TGF-β superfamily signaling is essential for tooth and hair morphogenesis and differentiation

ARTICLE IN PRESS

European Journal of Cell Biology 86 (2007) 781–799 www.elsevier.de/ejcb

TGF-b superfamily signaling is essential for tooth and hair morphogenesis and differentiation Borut Klopcica,1,2, Thorsten Maassa,1, Erik Meyera,3, Hans A. Lehrb, Daniel Metzgerc, Pierre Chambonc, Amrit Mannd, Manfred Blessingd, a

I. Medical Department, Section Pathophysiology, Johannes Gutenberg University, Mainz, Germany Institute of Pathology, CHUV, Lausanne, Switzerland c Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, Department of Physiological Genetics, Illkirch; Inserm, U596, CNSR, UMR7104, Universite´ Louis Pasteur, Strasbourg, France d Center for Biotechnology and Biomedicine, Faculty of Veterinary Medicine, Leipzig University, Deutscher Platz 5, D-04103 Leipzig, Germany b

Received 18 September 2006; received in revised form 9 March 2007; accepted 9 March 2007

Abstract Members of the transforming growth factor b (TGF-b) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-b family, notably TGF-b and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-b superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-b blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-b superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues. r 2007 Elsevier GmbH. All rights reserved. Keywords: Smad7; TGF-b; Activin; BMPs; Keratinocytes; Thymic T cells; Incisors; Tooth development; Hair follicle

Introduction Corresponding author. Tel.: +49 341 973 1220;

fax: +49 341 973 1229. E-mail address: [email protected] (M. Blessing). 1 These two authors contributed equally to this work. 2 Present address: School of Medicine and Pharmacology, University of Western Australia, Fremantle, Australia. 3 Present address: PHAST GmbH, Merck KGaA, Darmstadt, Germany. 0171-9335/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2007.03.005

Although most ectodermal organogenesis is initiated during the embryonic period, morphogenesis does continue after birth (Pispa and Thesleff, 2003). Several signaling networks control inductive interactions between epithelium and stroma during the development of skin appendages. Development of different organs is not

ARTICLE IN PRESS 782

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

only similar with respect to the morphological features of organogenesis but also the same groups of factors are involved in similar events during the formation of totally different organs (Hogan et al., 1994). The growth factors involved in many of these events include members of the fibroblast growth factor (FGF), TGFb, as well as hedgehog and Wnt families (Hogan et al., 1994; Jernvall et al., 1998; Yamaguchi and Rossant, 1995). The TGF-b family of growth factors is a large evolutionarily conserved family named after its first identified member TGF-b (Roberts et al., 1981). This superfamily consists of more than 35 members including TGF-bs, activins, inhibins, bone morphogenetic proteins (BMPs), and growth differentiation factors (GDFs) that are further divided into subfamilies (Chang et al., 2002; Kingsley, 1994). Members of the TGF-b family of growth factors play an important role in the regulation of differentiation, proliferation, apoptosis, and function of a wide variety of cells (Glick et al., 1993; Guo and Kyprianou, 1999; Motyl et al., 1998). In mice, BMP-4, a member of the BMP-subfamily, is expressed in the presumptive dental epithelium during early tooth morphogenesis. As the dental mesenchyme acquires odontogenic potential, BMP-4 expression shifts to the underlying mesenchymal condensations (Ohazama et al., 2005; Vainio et al., 1993). Antagonistic interactions between BMP and FGF signaling regulate loco-regional aspects of tooth formation (Laurikkala et al., 2003; Neubuser et al., 1997; Thesleff and Mikkola, 2002), and BMP-4-induced apoptosis in the enamel knot regulates cusp morphogenesis (Jernvall et al., 1998; Jernvall and Thesleff, 2000). BMP-4 is also expressed in the mesenchymal condensations at the sites of hair follicle morphogenesis in the epidermis (Botchkarev et al., 1999; Jones et al., 1991) and consequently, overexpression of BMP-4 in the skin of transgenic mice disturbs hair morphogenesis (Blessing et al., 1993). Activin-bA, a member of the TGF-b superfamily, is essential for the formation of incisors and mandibular molars (Ferguson et al., 1998, 2001), and inhibition of activin signaling disturbs cutaneous homeostasis (Bamberger et al., 2005; Munz et al., 1999). Previous studies have indicated that TGF-b and its cognate receptors are important regulators of early tooth development (Chai et al., 1994, 1999; Pelton et al., 1991). However, no tooth abnormalities but distinct craniofacial malformations were seen in any of the TGF-b isoform null-mutants, indicative of a functional overlap between various isoforms (Dickson et al., 1995; Sanford et al., 1997). Whereas mice with a targeted deletion of TGF-b exhibited epidermal hyperproliferation (Glick et al., 1993), epidermal overexpression of TGF-b lead either to increase or decrease of proliferation depending on the pattern of transgene expression

(Cui et al., 1995; Sellheyer et al., 1993; Wang et al., 1999). Blockade of TGF-b signaling by overexpressing a dominant negative type II TGF-b receptor in the basal compartment of the interfollicular epidermis and outer root sheath (ORS) of hair follicles did not lead to hyperproliferation or aberrant differentiation as the expression patterns of keratins 1, 5, 6 and 10 were not altered (Amendt et al., 1998). TGF-b superfamily members convey signals via type I and type II receptors and downstream effector molecules, termed Smads (Derynck and Feng, 1997; Massague, 1998). After ligand binding, the activated receptor/ ligand complex phosphorylates a receptor-regulated Smad protein (R-Smad), which then binds to the common partner (Co-Smad) Smad4 and subsequently translocates to the nucleus to eventually regulate the transcription of target genes (Heldin et al., 1997). Three of the five known R-Smads Smad1, 5 and 8 mediate BMP signal transduction whereas the other two, Smad2 and 3 mediate signaling of the activin and TGF-b subfamilies (Massague and Wotton, 2000; Miyazono et al., 2001; Ten Dijke et al., 2002). Smad-mediated signaling is in turn controlled through a negativefeedback mechanism mediated by inhibitory Smads (I-Smads: Smad6 and 7) (Imamura et al., 1997; Nakao et al., 1997). Early studies had postulated that I-Smads act by competing with R-Smads for the activated receptors (Hayashi et al., 1997; Souchelnytskyi et al., 1998), or with Smad4 for the phophorylated R-Smad (Hata et al., 1998). However, more recent studies have concentrated on the activation and recruitment of ubiquitin ligases Smurf-1 and -2 by I-Smads, hence leading to the degradation of activated receptors and of R-Smads (Ebisawa et al., 2001; Kavsak et al., 2000; Murakami et al., 2003). Smad6 transcription is activated by BMP signaling, and Smad7 is induced by TGF-b/activin signaling (Nagarajan et al., 1999; Nakao et al., 1997; Takase et al., 1998). This differential regulation suggested that Smad6 and Smad7 might be part of negative feedback loops to restrict the cellular response to BMP- and TGF-b/activin signaling, respectively. Yet, the function of Smad7 as an inhibitor of TGF-b and activin signaling (Ishisaki et al., 1999; Souchelnytskyi et al., 1998) may be more complicated, because is has been shown to inhibit BMP signaling as well (Casellas and Brivanlou, 1998). Smad7 can block TGF-b-induced growth inhibition and apoptosis on the one hand (Halder et al., 2005) and promote TGF-b-induced apoptosis in epithelial cells on the other (Landstrom et al., 2000). Smad7 expression has been shown to be upregulated in a study involving chemical carcinogenesis whereas R-Smads and the coSmad, Smad4 were lost (He et al., 2001). Overexpression of Smad7-induced malignant conversion in a murine model of squamous cell carcinoma (Liu et al., 2003). Constitutive overexpression of Smad7 under the control

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

of the keratin 5 promoter in epithelial tissues of mice, elicited delayed and aberrant hair follicle morphogenesis, severe epithelial hyperplasia in the skin and other stratified epithelia and thymic atrophy. Embryonic and perinatal lethality prevented the generation of transgenic lines (He et al., 2002). In order to be able to study the effect of overexpression of Smad7 on the organogenesis of epidermal appendages, we used a vector system for conditional overexpression of the transgene under the control of the bovine keratin 5 (K5) promoter with a loxP-flanked lacZ-reporter gene placed between the promoter and the smad7 cDNA (K5-lacZfl-smad7). In the absence of Smad7 induction, the transgene expression pattern could already be investigated with the help of reporter gene analysis without the possible risk of developing a lethal phenotype associated with Smad7 overexpression (He et al., 2002). Depending upon the mouse model used for the induction of Cre-recombinase-mediated excision of the reporter gene; onset of the Smad7 transgene can be controlled spatially and temporally. We crossed these mice with nestin-cre deleter mice where Cre-recombinase activity starts before gastrulation (Betz et al., 1996), and with K14-creERT2 transgenic mice (Li et al., 2000) in which expression of the transgene was induced in double transgenic adult mice by intraperitoneal injection of hydroxytamoxifen (4-OHT). The combination of these expression systems allowed us for the first time to resolve the problem of embryonic and perinatal lethality. The data presented in this study demonstrate that overexpression of Smad7 in embryonic state leads to severe defects in the organogenesis of teeth and hair. However, induction of Smad7 in adult mice also leads to differentiation-associated defects of skin and its appendages as well as incisors. Also, increased proliferation in epidermal keratinocytes, reduced Smad3 expression and changes in thymic T cell differentiation and survival were observed. These findings indicate that inhibition of TGF-b superfamily signaling, most notably TGF-b and activin signaling, not only disturbs morphogenesis, but also blocks differentiation in adult tissues.

