Abstract

EDA splice isoforms EDA-A1 and EDA-A2 belong to the TNF ligand family and regulate skin appendage formation by activating NF-kB- and JNK- promoted transcription. To analyze their action further, we conditionally expressed the isoforms as tetracycline (‘Tet’)-regulated transgenes in Tabby (EDA-negative) and wild-type mice. Expression of only the mEDA-A1 transgene had two types of effects during embryogenesis: (1) determinative effects on sweat glands and hair follicles. In Tabby mice, one type of hair follicle (‘guard hair’) was restored, whereas a second type, the dominant undercoat hair follicle (‘zigzag’) was not; furthermore, the transgene sharply suppressed zigzag hair formation in wild-type mice, with the overall numbers of back hair follicles remaining the same; and (2) trophic effects on sebaceous and Meibomian glands. Marked hyperplasia resulted from expansion of the sebocyte-producing zone in sebaceous glands, with particularly high expression of the transgene and the replication marker PCNA, and correspondingly high production of sebum. The phenotypic effects of mEDA-A1 on sebaceous glands, but not on hair follicles, were reversed when the gene was repressed in adult animals. The results thus reveal both initiating and trophic isoform-specific effects of the EDA gene, and suggest a possible balance of isoform interactions in skin appendage formation.

INTRODUCTION

As in many developmental processes, the formation of skin appendages involves a tissue-specific route to the activation of major signaling pathways. High specificity for skin appendage formation is based on ectodysplasin, the protein encoded by the X-linked EDA gene (1,2). It intervenes in skin appendage development as a novel TNF family ligand, activating NF-kB transcription factors through EDAR/XEDAR receptors, an EDARADD receptor adaptor and TRAF signal proteins (310). Consistent with the first steps in a coherent pathway, mutations in EDA cause the human disorder anhidrotic ectodermal dysplasia (‘EDA’; OMIM305100) and its mouse equivalent, ‘Tabby’, and mutations in the EDA receptor/adaptor genes cause similar phenotypes (614). Thus, the pathway is pivotal in skin appendage formation, but its details remain largely unknown.

In the complete absence of EDA expression, sweat glands are totally missing, some teeth are rudimentary or aberrant, and two types of back hair follicles, ‘guard’ and ‘zigzag’ are not formed. However, the third type of back hair, ‘medium’ (including awls and auchenes), is still formed in Tabby, and is even more numerous compared to wild-type littermates (1517).

Recent evidence suggests that a complex role for EDA derives in part from the action of several isoforms. We found that moderate levels of one of the longest isoforms, mEDA-A1, can largely restore missing appendages (15), with the accompanying selective augmentation of expression of the downstream NF-kB and JNK pathways and a number of newly identified downstream target genes (18). However, it was not clear when mEDA-A1 action was critical during mouse development (or thereafter). Also, because some appendages were not restored, it seemed possible that mEDA-A1 action was dose-dependent, or that formation of other appendages might require other isoforms.

To determine the timing, dose-dependence, and reversibility of EDA action, we generated both Tabby and wild-type mice in which transgenic mEDA-A1 or mEDA-A2 isoforms could be conditionally expressed up to high levels from a tetracycline-regulated promoter. Consistent with recent reports (19,20) (see below), we saw no effect of mEDA-A2 on its own; but mEDA-A1 alone had two kinds of effects on particular appendages: it commited precursor cells to form sweat glands and the longest (‘guard’) hair, and repressed the alternative pathway to form zigzag hair follicles; and in a trophic role, it provoked the proliferation of sebaceous gland progenitors. The sebaceous gland hypertrophy, but not the determination of hair follicles, was reversed when the gene was shut down after birth.

RESULTS

To facilitate the recognition of time- and isoform-dependent actions of EDA in embryonic and adult mice, we have used a controllable, double transgenic model.

Mice transgenic for Tet-regulated EDA/EDA pathway genes

The two longest EDA isoforms, EDA-A1 and EDA-A2, are the most common, and the only ones that include a TNF-ligand domain. Though differing by only two amino acids, they have distinct corresponding receptors, EDAR and XEDAR (79). Transgene constructs for the two mouse isoforms are diagrammed in Figure 1A, and were confirmed in animals by PCR genotyping. Expression of these constructs is negatively regulated by the Tet promoter, that is, EDA is repressed by the addition of doxicycline to cells or when fed to animals as a dietary supplement, and is expressed in doxicycline's absence. Appropriate regulation was confirmed by transient transfection assays in cell culture. Regulation of mRNA levels was confirmed in transgenic mice (Fig. 1B) by real-time RT–PCR with RNAs from skin samples (see Materials and Methods). Their level was increased about 1000-fold in the mEDA-A1 transgenic mice in either Tabby or wild-type background, and returned to basal levels when the mice were given food containing doxicycline (200 mg/kg). In comparable experiments, transgenic mEDA-A2 expression was 50-fold higher than in wild-type mice (Fig. 1B, A2 on) and also returned to basal levels in the presence of doxicycline. As shown in Figure 1C, for mEDA-A1, protein expression was apparent in the ‘Tet-’ condition (lanes 2 and 3), and was completely inhibited after the addition of 2 µg/ml doxicycline to culture medium (lanes 4 and 5). Thus, the gene can be selectively turned on or off at any stage of development or adulthood.

mEDA-A1 transgene restored guard hair, but not zigzag hair, in Tabby mice

Tabby mice bearing the mEDA-A1 transgene under tetracycline control showed striking phenotypes in the ‘on’ state (in the absence of doxicycline; see Materials and Methods). Table 1 summarizes the effects. Some partial restorative effects observed previously with lower-level expression of the isoform (15) were more complete with the higher levels attained here. They included fully restored hair behind the ears, and darker and thicker but somewhat ‘scruffy’ disordered coat hair [compare WT(mEDA-A1) and Ta(mEDA-A1) with WT and Ta in (A–D) of Fig. 2].

