal end of the main duct. Thereafter, theepithelium differentiates into saliva-producing terminal buds (acini)and transporting ducts (Borghese, 1950).

The factors regulating SMG morphogenesis have been studiedintensively 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, aswell the function of the epidermal growth factor (Egf) pathwayhave been well characterized in submandibular salivary glanddevelopment (Kashimata and Gresik, 1997; Hoffman et al., 2002;Koyama et al., 2008; Häärä et al., 2009). Fgf proteins and Egffamily ligands act in concert with extracellular matrix andbasement membrane components, such as fibronectin, collagen IV,matrix metalloproteinases and laminins (Sakai et al., 2003; Larsenet al., 2006; Tucker, 2007).

Much less is known about the roles of the other conserved signalfamilies that regulate the development of other ectodermal organs.In vitro studies have suggested that sonic hedgehog (Shh) signalingregulates epithelial duct formation in salivary glands (Hashizumeand Hieda, 2006) and also indicated involvement of Shh inbranching morphogenesis (Jaskoll et al., 2004a). One of the keyplayers in ectodermal organ formation is the Wnt/b-catenin(hereafter Wnt/b-cat) pathway. The canonical Wnt pathway wasrecently implicated in postnatal salivary gland development andregeneration (Hai et al., 2010), but practically nothing is knownabout its role during embryonic salivary gland development. Therole of Eda has been addressed in few studies that have indicatedthe stimulatory effect of Eda signaling on salivary glanddevelopment (see above). However, the pathogenesis behind thesalivary gland defects associated with HED, and the intracellularmediators and downstream targets of Eda, have not beencharacterized in detail.

To address the molecular mechanisms that lead to impairedfunction of salivary glands in individuals with HED, we analyzedsubmandibular salivary gland development in Eda mutant mice,both loss- and gain-of-function, followed by in vitromorphometrical and functional studies. We report that branching ishighly dependent on Eda activity and that NF-kB is an essentialmediator of Eda signaling. We provide evidence to indicate thatShh is a crucial downstream component of Eda/Edar/NF-kBpathway in the salivary gland and is required to mediate Eda-induced branching morphogenesis. Our data also reveal thatsuppression of mesenchymal Wnt/b-cat signaling leads todecreased SMG branching morphogenesis accompanied bydownregulation of Eda expression.

MATERIALS AND METHODSAnimalsThe following mouse strains and their genotyping have been describedearlier: K14-Eda (Mustonen et al., 2003), Eda deficient (Tabby) (Pispa etal., 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 tomonitor NF-kB activity. IkBaDN mice were in a C57Bl/6 background, andwere crossed with NF-kB REP mice for the analysis of NF-kB reporterexpression. BATgal mice (Maretto et al., 2003) were kindly provided byStefano 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 WalterBirchmeier (MDC, Berlin, Germany). Heterozygous Shh-CreGFP mice(Harfe et al., 2004) expressing green fluorescent protein under Shhpromoter, 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 b-catfl/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 culturesSubmandibular/sublingual salivary gland rudiments (referred to as SMGs)were cultivated in Trowell-type culture as described previously (Fliniauxet al., 2008; Häärä et al., 2009). In short, the explants were cultured in air-liquid interface on filters (Whatman Nuclepore, 0.1 mM) in Dulbecco’sminimum essential medium (DMEM) supplemented with 10% fetal calfserum (PAA laboratories, Pasching, Austria), 2 mM glutamine, 100 U/mlpenicillin and 100 mg/ml streptomycin. For cultures longer than 2 days,DMEM and F12 medium (1:1) was used and supplemented with 10% fetalcalf serum, ascorbic acid (0.075 g/l), glutamine and penicillin-streptomycin. The following proteins or inhibitors were used atconcentrations 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 microscopyand morphometric analyses, the samples were imaged under OlympusSZX9 stereomicroscope or under Olympus AX70 microscope. Data wereanalyzed by freely available ImageJ software (NCBI). All statisticalcomparisons were made between mutants and their littermate controls,except for Eda–/– mice, which were kept as a homozygous colony. Fornumber of explants analyzed, see figure legends and Fig. S1 in thesupplementary material.

HistologyThe tissues were fixed in 4% paraformaldehyde and taken through ethanolseries and xylene into paraffin and sectioned at 7 mm for histology. The sizeof the salivary gland epithelium of DermoCre+; b-cat–/fl mice and theirlittermate controls was measured from serial tissue sections: the area of themiddle (largest) cross-section (maxA) of the epithelium in each gland wasmeasured (in mm2) using ImageJ software and the diameter (d) of theglands was calculated from the number of sections times the thickness ofone 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 hybridizationThe embryonic tissues were fixed in 4% paraformaldehyde (PFA) for 24hours 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 previouslydescribed (Fliniaux et al., 2008) using probes specific to Wnt4, Wnt6 andWnt11 (Sarkar and Sharpe, 1999). Digoxigenin-labeled probes weredetected with BM Purple AP Substrate Precipitating Solution (BoehringerMannheim Gmbh, Germany). Radioactive in situ hybridization on paraffinsections was carried out according to standard protocols using 35S-UTPlabeling (Amersham) and probes specific to Shh, Dhh, Ptch1, Eda andEdar (Bitgood and McMahon, 1995; Laurikkala et al., 2002). Thequantification of Eda expression was carried out using ImageJ from dark-field images (threshold=200), measuring the mean luminosity in themesenchyme in each gland.

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X-gal stainingEmbryos were fixed for 30 minutes in 2% paraformaldehyde, 0.2%glutaraldehyde, washed three times for 10 minutes in Dulbecco’s PBS (pH7.5) containing 2 mM MgCl2 and 0.02% Nonidet P-40. Samples werestained overnight at room temperature with X-gal staining solution [1mg/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 post-fixed in 4% PFA overnight at +4°C, embedded in paraffin and sectioned at7 mm.

Quantitative PCRDissected salivary glands were placed into 350 ml lysis buffer of theRNeasy mini kit (Qiagen) containing 1% b-mercaptoethanol (Sigma). TotalRNA was isolated as specified according to the manufacturer’s instructionsand quantified using a nanodrop spectrophotometer. A minimum of eightsamples were analyzed in each experiment. 100 ng of total RNA wasreverse transcribed using 500 ng of random hexamers (Promega) and 100units of Superscript II (Invitrogen) following the manufacturer’sinstructions. Lightcycler DNA Master SYBR Green I (Roche) was usedwith a Lightcycler 480, and the software provided by the manufacturer wasused for analysis. The data were normalized against Ranbp gene (Ranbp1– Mouse Genome Informatics) (Fliniaux et al., 2008). Primer sequencesare available upon request. Gene expression was quantified by comparingthe sample data against a dilution series of PCR products of the gene ofinterest.