783

expression vector (Blessing et al., 1993) and inserted 50 of the b-globin intron. A 3.3-kb fragment containing the lacZ gene with a nuclear localization signal was excised from pPD16.43 (Fire et al., 1990) and inserted 50 of an 0.7-kb SmaI/KpnI fragment of hGH polyA which was previously cloned into pBR322*3. LoxP sites were generated using TV ¼ A, a targeting vector available in our lab, as template (Jahnen-Dechent et al., 1997). Primer pairs used for amplification contained restriction sites and were as follows: loxP-1-XbaI: 50 -CGAGTCTAGACTTGATTTTTATCT AATAACTTCG-30 , loxP-1-SmaI: 50 -TCTCG GGCCCGTCGACCTGCAGCCCGGGCTTATAAC-30 and loxP-2-XhoI: 50 -CGAGCTCGAGCTTGATTTTTA TCTAATAACTTCG-30 , loxP-2-Bsp1201: 50 -TCTCGGG CCCGTCGACCTGCAGCCCGGGCTTATAAC-30 . The products were excised with XbaI and SmaI, and XhoI and Bsp1201 and inserted 50 of the lacZ cDNA and 30 of the polyA site following lacZ cDNA. The LacZ gene flanked by loxP (lacZfl) sites was excised with XbaI and Bsp1201, endfilled using Klenow enzyme and inserted into the SmaI site located between the b-globin intron and the hGH polyA site of the keratin 5 vector. The cDNA for murine smad7 (Nakao et al., 1997) was derived from total liver RNA of FVB/NHSD mice by RT-PCR. One primer contained an SrfI site and a Kozak translation initiation consensus sequence (Kozak, 1987) followed by the ATG of the smad7 gene (50 -ATATGCCCGGGCCGCCACCATGTTCAGGAC CAAACGATCTCG-30 ) while the other primer contained an XhoI site located 30 of the stop codon of the smad7 gene (50 -TATACTCGAGCTACCGGCTGTTGAAGATGACC-30 ). The 1.3-kb product was digested with SrfI and XhoI and inserted between the 30 loxP site and the 30 hGH polyA site of the K5-lacZfl vector. The 11.7-kb K5-lacZfl-smad7 expression cassette was excised with NotI and inserted into the Bsp120I site of a pBR322*3 derivative containing two pairs of human globin HS4 insulators orientated in tandem conformation with a polylinker in between (Li and Stamatoyannopoulos, 1994).

Generation of transgenic mice

Material and methods Expression vector construction A 1.4-kb XhoI/KpnI fragment from a keratin10 expression vector (Werner et al., 1993) containing a rabbit b-globin intron and a human growth hormone gene (hGH) polyadenylation signal was cloned into pBR322*3, a 2.1-kb pBR322 derivative (Blessing et al., 1993). A 5.2-kb fragment containing upstream regulatory elements of the bovine keratin 5 promoter was excised from a keratin 5

The K5-lacZfl-smad7 expression cassette was excised with SgrAI and gel purified. Transgenic mice were generated essentially as described (Hogan et al., 1994). K5-lacZfl-smad7 transgenic animals were genotyped by PCR on ear biopsies using primers specific for smad7 cDNA (S7-900: 50 -ACAGCTCAATTGGGACAAC-30 ; NCBI Acc. No. AF015260 pos. 897–916 (Nakao et al., 1997)) and for the hGH polyA site, respectively (pArev: 50 -ATGCATGCCTGGAATCCC-30 ; NCBI Acc. No. NG_001334, pos. 7019–7002 (George et al., 1981)). Nestin-cre deleter mice were a kind gift of Dr. W. Mueller (GBF, Braunschweig, Germany) (Betz et al., 1996).

ARTICLE IN PRESS 784

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

Offspring of nestin-cre mice was analyzed by PCR using primers specific for the Cre-recombinase (creIa: 50 -GCACTGTTTCGACCAGGTT-30 ; GenBank Acc. No. X03453 pos. 900–919 and CreIIa: 50 -GCTAACCAGCGTTTTCGTTC-30 ; GenBank Acc. No. X03453 pos. 1099–1080 (Sternberg et al., 1986)). K5-dnTGFbRII transgenic mice were engineered in our lab and have been described previously (Amendt et al., 1998). K14-creERT2 mice have been described previously (Li et al., 2000). K14-creERT2 transgenic animals were genotyped by using a specific primer pair (K14: 50 -ATATGACAGCACTGATGAGGC-30 ; NCBI Acc. No. U11076, pos. 2135–2155 (Staggers et al., 1995) and K14-cre: 50 -GGTGTACGGTCAGTAAATTGG-30 ; NCBI Acc. No. X03453, pos. 509–489 (Sternberg et al., 1986)). All animals, unless mentioned otherwise, were maintained as hemizygotes on a FVB/NHSD background. Animal care was in accordance with the governmental and institutional guidelines.

4-Hydroxytamoxifen treatment Preparation of 4-hydroxytamoxifen (4-OHT, Sigma, Deisenhofen, Germany) solution has been described elsewhere (Metzger and Chambon, 2001). Seven-weekold, double transgenic mice (K5-lacZfl-smad7+/ X K14creERT2+/, dtg+) were injected with 0.1 mg 4-OHT (in 100 ml sunflower oil) per mouse i.p. for six consecutive days. Age-matched non-transgenic (wt), single transgenic for K5-lacZfl-smad7 (tg) and un-induced K5-lacZflsmad7+/  K14-creERT2+/ double transgenic (dtg) littermates were used as controls. The 4-OHT-induced experimental group and the un-induced control groups were all housed in separate cages.

DNA preparation and Southern-blot analysis Tissue was digested in proteinase K solution (100 mM Tris–HCl (pH 8.4), 200 mM NaCl, 10 mM EDTA, 0.2% SDS, 0.4 mg/ml proteinase K) and digested overnight at 56 1C. For PCR analysis, this solution was diluted 1:50 in H2O, and 2 ml were used per 50 ml reaction mix. For Southern blot analysis, the genomic DNA was extracted with phenol/chloroform, precipitated with isopropanol and re-dissolved in TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA). Southern blot analysis was performed essentially as described (Church and Gilbert, 1984). The probe was a 2.2-kb HindIII/SmaI restriction fragment of the expression vector containing 1.5 kb of the 50 upstream sequence of the bovine K5 promoter and the b-globin intron. The expected sizes for DNA digested with HindIII were 5.3 kb for a non-recombined vector unit and 3.3 kb for a recombined unit. A 500-bp BamHI/

BsmI restriction fragment of the bmp-4 locus was used as a probe for normalization (Winnier et al., 1995).

RT-PCR analysis Total RNA was isolated using Tri Reagent (Sigma). Reverse transcription was performed using a RevertAid H Minus First Strand cDNA Synthesis Kit (MBI) according to the manufacturer’s instructions. The resultant cDNA was amplified by PCR using a FailSafe PCR Kit (Epicentre) in 35 cycles (step 1: 95 1C for 5 min, step 2: (95 1C for 30 s, 58 1C for 40 s 72 1C for 60 s)  35, step 3: 72 1C for 5 min). Amplification products were run on a 1.5% agarose gel containing 0.1 mg/ml ethidiumbromide. Staining was assessed densitometrically using AIDA 3.44 (Raytest, Germany). b-Actin was used as housekeeping gene control and its intensity was defined as 100%. At least three animals per genotype were analyzed. Primer pairs used to detect transgene-derived Smad7 mRNA were K5-Xho: 50 -TGGATCTCGAGGTCGAGG-30 ; GenBank Acc. No. NM_011057 (Blessing et al., 1993) or RT-smad7-for: 50 -AGA GCG CTT TCC GCG TCC TG-30 ; GenBank Acc. No. Z32746 pos.: 1284–1303 (Casatorres et al., 1994) and S7revII-for: 50 AGCGCAGATCGTTTGG-30 ; pos. 26–11; GenBank Acc. No. AF015260 (Nakao et al., 1997). Detection of the Smad isoforms, TGF-b type II receptor, PDGF-B and b-actin was done using the following primer pairs: Smad1: Smad1-for: 50 -TTACGAGGAACCAAAACACTG-30 ; pos. 1189–1209, and Smad1-rev: 50 -TGGTGAGTTCATACACGGTCT-30 ; pos. 1619–1599, both GenBank Acc. No. NM_008539 (Yingling et al., 1996), Smad2: Smad2-for: 50 -GAAT TTGCTGCTCTTCTGGCTCAG-30 ; pos. 1273–1296, and Smad2-rev: 50 -GTCTTATGCCCAACAAGACATCA-30 ; pos. 1755–1733, both GenBank Acc. No. NM_010754 (Baker and Harland, 1996), Smad3: Smad3-for: 50 -GTGCCTCAGTGACAGTGCTAT-30 ; pos. 1235–1255, and Smad3-rev: 50 -GACCAAGTTGGATGGAGTTCT-30 ; pos. 1678–1658, both GenBank Acc. No. NM_016769 (Labbe et al., 1998), Smad4: Smad4-for: 50 -TGGACGTTCAGGTAGGAGAGA-30 ; pos. 1429–1449, and Smad4-rev: 50 -CCTTCACAAA GCTCATCCTGA-30 ; pos. 1959–1939, both GenBank Acc. No. NM_008540 (Anna and Devereux, 1997), Smad5: Smad5-for: 50 -AGATAATTCCCAGCCTATGGA-30 ; pos. 895–915, and Smad5-rev: 50 -AATCCAT GGTTGACTGACTGA-30 ; pos. 1389–1369, both GenBank Acc. No. NM_008541 (Meersseman et al., 1997), Smad6: Smad6-for: 50 -CAACCCCTACCACTTCAGC30 ; pos. 1083–1101, and Smad6-rev: 50 -CAGTATGCCAAAACCGATCT-30 ; pos. 1476–1457, GenBank Acc. No. NM_008542 (Imamura et al., 1997), Smad7: Smad7-for: 50 -AAGTCAAGAGGCTGTGTTGC-30 ;