The analysis of transgene effects was extended to quantitate hair follicles and subtypes of hair. Tabby male mice lack both guard (‘G’) and zigzag (‘Z’) hair, although the other major class, medium-length (‘M’) hair, is present at higher levels than in wild-type mice (15,16) (see examples in Fig. 3C). To assess possible differential effects, hair was harvested by shaving equivalent areas of back skin of wild-type, Tabby and mEDA-A1 transgenic mice and counting the numbers of G, M and Z hairs under a stereomicroscope. Wild-type mice have a population of hairs containing about 5.3±0.4% G, 27.0±0.9% M and 67.7±0.5% Z; in agreement with other recent studies (16,21), Tabby male mice have no G or Z hair, but more M hair is formed in Tabby than in wild-type mice, so that the overall number of hair follicles is the same (Fig. 3A).

In male mEDA-A1 transgenic Tabby mice, the number of M hairs remains comparable to Tabby, but G hair is restored to wild-type levels (6.6% of total hair). In contrast, however, no Z hairs at all were found on the backs of the six transgenic male mice from two independent transgenic lines (Fig. 3B). Interestingly, in these animals as well, although the composition of hair types was dramatically changed, the total hair numbers were unchanged in transgenic mouse skin, either in Tabby or in wild-type background (Fig. 3A and B).

Other subtle differences between wild-type and mEDA-A1 transgenic mice were also seen. Tail hairs were reduced in number and length, and the tail-specific ridges on skin surfaces, which are prominent in wild-type mice but absent in Tabby mice, are not restored in transgenic Tabby mice (Table 1). One other measured phenotype is, like Z hair, completely unrestored: Tabby mice are smaller than wild-type littermates, and male transgenic mice were as small as Tabby. At 5 months, the weights of transgenic and Tabby mice were about 80.0±1.7 and 76.9±1.8% of wild type, respectively (Table 1).

Zigzag hair formation was suppressed in wild-type mice carrying an mEDA-A1 transgene

The failure of Z hair follicle formation in mEDA-A1 transgenic Tabby mice was not simply a result of a lack of capacity of the A1 isoform to initiate those follicles. Greater complexity was revealed when the transgene was introduced into wild-type mice (which were identified by SSCP) (15). In the presence of a fully intact EDA gene, overall skin, tail, and sweat gland function were indistinguishable from mice lacking the transgene (Fig. 2). However, the distribution and numbers of hair follicle types on the backs of the transgenic animals were markedly affected. G hair numbers were somewhat increased (to 8.7% compared with 5–6% in wild-type); but strikingly, Z hair was reduced to about 6% (range 0–11%), at least 6-fold less than in wild-type (Fig. 3B). These results suggest that mEDA-A1 is not only unable to initiate Z hair follicles, but also suppresses their formation (see Discussion). Furthermore, Z hair was not restored when the A1 transgene was turned off in adult wild-type transgenic skin (data not shown). This is again consistent with a critical irreversible role for EDA in embryonic stages.

Restoration of sweat but not preputial glands by mEDA-A1

Like coat- and ear-associated hair, sweat glands were more fully developed with the mEDA-A1 transgene under tetracycline control. Positive results with the starch-iodine test (15) were confirmed in histological sections, exemplified in Figure 4 (‘S’ panels). Tabby footpad sections showed only dermis and expanded muscle tissue, but sections from Tabby animals bearing the mEDA-A1 transgene showed the restoration of clearly defined sweat glands, very similar to wild-type mice with or without the transgene.

In contrast, the supragonadal preputial gland, which secretes pheromones (22), is absent in Tabby mice, which retain only an associated fat pad, and is not at all restored by the mEDA-A1 transgene. Nor does an added transgene change the appearance of the gland of wild-type animals [Fig. 4 (‘P’ panels) shows both hematoxylin–eosin staining of sections and photographs of the dissected surface of the gland regions, with the fat pads outlined in black]. Thus, as in the case of hair follicles, mEDA-A1 has a selective determinative effect on some exocrine glands.

Hyperplastic trophic effect of transgenic mEDA-A1 on sebaceous and Meibomian glands

Histological studies revealed a hypertrophic effect of mEDA-A1 on several types of sebaceous glands. One, the Meibomian gland, is a variant sebaceous gland that is attached to the inner surface of the eyelid and produces lipid-rich secretions (22). It was lacking in Tabby mice and was not restored by an mEDA-A1 transgene (Fig. 4, panel M), which may account for the progressive blindness of the aging mice (noted in Table 1). However, the prominent gland of wild-type mice was consistently larger in the presence of an active mEDA-A1 transgene (Fig. 4, panel M).