RESULTSBranching depends on, and correlates with, EdaactivityTo study the effect of Eda signaling on branching morphogenesisof the salivary glands, we focused on the early stages of branchingat E13-E15, and used a well-established ex vivo culture system forsubmandibular salivary glands (SMG), which is known torecapitulate the branching pattern that occurs in vivo and allows anaccurate quantification of forming branches (Borghese, 1950; Patelet al., 2006). SMG development was compared between Eda loss-of-function (Eda deficient, Tabby), wild-type and Eda gain-of-function (K14-Eda) mice. The SMG rudiments were dissected atE13 and cultured for 2 days. The glands of Eda–/– mice weresimilar to wild type in appearance at the onset of branching, atE12.5-E13. However, after 2 days of culture, the epithelium wasless branched and smaller, with significantly fewer (~65%) endbuds compared with wild type (Fig. 1A,B,D). Overexpression ofEda under the K14 promoter led to a substantial increase inbranching and end bud formation (Fig. 1C,D).

Shh is a crucial target of Eda in the salivary glandShh has been shown to be involved in SMG development (Jaskollet al., 2004a), and there is evidence that Shh may be a directdownstream target of Eda in hair follicles (Cui et al., 2006;

Schmidt-Ullrich et al., 2006; Pummila et al., 2007). Therefore, weanalyzed the connection between Eda and Shh in the developingsalivary gland. Quantification of Shh transcripts in Eda null, controland K14-Eda transgenic SMGs at E14 revealed that the amount ofShh correlated with the Eda status (Fig. 2A). However, Shhexpression was not completely absent in Eda–/– glands, indicatingthat other factors besides Eda are likely to regulate Shh expression.Supplementation of Shh protein to cultured E13 and E14 SMG waspreviously shown to increase branching morphogenesis, whereashedgehog pathway antagonist cyclopamine suppressed branching(Jaskoll et al., 2004a). To test whether the effect of Shh signalingdepends 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 inEda-null explants, a significantly weaker response in control andno apparent effect in K14-Eda SMGs (Fig. 2B). Next, wecompared the ability of Shh and recombinant Eda protein (Fc-Eda500 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 notcomplete though, as the average number of end buds after 2 daysculture was less than in wild-type glands (see Fig. S1 in thesupplementary material). By contrast, treatment of E13 Shh+/–

salivary glands, which were less branched than wild type, for 2days with Eda protein, had no significant effect, yet Shh proteinrescued branching as expected (see Fig. S2 in the supplementarymaterial).

To test whether changes caused by differences in Eda expressionlevels were affected by suppression of hedgehog signaling,cyclopamine (5 mM) was added to salivary glands dissected fromE13 Eda–/–, control and K14-Eda embryos. Cyclopamine reversedexcessive branching in K14-Eda glands significantly and reducedbranching clearly in control glands, but had no effect on Eda–/–

glands (Fig. 2G-M). Thus, inhibition of hedgehog signaling bycyclopamine in wild-type and K14-Eda salivary glandsphenocopied Eda–/– salivary glands. Taken together, the effect ofboth enhancing and inhibiting hedgehog pathway was strictlydependent on Eda signaling activity.

If Shh was a downstream target of Eda in the developing salivarygland, Shh transcripts should be localized in the epithelium whereEdar expression and signaling activity is confined (Pispa et al.,2003) (see also below). Radioactive in situ hybridization with aShh-specific probe revealed low level of Shh expression in theepithelium of wild-type salivary glands, which was augmented inK14-Eda glands, but was similar to negative control in Eda–/–

glands (Fig. 3A-C). In addition, Ptch1, a transcriptional target ofShh, was expressed at low levels in developing SMGs (see Fig. S2in the supplementary material). To address whether other hedgehog

2683RESEARCH ARTICLEEctodysplasin and Wnt in salivary gland

Fig. 1. Eda regulates SMG branching morphogenesis. E13 SMGs were cultured for 2 days. (A-C) Severe reduction in epithelial branching wasseen in Eda–/– salivary glands compared with wild type, whereas overexpression of Eda under the keratin 14 promoter in K14-Eda mice led to asignificant 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.

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family members, desert (Dhh) and Indian hedgehog (Ihh), could beinvolved in SMG morphogenesis, we analyzed their expression byqRT-PCR at E13 and E14. Ihh expression levels were very low atboth stages, whereas the amount of Dhh transcripts exceeded thatof Shh (Fig. 3D). However, expression of Dhh was not localized tothe epithelium (Fig. 3E) and did not differ significantly betweenEda-null, wild-type and K14-Eda salivary glands (Fig. 3F). Takentogether, our results (Figs 2, 3) indicate that Shh is an importantdownstream mediator of Eda in SMG branching morphogenesis.These observations, however, do not exclude the possible role ofother hedgehog family members in salivary gland morphogenesis.

Eda signaling in SMG is mediated by NF-kBIn developing teeth and hair follicles, the effects of Eda aremediated 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 toinhibit 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 Edasignaling in the salivary gland. To study this obvious discrepancy,we first analyzed NF-kB activity with a NF-kB lacZ reportermouse (Bhakar et al., 2002; Pispa et al., 2008) in the developingsalivary gland. At all stages studied (E12-E17), we observed strongNF-kB signaling activity in the epithelium (Fig. 4A-E; see Fig. S3in the supplementary material), where also Edar was expressed(Fig. 4F,G) (Pispa et al., 2003). However, NF-kB activity was notdistributed equally throughout the epithelium, but was more intensein the outermost cell layer (Fig. 4B-E) adjacent to the mesenchymalsource 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 salivaryglands at any stage studied (E12-E17) (Fig. 4H,I; and data notshown), indicating that NF-kB signaling in SMG is entirelydependent on Eda. Furthermore, increased NF-kB reporter activitywas observed in K14-Eda transgenic embryos compared with theircontrol littermates (Fig. 4J; and data not shown). To study whetherEda can induce NF-kB activity in vitro, we treated Eda–/–; NF-kBreporter glands with different concentrations of Eda protein andfollowed the expression of the reporter. After 1 day of treatment, NF-kB 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 culturedunder the same conditions (Fig. 4N), and was limited to the marginsof the epithelium more strictly than in control glands. This suggeststhat the recombinant protein had a limited ability to penetrate throughthe salivary tissue ex vivo. This finding may also explains theincomplete rescue of Eda–/– salivary glands by the Eda protein (Fig.2C; see Fig. S1 in the supplementary material).