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

pos. 1954–1973, and Smad7-rev: 50 -TCTCATAGTCAAAAGCCTTGATGGAGAAA-30 ; pos. 2553–2525, GenBank Acc. No. NM_008543 (Nakao et al., 1997), TGF-b-RII: TGF-b-RII-for: 50 -AGCAGAAGCTGA GCCCGTCCTGGG-30 ; pos. 908–931, and TGF-bRII-rev: 50 -GGGGCCATGTATCTTGCCGTTCCC-30 ; pos. 1607–1584, both GenBank Acc. No. NM_009371 (Lawler et al., 1994), PDGF-B: PDGF-B-for: 50 ATGAATCGCTGCTGGGCGCTCTTC-30 , pos. 977– 1000, and PDGF-B-rev: 50 -TAGGGCTCGGAG GGTCTCCTTCA-30 , pos. 1701–1680, both GenBank Acc. No. NM_011057 (Bonthron et al., 1991), b-actin: b-actin-for: 50 -GTGGGCCGCCCTAGGCACCA-30 ; pos. 183–202 and b-actin-rev: 50 -TAGCCCTCGTAGATGGGCACA-30 ; pos. 586–565, both GenBank Acc. No. NM_007393 (Alonso et al., 1986).

Histochemistry Histochemistry was done on 7 mm-thick cryosections. For embryonal whole mounts, K5-lacZfl-smad7 mice were mated with nestin-cre deleter mice. Embryonal stage was determined from the time of appearance of the vaginal plug (E ¼ 0.5). Whole embryos were removed and washed in phosphate-buffered saline (PBS) before fixing. For b-galactosidase histochemistry, whole embryos or sections were stained with X-Gal (5-bromo-4chloro-3-indolyl b-D-galactoside) as described (Ramirez et al., 1994). For general histology, the cryosections were fixed in 4% formaldehyde in PBS for 20 min at room temperature and stained with hematoxylin and eosin routinely.

Immunohistochemistry Cryosections were cut at 7 mm thickness and processed as described previously (Breuhahn et al., 2000). The following antibodies were used in this study: rabbit polyclonal anti-keratin 14 antibody (1:800, Blessing et al., 1996), rabbit polyclonal anti-keratin 6 antibody (1:600, Blessing et al., 1996), mouse monoclonal anti-keratin 8.6 (cytokeratin 10) (1:800, Sigma), monoclonal anti-cytokeratin 4 (1:10, Progen Biotechnik, Heidelberg, Germany), rabbit polyclonal anti-filaggrin antibody (1:800, Covance, CA, USA), AP-conjugated goat anti-rabbit antibody (1:1000, Boehringer Mannheim, Germany), cyTM3-conjugated goat anti-rabbit antibody (1:1000, Dianova, Germany), and AP-conjugated sheep anti-mouse antibody (1:1000, Boehringer Mannheim). Where applicable, signal detection was done using Fast Red (Sigma) as substrate.

BrdUrd labeling BrdUrd-labeling experiments were performed using an ‘In Situ Cell Proliferation Kit’ (Roche) on 7 mm-thick

785

cryosections. Mice were injected i.p. with 30 mg of BrdUrd per gram body weight and sacrificed after a labeling period of 1 h. Fixation and processing of the samples were performed according to the manufacturer’s instructions. The total number of labeled and unlabeled nuclei in the basal layer of the interfollicular epidermis was determined and related to each other. Values were obtained from at least three animals per experimental group and at least 2000 cells were analyzed per animal.

Statistical analysis Mean7SEM are given. For comparison of groups, the non-parametric Mann–Whitney-U test was applied. A po0.005 was considered to be significant and a po0.001 was considered to be highly significant when compared to the wild-type and single transgenic group.

Results Generation of transgenic mice In order to elucidate the inhibitory role played by Smad7 in signaling by the TGF-b family of growth factors, which includes activin, BMPs and TGF-b isoforms, we constructed a vector system for conditional expression of Smad7 through Cre-mediated recombination (Fig. 1a). Eight transgenic founders were generated by microinjection of this expression cassette into the fertilized oocytes of FVB/NHSD mice, of which 7 transmitted the transgene to progeny leading to 7 transgenic lines. Mice of all transgenic lines appeared phenotypically normal and litter sizes were normal.

Analysis of lacZ reporter gene expression b-Galactosidase activity was detected in the sections from ear biopsies by staining with X-Gal for the pattern and strength of lacZ reporter gene expression. Transgenic animals of all lines expressed b-galactosidase to varying degrees in the epidermis and the outer root sheath (ORS) of the hair follicles. For further detailed characterization, two transgenic lines tg1 (K5-lacZflsmad7-line1) and tg2 (K5-lacZfl-smad7-line2) were selected. Line tg1 exhibited strong and continuous expression of lacZ in the epidermis and ORS of the hair follicles (Fig. 1b), whereas line tg2 exhibited a weaker and rather patchy expression of the reporter gene (Fig. 1c). Embryos of different developmental stages from the selected two transgenic lines were submitted to whole-mount X-Gal staining analysis (Fig. 1d–i). LacZ

ARTICLE IN PRESS 786

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

expression could first be detected on day 11.5 of gestation (Fig. 1d, e). In the embryos of line tg1, staining was observed in the lateral parts of the body surface (Fig. 1d, arrowheads), which include parts of the forming limbs and whisker-pads of the facial primordium (Fig. 1d, arrows). In line tg2, staining was much

weaker at this stage and was restricted to a stripe on the lateral body surface (Fig. 1e, arrowhead). At E12.5, the area of b-galactosidase activity was enlarged in both lines (Fig. 1f, g). At E13.5, most of the body lining expressed b-galactosidase with the exception of the cornea (Fig. 1h, arrows) and the interdigital fields of the

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

787

forming limbs (Fig. 1h, i, arrowheads). In comparison to line tg1, line tg2 exhibited a weaker staining in general. For analysis of the promoter activity in the oral cavity, X-Gal-stained embryos of both lines were embedded in paraffin and serial sections were analyzed (Fig. 2). At E11.5, b-galactosidase activity could be detected in the oral epithelium in line tg1 (Fig. 2a, arrowheads), while the low expressing line tg2 exhibited no reporter gene activity at this stage (Fig. 2b). By E12.5 most of the oral epithelium including the epithelial thickening of the forming tooth buds stained positive (Fig. 2c, arrowheads). Consistent with the whole-mount analysis, transgene expression in line tg2 was weaker and was first visible at E12.5 in the epithelial thickening of the forming tooth buds (Fig. 2d, arrowheads). At E13.5 the whole oral epithelium including the tongue and the epithelial component of the tooth buds showed strong staining in both lines with expression being higher in line tg1 (Fig. 2e, f, arrowheads). To evaluate whether the transgene is expressed during ameloblast differentiation, cryosections of embryos at E17.5 and/or newborn pups from both lines were analyzed. At this stage, ameloblasts and odontoblasts have started to secrete material of the tooth matrix. X-Gal staining showed strong expression in the ameloblast layer at day 17.5 (data not shown) and in newborn pups (Fig. 2g, arrowheads). In the odontoblast layer, no b-galactosidase activity could be detected (Fig. 2g, arrows).

Cre-recombinase activity in nestin-cre mice starts before gastrulation (Betz et al., 1996), recombination is complete by the onset of K5 promoter activity, which was reported between 9.5 and 10.5 days of gestation (Byrne et al., 1994). Offspring was screened for the presence of the K5 construct and/or the Cre construct by PCR (data not shown). Cre-mediated recombination in 6-week-old nestin-cre/K5-lacZfl-smad7 double transgenic animals was confirmed by Southern-blot analysis (Fig. 3a). In order to reveal the state of recombination in keratinocytes, ear and back skin of double transgenic animals of both lines was analyzed for lacZ expression (Fig. 3c–e). Consistent with the results of the Southernblotting experiments, no residual b-galactosidase expression could be detected by staining with X-Gal in adult nestin-cre/K5-lacZfl-smad7 double transgenic animals (Fig. 3d, e). In order to detect the induced transcripts of Smad7, RT-PCR with a combination of primers specific for transgene-derived smad7 transcripts was performed on total RNA derived from back skin of double transgenic animals (Fig. 3b). A 197-bp band indicative of correctly spliced transgene-derived smad7 transcripts, was detected in both lines of double transgenic animals (Fig. 3b, lanes 2 and 4).