An equivalent or even more marked change was seen in sebaceous glands of transgenic mouse skin, and was analyzed further. Sebaceous glands were 200–300% larger than in wild-type or Tabby mice. The size of individual sebocytes was the same (Fig. 5A–C), but the numbers were increased, with a correspondingly excessive production of sebum (Fig. 5D–F). Normal G follicles have two associated sebaceous glands; M follicles have only one (21). Hyperplasia was obvious in both glands associated with each de novo G follicle (Fig. 5), and also in the single sebaceous gland associated with each M follicle. Thus, the effect is exerted distal to the commitment to formation of hair follicle subtypes. Furthermore, because functional sebaceous glands are formed on M hair follicles in the absence of active EDA in Tabby mice (Fig. 5B and E), the effect of the mEDA-A1 transgene is apparently trophic rather than morphogenetic.

The larger sebaceous glands could result from either a slower turnover of sebocytes or an increased formation and flux of sebocytes through their life cycle. An effect on the formation of sebocytes was consistent with increased numbers of multiple immature sebocytes, which are sparse in wild-type or Tabby, but are numerous in the transgenic animals (Fig. 5C, circled, diagrammed in Fig. 6B). [Interestingly, the mEDA-A1 transgene is highly expressed in the bottom generative layer of sebaceous glands (Fig. 6A).]

Immunostaining with the proliferation marker PCNA further supported increased formation of mature sebocytes as the basis for the hypertrophy. PCNA is, like mEDA-A1, highly expressed in the generative layer, and is down-regulated when transgene expression is shut off (circled region in Fig. 6C–F). Thus, the large sebaceous glands in transgenic mice probably result from EDA activation of hyperproliferation of sebocyte progenitors located at the base of mouse sebaceous glands. In good agreement with this scenario, sebum production, visualized by Oil Red O staining, gradually increases from the lower, proliferating layer to the upper differentiated zone of transgenic sebaceous glands, i.e. along the proliferation/maturation axis (Figs 5F and 6B).

The reversible Tet-regulation of the transgene permitted us to adduce more evidence regarding a trophic effect. When transgene function was shut down through the addition of 200 mg/kg doxicycline to the normal diet of 3-month-old transgenic mice, we found that the hyperplasia of sebaceous glands was reversed (Fig. 5C). We confirmed relatively complete shutdown of transgene expression in transgenic skin by quantitative real-time RT–PCR (Fig. 1B, A1 off). Histological examination showed that hyperplasia of sebaceous glands was partially reversed within one month of the shutoff of the gene, and after 3 months effects were more pronounced; the remaining sebaceous glands decreased in size and were comparable to the size of those in wild-type mice (insert in Fig. 5C). This suggests a requirement for continuous expression of the mEDA-A1 isoform for sebaceous gland hyperplasia.

Although sebaceous glands diminished in size after doxicycline administration, the number of guard hairs remained unchanged (data not shown). This suggests that the A1 isoform exerts its primary effect on guard hair synthesis, with little or no influence on its maintenance. However, the upright, rather perpendicular follicular angle to the skin's surface in transgenic animals is completely reverted to the wild-type angle (insert in Fig. 5C). It is speculatively possible that the enlarged sebaceous glands distort the angle of the hair shaft.

Induction of mEDA-A1 expression in adult Tabby mice did not rescue Tabby phenotypes

The time at which mEDA-A1 affects various skin appendages could also be tested through controlled expression of the transgene. When mice were given doxicycline throughout pregnancy, male transgenic progeny were essentially identical to Tabby littermates. In particular, they showed the same lack of sweat glands, tail hair, guard hair and zigzag hair; subsequent withdrawal of doxicycline from food (at 6 weeks of age) and resumption of A1 expression showed that these mice remained like their Tabby littermates after 2, 4, 6, 8 or 10 weeks of re-expression (data not shown). These results are consistent with requisite EDA function in early embryonic life.

mEDA-A2 transgene failed to complement Tabby phenotypes

Because the mEDA-A2 isoform also activates NF-kB transcription factors through its own receptor, XEDAR (8), during skin appendage development, we expected that it might function analogously to EDA-A1 as a transgene (15). Instead, Tabby mice bearing the mEDA-A2 transgene were indistinguishable from transgene-less Tabby littermates; and transgenic wild-type mice were like wild-type mice (Table 1). mEDA-A2 transgene expression was 50-fold higher in transgenic skin compared with wild-type (Fig. 1B). The corresponding level of protein, which is low in wild-type and transgenic animals (20), was not directly measured, so that inferences are indirect; in ongoing work, real time RT–PCR assays showed that the EDA-A2 XEDAR receptor was slightly higher in transgenic skin, and the findings listed in Table 1 are consistent with recently reported negative results (19,20).

DISCUSSION

In the transgenic Tabby mice that we studied originally (15), in spite of the use of the ubiquitous CMV-promoter, mEDA-A1 partly rescued the Tabby phenotype without causing detectable abnormalities in the skin or elsewhere. It seemed possible that other isoforms of EDA might be involved in rescue of some features, or, alternatively, because the CMV-mEDA-A1 isoform expression was lower than wild-type levels, low expression of the transgene might account for the limited rescue (and possibly the absence of overt toxic effects). Thus we generated Tet-EDA-A1 transgenic mice and, for comparison, Tet-mice with transgenic A2, -B and -C isoforms. The Tet-EDA transgenic system provides high levels of expression of EDA isoforms in the skin epithelium that are sharply controlled by doxicycline. Correspondingly sharp phenotypic changes, again without overt toxicity, were observed with mEDA-A1 alone. We focus here on two types of observed effects, determinative and trophic.