Fig. 2. Shh mediates the effects of Eda in developing SMG. (A) Thein vivo steady-state expression level of Shh was quantified by qRT-PCR.The amount of Shh mRNA was reduced in E14 Eda–/– salivary glands andincreased in K14-Eda glands compared with wild type. (B-M) E13 SMGswere 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 K14-Eda SMGs. Shh protein induced a nearly 1.5-fold increase in the numberof 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 orEda protein. (D-F) Shh (n=13) and Eda (n=12) proteins increasedbranching of Eda–/– SMGs to the same extent. (G-L) Effect of thehedgehog pathway inhibitor cyclopamine (5mM) on Eda–/– (n=10), wild-type (n=6) and K14-Eda (n=6) SMGs. Branching was strongly reduced inwild-type and K14-Eda glands, while Eda–/– SMGs did not respond tocyclopamine. (M) Number of end buds±s.e.m. for data shown in G-L.*P<0.05, **P<0.01.

Fig. 3. Expression of hedgehog family members in developingSMGs. (A-C) Expression of Shh was analyzed by radioactive in situhybridization at E14. Expression of Shh in SMG epithelium (arrows)correlated with the expression level of Eda, and pronouncedupregulation of Shh was detected in K14-Eda SMGs. (D) Expression ofShh, Dhh and Ihh at E13 and E14 was determined by qRT-PCR. Data aremean+s.e.m. (E) Radioactive in situ hybridization revealed that Dhhexpression did not localize to the epithelium. (F) Quantification of Dhhtranscripts by qRT-PCR in Eda–/–, wild-type and K14-Eda SMGs showedthat expression of Dhh did not correlate with the Eda pathway activityat E14. Data are mean+s.e.m.

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In order to study the importance of NF-kB signaling in salivarygland branching morphogenesis, we analyzed IkBaDN mice, whichlack NF-kB activity (Schmidt-Ullrich et al., 2001). Suppression ofNF-kB signaling in these mice is achieved by ubiquitousexpression of the transdominant super-repressor IkBaDN. Analysisof NF-kB reporter expression confirmed that NF-kB activity wascompletely inhibited in SMGs of IkBaDN embryos (Fig. 5A-C).Importantly, loss of NF-kB activity led to a similar branchingdefect as seen in Eda-null mice (Fig. 5D,E,H; compare to Fig. 1).To test whether overexpression of Eda can induce branchingindependently of NF-kB activity, we crossed IkBaDN mice withK14-Eda mice and analyzed the branching pattern of culturedSMGs. IkBaDN and compound IkBaDN; K14-Eda mutantsshowed a similar branching defect, with reduced branchingcompared with wild-type and K14-Eda littermates (Fig. 5D-H).These findings show that Eda-induced branching is completelydependent on NF-kB in developing salivary glands.

Wnt activity is restricted to the mesenchymeduring early stages of salivary gland developmentIn many ectodermal appendages, canonical Wnt activitycolocalizes with Edar/NF-kB activity in the epithelium and studieson 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 pathwayhas been placed upstream of Edar, which in turn is thought toregulate expression of Wnt ligands (Zhang et al., 2009). To addresswhether a similar interplay between the two pathways operates alsoin developing salivary glands, we analyzed the localization of Wntactivity using the Axin2-lacZ reporter mice (Lustig et al., 2002).At E13-E14, Axin2-lacZ expression was entirely confined to themesenchyme surrounding the branching epithelium and wasstrikingly different from the NF-kB lacZ reporter expression (Fig.6A,A�,A�,B,B�,B�; compare to Fig. 4A-E). Only at E15, when themain salivary duct starts to differentiate and forms columnarepithelium, was Axin2-lacZ detected in the ductal epithelium inaddition to mesenchyme (Fig. 6C,C�,C�). From E16 onwards, Wntactivity was exclusively epithelial and localized to the developingducts (Fig. 6D,D�,D�). Side-by-side comparison of Axin2 and NF-kB reporter tissues confirmed mirror-image Wnt and NF-kBactivities (Fig. 6E,F), and even when Wnt activity was present inthe epithelium, it was strictly limited to the ducts, whereas NF-kBactivity was localized to the branching buds and alveoli (Fig.6G,H). Analysis of two other Wnt/b-catenin reporters, TOP-gal andBAT-gal (DasGupta and Fuchs, 1999; Maretto et al., 2003), did notreveal any canonical Wnt activity in the branching epithelium at


Fig. 4. NF-kB activity is dependent onEda signaling. NF-kB reporter activitywas analyzed by b-gal whole-mountstaining in developing SMGs. (A) b-Galactivity (blue) was localized to theepithelium throughout prenatal salivarygland development. (B-E) Sections ofwhole-mount stained tissues show thatNF-kB reporter expression was highest inthe epithelial cell layer closest to themesenchyme. C and E are highermagnifications of B and D, respectively.(F,G) Expression of Edar was localized tothe epithelium by radioactive in situhybridization. (H-J) In vivo, NF-kB reporterexpression was absent in Eda–/– SMGs andincreased in K14-Eda transgenic embryos.(K-N) E13 Eda–/–;NF-kB REP or wild-typeNF-kB REP salivary glands were culturedfor 1 day. (K) No NF-kB activity wasobserved in Eda–/– salivary glands (arrow).(L,M) Application of recombinant Eda toEda–/– explants induced the reporterexpression in a dose-dependent manner.Arrows indicate NF-kB reporterexpression. (N) NF-kB reporter expressionwas higher in cultured wild-type SMGsthan in Eda-treated Eda–/– explants.Compare N with L and M. Arrows indicateNF-kB reporter expression. Scale bars: 500mm for A,B,D,F,H-N; 100mm forC,E,G.

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E13 to E15 either (see Fig. S4 in the supplementary material).Thus, the complementary activities of NF-kB and Axin2 reportersin epithelium and mesenchyme, respectively, suggest that thesituation in salivary glands may differ from other ectodermalappendages.