Constitutive expression of Smad7 during embryogenesis

Since both parental animals were hemizygous for the transgenic locus, 25% of the offspring derived from the matings was expected to be double transgenic. However, when animals of line tg1 were mated with nestin-cre deleter mice, only two pups out of 92 were found to be double transgenic after weaning (expected: 23). The fact

For constitutive Smad7 overexpression under control of the K5 promoter, K5-lacZfl-smad7 mice of lines tg1 and tg2 were crossed to nestin-cre deleter mice. Since

Survival of nestin-cre/K5-lacZfl-smad7 double transgenic mice

Fig. 1. Generation of K5-lacZfl-smad7 transgenic mice and characterization of the lacZ reporter gene activity. (a) Structure of the K5-lacZfl-smad7 transgene. A 5.2-kb fragment containing upstream regulatory regions of the bovine keratin 5 gene is located 50 of a rabbit b-globin intron, followed by a lacZ cDNA with nuclear localization signal and a polyadenylation (polyA) signal from the gene for human growth hormone (hGH). LacZ cDNA and polyA signal are flanked by two loxP-recognition sites for Crerecombinase. The cDNA for murine Smad7 and another hGH polyA signal were cloned 30 of the second loxP site. The whole expression cassette is flanked by two pairs of human 50 HS-4 insulators (upper panel). Expression of Cre-recombinase in mice transgenic for K5-lacZfl-smad7 deletes the lacZ reporter gene and puts the smad7 cDNA under control of the promoter (lower panel). The probe used to confirm the recombination by Southern blot (black bar) and primer pair used to genotype the transgenic offspring (arrowheads) are shown in the upper panel. The primer pair used to detect Smad7 expression by RT-PCR is depicted in the lower panel (arrowheads). (b, c) Reporter gene expression in adult skin. Cryosections prepared from the ear biopsies were stained with X-Gal. Animals of line tg1 (b) exhibit strong and continuous b-galactosidase activity throughout the basal layer of the epidermis (arrows) including the ORS of hair follicles (arrowheads). Animals of line tg2 (c) show a weaker expression with large patches of positive cells in the epidermis (arrows) and ORS (arrowheads). (d-i) Reporter gene expression in embryos. Whole K5lacZfl-smad7 transgenic embryos of developmental stages E11.5 (d, e), E12.5 (f, g) and E13.5 (h, i) from transgenic lines tg1 (d, f, h) and tg2 (e, g, i) were stained with X-Gal. At E11.5 K5 promoter activity could be detected laterally along the body in both transgenic lines (d, e; black arrowheads). In line tg1, staining spreads anteriorly to the forming whisker pad (d, e; black arrows). At E12.5 lateral b-galactosidase-positive areas are larger while staining is still reduced dorsally (f, g; white arrowheads) and in the head area (white arrows). Reporter gene expression in line tg2 can be detected now in the developing whisker pad (g; black arrow). At E13.5 almost the entire embryo is stained in both transgenic lines indicating K5 promoter activity in the epidermis throughout the body (h, i). No b-galactosidase activity is found in apical areas of the limbs (h, i; white arrowheads) and the forming cornea (white arrows). Bars: 200 mm (b, c); 1 mm (d–i).

ARTICLE IN PRESS 788

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

Fig. 2. Reporter gene expression in the oral cavity. (a–f) Paraffin sections of whole-mount X-Gal-stained K5-lacZfl-smad7 embryos of developmental stages E11.5 (a, b), E12.5 (c, d) and E13.5 (e, f) from transgenic lines tg1 (a, c, e) and tg2 (b, d, f). At E11.5 K5 promoter activity begins in the oral epithelium of line tg1 (a; arrowheads). In line tg2 no promoter activity could be detected in the oral cavity at that stage (b). At E12.5, K5 promoter activity could be detected in both transgenic lines at sites, where the oral epithelium thickens as tooth formation is initiated (c, d; arrowheads) although expression is much stronger in line tg1 than in tg2. At E13.5 b-galactosidase could be detected throughout the epithelial lining of the oral cavity in both transgenic lines including the tooth buds and the tongue epithelium. (e, f; arrowheads). (g) Reporter gene expression in tooth germs. Cryosection of a forming molar tooth of a newborn pup from transgenic line tg1 (differential interference-contrast image). Cells of the epithelium-derived enamel organ, especially the cylindrical ameloblasts (arrowheads) express b-galactosidase. Cells of the mesenchyme-derived pulpa with the epithelium-like odontoblast layer (arrows) show no K5 promoter activity. (eo) enamel organ; (oc) oral cavity; (pu) pulpa; (tb) tooth buds; (to) tongue; Bars:100 mm.

that no remains of dead pups were found in the cages at this stage indicated that most double transgenic animals died in utero or shortly after birth. Crossing of line tg2 with deleter mice showed the expected ratios of double transgenics. Yet, the double transgenic animals of line tg2 exhibited significantly reduced size and weight. Most of the animals died at an age of 4–6 weeks. From these observations it can be concluded, that locus recombination and overexpression of Smad7 during embryogenesis leads to embryonal and perinatal mortality in a dose-dependent manner.

Smad7 trangene expression leads to coat abnormalities Double transgenic mice of both lines showed coat abnormalities, which lead to a disheveled appearance

(Fig. 4a). In particular, size and morphology of tail- and whisker hair shafts were aberrant (Fig. 4b). Histological analysis of the tail tip of transgenic animals revealed that hair shafts were reduced in size and length (Fig. 4c, arrowheads) as compared to the control animals (Fig. 4d). However, the density of the hair follicles was found to be normal (Fig. 4c, arrows).

Smad7 expression leads to developmental defects in tooth formation In addition to hair defects, all nestin-cre/K5-lacZflsmad7 double transgenic animals had characteristic tooth abnormalities (Fig. 5a,b) in comparison to the wild-type controls (Fig. 5c). Both surviving animals derived from crossing of tg1 animals with deleter mice had deep furrows in incisors of the lower jaw (Fig. 5f, arrowheads).

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

789

Fig. 3. Detection of Cre-mediated excision of the reporter gene. (a) Detection of Cre-mediated recombination in nestin-cre/K5lacZfl-smad7 double transgenic mice by Southern blot. Genomic DNA from spleen was digested with HindIII and separated on a 1% agarose gel. (lane 1) K5-lacZfl-smad7 transgenic animal of line tg2; (lane 2) nestin-cre/K5-lacZfl-smad7 double transgenic animal of line tg2; (lane 3) K5-lacZfl-smad7 transgenic animal of line tg1; (lane 4) nestin-cre/K5-lacZfl-smad7 double transgenic animal of line tg1; (lane 5) nestin-cre transgenic animal (upper panels). Upon hybridization with transgene-specific probe, un-recombined loci showed a restriction fragment of 5.3 kb length (lanes 1 and 3). Recombined loci were identified by a 3.3-kb restriction fragment (lanes 2 and 4). Hybridization with probe of a single copy gene as loading control (lower panels). (b) Detection of smad7-transgene expression by RT-PCR. (Lane 1) K5-lacZfl-smad7 transgenic animal of line tg1; (lane 2) nestin-cre/K5-lacZfl-smad7 double transgenic animal of line tg1; (lane 3) K5-lacZfl-smad7-transgenic animal of line tg2; (lane 4) nestin-cre/K5-lacZfl-smad7 double transgenic animal of line tg2 (upper panel). In nestin-cre/K5-lacZfl-smad7 double transgenic animals of both transgenic lines (lanes 2 and 4), a 197-bp band is detected for a correctly spliced transgene-derived smad7 mRNA, whereas in K5-lacZfl-smad7 transgenic animals of both lines (lanes 1 and 3), no specific signal is detected. Detection of a 725-bp band specific for PDGF-B as control reaction (lower panel). (c-e) Reporter gene expression in nestin-cre/K5-lacZfl-smad7 double transgenic mice. X-Gal staining of cryosections from ear biopsies. (c) K5-lacZfl-smad7 transgenic animal of line tg1 as positive control; (d) nestin-cre/K5-lacZfl-smad7 double transgenic animal of line tg1; (e) nestin-cre/K5-lacZfl-smad7 double transgenic animal of line tg2. No b-galactosidase activity was detected in the epidermis of nestin-cre/K5-lacZfl-smad7 double transgenic animals (d, e; arrowheads). Bars: 200 mm.

Fig. 4. Fur abnormalities in nestin-cre/K5-lacZfl-smad7 double transgenic mice. (a) A double transgenic animal with an uneven fur with bald spots (black arrows, left hand side) as compared to its control littermate (right hand side). (b) Deformation of hair shafts in nestin-cre/K5-lacZfl-smad7 double transgenic mice. Nestin-cre/K5-lacZfl-smad7 double transgenic animals of line tg2 (upper left panel) exhibit hardly any or deformed whiskers at 4 weeks of age as compared to their littermate controls (upper right panel). Also hair shafts are degenerated and reduced in size at the tail tip of nestin-cre/K5-lacZfl-smad7 double transgenic animals (lower left panel; arrows) when compared to littermate controls (lower right panel; arrows). (c, d) Histological analysis of hair follicles. Paraffin sections from tail skin of a nestin-cre/K5-lacZfl-smad7 double transgenic animal of line tg2 (c) and of control littermate (d) were stained with hematoxylin and eosin. Note the deformation in the hair follicles and reduction in the size of hair shafts in nestin-cre/ K5-lacZfl-smad7 double transgenic animals (c; arrowheads) as compared to littermate control (d; arrows). Bars: 100 mm.

