Materials and Methods

Reagents
Primary antibodies used were against: E-cadherin (M. Takeichi, Kyoto University, Japan); α-catenin, β-catenin, pMAPK, tubulin (Sigma, St. Louis, Missouri, United States), Ajuba (G. Longmore, Washington University, St. Louis, Missouri, United States); β4 integrin/CD104 (BD Pharmingen, San Diego, California, United States), laminin 5 (R. Burgeson, Harvard University, Cambridge, Massachusetts, United States), K5, K1, loricrin (Fuchs Lab), involucrin, fillagrin (Covance, Berkeley, California, United States), MAPK, pSMAD2 (Cell Signaling, Beverly, Massachusetts, United States); Grb-2 (Santa Cruz Biotech, Santa Cruz, California, United States); P-cadherin (Zymed Laboratories, South San Francisco, California, United States); HA (Roche Biochemicals), vimentin (Chemicon, Temecula, California, United States), Ki67 (Novo Castra, Newcastle Upon Tyne, United Kingdom), keratin 6 (P. Coulombe, John Hopkins University, Baltimore, Maryland, United States), cyclin D (Oncogene, San Diego, California, United States), and TGF-β2 (L. Gold, New York University, New York, New York, United States). FITC-, Texas Red-, or HRP-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, Pennsylvania, United States). Biotinylated secondary antibodies were from Vector Labs (Burlingame, California, United States). Dilutions were according to the manufacturer's recommendation. The Snail antibody was generated in Guinea pigs by inoculating them with the N-terminal sequence of murine Snail fused to GST (Covance, Princeton, New Jersey, United States). Recombinant human TGF-β2 was purchased from R&D (Minneapolis, Minnesota, United States). Heat inactivated TGF-β2 was generated by heating the recombinant protein at 100 °C for 10 min.

Mice
The K14-Snail Tg mouse was generated by digesting the pcDNA3-mm Snail-HA plasmid (G. de Herreros, Universitat Pompeu, Fabra, Barcelona, Spain) with BamHI and NotI and subcloned into the K14 vector [49]. The linearized construct was injected into the nucleus of embryos from CD1 mice. The K14-Smad 2 Tg mouse was reported in Ito et al., 2001. The TGF-β2 knockout (KO) mouse was described in [34]. The shh KO mouse [38] and TOPGal mouse [20] have previously been reported.

Western blot and immunoprecipitation
Protein extracts from primary keratinocytes were generated either by lysing cells in lysis buffer (1% NP-40, 1% sodium deoxycholate, 20 mM Tris-Cl [pH 7.4], 140 mM NaCl containing 1 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and protease inhibitors) or directly in Laemmli bβuffer and boiled. For skin tissue: Frozen tissue was pulverized in a liquid nitrogen-cooled Gevebesmascher and the powder scraped into a chilled microcentrifuge tube. RIPA buffer (1% Triton X-100 in PBS with 10 mM EDTA, 150 mN NaCl, 1% sodium deoxycholate, and 0.1% SDS) and protease inhibitors or Laemmli buffer was added. The cell suspension was sonicated three times for 15 s and centrifuged at 14,000 rpm at 4 °C. The supernatant was separated from the pellet and used in the experiments. Extracts subjected to immunoprecipitation were precleared with Protein G Sepharose (Amersham, Piscataway, New York, United States) and incubated with antibody with rocking overnight at 4 °C. Protein G Sepharose was added and samples were incubated for 1 h at 4 °C with rocking. Samples were washed three times for 5 min each in lysis buffer, and the Protein G Sepharose-antibody-antigen pellet was resuspended in Laemmli buffer and boiled for 10 min. Samples were run on SDS-PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell Bioscience, Keene, New Hampshire, United States). Western blot signals were developed using the enhanced chemiluminescence kit from Amersham

Cell culture
Primary keratinocytes were culture in low-calcium medium as previously described [4]. Transient transfections were carried out with FuGENE6 reagent (Roche, Indianapolis, Indiana, United States) according to the manufacturer's protocol. Measurement of β-galactosidase or luciferase levels in promoter activity studies were carried out with the Galacto-Lite assay kit (TROPIX, Bedford, Massachusetts, United States) and the Dual luciferase (Promega, Madison, Wisconsin, United States), respectively. Runella luciferase was cotransfected into cells to correct for transfection efficiency. Experiments were done in triplicate and repeated at least three times. Measurements were done on a luminometer (MGM Instruments, Hamden, Connecticut, United States). For experiments measuring phosphorylation of MAPK, keratinocytes were serum starved for 3 h prior to harvesting of cells by incubation in medium lacking serum. Treatment of cells with Wnt- and noggin-conditioned medium was previously described [4].