Inhibition of mesenchymal Wnt/b-cateninsignaling impairs SMG branching morphogenesisTo assess the significance of mesenchymal Wnt signaling in theearly branching morphogenesis of SMG, we analyzed the effect ofWnt inhibition in vitro and in vivo. First, we treated E13 wild-typesalivary glands for 2 days with the Wnt signaling inhibitorsXAV939 (Huang et al., 2009) or CKI-7 (Peters et al., 1999;Fliniaux et al., 2008) (Fig. 7A-D). As the reporter studies indicatedthat there was no canonical Wnt signaling in the epithelium at thesestages, these inhibitors were expected to affect mesenchymal Wntactivity only. Twenty-four hour treatment with XAV939 (10 mM or100 mM) did not have any obvious effect, yet 2 day treatment ledto a significant reduction in epithelial branching and growth (Fig.7E), which resembled the Eda-null and IkBaDN mutantphenotypes. Under these conditions, XAV939 inhibited Wntsignaling, visualized by reduced lacZ expression in Axin2-lacZSMGs (see Fig. S5 in the supplementary material). In addition,treatment with 100 mM CKI-7 caused a decrease in the number ofend buds (Fig. 7F). To validate these in vitro findings, we analyzedconditional b-catenin-null mice, DermoCre+; b-cat–/fl mice, inwhich b-catenin is deleted using Cre expressed in the mesenchyme.Analysis of lacZ expression in DermoCre mice crossed with theRosa26 reporter mice confirmed widespread mesenchymal Creactivity in SMG at E14 (data not shown). SMGs of DermoCre+; b-cat–/fl were analyzed at E13 to E15, and showed a significantlysmaller size and reduced branching as compared with control

littermates (Fig. 7G-I). Collectively, these results imply thatmesenchymal Wnt signaling is required for the growth andbranching of the submandibular salivary gland. The identity of theWnt 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 expressedin the mesenchyme (see Fig. S4 in the supplementary material).

Lef1, a transcription factor mediating Wnt/b-cat signaling, hasbeen suggested to regulate the expression of Eda, and aconserved Lef1-binding site is found in the promoter region ofEda (Durmowicz et al., 2002; Laurikkala et al., 2001; Srivastavaet al., 1997); therefore, we tested whether mesenchymalexpression of Eda in developing SMG was affected by inhibitionof mesenchymal Wnt signaling. Twenty-four hour treatment ofE13 SMGs with 100 mM XAV939 led to a notable, butstatistically non-significant, reduction in the amount of EdamRNA (data not shown). Importantly, in situ hybridizationrevealed that expression of Eda was downregulated in vivo insalivary glands of DermoCre+; b-cat–/fl embryos compared withtheir control littermates (Fig. 7J-L). These findings led us tohypothesize that Eda could be an important downstreammediator of the Wnt/b-cat pathway in developing SMGs. If thiswas the case, exogenous Eda protein should overcome thebranching defect caused by suppressed Wnt/b-cat activity. Totest this, we cultured control and XAV939-treated E13 salivaryglands for 2 days in the presence of recombinant Eda protein(Fig. 7M-P). We observed that Fc-Eda had a significant restoringeffect on branching of XAV939-treated SMG explants, thenumber of end buds being normalized close that of controlexplants (Fig. 7O-Q). Moreover, the ability of Eda to stimulatebranching was substantially higher in XAV939-treated glandscompared with control glands (Fig. 7R).


Fig. 5. NF-kB activity is required for Eda-mediatedbranching morphogenesis. (A-C) Expression of the NF-kBreporter was lost in the IkBaDN background. (D-G) E13SMGs were cultured for 2 days. SMGs of IkBaDN embryos(n=14) displayed reduced branching compared with controllittermates (n=6), while branching was increased in K14-Edalittermates (n=10). Eda-induced branching was completelysuppressed in compound I-kBaDN; K14-Eda mutants (n=9).Scale bars: 500mm. (H) Mean number of end buds+s.e.m.for data shown in D-G. ***P<0.001.

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DISCUSSIONOwing to the apparent phenotypic similarity of Eda mutant (Tabby)mice and humans carrying inactivating mutations in Eda, the mousemodels have turned out to be highly useful tools with which tounravel the molecular pathogenesis of HED. Thus far, the glandularaspects of HED have not been studied intensively, although over 20different exocrine glands are known to be affected in Eda mutantmice and/or individuals with HED (Clarke et al., 1987; Grüneberg,1971). In general, the glands are either missing or hypoplastic. Thefirst case is exemplified by sweat glands, tracheal submucosal glandsand minor salivary glands where a lack of the initial outgrowth leadsto 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 isrequired either for epithelial growth, branching and/orcytodifferentiation (Grüneberg, 1971). Here, we report acomprehensive morphological and molecular analysis of salivarygland development in mice mutant for the Eda/NF-kB pathway usingthe ex vivo SMG explant culture system. We show that loss of Edacompromised SMG growth and branching, whereas excess of Edaled to highly accelerated branching morphogenesis. We propose thatthe diminished branching of embryonic Eda-null glands eventuallyleads to smaller salivary glands with reduced epithelial surface arearesulting in decreased saliva production seen in individuals withHED. In addition, the saliva composition is altered in individuals

with HED (Lexner et al., 2007), but it is currently unclear whetherthis reflects a functional defect in mature salivary glands or whetherthe development of distinct salivary glands (sublingual,submandibular and parotid), which are known to produce unequaltypes of saliva (Tucker, 2007), is differentially affected by the lackof 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 forSMG morphogenesis. Furthermore, the ability of Eda to regulatesalivary gland development relied on intact NF-kB activity.Analysis of NF-kB reporter mice revealed that activation of NF-kB is entirely dependent on Eda during embryonic SMGdevelopment and not, for example, on TNFa, as recently suggested(Melnick et al., 2009). Previous studies have shown thatsupplementation of TNFa substantially increases epithelial cellproliferation and branching of cultured SMGs (Melnick et al.,2001b), and in light of our findings it is possible that as a powerfulinducer of NF-kB, exogenous TNFa may have mimicked the effectof Eda, as previously shown in embryonic Eda–/– skin explantswhere TNFa rescued primary hair placode formation (Schmidt-Ullrich et al., 2006). Contrary to previous speculations (Jaskoll etal., 2003), analysis of the NF-kB reporter mice indicated that theEda/NF-kB pathway is active already at the earliest developmentalstages, although our data show that it is dispensable for initiation


Fig. 6. Wnt reporter activity does notcolocalize with NF-kB activity at anystage. (A-D�) Wnt/b-cat activity indeveloping SMGs was assessed usingAxin2LacZ/+ embryos. (A-B�) b-Gal activitylocalized to the mesenchyme at E13-E14;compare with NF-kB reporter expression inFig. 4A-E. (C-C�) At E15, Axin2-lacZexpression was detected both in themesenchyme and the main epithelial duct.(D-D�) At E16 Axin2-lacZ was solelyobserved in developing ducts. Black arrowshighlight Axin2-lacZ expression, whitearrows its absence. (E-H) Comparison ofAxin2-lacZ and the NF-kB reporter activitiesin wholemounts revealed non-overlappingexpression at E15 and E16. At E16, theducts (broken line in H) were positive forWnt but not for NF-kB reporter, whereasthe developing alveoli were positive for NF-kB but not for Wnt activity. Scale bars: 500mm for A-D and A�-D�; 100mm for A�-D�.