ARTICLE IN PRESS 790

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

Histological analysis revealed that the defect (Fig. 5a,b) was in fact one incisor with two shafts that were fused at their bases (Fig. 5d,f). Organization of tooth-forming tissues in the tooth base was impaired, too. Furrows and duplications resulted from interrupted ameloblast layers (Fig. 5d, f, arrowheads). Dentin organization was also disturbed in the affected areas (Fig. 5f, arrows). Also, incisors displayed an unusual bright appearance indicating an impaired mineralization process in at least one of the two mineralized layers of the tooth (Fig. 6a,b). Consequently, affected teeth were worn out faster and broke off regularly (Fig. 6b). Histological

examination of 6-week-old double transgenic animals of line tg2 revealed that dentin of incisors had an impaired mineralization pattern with inflammatory infiltrates (Fig. 6d,f). Dentin defects, infiltration and, ultimately, breaking of the teeth are thought to be a secondary effect caused by suboptimal protection of the tooth structure due to a defective enamel layer.

Conditional expression of Smad7 in adult mice In order to bypass the embryonic and perinatal lethality observed in nestin-cre/K5-lacZfl-smad7 double

Fig. 5. Aberrant lower incisors in nestin-cre/K5-lacZfl-smad7 double transgenic animals. (a–c) Affected teeth in double transgenic animals from line tg1 (a) and tg2 (b) are deeply furrowed on their surface (a; arrowheads) or have duplicated shafts (a, b; arrows) when compared to littermates (c). (d–g) Histological analysis of aberrant lower incisors. Paraffin sections of demineralized heads were stained with hematoxylin and eosin. Basal region of lower right incisor of nestin-cre/K5-lacZfl-smad7 double transgenic animal from line tg1 (d, magnified in f) and wild-type control (e, magnified in g). Affected teeth have a discontinuous ameloblast layer (d; arrowheads) and the enamel gap, an artefact of the missing demineralized enamel layer, is interrupted by the irregularly organized dentin layer (f; arrows). In control littermates, ameloblasts and enamel gap are continuously organized on the front of the tooth (e; arrowheads). (am) ameloblasts; (de) dentin layer; (en) enamel gap; (od) odontoblasts. Bars: 200 mm.

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

transgenic animals, we mated K5-lacZfl-smad7 transgenics from both lines with K14-creERT2 animals. Ageand sex-matched un-induced double transgenic animals (dtg), K5-lacZfl-smad7 single transgenics (tg) and wildtype littermates (wt) were used as controls to 4-OHTinduced animals (dtg+). Eight weeks post-induction of

791

Cre-recombinase activity, significantly reduced expression of the lacZ reporter gene could be observed in the skin of line tg1 (Fig. 7a, dtg1+). Also, un-induced double transgenic animals of this line exhibited some reduction in the lacZ staining indicative of a basal Cre-recombinase activity even without induction

Fig. 6. Aberrant upper incisors in nestin-cre/K5-lacZfl-smad7 double transgenic animals. (a–c) In double transgenic animals of line tg2, the characteristic yellow color of a regularly mineralized enamel is partly absent (a, white arrowheads). Note the difference in tooth length in the affected area (a, white arrows). In heavily affected animals (b), teeth are completely bright. Tooth length is reduced and the root channels are visible (b, black arrows). Control littermates (c) exhibit a uniform yellow color. (d–g) Histological analysis of aberrant upper incisors. Paraffin sections of demineralized heads were stained with hematoxylin and eosin. In severely affected double transgenic animals of line tg2 (d, magnified in f), after 6 weeks the tooth has broken off (d; arrows). The tooth architecture is heavily disorganized with largely demineralized dentin including inflammatory cell infiltrates (f; arrowheads) whereas wild-type littermates have uniformly mineralized dentin without cellular infiltrates (e, magnified in g). Bars: 200 mm (a–c); 100 mm (d–g).

ARTICLE IN PRESS 792

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

(Fig. 7a, dtg1). Further, in thymus, diminished bgalactosidase activity was observed in induced and to a lesser extent in un-induced animals (Fig. 7a). Double transgenic animals of line tg2, which showed a patchy expression pattern of the reporter gene, exhibited comparable reduction in the b-galactosidase activity after induction with 4-OHT, though this was not as

pronounced as in double transgenic animals of line tg1 (data not shown). Cre-recombinase-mediated excision of the reporter gene lead to Smad7 expression in the skin, which was upregulated by a factor of 1.6 in transgenic line tg1 (Fig. 7b, lanes 2). The relative level of induced Smad7 expression in comparison to a housekeeping gene

Fig. 7. Hydroxytamoxifen-induced Cre-mediated recombination and expression of Smad7. (a) 4-OHT-induced Cre-mediated recombination. Skin and thymus cryosections from wild-type (wt), K5-lacZfl-smad7 single transgenics (tg1), un-induced K14creERT2/K5-lacZfl-smad7 double transgenics (dtg1) and induced K14-creERT2/K5-lacZfl-smad7 double transgenics (dtg1+) were stained with X-Gal for reporter gene expression. Note the reduced staining in dtg1 animals and almost absence of reporter gene expression in the dtg1+ group (arrowheads). E, epidermis; D, dermis; Hf, hair follicle. (b) RT-PCR analysis of smad7-transgene expression. Thymus RNA (lane 1), skin RNA (lane 2) of the respective groups. b-Actin was used as control. (c, d) Gross phenotype of hydroxytamoxifen-induced K14-creERT2/K5-lacZfl-smad7 double transgenics. Eight weeks post-induction of Cre-mediated Smad7 expression, K14-creERT2/K5-lacZfl-smad7 double transgenics exhibited severe fur and whisker abnormalities (c, dtg1+). dtg1+ transgenics were also characterized by pronounced weight loss (c, dtg1+). Un-induced K14-creERT2/K5-lacZfl-smad7 double transgenics did not differ much from the wt or tg1 animals (c). Additionally, hydroxytamoxifen-induced K14-creERT2/K5-lacZflsmad7 double transgenics exhibited pronounced aberrations in the development of incisors as compared to the control groups (d).

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

(b-actin) in skin was twice as high in line 1 as compared to line 2 (data not shown). Also, in un-induced double transgenic animals, Smad7 expression was upregulated resulting from a basal level of Cre-recombinase activity of the K14-creERT2 inductor system. Upon induction, Smad7 was also upregulated in the thymi of line tg1 by a factor of 3 (Fig. 7b, lanes 1).

Smad7 expression in adult mice leads to coat and teeth abnormalities Induced double transgenic animals of line tg1 exhibited a strong coat abnormality, so that these

793

animals appeared disheveled (Fig. 7c, dtg1+). Also the whiskers were distorted (Fig. 7c). A slight fur phenotype could be observed in the un-induced double transgenic animals of this line (Fig. 7c, dtg1). Additionally, the induced animals of line tg1 exhibited significant alterations in the teeth, too. Not only were the incisors bright white in this line, they were also fragile and they broke off regularly or were worn out fast (Fig. 7d). Interestingly, line tg2 exhibited no alterations in fur, whiskers or teeth after induction of Smad7 expression in comparison to the control animals (data not shown). Four weeks after induction of recombination, significant weight loss was observed in induced transgenic mice of line tg1. At 8 weeks post-induction these animals

Fig. 8. Hematoxylin and eosin staining of skin cryosections exhibited relatively thin and flattened keratinocytes in the basal layer of the epidermis of hydroxytamoxifen-induced K14-creERT2/K5-lacZfl-smad7 double transgenics whereas the suprabasal layers are partly absent (dtg1+, white arrows). Also, the stratum corneum exhibited pronounced thickening (dtg1+, arrowheads). E, epidermis; D, dermis; Hf, hair follicle. Bars: 500 mm. (b) Epidermal proliferation as determined by BrdUrd labeling. Hydroxytamoxifen-induced K14-creERT2/K5-lacZfl-smad7 double transgenic animals exhibited a significant increase in the proliferation index in the basal layer of the interfollicular epidermis in comparison to the wt, tg1 and dtg1 animals. (c) Expression profiling of the endogenous components of TGF-b superfamily signaling via RT-PCR. At least three animals per genotype and line were analyzed. Endogenous b-actin was used as control and standard. Hydroxytamoxifen-induced double transgenics (dtg1+) exhibited significant reduction in Smad3 expression.

ARTICLE IN PRESS 794

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

weighed half as much as their un-induced counterparts and 40% of the induced animals of line tg1 had died by this time point (data not shown). No weight loss and mortality was observed in line tg2 (data not shown). On a histological level, the thickness of the stratum corneum in the induced double transgenic mice of line tg1 was increased by 70% in comparison to the total epidermis thickness (Fig. 8a, arrowheads). Also, the proliferation rate in the interfollicular keratinocytes in the basal layer of the epidermis in these animals was significantly higher though no epidermal hyperplasia was observed (Fig. 8a,b). However, bromodeoxyuridine (BrdUrd) labeling was restricted to the basal layer of the epidermis. RT-PCR analysis of different Smad isoforms as well as TGF-bRII showed that in induced double transgenic animals, expression of Smad3 was reduced by 80% as compared to the control animals (Fig. 8c). No significant differences in the expression levels of these genes

after the induction of Smad7 expression were observed in line tg2 (data not shown). In order to explore the effect of Smad7 overexpression on keratinocyte differentiation, expression of differentiation markers was analyzed in the skin of induced and un-induced animals of line tg1, K5-dnTGFb-RII transgenics (Amendt et al., 1998) as well as control animals (Fig. 9). Keratin 14 could be detected in the interfollicular epidermis as well as in the ORS of the hair follicles in all the transgenic and wild-type animals (Fig. 9, left panel). Keratin 6 was expressed in the hair follicles and not in the interfollicular epidermis in all genotypes analyzed (Fig. 9, right panel). Keratin 10 was normally expressed in wild-type, un-induced K14creERT2/tg1 double transgenics as well as K5-dnTGFbRII transgenics but was completely absent in the epidermis of 4-OHT-induced K14-creERT2/tg1 double transgenics (Fig. 9, middle panel). Also, expression of filaggrin, a later differentiation marker, was altered in the 4-OHT-induced K14-creERT2/tg1 double

Fig. 9. Analysis of the state of keratinocyte differentiation. Expression patterns of keratins K14, K10 and K6 were analyzed using specific antibodies and Fast Red as chromogen substrate. No differences in the expression pattern of K14 (left column), and K6 (right column) could be observed in wild-type (Wt), K5-dnTGF-bRII transgenics (K5-dnRII), K5-lacZfl-smad7 transgenics of line tg1 (Tg1), un-induced K14-creERT2/K5-lacZfl-smad7 double transgenics of line tg1 (dTg1), and in hydroxytamoxifen-induced K14creERT2/K5-lacZfl-smad7 double transgenics of line tg1 (dTg1+). Expression of K10 (middle column) was, however, completely absent in hydroxytamoxifen-induced double transgenics of line tg1 (dTg1+). Bars: 200 mm.