Positive for sweat but not preputial glands, and G but not Z follicle formation

Tet regulation permits control of EDA expression over a wide range. Many phenotypic effects of mEDA-A1 expressed up to 1000-fold greater than wild-type levels were qualitatively comparable to those seen earlier with levels about 60–90% of wild-type (e.g. the restoration of sweat glands [Fig. 4, top panels, and cf. Table 1 in Srivastava et al. (15)]. In general, determinative effects were selective at all levels of expression. Thus, unlike sweat glands, exocrine preputial glands were not restored even at high levels of EDA-A1. However, quantitation and additional histological and timed observations revealed additional features of mEDA-A1 action. Strikingly, in the presence or absence of high levels of the isoform, the absolute number of hair follicles per square cm of back skin was the same in Tabby, wild-type, and transgenic mice; but the proportions of subtypes were changed (Fig. 3). In the presence of mEDA-A1, G hair was fully restored to Tabby mice, and relatively high numbers of M follicles were still retained, but the dominant undercoat hair, Z, was still missing.

Overexpression of the mEDA-A1 transgene in a wild-type background revealed that it not only failed to induce Z hair follicles, but also could repress their formation. [The low expression from the CMV promoter showed no comparable effect (15).] The reduction of Z follicles was compensated by an increase of M follicles to maintain the constant overall number of follicles. Thus, mEDA-A1 at high levels may block a transition at E17, when Z follicles are formed. Perhaps EDA-A1 alters the commitment of follicles to Z form, or a negative trophic effect on follicles causes them to revert to a ‘default M pathway’. Additional information should come from examination of embryonic stages in the Tet-mEDA-A1 transgenic mice, although the experiments are cumbersome, because only a few male progeny of crosses contain the Tet gene along with the Tabby mutation and the transgene.

It is notable that expression of mEDA-A1 did not change overall hair follicle number and had no effect on follicle formation or structure after birth. Nor did the expression of mEDA-A1 (or mEDA-A2) lead to tumorigenesis, as has been reported for hair follicle morphogens such as transgenic Wnt, SHH, Notch and FGFs (2325). Mutant and knockout mouse studies indicate that different types of hair have different requirements for the Wnt and EDA pathways as well as for BMPs, Noggin and SHH (11,12,2632). Anomalies in these mutants might reflect redundant roles for Wnt and EDA pathways, or alterations of a single gene network with various compensatory mechanisms active at different times during development. However, the restriction of activity of the EDA pathway, but not the Wnt pathway, to embryo-fetal action, coupled with the lack of tumorigenicity of EDA, suggests that the pathways are not redundant, but affect distinct features of hair follicles in the context of a single gene network.

Trophic effect on sebaceous glands

A more clearly trophic, and positive, effect of mEDA-A1 was seen in sebaceous glands. Meibomian glands provide an instance in which determinative and trophic effects of mEDA-A1 are clearly separated. The transgene did not restore the gland to Tabby animals, but it increased the size of glands in wild-type mice (Fig 4, ‘M’ panels). In a comparable way, the glands associated with G follicles obviously depend secondarily for their formation on mEDA-A1, but the augmentation of expression from the Tet-regulated promoter led to proliferation of sebocytes in wild-type as well as Tabby animals. It is of interest that, although M follicles and their single associated sebaceous glands form in Tabby mice in the complete absence of EDA, the glands nevertheless respond to high levels of mEDA-A1 with hyperplasia. Strikingly, multiple proliferating cells in transgenic sebaceous glands, which were sparse in wild or Tabby mice, formed a ‘tail-like’ structure in the bottom layer where the A1 transgene is most highly expressed (Fig. 6). We infer that the layer contains progenitor or partially committed stem cells for sebocytes.

Consistent action of mEDA-A1 provided as transgene or protein ligand

Two publications that appeared during the preparation of this report used either injection of recombinant mEDA-A1 protein, containing a heterologous multimerization domain, into Tabby mice (19) or promotion of mEDA-A1 from a skin-specific keratin 14 (K14) promoter in wild-type mice (20) to study its effects. Possibly resulting from the different modes of provision of the protein, there are some inconsistencies in the results. For example, free ligand—in contrast to mEDA-A1 transgene (Fig. 4, ‘M’ panels)—supported the restoration of Meibomian glands, and numbers of molars were somewhat variable between approaches, but other phenotypes that were scored in different studies (Table 1) were generally in accord.

The results with soluble mEDA-A1 over a range of administered doses have practical implications for possible intervention (19), because the lack of toxic effects and the correction of the Tabby phenotype were very similar to that reported for the CMV-promoted isoform (15). Also, in the study with K14 promotion in a wild-type background, which would be expected to magnify any toxic effects, high expression in the epidermal basal layer produced only mild anomalies (occasional supernumerary molars or nipples, enamel hypoplasia, absence of zigzag hair, and enlarged sebaceous glands).

In the double transgenic model that we describe here, high levels of Tet-promoted mEDA-A1 were studied in both Tabby and wild-type mice. In contrast to the studies with protein ligand or K14 promotion, Tet control permitted the gene to be selectively turned on or off at invervals in adult or embryonic mice. High levels of embryonic expression correlated with more complete rescue of some features of the Tabby phenotype (e.g. sweat glands, guard hair, molars) than we had seen with CMV-promoted isoform. As in the study with K14-promoted expression, anomalies of phenotype were mild, but notably zigzag hair was again suppressed and sebaceous glands were enlarged. In addition, we found that the suppression of zigzag hair occurs with the maintenance of a conserved overall number of hair follicles, and, using the conditional promoter, that the effects of mEDA-A1 on hair follicles required expression in utero (19), and were thereafter irreversible when the transgene was turned off. We also found that sebaceous gland hypertrophy was due to increased progenitor cell multiplication, and the trophic effect was reversible when the transgene is later repressed.