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of SMG morphogenesis. The similarity of the SMG phenotypes ofEda-null and IkBaDN mutant mice and the absence of any residualNF-kB signaling activity in both mutants demonstrate that lack ofan early phenotype is not due to redundancy with another pathwayactivating NF-kB.

Shh is an important mediator of Eda signaling inthe developing salivary glandDownstream target genes of the Edar/NF-kB pathway have beenunder intensive study during recent years. Shh, Wnt10a andWnt10b, Wnt pathway inhibitor Dkk4, and BMP inhibitorsCcn2/Ctgf (CCN family protein 2/connective tissue growth factor)and follistatin have been shown to be downstream of the Edapathway 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 suspectedtranscriptional targets of Eda, Shh is so far the only one studied inthe context of salivary gland development (Jaskoll et al., 2004a).Here, we report that the response of cultured SMGs to themanipulation of the hedgehog pathway, either by application ofShh protein or by inhibition with cyclopamine, is crucially

dependent on Eda. These data combined with the fact that theexpression level of Shh positively correlated with Eda signalingactivity in vivo, implicates Shh as a key mediator downstream ofEda in developing salivary glands. While our manuscript was underreview, Wells et al. (Wells et al., 2010) reported the rescue of anEdar-deficient SMG branching defect (which was similar to thatseen 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 notentirely clear, Fgf8, a potent mitogen that is essential for the growthof SMG (Jaskoll et al., 2004b), has been proposed to act downstreamof Shh, as exogenous Fgf8 was shown to rescue the growth defect ofcyclopamine-treated SMGs (Jaskoll et al., 2004a). In line with this,qRT-PCR analysis of Eda-null SMGs at later stages (E16 tonewborn) indicated that both Shh and Fgf8 were downregulated(Melnick et al., 2009). Wells et al. (Wells et al., 2010) reported thattreatment with Fgf8 protein did not rescue the branching defect ofEdar mutant SMGs, but did increase bud surface area. It is likely thatmany signaling molecules, acting either in the same or in parallelpathways, function as proliferative cues to promote epithelial growthand branching of SMG (Patel et al., 2006).


Fig. 7. Suppression of mesenchymal Wnt/b-cat activityreduces branching and size of the SMG. (A-D) E13 SMGswere cultured for 2 days in the presence of tankyrase inhibitorXAV939 or casein kinase inhibitor CKI-7. XAV939 [10mM(n=16) and 100mM (n=6)] reduced branching by 40%compared with controls; 100mM CKI-7 also had a similareffect. (E) Mean number of end buds±s.e.m. of SMGs culturedfor 1 or 2 days in the presence of XAV939. (F) Mean numberof end buds+s.e.m. of SMGs cultured for 2 days in thepresence of CKI-7. (G,H) Histology of the salivary glands ofDermoCre+; b-cat–/fl embryos at E14. Deletion ofmesenchymal b-catenin led to an overall smaller size andfewer branches compared with control littermates. Scale bars:500mm. (I) Quantification of the epithelial size and thenumber of end buds from serial sections in DermoCre+; b-cat–/fl embryos at E13 to E15 (n=3 for E13; n=6 for E14, andn=3 for E15) in comparison with control littermates. Values forcontrol SMGs were set at 1 (broken line). Data aremean+s.e.m. (J,K) Radioactive in situ hybridization showedreduced expression of Eda in DermoCre+; b-cat–/fl salivaryglands compared with control littermates at E14. (L) In situhybridization signal of mesenchymal Eda expression wasquantified in E14 DermoCre+; b-cat–/fl (n=4) and controlsalivary glands (n=4), four sections per sample were analyzed.Data are mean+s.e.m. (M-P) E13 salivary glands were culturedfor 2 days in the presence or absence of 500 ng/ml ofrecombinant Eda and 10mM XAV939. Eda protein restored theXAV939-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) wassignificantly higher in XAV939-treated (n=15) salivary glandscompared with untreated (n=6) ones. Data are mean+s.e.m.***P<0.001, **P<0.01, *P<0.05.

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It is conceivable that Shh is not the only important target geneof the Eda pathway in the salivary gland. Besides Shh, exogenousEda protein induces the expression of follistatin in culturedembryonic skin explants (Pummila et al., 2007). Although thefunction of endogenous follistatin in salivary gland development isunknown, it may have a role as an antagonist of activin, whichcauses a severe disruption of SMG morphogenesis when appliedex vivo (Ritvos et al., 1995). With increasing knowledge of thedownstream events of the Eda pathway (Mikkola, 2009), noveltarget genes and their functional relevance are likely to beuncovered and will be tested in the future.

Mesenchymal Wnt signaling is required for Edaexpression and SMG branching morphogenesisZhang et al. (Zhang et al., 2009) recently showed a sequentialinterdependency between the Wnt and Eda pathways in developinghair follicles: Wnt/b-cat signaling is essential for NF-kB activationwhereas Edar/NF-kB is thereafter required to strengthen andmaintain Wnt/b-cat activity. The link between the two pathways isfurther supported by the conspicuous similarities of individualscarrying mutations in Eda/Edar/Edaradd or in Wnt10a (Adaimy etal., 2007; Cluzeau et al., 2011) (OMIM 257980), which is one ofthe proposed targets of Edar/NF-kB (Zhang et al., 2009).Moreover, the transcriptional targets of the two pathways arepartially overlapping (Fliniaux et al., 2008; Zhang et al., 2009), andin developing hair, tooth and mammary buds, high Wnt/b-cat andNF-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 andNF-kB reporter expression patterns in developing salivary glands,suggesting that unlike in hair follicles, Wnt/ b-cat and Eda/NF-kBmay not collaborate in regulation of target gene expression inSMG.

The role of Wnt/b-cat signaling in branching morphogenesis hasnot been studied in detail in cutaneous glands, with the exceptionof mammary glands (for a review, see Boras-Granic andWysolmerski, 2008). High epithelial Wnt reporter activity is seenduring onset of embryonic branching morphogenesis (Chu et al.,2004), and suppression of canonical Wnt activity via deletion ofthe co-receptor Lrp6 compromises branching development(Lindvall et al., 2009). In developing salivary glands, we did notdetect Wnt pathway activity in branching buds or alveoli with anyof the three reporters analyzed. The reporter studies seem tosuggest that epithelial Wnt signaling activity does not regulatebranching morphogenesis in the SMGs, but may instead beinvolved in ductal differentiation and lumen formation.

