Monitoring Wnt/β-Catenin Signaling in Skin

Abstract

Wnt signaling through β-catenin plays a crucial role in skin development and homeostasis. Disruption or hyperactivation of this pathway results in skin defects and diseases (Lim and Nusse, Cold Spring Harb Perspect Biol 5(2), 2013). Monitoring Wnt signaling in skin under normal and abnormal conditions is therefore critical to understand the role of this pathway in development and homeostasis.

In this chapter, we provide methods to detect Wnt/β-catenin (canonical) signaling in the skin. We present a comprehensive list of Wnt reporter mice and detail the processing of skin tissue to detect reporter genes. From this list, we focus on the three most recent lines that, according to reports, are the most sensitive in skin. Additionally, we describe a protocol to detect nuclear β-catenin, a hallmark of active Wnt signaling, although this technique should be used with caution due to its limited sensitivity. The techniques outlined below will be useful for detecting active Wnt signaling in skin.

1 Introduction

The skin is a self-renewing tissue made up of the stratified epidermis, its appendages and the underlying dermis [1]. The stratified epidermis, which provides protective barrier function of the skin, continually sheds its outermost layer. Cells in the basal layer proliferate, move upward and differentiate to replenish the outermost layer. Whereas the stratified epidermis self-renews continuously, hair follicles undergo periodic degeneration and regeneration, depending on signals from a specific dermal niche, the dermal papilla [2]. In response to wounding, stem cells from both the stratified epidermis and hair follicles contribute to repair of the epidermis [3–9].

Canonical Wnt signaling plays a critical role in skin epidermal development and homeostasis [10]. During morphogenesis, Wnt/β-catenin signaling is required in both dermal and epidermal cells to specify formation of the hair placode and the dermal papilla precursor, the dermal condensate [11]. Subsequently, Wnt/β-catenin signaling is essential in both hair follicular progenitor cells and the dermal papilla for hair regeneration to occur [12–15]. Under homeostatic conditions, Wnt/β-catenin controls normal proliferation of the basal cells of the stratified epidermis [16, 17].

Given the crucial role of Wnt/β-catenin signaling in skin biology, monitoring Wnt signaling in the skin is of vital interest to skin researchers. Because canonical Wnt signaling results in the translocation of β-catenin into the nucleus, detection of nuclear β-catenin has been used to demonstrate active Wnt signaling. However, due to the low sensitivity of the assay, nuclear β-catenin may reflect only very high Wnt activity. A more sensitive method is based on the detection of Wnt target genes. Wnt target genes are transcribed when nuclear β-catenin binds to TCF/LEF, which bind to TCF/LEF binding sites of the promoters of Wnt target genes, and transactivate their transcription. In order to detect Wnt activity, a series of Wnt reporter mice have been developed. These mice can be categorized into two groups according to the types of promoter. One group expresses reporter gene lacZ or green fluorescent protein (GFP) or its variants under the promoter containing multimerized TCF /LEF binding sites [18–25]. The other group contains the reporter gene knock-in under the control of the endogenous promoter of the Wnt target gene Axin2, or inducible cre-recombinase (creERT2) knocked into the Axin2 locus and used with Rosa26 reporter lines [17, 26–30].

In this chapter, we summarize the current Wnt reporter strains used to monitor canonical Wnt signaling and provide methods for detecting different reporter genes in the skin. We also provide an immunostaining protocol for detecting nuclear β-catenin.

2 Materials

2.1 Wnt Reporter Mice

See Table 1. We used wild-type C57BL/6 mice, TCF/LEF:H2BGFP mice [25], BAT-GAL mice [20], and Axin2^lacZ mice [27] as specific examples in this chapter. Low Wnt activity is detected in basal cells of the epidermis only in TCF/LEF:H2B-GFP mice and Wnt reporter mice with Axin2 promoter [16, 17].

Table 1.