Taken together, the various assay systems suggest that the specificity of EDA action, and its activity across a wide range of levels without toxicity, is probably due to regulation through specific downstream effectors, including the receptor EDAR.

Requirement for other isoforms, and determinative versus trophic action

How can one now rationalize the features of the Tabby mouse that are not corrected by mEDA-A1? They include the somewhat smaller size of Tabby mice (Table 1) and the failure to restore preputial glands (Fig. 4, middle panels ‘P’). The complete gene, in contrast to mEDA-A1, obviously corrects these deficiencies and also, for example, does not inhibit Z hair follicle formation. The differences could most simply result from the absence of balanced expression of other EDA isoforms when only mEDA-A1 is supplied, or when it is in excess in wild-type mice. We (like 19,20) find no restoration of hair follicles or other skin appendages by mEDA-A2 alone, and no pathogenetic mutations have been reported in mEDA-A2 or its receptor (4,33,34). Thus, a pathway initiated by EDA-A2 may have no leading role in skin appendage formation (34). Nevertheless, mEDA-A2 itself can also stimulate NF-kB transcription factors in vitro, and it is suggestively selectively expressed at the time when Z hair is produced (35). Similarly, other isoforms, mEDA-B and mEDA-C (2), expressed under Tet control (work in progress) have thus far themselves shown no phenotypic effects. But the study of time- and dose-dependent expression of Tet-regulated isoforms acting in conjunction may permit a more complete definition of important interactions.

Finally, although studies have generally focused on other skin appendages where EDA has a determinative effect, the hypertrophic effect on sebaceous glands adds a possible proliferative action to its potential roles. The rate of formation of sebocytes can apparently be increased by EDA action (Figs 5 and 6). It is provocative that signaling through beta-catenin, which interacts with the EDA pathway, has now been independently implicated in sebaceous tumors and the proliferation of undifferentiated sebocytes (36). Furthermore, the sebocytes stimulated by mEDA-A1 are productively making sebum. The process, presumably mediated by TNF downstream of EDA, may be speculatively involved in acne or other instances of altered sebum production, and could be a target for intervention.

MATERIALS AND METHODS

Generation of transgene constructs

pTRE-mEDA-A1 construct

The construct is schematized in Figure 1A. A 1.2 kb cDNA fragment spanning the entire open reading frame (ORF) of mouse mEDA-A1 was generated via PCR, using a full-length mEDA-A1 cDNA (GenBank no. AF016628) as template, and cloned into the pPCR-Script Amp SK (+) cloning plasmid (Stratagene, West Cedar Creek, TX, USA). The targeted sense primer sequence for PCR was 5′-GTAGCCACCATGGGCTACCCA GAGGTAG-3′, containing a strong Kozak consensus ribosome binding site (underlined) (37), and the antisense primer was 5′-CATGGGCCAGGATGGAATG-3′. The amplified cDNA fragment included the ORF and 26 bp of the 3′-untranslated region. The cDNA fragment was ligated into the pTRE vector (Clontech, Palo Alto, CA, USA). Orientation of the cDNA insert was confirmed by PvuII digestion and subsequent electrophoresis, followed by sequencing on an ABI Prism 377 DNA Sequencer. Primer 1 for sequencing was complementary to the pTRE vector, 100 bp upstream of translation start site (5′-CGCCTGGAGACGCCATCC-3′). Primer 2 starts at the 333 bp position, in the mEDA-A1 ORF (5′-CAACAGCAGCCTTTGGAGCCG-3′), and primer 3 begins 718 bp into the ORF (5′-AAAACTGGAACTCGGGAAAATCAGCC-3′). A linear XhoI/HindIII fragment from the pTRE-mEDA-A1 construct was then purified by Elutip-D column chromatography (Schleicher and Schuell, Keene, NH, USA), resuspended in pre-filtered 8 mM Tris–HCl, pH 7.5, 0.1 mM EDTA buffer, and microinjected into pronuclei of one-cell inbred C57BL/6J mouse embryos (the genetic background of Tabby〈6J〉).

pTRE-mEDA-A2 construct

The mEDA-A2 isoform is only six nucleotides shorter than the A1 isoform (35), so that we could use high fidelity PCR amplification with a Quickchange site-directed mutagenesis kit (Stratagene) to generate the pTRE-mEDA-A2 construct. The sense primer lacking the 6 nucleotides specific for the A1 isoform was 5′-GCACCTACTTCATCTATAGTCAGGTCTACTACATCAACTTCACTG-3′, and the antisense primer was 5′-CAGTGAAGTTGATGTAGTAGACCTGACTATAGATGAAGTAGGTGC-3′. After PCR (20 cycles with 95°C, 0.5 min, 55°C, 1 min, 68°C, 10 min), the PCR template pTRE-mEDA-A1 (methylated) was eliminated by DpnI enzyme digestion, and the PCR product pTRE-mEDA-A2 was propagated in E. coli. The pTRE-mEDA-A2 sequence was verified by sequencing and microinjected as described above.