Numerous studies have revealed the importance of epithelial-mesenchymal interactions in salivary gland development, andclassic tissue recombination experiments suggest that the branchingpattern is controlled largely by signals from the mesenchyme (Patelet al., 2006; Tucker, 2007). Our results indicate that mesenchymalWnt/b-cat activity regulates SMG morphogenesis. Inhibition ofWnt signaling either in vivo or in vitro produced smaller glandswith fewer end buds. This function may be transient though, asmesenchymal expression of Axin2-lacZ reporter vanished by E16.The mechanism for how mesenchymal Wnt/b-cat signalingregulates epithelial growth and branching has yet to be clarified,but the Wnt target genes may include paracrine factors such assome of the many Fgfs (Hoffman et al., 2002; Patel et al., 2006;Steinberg et al., 2005). Our data suggest that transcriptionalregulation of Eda may be one important function of mesenchymalWnt/b-cat signaling, and that Eda in turn acts as a mesenchymalcue to regulate epithelial branching via NF-kB and Shh (Fig. 8).

AcknowledgementsWe thank Dr Walter Birchmeier and Dr Stefano Piccolo for mice; Dr PascalSchneider for Fc-Eda-A1; and Merja Mäkinen, Riikka Santalahti and RaijaSavolainen for excellent technical assistance. The work was financiallysupported by the Academy of Finland, Sigrid Jusélius Foundation and ViikkiGraduate School in Molecular Biosciences.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.057711/-/DC1

ReferencesAdaimy, L., Chouery, E., Megarbane, H., Mroueh, S., Delague, V., Nicolas, E.,

Belguith, H., de Mazancourt, P. and Megarbane, A. (2007). Mutation inWNT10A is associated with an autosomal recessive ectodermal dysplasia: theodonto-onycho-dermal dysplasia. Am. J. Hum. Genet. 81, 821-828.

Bhakar, A. L., Tannis, L. L., Zeindler, C., Russo, M. P., Jobin, C., Park, D. S.,MacPherson, S. and Barker, P. A. (2002). Constitutive nuclear factor-kB activityis required for central neuron survival. J. Neurosci. 22, 8466-8475.

Bitgood, M. J. and McMahon, A. P. (1995). Hegehod and bmp genes arecoexpressed at many diverse sites of cell-cell interaction in the mouse embryo.Dev. Biol. 172, 126-138.

Blecher, S. R., Debertin, M. and Murphy, J. S. (1983). Pleiotropic effect of Tabbygene on epidermal growth factor-containing cells of mouse submandibulargland. Anat. Rec. 207, 25-29.

Boras-Granic, K. and Wysolmerski, J. J. (2008). Wnt signaling in breastorganogenesis. Organogenesis 4, 116-122.

Borghese, E. (1950). The development in vitro of the submandibular andsublingual glands of Mus Musculus. J. Anat. 84, 287-302.

Chang, S. H., Jobling, S., Brennan, K. and Headon, D. J. (2009). Enhanced Edarsignalling has pleiotropic effects on craniofacial and cutaneous glands. PLoSONE 4, e7591.

Chu, E. Y., Hens, J., Andl, T., Kairo, A., Yamaguchi, T. P., Brisken, C., Glick, A.,Wysolmerski, J. J. and Millar, S. E. (2004). Canonical WNT signaling promotesmammary placode development and is essential for initiation of mammary glandmorphogenesis. Development 131, 4819-4829.


Fig. 8. Regulation of salivary gland morphogenesis by Edasignaling and its integration with Wnt and Shh pathways. Edaexpression is restricted to the SMG mesenchyme and is downstream ofWnt/b-cat signaling. Eda is synthesized as an integral membraneprotein and its cleavage is required for biological activity. Edatranslocates to the epithelial compartment and binds to its receptorEdar, which ultimately leads to the activation of the transcription factorNF-kB. The degradation of the repressor IkBa promotes the transport ofNF-kB to the nucleus where it induces the expression of Shh and othertarget genes essential for epithelial growth and branching.

DEVELO

PMENT

2690

Clarke, A., Phillips, D. I., Brown, R. and Harper, P. S. (1987). Clinical aspects ofX-Linked hypohidrotic ectodermal dysplasia. Arch. Dis. Child. 62, 989-996.

Cluzeau, C., Hadj-Rabia, S., Jambou, M., Mansour, S., Guigue, P., Masmoudi,S., Bal, E., Chassaing, N., Vincent, M. C., Viot, G. et al. (2011). Only fourgenes (EDA1, EDAR, EDARADD, and WNT10A) account for 90% ofhypohidrotic/anhidrotic ectodermal dysplasia cases. Hum. Mutat. 32, 70-77.

Cui, C. Y., Hashimoto, T., Grivennikov, S. I., Piao, Y., Nedospasov, S. A. andSchlessinger, D. (2006). Ectodysplasin regulates the lymphotoxin-b pathway forhair differentiation. Proc. Natl. Acad. Sci. USA 103, 9142-9147.

DasGupta, R. and Fuchs, E. (1999). Multiple roles for activated LEF/TCFtranscription complexes during hair follicle development and differentiation.Development 126, 4557-4568.

Durmowicz, M. C., Cui, C. Y. and Schlessinger, D. (2002). The EDA gene is atarget of, but does not regulate Wnt signaling. Gene 285, 203-211.

Farley, F. W., Soriano, P., Steffen, L. S. and Dymecki, S. M. (2000). Widespreadrecombinase expression using FLPeR (Flipper) mice. Genesis 28, 106-110.

Fliniaux, I., Mikkola, M. L., Lefebvre, S. and Thesleff, I. (2008). Identificationof dkk4 as a target of Eda-A1/Edar pathway reveals an unexpected role ofectodysplasin as inhibitor of Wnt signalling in ectodermal placodes. Dev. Biol.320, 60-71.

Gaide, O. and Schneider, P. (2003). Permanent correction of an inheritedectodermal dysplasia with recombinant EDA. Nat. Med. 9, 614-618.

Grüneberg, H. (1971). The glandular aspects of the Tabby syndrome in themouse. J. Embryol. Exp. Morphol. 25, 1-19.

Häärä, O., Koivisto, T. and Miettinen, P. J. (2009). EGF-receptor regulatessalivary gland branching morphogenesis by supporting proliferation andmaturation of epithelial cells and survival of mesenchymal cells. Differentiation77, 298-306.