Wnt reporter mice

Transgenic reporter mice with promoter containing multimerized TCF/LEF binding sites
	Promoter	References
TOP-GAL	3 TCF/LEF binding sites   with c-fos minimal promoter	DasGupta and Fuchs, 1999 [18]
TOP-lacZ	3 TCF/LEF binding sites   with TK minimal promoter	Staal et al., 2001 [19]
BAT-GAL	7 TCF/LEF binding sites with   siamois minimal promoter	Maretto et al., 2003 [20]
TCF/LEF-lacZ	6 TCF/LEF binding sites with   hsp68 minimal promoter	Mohamed et al., 2004 [21]
BAT-lacZ	8 TCF/LEF binding sites with   minimal siamois promoter	Nakaya et al., 2005 [22]
Ins-TOPGAL	6 TCF/LEF binding sites   with TK minimal promoter	Moriyama et al., 2007 [23]
Ins-TOPEGFP	6 TCF/LEF binding sites   with TK minimal promoter	Moriyama et al., 2007 [23]
LEF-EGFP	7 TCF/LEF binding sites with c-fos   minimal promoter	Currier et al., 2010 [24]
TCF/LEF:H2B-GFP	6 TCF/LEF binding sites with   hsp68 minimal promoter	Ferrer-Vaquer et al., 2010 [25]
Reporter mice with Wnt target gene promoter (Axin2)
Axin2-EGFP	5.6 kb genomic fragment containing   promoter and first intron of Axin2	Jho et al., 2002 [26]
Axin2lacZ	Endogenous Axin2 promoter	Lustig et al., 2002 [27]
Axin2creERT2;Rosa26RmTmG	Endogenous Axin2 promoter	van Amerongen et al., 2012;   Muzumdar et al., 2007 [28, 29]
Axin2creERT2;Rosa26RlacZ	Endogenous Axin2 promoter	van Amerongen et al., 2012;   Soriano 1999 [28, 30]
Axin2creERT/tdT;Rosa26RmTmG	Endogenous Axin2 promoter	Choi et al., 2013; Muzumdar   et al., 2007 [17, 29]

2.2 Tissue Embedding Components

1. Electric clipper.

2. 4-in. scissors.

3. 4-in. dissecting forceps.

4. Razor blade.

5. Brown paper towels.

6. Cryomolds 25 × 20 mm.

7. O.C.T. compound.

8. Dry ice block.

2.3 Tissue Fixation Reagents and Components

1. 30 % sucrose solution. Filtrate with 0.45 um filter. Store at 4 °C.

2. 10 % sucrose solution. Filtrate with 0.45 um filter. Store at 4 °C.

3. Phosphate-buffered saline (PBS).

4. EMS 16 % paraformaldehyde (PFA) in 10 mL glass ampules.

5. Falcon 5 mL 12 × 75 mm snap-cap polypropylene tubes.

6. Aluminum foil.

7. Shaker.

2.4 Tissue Sectioning and Staining Components

1. Cryostat.

2. Adhesion Micro Slides.

3. Microscope cover glass.

4. Hematoxylin 2.

5. Glass staining jar.

6. 10 % Triton X-100 solution.

7. 20 % bovine serum albumin (BSA). Filtrate with 0.22 um filter, aliquot, and store at −20 °C.

8. Gelatin from cold water fish skin (Sigma).

9. 10 % normal donkey serum (NDS).

10. PBSGT buffer: 2 % fish gelatin, 0.2 % Triton, in PBS.

11. Blocking buffer: PBSGT buffer with 2 % BSA with 10 % NDS.

12. Staining buffer: PBSGT buffer with 1 % BSA with 5 % NDS.

13. Hoechst 33342.

14. ProLong Gold Antifade (Molecular Probes, Thermofisher).

15. Nail polish.

Additional components for immunohistochemistry staining of β-catenin using M.O.M kit

1. Citrus clearing solvent as xylene substitute.

2. Ethanol series: 100, 95, 85, 70, 50 %.

3. 10 mM citric acid buffer, pH 6.0.

4. 0.3 % Hydrogen peroxidase (H₂O₂). Prepare fresh from 30 % hydrogen peroxide.

5. Mouse on Mouse (M.O.M. ™) basic kit (Vector).

6. Avidin/Biotin blocking kit.

7. M.O.M blocking buffer with Avidin: Add 45 µL of IgG reagent (from M.O.M. kit), four drops of avidin solution (from Avidin/Biotin Blocking kit) to 1 mL PBSGT buffer.