Cell transfection and western blotting

The expression competence of constructs was confirmed by transient transfections into the MCF-7 Tet-off cell line (Clontech). Three micrograms of construct were mixed with 30 µl of DOTAP liposomal transfection reagent (Boehringer Mannheim, Indianapolis, IN, USA) and transfected into cells in a 6 cm culture dish. Cells were cultured either in medium lacking or containing 2 µg/ml doxicycline for up to 48 h, and were then dissolved in Laemmli gel sample buffer. Proteins were fractionated in 10% SDS/polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane. Antibody raised to the C-terminal peptide of EDA (38) was used as a primary antibody and the reactive bands were detected via an Enhanced Chemiluminescence kit (ECL, Amersham Life Sciences, Little Chalfont, Bucks, UK).

Generation of transgenic mice

Microinjected embryos were implanted into pseudo-pregnant female mice and DNAs from tail samples of progeny were genotyped for presence of the transgene by PCR. Potential founders were mated to C57BL/6J mice to identify those passing the transgene. The transgene-positive male mice (two independent lines for each construct) were mated with heterozygous Tabby females (C57BL/6J-Aw-j-Ta6j strain, Jackson Laboratory, Bar Harbor, ME, USA). Tabby females carrying the transgene (genotyped by PCR and SSCP) were then mated with confirmed Tet-off male mice [C57BL/6J-TgN (MMTVtTA), Jackson Laboratory] (39) to activate the mEDA-A1/mEDA-A2 transgene. Phenotypes were recorded from the final cross's progeny, i.e. double transgenic offspring under Tabby or wild-type background. Doxicycline (200 mg/kg in Purina mouse chow, Bio-Serv, Frenchtowne, NJ, USA) was administered to transgenic progeny or final breeders to turn off transgene expression selectively.

Genotyping and genetic background detection

Genotyping was done by PCR with two transgene-specific primer sets, both of which were used for progeny from both mEDA-A1 and -A2 lines. Primer set 1: sense primer, 5′-GCTACCTAGAGTTGCGGTCCG-3′; antisense primer, 5′-CCCACCTGGCCCTCCTGGTCCTCA-3′ (spanning intron I of the mEDA gene and amplify 330 bp products from the transgene, Fig. 2E, upper panel). Cycling conditions: denaturation at 95°C for 3 min, 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1 min. Primer set 2: sense primer, 5′-CTGGAACTCGGGAAAATCAG-3′ (in exon 5 of mEDA); antisense primer, 5′-TGGTGTGCTTGCTTGCTCATATTG-3′ (in exon 8, amplifying a 411 bp product only from the transgene). Cycling conditions: denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 2 min.

To detect the presence of the Tet-off transgene, which encodes the tetracycline-controlled transactivator (tTA), we used the following primer set: sense primer, 5′-CTGATCTGAGCTCTGAGTG-3′; antisense primer, 5′-GCAAAAGTGAGTATGGTGCC-3′, which amplifies a 300 bp product from the Tet-off transgene. Cycling conditions were denaturation at 94°C for 1.5 min followed by 35 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s.

The genetic background of transgenic progeny was determined through the use of the Incorporation PCR SSCP (single-strand conformational polymorphism) method (15). Briefly, a 387 bp product was amplified in the presence of 1 µCi of [α-32P]dCTP with the primer set (sense, 5′-GCTACCTAGAGTTGCGGTCCG-3′; antisense, 5′-AACCTGACCTGGACAACCTCT-3′), and the 32P-labeled product was denatured and analyzed by electrophoresis. The mutant allele is one nucleotide shorter than wild-type, producing an amplification product that moves correspondingly faster in electrophoresis.

Quantitative analysis of transgene expression in transgenic skin

One-step quantitative real-time RT-PCR with Taqman probes and primers (ABI Prism 7700 Sequence Detection System, Applied Biosystems, Foster, CA, USA) was performed to detect the transcription level of transgenes and control genes (18). Total RNAs were isolated from back skin of adult wild-type, transgenic, and Tabby male mice with Trizol (Gibco, Grand Island, NY, USA), and were treated with a ‘DNA free kit’ (Ambion, Austin, TX, USA), eliminating genomic DNA contamination. Total RNAs from 19.5 d.p.c. wild-type C57BL mouse embryos were used to generate a standard curve. PCR was carried out according to the manufacturer's protocols. Reactions were normalized to GAPDH expression levels. Probe/primer set for A1 and A2 transgenes: probe, 5′-CAGGACCGGCACCAGATGGCC-3′; sense primer, 5′-TTGGAGCCGGGAGAAGATC-3′, antisense primer, 5′-CAGAATATGCCTTTTCATCAGGAA-3′. Probes and primers for EDA pathway genes were described in Cui et al. (18).

Analysis of hair subtypes and sebaceous gland function

To assess transgene effects on hair type, we cut hair from equivalent areas of back skin of mice and enumerated the easily-distinguishable hair types (16) under a stereomicroscope. Guard (G; Tylotrich) hair is long, straight and dark; medium size hair (M; including awls and auchenes) is yellowish, thinner and either straight or slightly bent; zigzag hair (Z) is yellowish and sharply angled. Hairs were counted from males including four wild-type, three Tabby, six A1 transgenic in Tabby background, five A1 transgenic in wild-type background, and three A2 transgenic in Tabby background. At least 350 hairs were counted from each mouse.