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

795

Fig. 10. Analysis of filaggrin expression. Expression pattern of a late differentiation marker, filaggrin, in the suprabasal keratinocytes was analyzed using specific antibodies. Whereas suprabasal keratinocytes of line K5-lacZfl-smad7 exhibited a normal filaggrin expression pattern (a), the expression pattern of filaggrin was rather patchy (arrowheads) in hydroxytamoxifen-induced K14-creERT2/K5-lacZfl-smad7 double transgenics of line tg1 (b). No difference in the expression pattern of keratin 14, including the persistence into suprabasal layers, was observed in K5-lacZfl-smad7 transgenics (c) and hydroxytamoxifen-induced K14-creERT2/ K5-lacZfl-smad7 double transgenics of line tg1 (d). Arrowheads point to the epidermis (E). D: dermis. Bars: 100 mm.

transgenics (Fig. 10b) as compared to the K5-lacZflsmad7 single transgenics (Fig. 10a) or un-induced K14-creERT2/tg1 double transgenics and wild-type littermates (data not shown). However, persistence of K14 in suprabasal layers could be observed in both (Fig. 10c,d). Staining for keratins 4/13, markers of mucosal differentiation, remained negative for all genotypes analyzed (data not shown).

Discussion Members of the TGF-b superfamily regulate cell proliferation, differentiation and apoptosis, controlling not only the development but also maintenance of most tissues (Glick et al., 1993); Guo and Kyprianou, 1999; Motyl et al., 1998). Smad7 is a potent inhibitor of many members of the TGF-b family in vivo. Smad7 is induced by TGF-b/activin signaling, indicating that Smad7 is part of a negative feedback loop to restrict the cellular response to TGF-b/activin signaling (Nagarajan et al., 1999; Nakao et al., 1997; Takase et al., 1998). Function of Smad7 as an inhibitor of TGF-b and activin signaling (Ishisaki et al., 1999; Souchelnytskyi et al., 1998) may be more complicated because is has been shown to inhibit BMP signaling as well (Casellas and Brivanlou, 1998). Mice constitutively overexpressing Smad7 under the control of a keratin 5 promoter in epithelial tissues exhibited delayed and aberrant hair follicle morphogenesis, severe epithelial hyperplasia in the skin and other stratified epithelia, and thymic atrophy leading to embryonic and perinatal lethality (He et al., 2002). In the present study we established a Cre/loxP-based model allowing spatial and temporal control of the onset of Smad7 expression. Reporter gene analysis in the seven transgenic lines using X-Gal staining exhibited significant variations in the level of reporter gene expression. Such variations in adult tissues have been described for lacZ under control of bovine K5 sequences (Ramirez et al., 1994, 2001).

The spatial and temporal distribution of reporter gene expression in embryos was in general similar to that achieved using 5- to 6-kb upstream sequences of the bovine or human K5 genes (Byrne et al., 1994; Ramirez et al., 1994). Both transgenic lines analyzed in our study, exhibited b-galactosidase expression as early as E11.5. Ramirez and coworkers described K5 immunoreactivity in the mesenchymally derived odontoblasts of the tooth germ at E17.5 (Ramirez et al., 1994). By contrast, no b-galactosidase activity was found in mesenchymal components of the tooth germ including the odontoblast layer in our study, which might be due to the use of insulators (Potts et al., 2000). Cells of the epithelially derived enamel organ including the ameloblasts, however, exhibited a strong expression of the transgene. K5-lacZfl-smad7 transgenic mice were mated with Cre-deleter mice to induce the embryonic expression of Smad7. Deletion of loxP-flanked alleles have been shown to occur between 3.5 and 10.5 days post-coitum (dpc) when nestin-cre mice were used for the Cremediated recombination (Betz et al., 1996), whereas earliest K5 promoter activity has been reported for 10.5 dpc (Byrne et al., 1994). Double transgenic animals showed whisker and body hair defects. Additionally, severe defects in tooth development were observed which probably lead to embryonic and perinatal death in the high expressing line and to a wasting syndrome and eventual death in a second line with a weaker expression of Smad7. Similar defects in hair follicle development have also been shown in transgenic mice overexpressing Smad7 constitutively under control of the bovine K5 promoter, although the transgenic mice exhibited early mortality which correlated to the expression levels of the transgene (He et al., 2002). In our study, only the higher expressing line showed high mortality during development. In contrast to the perinatal lethality of all newborn K5.smad7 transgenics in the study by He et al. (2002), we were able to establish transgenic lines. This less severe phenotype could be due to different Smad7 expression levels due to integration

ARTICLE IN PRESS 796

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

position effects or to strain background. Also, tooth formation is already initiated at 10.5 dpc and the odontogenic potential switches from epithelium to mesenchyme (Thesleff et al., 1995), which indicates towards the role of Smad7 in tooth organogenesis and morphogenesis. Overexpression of Smad7 in this early phase could interfere with BMP-4 as a mediator of tooth bud induction, since BMP-4 is expressed in the epithelial thickening and switches to the condensing mesenchyme as odontogenic potential is acquired (Vainio et al., 1993). BMP-4 has also been discussed as a negative regulator of enamel knot size by induction of apoptosis in this area of the tooth bud epithelium (Jernvall et al., 1998). Smad7 may also interfere with activin signaling (Massague, 1998), and inactivation of activin signaling in knock-out mice leads to defects in incisor and mandibular molars (Ferguson et al., 1998). In order to analyze the effect of Smad7 overexpression in adult mice, we used conditional tamoxifeninducible Cre-recombinase-mediated excision of the reporter gene. K5-lacZfl-smad7 transgenic mice were mated with K14-creERT2 mice (Li et al., 2000), and Smad7 expression was induced when the animals were 6 weeks old. While no adverse effects of tamoxifen were observed, double transgenic animals of the high expressing line, started exhibiting a weak coat phenotype at an age of 6–8 weeks, even without induction of the Cre-recombinase activity, suggesting a leakiness of the K14-creERT2 system. Since previous studies detected no background recombinase activity in K14-creERT2 mice in the absence of OHT administration, the background activity seen in this study might be related to different genetic backgrounds, or to a higher accessibility of cre to the LoxP sites present in the K5-lacZfl-smad7 transgene (Indra et al., 2000). While the mice of the high expressing line developed severe abnormalities of whiskers, hair and teeth, comparable to the phenotype observed in the previous setting in this study, the second line with low expression of Smad7, did not develop a consistent phenotype, indicating that negative regulation of TGF-b signaling by Smad7 is dose dependent. Ito and coworkers have also shown that the negative regulation of TGF-b signaling is mediated by intracellular Smads in a dosedependent manner. In their study, attenuation of Smad7 had resulted in a significant inhibition of embryonic tooth development with increased apoptotic activity within the enamel organ epithelium (Ito et al., 2001). Induction of Smad7 by 4-OHT in adult mice affected the differentiation process in the skin and teeth. As expected, no comparable morphogenetic defects were observed in the teeth, as observed in the previous settings in the present study. Rather, we noted a characteristic disappearance of the enamel layer. Also, in skin differentiation- rather than morphogenesisassociated defects were observed. Induction of Smad7

lead to enhanced keratinocyte proliferation and defects in the differentiation of keratinocytes as exhibited by the complete absence of keratin 10. However, none of the mice with either interrupted TGF-b signaling (Amendt et al., 1998; Wang et al., 1999) or with interrupted activin signaling (Wankell et al., 2001) exhibited loss of keratin 10 without the induction of alternative keratins 6/16 and an altered pattern of filaggrin expression, a later stage differentiation marker for keratinocytes. We therefore propose that Smad7 also inhibits later stages of keratinocyte differentiation. However, due to the use of a basal cell-specific promoter, effects on keratinocyte differentiation are not entirely penetrant in the suprabasal layers. In a similar model (Han et al., 2006) it had been shown that conditional epidermal expression of Smad7 in keratinocytes also lead to aberrant hair follicle and sebaceous gland morphogenesis due to induced b-catenin degradation. This might well be the reason for disturbed differentiation in the interfollicular epidermis in our model since it has been shown that b-catenin function is essential for Ca2+-dependent induction of keratinocyte differentiation (Xie and Bikle, 2007). In summary, blockade of TGF-b superfamily signaling, most notably activin and TGF-b, results in failure of adult ameloblasts to synthesize an appropriate enamel layer and in failure of expression of early markers of terminal differentiation in keratinocytes.