8. M.O.M. Diluent Buffer with Biotin: Add 100 µL of Protein Concentrate reagent (from M.O.M. kit), four drops of biotin solution (from Avidin/Biotin Blocking kit) to 1 mL PBSGT buffer.

9. M.O.M. Diluent Buffer without Biotin: Add 80 µL of Protein Concentrate reagent (from M.O.M. kit) to 1 mL PBSGT buffer.

10. Vectastain Elite ABC reagent: Prepare fresh. Add one drop of Reagent A to 1.25 mL PBS, mixing well, then adding one drop of Reagent B, then mixing well again. Allow ABC mixture to stand for 30 min at room temperature before use.

11. ImmPACT DAB Peroxidase (HRP) substrate: Prepare fresh. Add one drop of concentrated DAB to 1 mL of the provided diluent buffer.

12. Clarifier 2.

13. Bluing reagent.

14. Permount Mounting Medium.

15. Coplin staining jar.

16. Hybridization oven.

17. Microwave oven.

2.5 Primary and Secondary Antibodies

1. Rabbit α GFP (Molecular Probes, G10362, dilution 1:250).

2. Rabbit α mouse keratin 5 primary antibody.

3. Rat α β-4 integrin (BD, 553745, dilution 1:200).

4. Mouse α β-catenin monoclonal antibody (Sigma, 15B8, dilution 1:500).

5. AF488-conjugated donkey α rabbit secondary antibody.

6. RRX-conjugated donkey α rabbit secondary antibody (2° Abs: affinity-purified whole IgG, cross-adsorbed).

7. RRX-conjugated donkey α rat secondary antibody (2° Abs: affinity-purified whole IgG, cross-adsorbed).

2.6 X-gal Staining

1. 25 % glutaraldehyde solution.

2. VECTOR Nuclear Fast Red solution (Vector Lab).

3. 20 mg/mL X gal stock: Dissolve X-gal in N′N′ dimethylformamide (DMF). Store at −20 °C protect from light.

4. 1 M Na-phosphate solution.

5. 1 M MgCl₂.

6. 50 mM K₃Fe(CN)₆. Store at 4 °C, protect from light.

7. 50 mM K₄Fe(CN)₆. Store at 4 °C, protect from light.

8. 10 % Na deoxycholate.

9. 10 % NP40.

10. 80 % Glycerol.

11. X-gal staining solution: 100 mM Na-phosphate (pH 7.3), 1.3 mM MgCl₂, 3 mM K₃Fe(CN)₆, 3 mM K₄Fe(CN)₆, 0.01 % Na deoxycholate, 0.2 % NP40, 2 mg/mL X-Gal.

2.7 Microscopy and Imaging Setup

1. Transmitted light microscope.

2. Fluorescence microscope.

3 Methods

We describe here the use of two types of Wnt reporter mice to monitor the activity of Wnt/β-catenin signaling: TCF /LEF:H2B- GFP [25] and BAT-GAL [20] or Axin2^lacZ [27]. The reporter genes are either GFP- or β-galactosidase-based. TCF/LEF:H2B-GFP is a transgenic line expressing histone H2B fused with GFP under a promoter containing 6 TCF/LEF binding sites [25]. BAT- GAL is a transgenic line expressing lacZ reporter gene under a promoter containing 6 TCF /LEF binding sites [20]. Axin2^lacZ line contains the lacZ gene inserted into the Axin2 locus [27]. Immunofluorescent detection of GFP and immunohistochemical detection of β-galactosidase activity are both performed on fresh cryosections. To visualize GFP signal in situ, pre-fixation (fixation before tissue embedding) is recommended to preserve the GFP signal.

3.1 Harvesting Backskin Tissue

1. Sacrifice the mice according to institutional guidelines (see Note 1) and closely shave all hair on the back with an electric shaver.

2. Mark the midline from neck to tail with a marker. Lift the backskin at the back of the neck with the fine forceps and make an incision. Insert the scissors underneath the skin and cut a rectangle shape from upper back to the lower back. Gently peel the entire backskin away from the body (Fig. 1).