Constructs
The 2.2-kb murine Snail promoter was generated by PCR using a forward primer with an XbaI linker sequence, 5′- TCTAGAATTGTTTGCTGCTGTATGGTCTTC-3′, along with a reverse primer with a BglII linker sequence, 5′- AGATCTGTTGGCCAGAGCGACCTAG- GTAG-3′, and mouse genomic DNA as a template. The PCR product was purified with the Gel Extraction Kit (Qiagen, Valencia, California, United States) and ligated into pCRII-TOPO TA vector (Invitrogen, Carlsbad, California, United States). The promoter was verified by sequencing and digested with XbaI and BglII and subcloned into the pβ-gal BASIC vector (BD Biosciences Clontech, Palo Alto, California, United States). The point mutations in the SMAD binding element was generated with the Quik-Change Kit (Stratagene, La Jolla, California, United States) using the forward primer 5′- GGGCGGGCTTAGGTGTTTTCATTTACTCTTGAGGAAAAGCTTGGC-3′ and the reverse primer 5′- GCTTTT- CCTCAAGAGTAAATGAAAACACCTAAGCCCGCCCTGCCC-3′. The probes for the Snail in situ hybridization were generated against the 3′ UTR by PCR using the forward primer 5′- ACCTTCTCCCGCATGTCCTTGCTCC-3′ and the reverse primer 5′- CTGCTGAGGCATGGTTACAGCTGG-3′, and genomic DNA as a template. The PCR product was gel purified and ligated into pCRII-TOPO TA vector. The pre-LIM domain of Ajuba was generated essentially as described [9], but was fused to GFP by subcloning from the pEGFP-N1 20 vector (BD Biosciences Clontech)

In situ hybridization
The pCRII-TOPO TA vector containing a region of the 3′ UTR of Snail was used as a template to generate digoxigenin-labeled sense and antisense riboprobes (Roche). The respective probes were obtained by XhoI and BamH1 digestions. In situ hybridizations were performed on 10-μm thick sections of E17.5 mouse embryos. The sections were fixed with 4% PFA for 10 min at room temperature, prehybridized at room temperature for 4.5 h, hybridized with the probe (2 μg/ml) at 55 °C for 12–14 h, blocked with 10% NGS, and treated with anti-dig Fab-AP antibody (Roche #1093274) at a 1:2,500 dilution for 3 h. The sections were incubated with NBT and BCIP until adequate signal was detected.

Immunofluorescence and immunohistochemistry
Tissue samples for immunofluorescence were frozen in OCT and sectioned 10 μm thick on a cryostat. Sections were fixed in 4% paraformaldehyde for 10 min at room temperature, blocked, and stained with antibodies. Tissue samples for immunohistochemistry were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Samples were sectioned on a microtome (10 μm thick) and rehydrated prior to staining with antibody. Samples stained with Snail, pMAPK, pSmad2, and cyclin D were antigen unmasked with 10 mM sodium citrate (pH 6) in an Antigen Retriever 2100 (Pickcell Laboratories, Leiden, Netherlands). The DAB substrate kit (Vector Labs) was used according to manufacturer's instructions to develop the signal.

RT-PCR
RNA was extracted from keratinocytes or skin tissue with Trizol (Invitrogen) according to the manufacturer's protocol. cDNA was generated using oligo-dT primers and the Superscript II kit (Invitrogen). The primers used for PCR were Snail forward: 5′- CAGCTGGCCAGGCTCTCGGT-3′; Snail reverse: 5′- GCGAGGGCCTCCGGAGCA-3′; GAPDH forward 5′- CGTAGACAAAATGGTGAAGGTCGG-3′; and GAPDH reverse: 5′- AAGCAGTTGGTGGTGCAGGATG-3′.