Acknowledgments The authors would like to thank Claudia Braun and Antonietta Rastiello for excellent technical assistance and Dr. Belinda Knight for critical reading of the manuscript. This work was funded by the Free State of Saxony and EFRE project no. 4212 105-12/01.

References Alonso, S., Minty, A., Bourlet, Y., Buckingham, M., 1986. Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warmblooded vertebrates. J. Mol. Evol. 23, 11–22. Amendt, C., Schirmacher, P., Weber, H., Blessing, M., 1998. Expression of a dominant negative type II TGF-beta receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development. Oncogene 17, 25–34. Anna, C.H., Devereux, T.R., 1997. Sequence and chromosomal mapping of the mouse homolog (Madh4) of the human DPC4/MADH4 gene. Mamm. Genome 8, 443–444. Baker, J.C., Harland, R.M., 1996. A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway. Genes Dev. 10, 1880–1889. Bamberger, C., Scharer, A., Antsiferova, M., Tychsen, B., Pankow, S., Muller, M., Rulicke, T., Paus, R., Werner, S.,

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

2005. Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes. Am. J. Pathol. 167, 733–747. Betz, U.A., Vosshenrich, C.A., Rajewsky, K., Muller, W., 1996. Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination. Curr. Biol. 6, 1307–1316. Blessing, M., Nanney, L.B., King, L.E., Jones, C.M., Hogan, B.L., 1993. Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles. Genes Dev. 7, 204–215. Blessing, M., Schirmacher, P., Kaiser, S., 1996. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions. J. Cell Biol. 135, 227–239. Bonthron, D.T., Sultan, P., Collins, T., 1991. Structure of the murine c-sis proto-oncogene (Sis, PDGFB) encoding the B chain of platelet-derived growth factor. Genomics 10, 287–292. Botchkarev, V.A., Botchkareva, N.V., Roth, W., Nakamura, M., Chen, L.H., Herzog, W., Lindner, G., McMahon, J.A., Peters, C., Lauster, R., McMahon, A.P., Paus, R., 1999. Noggin is a mesenchymally derived stimulator of hairfollicle induction. Nat. Cell Biol. 1, 158–164. Breuhahn, K., Mann, A., Muller, G., Wilhelmi, A., Schirmacher, P., Enk, A., Blessing, M., 2000. Epidermal overexpression of granulocyte-macrophage colony-stimulating factor induces both keratinocyte proliferation and apoptosis. Cell Growth Differ 11, 111–121. Byrne, C., Tainsky, M., Fuchs, E., 1994. Programming gene expression in developing epidermis. Development 120, 2369–2383. Casatorres, J., Navarro, J.M., Blessing, M., Jorcano, J.L., 1994. Analysis of the control of expression and tissue specificity of the keratin 5 gene, characteristic of basal keratinocytes. Fundamental role of an AP-1 element. J. Biol. Chem. 269, 20489–20496. Casellas, R., Brivanlou, A.H., 1998. Xenopus Smad7 inhibits both the activin and BMP pathways and acts as a neural inducer. Dev. Biol. 198, 1–12. Chai, Y., Mah, A., Crohin, C., Groff, S., Bringas Jr., P., Le, T., Santos, V., Slavkin, H.C., 1994. Specific transforming growth factor-beta subtypes regulate embryonic mouse Meckel’s cartilage and tooth development. Dev. Biol. 162, 85–103. Chai, Y., Zhao, J., Mogharei, A., Xu, B., Bringas Jr., P., Shuler, C., Warburton, D., 1999. Inhibition of transforming growth factor-beta type II receptor signaling accelerates tooth formation in mouse first branchial arch explants. Mech. Dev. 86, 63–74. Chang, H., Brown, C.W., Matzuk, M.M., 2002. Genetic analysis of the mammalian transforming growth factorbeta superfamily. Endocrine Rev. 23, 787–823. Church, G.M., Gilbert, W., 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991–1995. Cui, W., Fowlis, D.J., Cousins, F.M., Duffie, E., Bryson, S., Balmain, A., Akhurst, R.J., 1995. Concerted action of

797

TGF-beta 1 and its type II receptor in control of epidermal homeostasis in transgenic mice. Genes Dev. 9, 945–955. Derynck, R., Feng, X.H., 1997. TGF-beta receptor signaling. Biochim. Biophys. Acta 1333, F105–F150. Dickson, M.C., Martin, J.S., Cousins, F.M., Kulkarni, A.B., Karlsson, S., Akhurst, R.J., 1995. Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development 121, 1845–1854. Ebisawa, T., Fukuchi, M., Murakami, G., Chiba, T., Tanaka, K., Imamura, T., Miyazono, K., 2001. Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J. Biol. Chem. 276, 12477–12480. Ferguson, C.A., Tucker, A.S., Christensen, L., Lau, A.L., Matzuk, M.M., Sharpe, P.T., 1998. Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition. Genes Dev. 12, 2636–2649. Ferguson, C.A., Tucker, A.S., Heikinheimo, K., Nomura, M., Oh, P., Li, E., Sharpe, P.T., 2001. The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development. Development 128, 4605–4613. Fire, A., Harrison, S.W., Dixon, D., 1990. A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93, 189–198. George, D.L., Phillips III., J.A., Francke, U., Seeburg, P.H., 1981. The genes for growth hormone and chorionic somatomammotropin are on the long arm of human chromosome 17 in region q21 to qter. Hum. Genet. 57, 138–141. Glick, A.B., Kulkarni, A.B., Tennenbaum, T., Hennings, H., Flanders, K.C., O’Reilly, M., Sporn, M.B., Karlsson, S., Yuspa, S.H., 1993. Loss of expression of transforming growth factor beta in skin and skin tumors is associated with hyperproliferation and a high risk for malignant conversion. Proc. Natl. Acad. Sci. USA 90, 6076–6080. Guo, Y., Kyprianou, N., 1999. Restoration of transforming growth factor beta signaling pathway in human prostate cancer cells suppresses tumorigenicity via induction of caspase-1-mediated apoptosis. Cancer Res. 59, 1366–1371. Halder, S.K., Beauchamp, R.D., Datta, P.K., 2005. Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Exp. Cell Res. 307, 231–246. Han, G., Li, A.G., Liang, Y.Y., Owens, P., He, W., Lu, S., Yoshimatsu, Y., Wang, D., Ten Dijke, P., Lin, X., Wang, X.J., 2006. Smad7-induced beta-catenin degradation alters epidermal appendage development. Dev. Cell 11, 301–312. Hata, A., Lagna, G., Massague, J., Hemmati-Brivanlou, A., 1998. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. Genes Dev. 12, 186–197. Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y.Y., Grinnell, B.W., Richardson, M.A., Topper, J.N., Gimbrone Jr., M.A., Wrana, J.L., Falb, D., 1997. The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell 89, 1165–1173.

ARTICLE IN PRESS 798

B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

He, W., Cao, T., Smith, D.A., Myers, T.E., Wang, X.J., 2001. Smads mediate signaling of the TGFbeta superfamily in normal keratinocytes but are lost during skin chemical carcinogenesis. Oncogene 20, 471–483. He, W., Li, A.G., Wang, D., Han, S., Zheng, B., Goumans, M.J., Ten Dijke, P., Wang, X.J., 2002. Overexpression of Smad7 results in severe pathological alterations in multiple epithelial tissues. EMBO J. 21, 2580–2590. Heldin, C.H., Miyazono, K., ten Dijke, P., 1997. TGF-beta signalling from cell membrane to nucleus through Smad proteins. Nature 390, 465–471. Hogan, B.L., Blessing, M., Winnier, G.E., Suzuki, N., Jones, C.M., 1994. Growth factors in development: the role of TGF-beta related polypeptide signalling molecules in embryogenesis. Development (Suppl.), 53–60. Imamura, T., Takase, M., Nishihara, A., Oeda, E., Hanai, J., Kawabata, M., Miyazono, K., 1997. Smad6 inhibits signalling by the TGF-beta superfamily. Nature 389, 622–626. Indra, A.K., Li, M., Brocard, J., Warot, X., Bornert, J.M., Gerard, C., Messaddeq, N., Chambon, P., Metzger, D., 2000. Targeted somatic mutagenesis in mouse epidermis. Horm. Res. 54, 296–300. Ishisaki, A., Yamato, K., Hashimoto, S., Nakao, A., Tamaki, K., Nonaka, K., ten Dijke, P., Sugino, H., Nishihara, T., 1999. Differential inhibition of Smad6 and Smad7 on bone morphogenetic protein- and activin-mediated growth arrest and apoptosis in B cells. J. Biol. Chem. 274, 13637–13642. Ito, Y., Zhao, J., Mogharei, A., Shuler, C.F., Weinstein, M., Deng, C., Chai, Y., 2001. Antagonistic effects of Smad2 versus Smad7 are sensitive to their expression level during tooth development. J. Biol. Chem. 276, 44163–44172. Jahnen-Dechent, W., Schinke, T., Trindl, A., Muller-Esterl, W., Sablitzky, F., Kaiser, S., Blessing, M., 1997. Cloning and targeted deletion of the mouse fetuin gene. J. Biol. Chem. 272, 31496–31503. Jernvall, J., Thesleff, I., 2000. Reiterative signaling and patterning during mammalian tooth morphogenesis. Mech. Dev. 92, 19–29. Jernvall, J., Aberg, T., Kettunen, P., Keranen, S., Thesleff, I., 1998. The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125, 161–169. Jones, C.M., Lyons, K.M., Hogan, B.L., 1991. Involvement of bone morphogenetic protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111, 531–542. Kavsak, P., Rasmussen, R.K., Causing, C.G., Bonni, S., Zhu, H., Thomsen, G.H., Wrana, J.L., 2000. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol. Cell 6, 1365–1375. Kingsley, D.M., 1994. The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 8, 133–146. Kozak, M., 1987. An analysis of 50 -noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8148. Labbe, E., Silvestri, C., Hoodless, P.A., Wrana, J.L., Attisano, L., 1998. Smad2 and Smad3 positively and negatively regulate TGF beta-dependent transcription through the