3. Place the skin onto a paper towel with the epidermis side up and the dermis on top of the towel. Use forceps to spread the skin so that midline is straight. When working with multiple mice, align all pieces of skin in the same head-to-tail orientation (see Note 2).

4. Use a razor blade to cut the skin attached to the paper towel at the midline and on two sides parallel to the midline to yield two strips of skin 0.5 cm × 2.5 cm (see Note 3).

5. With GFP reporter mice proceed to the tissue processing with prefixation Subheading 3.2. When using lacZ reporter mice proceed to the embedding Subheading 3.3.

Fig. 1.

Stepwise illustration of backskin tissue harvesting. The mouse backskin is closely shaved before being marked at the midline from the neck to the base of the tail. An incision is made across the upper back and then along the two sides parallel to the midline. The skin is gently peeled off with the final cut across the back above the hind legs. The skin is spread on a brown paper towel with the midline aligned straight. The skin then can be cut into strips for processing

3.2 Prefixation of Tissue for GFP Preservation

For GFP reporter mice, prefixation of tissue prevents GFP signal loss. However, in the TCF /LEF: H2B-GFP [25] the GFP is fused with histone H2B, which renders the GFP much more stable, and therefore with this mouse line the prefixation step is optional. Prefixation is required for all other GFP reporter lines.

1. Transfer the skin strips to an aluminum foil-wrapped Falcon 5 mL tube to protect it from light throughout the whole procedure. All incubation steps below are performed with gentle rocking on the shaker.

2. Fix with fresh 4 % PFA for 2 h. Ensure the strip is fully immersed (see Note 4). Discard PFA as hazardous waste.

3. Wash with PBS 2–3 times, 5 min each wash.

4. Soak tissues in 10 % sucrose and incubate for 2 h. Aspirate.

5. Soak tissues in 30 % sucrose and incubate overnight at 4 °C in the dark.

6. The next day, equilibrate with 1:1 O.C.T. slurry (30 % sucrose: O.C.T. = 1:1) for 6 h. O.C.T. slurry can be made by damping out half of the sucrose and adding O.C.T. for 1:1 mixture. Proceed to embedding backskin in O.C.T. for cryosectioning (Subheading 3.3).

3.3 Embedding Backskin in OCT Block

1. Fill the cryomold with O.C.T.

2. Hold both ends of the skin strip with fine forceps, and place the skin strip with the midline touching the bottom of O.C.T.-filled cryomold (Fig 2a). Use fine forceps to hold the skin straight and to make sure the skin freezes exactly perpendicular to the bottom of the block (see Note 5). Hold the skin straight while placing the O.C.T. block on the dry ice to freeze (see Note 6).

3. Transfer the frozen O.C.T. block to prechilled cryostat for sectioning or store at −80 °C.

Fig. 2.

Illustration of skin embedding to acquire longitudinal sections of hair follicles. (a) Skin strip is placed into O.C.T. filled cryomold with the midline facing and touching the bottom of the cryomold. (b) Cartoon depicting a good day-28 skin section with an intact hair follicle

3.4 Cryosectioning

1. Follow cryostat instruction (see Note 7) to section tissue in 8 µm (range: 6–12 µm), two sections per slide.

2. Air-dry the first collected section on the slide and counterstain with hematoxylin solution for 1 min.

3. Gently wash off excess staining solution with tap water. Visualize hair follicles under a light microscope and adjust block angle until intact hair follicles can be detected (Fig. 2b, see Note 8).

4. Collect sections, air dry for 20 min (range: 15–30 min), and freeze at −80 °C for long-term storage.

3.5 Visualizing GFP Signal in GFP Reporter Mouse Skin

1. GFP can be visualized directly under a fluorescence microscope (see Note 9). If stored at −80 °C, thaw at room temperature for 10 min before use. To colocalize GFP signal with epidermal markers (or other markers), proceed to the next step (see Note 10). If GFP signal is weak, proceed to next step and immunostain for GFP expression (Fig. 3a).

2. Use a pap pen to draw a circle around the skin tissue to create a hydrophobic barrier.

3. Transfer the slide to an aluminum foil-wrapped, humidified staining chamber (see Note 11) and incubate with blocking buffer (150–200 µL/section in general) for 1 h.