Hai, B., Yang, Z., Millar, S. E., Choi, Y. S., Taketo, M. M., Nagy, A. and Liu, F.(2010). Wnt/b-catenin signaling regulates postnatal development andregeneration of the salivary gland. Stem Cells Dev. 19, 1793-1801.

Harfe, B. D., Scherz, P. J., Nissim, S., Tian, H., McMahon, A. P. and Tabin, C. J.(2004). Evidence for an expansion-based temporal Shh gradient in specifyingvertebrate digit identities. Cell 118, 517-528.

Hashizume, A. and Hieda, Y. (2006). Hedgehog peptide promotes cellpolarization and lumen formation in developing mouse submandibular gland.Biochem. Biophys. Res. Commun. 339, 996-1000.

Headon, D. J. and Overbeek, P. A. (1999). Involvement of a novel Tnf receptorhomologue in hair follicle induction. Nat. Genet. 22, 370-374.

Headon, D. J., Emmal, S. A., Ferguson, B. M., Tucker, A. S., Justice, M. J.,Sharpe, P. T., Zonana, J. and Overbeek, P. A. (2001). Gene defect inectodermal dysplasia implicates a death domain adapter in development. Nature414, 913-916.

Hoffman, M. P., Kidder, B. L., Steinberg, Z. L., Lakhani, S., Ho, S., Kleinman,H. K. and Larsen, M. (2002). Gene expression profiles of mouse submandibulargland development: FGFR1 regulates branching morphogenesis in vitro throughBMP- and FGF-dependent mechanisms. Development 129, 5767-5778.

Huang, S. M., Mishina, Y. M., Liu, S., Cheung, A., Stegmeier, F., Michaud, G.A., Charlat, O., Wiellette, E., Zhang, Y., Wiessner, S. et al. (2009). Tankyraseinhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614-620.

Huelsken, J., Vogel, R., Brinkmann, V., Erdmann, B., Birchmeier, C. andBirchmeier, W. (2000). Requirement for b-catenin in anterior-posterior axisformation in mice. J. Cell Biol. 148, 567-578.

Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G. and Birchmeier, W. (2001).b-catenin controls hair follicle morphogenesis and stem cell differentiation in theskin. Cell 105, 533-545.

Jaskoll, T., Zhou, Y. M., Trump, G. and Melnick, M. (2003). Ectodysplasinreceptor-mediated signaling is essential for embryonic submandibular salivarygland development. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 271, 322-331.

Jaskoll, T., Leo, T., Witcher, D., Ormestad, M., Astorga, J., Bringas, P., Jr,Carlsson, P. and Melnick, M. (2004a). Sonic hedgehog signaling plays anessential role during embryonic salivary gland epithelial branchingmorphogenesis. Dev. Dyn. 229, 722-732.

Jaskoll, T., Witcher, D., Toreno, L., Bringas, P., Moon, A. M. and Melnick, M.(2004b). FGF8 dose-dependent regulation of embryonic submandibular salivarygland morphogenesis. Dev. Biol. 268, 457-469.

Kashimata, M. and Gresik, E. W. (1997). Epidermal growth factor system is aphysiological regulator of development of the mouse fetal submandibular glandand regulates expression of the a6-integrin subunit. Dev. Dyn. 208, 149-161.

Kere, J., Srivastava, A. K., Montonen, O., Zonana, J., Thomas, N., Ferguson,B., Munoz, F., Morgan, D., Clarke, A., Baybayan, P. et al. (1996). X-linkedanhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a noveltransmembrane protein. Nat. Genet. 13, 409-416.

Kondo, S., Kuwahara, Y., Kondo, M., Naruse, K., Mitani, H., Wakamatsu, Y.,Ozato, K., Asakawa, S., Shimizu, N. and Shima, A. (2001). The Medaka rs-3locus required for scale development encodes ectodysplasin-A receptor. Curr.Biol. 11, 1202-1206.

Koyama, N., Hayashi, T., Ohno, K., Siu, L., Gresik, E. W. and Kashimata, M.(2008). Signaling pathways activated by epidermal growth factor receptor orfibroblast growth factor receptor differentially regulate branching

morphogenesis in fetal mouse submandibular glands. Dev. Growth Differ. 50,565-576.

Kunisada, M., Cui, C. Y., Piao, Y., Ko, M. S. and Schlessinger, D. (2009).Requirement for Shh and Fox family genes at different stages in sweat glanddevelopment. Hum. Mol. Genet. 18, 1769-1778.

Larsen, M., Wei, C. and Yamada, K. M. (2006). Cell and fibronectin dynamicsduring branching morphogenesis. J. Cell Sci. 119, 3376-3384.

Laurikkala, J., Mikkola, M., Mustonen, T., Aberg, T., Koppinen, P., Pispa, J.,Nieminen, P., Galceran, J., Grosschedl, R. and Thesleff, I. (2001). TNFsignaling via the ligand-receptor pair ectodysplasin and Edar controls thefunction of epithelial signaling centers and is regulated by Wnt and activinduring tooth organogenesis. Dev. Biol. 229, 443-455.

Laurikkala, J., Pispa, J., Jung, H. S., Nieminen, P., Mikkola, M., Wang, X.,Saarialho-Kere, U., Galceran, J., Grosschedl, R. and Thesleff, I. (2002).Regulation of hair follicle development by the TNF signal ectodysplasin and itsreceptor Edar. Development 129, 2541-2553.

Lexner, M. O., Bardow, A., Hertz, J. M., Almer, L., Nauntofte, B. andKreiborg, S. (2007). Whole saliva in X-Linked hypohidrotic ectodermaldysplasia. Int. J. Paediatr. Dent. 17, 155-162.

Lindvall, C., Zylstra, C. R., Evans, N., West, R. A., Dykema, K., Furge, K. A.and Williams, B. O. (2009). The Wnt co-receptor Lrp6 is required for normalmouse mammary gland development. PLoS ONE 4, e5813.

Liu, F., Chu, E. Y., Watt, B., Zhang, Y., Gallant, N. M., Andl, T., Yang, S. H., Lu,M. M., Piccolo, S., Schmidt-Ullrich, R. et al. (2008). Wnt/b-catenin signalingdirects multiple stages of tooth morphogenesis. Dev. Biol. 313, 210-224.

Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van deWetering, M., Clevers, H., Schlag, P. M., Birchmeier, W. et al. (2002).Negative feedback loop of Wnt signaling through upregulation ofconductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol. 22, 1184-1193.

Maretto, S., Cordenonsi, M., Dupont, S., Braghetta, P., Broccoli, V., Hassan,A. B., Volpin, D., Bressan, G. M. and Piccolo, S. (2003). Mapping Wnt/b-catenin signaling during mouse development and in colorectal tumors. Proc.Natl. Acad. Sci. USA 100, 3299-3304.