forkhead DNA-binding protein FAST2. Mol. Cell 2, 109–120. Landstrom, M., Heldin, N.E., Bu, S., Hermansson, A., Itoh, S., ten Dijke, P., Heldin, C.H., 2000. Smad7 mediates apoptosis induced by transforming growth factor beta in prostatic carcinoma cells. Curr. Biol. 10, 535–538. Laurikkala, J., Kassai, Y., Pakkasjarvi, L., Thesleff, I., Itoh, N., 2003. Identification of a secreted BMP antagonist, ectodin, integrating BMP, FGF, and SHH signals from the tooth enamel knot. Dev. Biol. 264, 91–105. Lawler, S., Candia, A.F., Ebner, R., Shum, L., Lopez, A.R., Moses, H.L., Wright, C.V., Derynck, R., 1994. The murine type II TGF-beta receptor has a coincident embryonic expression and binding preference for TGF-beta 1. Development 120, 165–175. Li, Q., Stamatoyannopoulos, J.A., 1994. Position independence and proper developmental control of gamma-globin gene expression require both a 50 locus control region and a downstream sequence element. Mol. Cell. Biol. 14, 6087–6096. Li, M., Indra, A.K., Warot, X., Brocard, J., Messaddeq, N., Kato, S., Metzger, D., Chambon, P., 2000. Skin abnormalities generated by temporally controlled RXRalpha mutations in mouse epidermis. Nature 407, 633–636. Liu, X., Lee, J., Cooley, M., Bhogte, E., Hartley, S., Glick, A., 2003. Smad7 but not Smad6 cooperates with oncogenic ras to cause malignant conversion in a mouse model for squamous cell carcinoma. Cancer Res. 63, 7760–7768. Massague, J., 1998. TGF-beta signal transduction. Annu. Rev. Biochem. 67, 753–791. Massague, J., Wotton, D., 2000. Transcriptional control by the TGF-beta/Smad signaling system. EMBO J. 19, 1745–1754. Meersseman, G., Verschueren, K., Nelles, L., Blumenstock, C., Kraft, H., Wuytens, G., Remacle, J., Kozak, C.A., Tylzanowski, P., Niehrs, C., Huylebroeck, D., 1997. The Cterminal domain of Mad-like signal transducers is sufficient for biological activity in the Xenopus embryo and transcriptional activation. Mech. Dev. 61, 127–140. Metzger, D., Chambon, P., 2001. Site- and time-specific gene targeting in the mouse. Methods 24, 71–80. Miyazono, K., Kusanagi, K., Inoue, H., 2001. Divergence and convergence of TGF-beta/BMP signaling. J. Cell. Physiol. 187, 265–276. Motyl, T., Grzelkowska, K., Zimowska, W., Skierski, J., Wareski, P., Ploszaj, T., Trzeciak, L., 1998. Expression of bcl-2 and bax in TGF-beta 1-induced apoptosis of L1210 leukemic cells. Eur. J. Cell Biol. 75, 367–374. Munz, B., Smola, H., Engelhardt, F., Bleuel, K., Brauchle, M., Lein, I., Evans, L.W., Huylebroeck, D., Balling, R., Werner, S., 1999. Overexpression of activin A in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair. EMBO J. 18, 5205–5215. Murakami, G., Watabe, T., Takaoka, K., Miyazono, K., Imamura, T., 2003. Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads. Mol. Biol. Cell 14, 2809–2817. Nagarajan, R.P., Zhang, J., Li, W., Chen, Y., 1999. Regulation of Smad7 promoter by direct association with Smad3 and Smad4. J. Biol. Chem. 274, 33412–33418.

ARTICLE IN PRESS B. Klopcic et al. / European Journal of Cell Biology 86 (2007) 781–799

Nakao, A., Afrakhte, M., Moren, A., Nakayama, T., Christian, J.L., Heuchel, R., Itoh, S., Kawabata, M., Heldin, N.E., Heldin, C.H., ten Dijke, P., 1997. Identification of Smad7, a TGFbeta-inducible antagonist of TGFbeta signalling. Nature 389, 631–635. Neubuser, A., Peters, H., Balling, R., Martin, G.R., 1997. Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90, 247–255. Ohazama, A., Tucker, A., Sharpe, P.T., 2005. Organized tooth-specific cellular differentiation stimulated by BMP4. J. Dent. Res. 84, 603–606. Pelton, R.W., Saxena, B., Jones, M., Moses, H.L., Gold, L.I., 1991. Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J. Cell Biol. 115, 1091–1105. Pispa, J., Thesleff, I., 2003. Mechanisms of ectodermal organogenesis. Dev. Biol. 262, 195–205. Potts, W., Tucker, D., Wood, H., Martin, C., 2000. Chicken beta-globin 5’HS4 insulators function to reduce variability in transgenic founder mice. Biochem. Biophys. Res. Commun. 273, 1015–1018. Ramirez, A., Bravo, A., Jorcano, J.L., Vidal, M., 1994. Sequences 5’ of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development. Differentiation 58, 53–64. Ramirez, A., Milot, E., Ponsa, I., Marcos-Gutierrez, C., Page, A., Santos, M., Jorcano, J., Vidal, M., 2001. Sequence and chromosomal context effects on variegated expression of keratin 5/lacZ constructs in stratified epithelia of transgenic mice. Genetics 158, 341–350. Roberts, A.B., Anzano, M.A., Lamb, L.C., Smith, J.M., Sporn, M.B., 1981. New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues. Proc. Natl. Acad. Sci. USA 78, 5339–5343. Sanford, L.P., Ormsby, I., Gittenberger-de Groot, A.C., Sariola, H., Friedman, R., Boivin, G.P., Cardell, E.L., Doetschman, T., 1997. TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development 124, 2659–2670. Sellheyer, K., Bickenbach, J.R., Rothnagel, J.A., Bundman, D., Longley, M.A., Krieg, T., Roche, N.S., Roberts, A.B., Roop, D.R., 1993. Inhibition of skin development by overexpression of transforming growth factor beta 1 in the epidermis of transgenic mice. Proc. Natl. Acad. Sci. USA 90, 5237–5241. Souchelnytskyi, S., Nakayama, T., Nakao, A., Moren, A., Heldin, C.H., Christian, J.L., ten Dijke, P., 1998. Physical and functional interaction of murine and Xenopus Smad7 with bone morphogenetic protein receptors and transforming growth factor-beta receptors. J. Biol. Chem. 273, 25364–25370.

799

Staggers, W.R., Paterson, A.J., Kudlow, J.E., 1995. Sequence of the functional human keratin K14 promoter. Gene 153, 297–298. Sternberg, N., Sauer, B., Hoess, R., Abremski, K., 1986. Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation. J. Mol. Biol. 187, 197–212. Takase, M., Imamura, T., Sampath, T.K., Takeda, K., Ichijo, H., Miyazono, K., Kawabata, M., 1998. Induction of Smad6 mRNA by bone morphogenetic proteins. Biochem. Biophys. Res. Commun. 244, 26–29. Ten Dijke, P., Goumans, M.J., Itoh, F., Itoh, S., 2002. Regulation of cell proliferation by Smad proteins. J. Cell. Physiol. 191, 1–16. Thesleff, I., Mikkola, M., 2002. The role of growth factors in tooth development. Int. Rev. Cytol. 217, 93–135. Thesleff, I., Vaahtokari, A., Partanen, A.M., 1995. Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int. J. Dev. Biol. 39, 35–50. Vainio, S., Karavanova, I., Jowett, A., Thesleff, I., 1993. Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75, 45–58. Wang, X.J., Liefer, K.M., Tsai, S., O’Malley, B.W., Roop, D.R., 1999. Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis. Proc. Natl. Acad. Sci. USA 96, 8483–8488. Wankell, M., Munz, B., Hubner, G., Hans, W., Wolf, E., Goppelt, A., Werner, S., 2001. Impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis. EMBO J. 20, 5361–5372. Werner, S., Weinberg, W., Liao, X., Peters, K.G., Blessing, M., Yuspa, S.H., Weiner, R.L., Williams, L.T., 1993. Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J. 12, 2635–2643. Winnier, G., Blessing, M., Labosky, P.A., Hogan, B.L., 1995. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105–2116. Xie, Z., Bikle, D.D., 2007. The recruitment of phosphatidylinositol 3-kinase to the E-cadherin-catenin complex at the plasma membrane is required for calcium-induced phospholipase C-gamma 1 activation and human keratinocyte differentiation. J. Biol. Chem. 282, 8695–8703. Yamaguchi, T.P., Rossant, J., 1995. Fibroblast growth factors in mammalian development. Curr. Opin. Genet. Dev. 5, 485–491. Yingling, J.M., Das, P., Savage, C., Zhang, M., Padgett, R.W., Wang, X.F., 1996. Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth. Proc. Natl. Acad. Sci. USA 93, 8940–8944.