For total hair number count, we used three skin samples from each strain. Skin samples were taken with a 6 mm Dermal biopsy punch (Miltex Instrument, Bethpage, NY, USA), fixed in 4% paraformaldehyde, and embedded in paraffin. Ten-micron sections were cut parallel to the skin surface with a microtome and stained with hematoxylin and eosin (Sigma). Hair numbers were count from three random areas (∼2.4 mm2) of each section and scored at 20× magnification.

Oil-Red O staining was used to test for sebaceous gland function. Frozen sections were stained with 0.5% Oil-Red O in 2-propanol (Sigma) at 37°C for 30 min, followed by washes with 70% 2-propanol and H2O. Sections were counterstained with hematoxylin (Sigma).

Histology, immunohistochemistry and in situ hybridization

Samples freshly harvested from control and experimental animals were fixed in 4% paraformaldehyde and 1% glutaraldehyde in phosphate-buffered saline overnight at 4°C. Fixed samples were paraffin embedded, and sections were stained with eosin/hematoxylin (Sigma) and observed in an Axiovert 200 microscope.

For immunohistochemistry (Fig. 6), sections were heated in 10 mM citrate buffer at 121°C for 5 min, then incubated with monoclonal antibody against proliferating cell nuclear antigen (PCNA) (NeoMarkers, Fremont, CA, USA) overnight at 4°C and stained with a streptavidin–biotin peroxidase system according to the manufacturer's recommendation (DAKO LSAB+ kit, Carpinteria, CA). Sections were counterstained with hematoxylin.

For in situ hybridization, a 411 bp PCR product from EDA transcript (primer set 2, above) was cloned into the pCR4-topo vector (Invitrogen, Carlsbad, CA, USA) and was labeled with digoxygenin-UTP (Roche, Indianapolis, IN, USA). Labeled probes (400 ng/ml in concentration) were hybridized with 4% paraformaldehyde fixed frozen sections at 65°C overnight. Following washing, alkaline phosphatase-conjugated anti-digoxygenin antibody (Roche) was applied for overnight incubation. Detection was performed with NBT/BCIP solution (Roche) for 24 h.

ACKNOWLEDGEMENTS

The authors particularly thank Eric Douglass for animal management, Ramaiah Nagaraja, Minoru Ko and Paul Waeltz for critical reading of the manuscript, Carol Borgmeyer and Dave Donovan for microinjection and technical assistance, Dan Rowley, Shengyuan Luo and Mohamed Mughal for genotyping and Yulan Piao for providing E19.5dpc whole embryo RNA and technical assistance.

*

To whom correspondence should be addressed at: Laboratory of Genetics, NIH/National Institute on Aging, 333 Cassell Dr., Suite 3000, Baltimore, MD 21224, USA. Tel: +1 4105588337; Fax: +1 4105588331; Email: schlessingerd@grc.nia.nih.gov

Figure 1. Constructs, expression, and regulation of transgenic mEDA-A1 and mEDA-A2. (A) The coding regions of mEDA-A1 and mEDA-A2 cDNAs were inserted between TRE/PminCMV promoter and SV40 poly A region (see Materials and Methods). The tetracycline-controlled transactivator (tTA) binds to TRE region of mEDA transgenes and activates transcription. (B) Quantitative RT–PCR shows elevated expression of mEDA-A1 and mEDA-A2 in transgenic mouse skin, and shut-down of transgene expression by doxicycline chow administration. Note exponential scale of expression; the Tabby mutation is a single base change, so that Ta and WT mRNA levels are equal. (C) EDA protein expression from transient transfection of mEDA-A1 into MCF-7 Tet-off cell line. Tet- condition (lanes 2 and 3) and shut-down in Tet+ condition (lanes 4 and 5). Lane 1, transfected with pTRE vector only and cultured under Tet− condition. Two bands about 46 kDa are glycosylated EDA protein (40).

Figure 1. Constructs, expression, and regulation of transgenic mEDA-A1 and mEDA-A2. (A) The coding regions of mEDA-A1 and mEDA-A2 cDNAs were inserted between TRE/PminCMV promoter and SV40 poly A region (see Materials and Methods). The tetracycline-controlled transactivator (tTA) binds to TRE region of mEDA transgenes and activates transcription. (B) Quantitative RT–PCR shows elevated expression of mEDA-A1 and mEDA-A2 in transgenic mouse skin, and shut-down of transgene expression by doxicycline chow administration. Note exponential scale of expression; the Tabby mutation is a single base change, so that Ta and WT mRNA levels are equal. (C) EDA protein expression from transient transfection of mEDA-A1 into MCF-7 Tet-off cell line. Tet- condition (lanes 2 and 3) and shut-down in Tet+ condition (lanes 4 and 5). Lane 1, transfected with pTRE vector only and cultured under Tet− condition. Two bands about 46 kDa are glycosylated EDA protein (40).

Figure 2. Appearance of mEDA-A1 transgenic mice. A1 transgenic mice in Tabby background [Ta(mEDA-A1)] showed darker, longer, disoriented hair and restored hair patches behind ears. Transgenic mice in wild-type background were similar. Photographs by Olympus C-700 digital camera.

Figure 2. Appearance of mEDA-A1 transgenic mice. A1 transgenic mice in Tabby background [Ta(mEDA-A1)] showed darker, longer, disoriented hair and restored hair patches behind ears. Transgenic mice in wild-type background were similar. Photographs by Olympus C-700 digital camera.

Figure 3. Total hair number and ratio of hair subtypes in mEDA-A1 transgenic mice. (A) Hair number in equivalent areas of back skin. Three independent mice for each strain, and three independent areas for each mouse, were counted. (B) Relative proportions of G, M and Z hair. (C) Three major hair subtypes from wild-type mice. Tabby male mice lack G and Z hairs.