Melnick, M., Chen, H., Zhou, Y. and Jaskoll, T. (2001a). The functional genomicresponse of developing embryonic submandibular glands to NF-kB inhibition.BMC Dev. Biol. 1, 15.

Melnick, M., Chen, H., Zhou, Y. and Jaskoll, T. (2001b). Embryonic mousesubmandibular salivary gland morphogenesis and the TNF/TNF-R1 signaltransduction pathway. Anat. Rec. 262, 318-330.

Melnick, M., Phair, R. D., Lapidot, S. A. and Jaskoll, T. (2009). Salivary glandbranching morphogenesis: a quantitative systems analysis of the Eda/Edar/NF-kBparadigm. BMC Dev. Biol. 9, 32.

Mikkola, M. L. (2009). Molecular aspects of hypohidrotic ectodermal dysplasia.Am. J. Med. Genet. A 149A, 2031-2036.

Mustonen, T., Pispa, J., Mikkola, M. L., Pummila, M., Kangas, A. T.,Pakkasjarvi, L., Jaatinen, R. and Thesleff, I. (2003). Stimulation ofectodermal organ development by Ectodysplasin-A1. Dev. Biol. 259, 123-136.

Nordgarden, H., Mustonen, T., Berner, H. S., Pispa, J., Ilmonen, M., Jensen, J.L., Storhaug, K., Thesleff, I. and Lyngstadaas, S. P. (2004). Ectodysplasin-A1promotes epithelial branching and duct formation in developing submandibularglands. Oral Biosci. Med. 1, 195-206.

Patel, V. N., Rebustini, I. T. and Hoffman, M. P. (2006). Salivary gland branchingmorphogenesis. Differentiation 74, 349-364.

Peters, J. M., McKay, R. M., McKay, J. P. and Graff, J. M. (1999). Casein kinase Itransduces Wnt signals. Nature 401, 345-350.

Pispa, J., Jung, H. S., Jernvall, J., Kettunen, P., Mustonen, T., Tabata, M. J.,Kere, J. and Thesleff, I. (1999). Cusp patterning defect in Tabby mouse teethand its partial rescue by FGF. Dev. Biol. 216, 521-534.

Pispa, J., Mikkola, M. L., Mustonen, T. and Thesleff, I. (2003). Ectodysplasin,Edar and TNFRSF19 are expressed in complementary and overlapping patternsduring mouse embryogenesis. Gene Expr. Patterns 3, 675-679.

Pispa, J., Pummila, M., Barker, P. A., Thesleff, I. and Mikkola, M. L. (2008).Edar and Troy signalling pathways act redundantly to regulate initiation of hairfollicle development. Hum. Mol. Genet. 17, 3380-3391.

Pummila, M., Fliniaux, I., Jaatinen, R., James, M. J., Laurikkala, J., Schneider,P., Thesleff, I. and Mikkola, M. L. (2007). Ectodysplasin has a dual role inectodermal organogenesis: inhibition of Bmp activity and induction of Shhexpression. Development 134, 117-125.

Rawlins, E. L. and Hogan, B. L. (2005). Intercellular growth factor signaling andthe development of mouse tracheal submucosal glands. Dev. Dyn. 233, 1378-1385.

Ritvos, O., Tuuri, T., Erämaa, M., Sainio, K., Hilden, K., Saxen, L. and Gilbert,S. F. (1995). Activin disrupts epithelial branching morphogenesis in developingglandular organs of the mouse. Mech. Dev. 50, 229-245.

Sakai, T., Larsen, M. and Yamada, K. M. (2003). Fibronectin requirement inbranching morphogenesis. Nature 423, 876-881.

Sarkar, L. and Sharpe, P. T. (1999). Expression of Wnt signaling pathway genesduring tooth development. Mech. Dev. 85, 197-200.


DEVELO

PMENT

Schmidt-Ullrich, R., Aebischer, T., Hulsken, J., Birchmeier, W., Klemm, U. andScheidereit, C. (2001). Requirement of NF-kB/Rel for the development of hairfollicles and other epidermal appendices. Development 128, 3843-3853.

Schmidt-Ullrich, R., Tobin, D. J., Lenhard, D., Schneider, P., Paus, R. andScheidereit, C. (2006). NF-kB transmits Eda A1/EdaR signalling to activate Shhand cyclin D1 expression, and controls post-initiation hair placode down growth.Development 133, 1045-1057.

Srivastava, A. K., Pispa, J., Hartung, A. J., Du, Y., Ezer, S., Jenks, T., Shimada,T., Pekkanen, M., Mikkola, M. L., Ko, M. S. et al. (1997). The Tabbyphenotype is caused by mutation in a mouse homologue of the EDA gene thatreveals novel mouse and human exons and encodes a protein (Ectodysplasin-A)with collagenous domains. Proc. Natl. Acad. Sci. USA 94, 13069-13074.

Steinberg, Z., Myers, C., Heim, V. M., Lathrop, C. A., Rebustini, I. T., Stewart,J. S., Larsen, M. and Hoffman, M. P. (2005). FGFR2b signaling regulates exvivo submandibular gland epithelial cell proliferation and branchingmorphogenesis. Development 132, 1223-1234.

Tucker, A. (2007). Salivary gland development. Semin. Cell Dev. Biol. 18, 237-244.Wells, K. L., Mou, C., Headon, D. J. and Tucker, A. S. (2010). Recombinant EDA

or Sonic hedgehog rescue the branching defect in Ectodysplasin A pathwaymutant salivary glands in vitro. Dev. Dyn. 239, 2674-2684.

Wells, K. L., Mou, C., Headon, D. J. and Tucker, A. S. (2011). Defects andrescue of the minor salivary glands in Eda pathway mutants. Dev. Biol. 349, 137-146.

Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E. N., Towler, D. A. andOrnitz, D. M. (2003). Conditional inactivation of FGF receptor 2 reveals anessential role for FGF signaling in the regulation of osteoblast function and bonegrowth. Development 130, 3063-3074.

Zhang, Y., Tomann, P., Andl, T., Gallant, N. M., Huelsken, J., Jerchow, B.,Birchmeier, W., Paus, R., Piccolo, S., Mikkola, M. L. et al. (2009). Reciprocalrequirements for EDA/EDAR/NF-kB and Wnt/b-catenin signaling pathways in hairfollicle induction. Dev. Cell 17, 49-61.


DEVELO

PMENT

ectodysplasin and wnt pathways are required for salivary ... · the developing submandibular...

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