Figure 3. Total hair number and ratio of hair subtypes in mEDA-A1 transgenic mice. (A) Hair number in equivalent areas of back skin. Three independent mice for each strain, and three independent areas for each mouse, were counted. (B) Relative proportions of G, M and Z hair. (C) Three major hair subtypes from wild-type mice. Tabby male mice lack G and Z hairs.

Figure 4. Differential effects of mEDA-A1 on three types of gland. (S) Determinative effect restoring sweat glands to Tabby [Ta(mEDA-A1)] mice with no additional effect in a wild-type [WT(mEDA-A1)] background. Hematoxylin-eosin staining. (P) Lack of restoration of preputial gland to Tabby mice, with no additional effect in wild-type. Paired subpanels show hematoxylin-eosin stained sections (left) and digital photographs of the surface of the gland region (right). The dashed black line outlines the overlying fat pad. (M) Lack of restoration of Meibomian gland to Ta, but trophic effect on gland in wild-type mice. Scale bars, 0.1 mm.

Figure 4. Differential effects of mEDA-A1 on three types of gland. (S) Determinative effect restoring sweat glands to Tabby [Ta(mEDA-A1)] mice with no additional effect in a wild-type [WT(mEDA-A1)] background. Hematoxylin-eosin staining. (P) Lack of restoration of preputial gland to Tabby mice, with no additional effect in wild-type. Paired subpanels show hematoxylin-eosin stained sections (left) and digital photographs of the surface of the gland region (right). The dashed black line outlines the overlying fat pad. (M) Lack of restoration of Meibomian gland to Ta, but trophic effect on gland in wild-type mice. Scale bars, 0.1 mm.

Figure 5. Hyperplasia of sebaceous glands in mEDA-A1 transgenic mice. Panels represent hematoxylin staining, with Oil Red O staining added for (D–F), for wild-type, Tabby, and transgenic Tabby skin. Sebaceous gland hyperplasia and follicle angle in de novo guard hair follicle were reversed by doxicycline food application in transgenic Tabby mice (C, insert). Accumulation of young sebocytes in the bottom of transgenic sebaceous glands (C, circled). Oil-Red O staining for sebum. Scale bars, 0.05 mm.

Figure 5. Hyperplasia of sebaceous glands in mEDA-A1 transgenic mice. Panels represent hematoxylin staining, with Oil Red O staining added for (D–F), for wild-type, Tabby, and transgenic Tabby skin. Sebaceous gland hyperplasia and follicle angle in de novo guard hair follicle were reversed by doxicycline food application in transgenic Tabby mice (C, insert). Accumulation of young sebocytes in the bottom of transgenic sebaceous glands (C, circled). Oil-Red O staining for sebum. Scale bars, 0.05 mm.

Figure 6. EDA expression and higher replication in sebocytes of transgenic mice. (A) In situ hybridization shows high expression of mEDA-A1 transgene in the lower portion of sebaceous glands [Ta(mEDA-A1)]. (B) Schematic of sebaceous gland proliferation and differentiation in transgenic mice. (C, D) Control PCNA staining of wild-type (C) and Tabby (D) sebaceous glands. PCNA is elevated in sebaceous glands of Ta(mEDA-A1) mice (E), and down-regulated when transgene is off (F). Immunostaining as in Materials and Methods. Scale bars, 0.05 mm.

Figure 6. EDA expression and higher replication in sebocytes of transgenic mice. (A) In situ hybridization shows high expression of mEDA-A1 transgene in the lower portion of sebaceous glands [Ta(mEDA-A1)]. (B) Schematic of sebaceous gland proliferation and differentiation in transgenic mice. (C, D) Control PCNA staining of wild-type (C) and Tabby (D) sebaceous glands. PCNA is elevated in sebaceous glands of Ta(mEDA-A1) mice (E), and down-regulated when transgene is off (F). Immunostaining as in Materials and Methods. Scale bars, 0.05 mm.

Table 1.

Gross phenotypes of mEDA-A1 transgenic mice in Tabby or wild-type background

Gross phenotypes WT Ta Ta(A1) WT(A1) Ta(A2) 
Hair coat Dark, shiny, ordered Yellowish, short, thin Dark, long, angled Dark, long, angled Yellowish, short, thin 
Bald patch behind ear − − − 
Tail hair − +/− − 
Tail skin ridges − − − 
Vibrissae 
Sweat glands − − 
Preputial glands − − − 
Meibomian glands − − − 
Blindness by 9 months − − 
Teeth (molars) 2–3 2–3 
Weight (%)a 100 77 80 101 82 
Gross phenotypes WT Ta Ta(A1) WT(A1) Ta(A2) 
Hair coat Dark, shiny, ordered Yellowish, short, thin Dark, long, angled Dark, long, angled Yellowish, short, thin 
Bald patch behind ear − − − 
Tail hair − +/− − 
Tail skin ridges − − − 
Vibrissae 
Sweat glands − − 
Preputial glands − − − 
Meibomian glands − − − 
Blindness by 9 months − − 
Teeth (molars) 2–3 2–3 
Weight (%)a 100 77 80 101 82 

(+) Yes or normal; (−) no or missing; (+/−) partial.

aFour male mice [three for Ta-(A2)], 5 months old, were weighed for each strain.

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