4. Add primary antibody (α β-4 integrin 1:200 and optional α GFP 1:250 diluted in staining buffer) and incubate for 2 h at room temperature or overnight (16–24 h) at 4 °C in humidified chamber.

5. The next day, wash with PBS in the staining jar, three times, 5 min each time.

6. Add fluorophore-conjugated secondary antibody (AF488 1:150 and RRX 1:200 diluted in staining buffer) for 45 min in the humidified chamber.

7. Wash in PBS briefly.

8. Counterstain in Hoechst 33342 (1:2000 dilution in PBS) at room temperature for 2 min.

9. Wash with PBS three times, 10 min total.

10. Aspirate excess buffer and mount the section with ProLong Gold antifade and cure overnight at room temperature in the dark.

11. Seal the slide with nail polish and air-dry for 30 min before imaging with a fluorescence microscope. If immediate imaging is not possible, slides can be stored at −20 °C (see Note 9).

Fig. 3.

Detecting Wnt/β-catenin signaling. (a) Fluorescence image of endogenous GFP signal in the hair follicle of 28-day-old TCF /LEF:H2B-GFP mouse, with nuclei counterstained with Hoechst. β4-integrin co-staining in red labels the basement membrane, delineating the interface between epithelial cells and the dermal papilla. X-Gal staining of 28-day-old (b) wild-type or Axin2^lacZ and (c) BAT-GAL mice. Skins were counterstained with Nuclear Fast Red to mark nuclei. Blue signal in the precortex of hair follicle indicates Wnt activity. (d) Immunostaining of β-catenin in 28-day-old wild-type mouse skin. Arrowheads point to nuclear β-catenin. Scale bar denotes 50 µm

3.6 X-gal Staining of lacZ Reporter Mouse Skin

1. Thaw slides from −80 °C for 10 min at room temperature (see Note 12).

2. Fix in freshly prepared 0.1 % glutaraldehyde in PBS for 2 min.

3. Wash with PBS in the staining jar at least five times, 5 min per wash.

4. Prepare X-Gal staining solution fresh by adding X-Gal (2 mg/mL) to pre-warmed base solution.

5. Incubate the slides at 37 °C in the hybridization oven. Cover the jar with slides with aluminum foil. Active β-galactosidase digests X-Gal as a substrate and converts it into a blue precipitate. Check the color development from 2 to 3 h of staining and let it go overnight if necessary.

6. Wash with PBS three times, 5 min each.

7. Rinse with distilled water.

8. Counterstain with Nuclear Fast Red for 5 min.

9. Wash with running water for 10 min.

10. Mount slides with 80 % glycerol and seal with nail polish.

11. Acquire images with a light microscope (Fig. 3b, c).

3.7 Immunostaining for Nuclear β-catenin

3.7.1 Dehydration and Antigen Retrieval of FFPE Sample

Use 5 µm skin sections from a formaldehyde-fixed paraffinembedded (FFPE) block (see Note 13).

1. Dewax paraffin sections by placing slides in a 60 °C prewarmed citrus solvent and incubate in the hybridization oven at 60 °C for 10 min.

2. Rehydrate the slides by sequential incubation in an alcohol series, 3 min each step: 100 % ethanol, 100 % ethanol, 95 % ethanol, 85 % ethanol, 70 % ethanol, 50 % ethanol.

3. Rinse the slides twice briefly in distilled water and transfer to the Coplin jar.

4. Antigen unmasking is achieved using the heat-induced epitope retrieval (HIER) method:

   • Fill the jar to the top with citric acid buffer (pH 6.0).
   • Boil in a microwave at 500 W (50 % power in regular microwave) for three rounds, 5 min each (15 min in total).
   • Between each round, refill citric buffer to the top to prevent the sections from drying. Set the jar on the benchtop and chill for 20 min until it reaches room temperature.

3.7.2 Blocking and Antibody Staining with M.O.M Kit

1. Briefly wash slides with distilled water.

2. Block endogenous peroxidase activity by incubating slides in 0.3 % H₂O₂ solution for 15 min in the glass staining jar (see Note 14).

3. Wash with PBS three times, 5 min each.

4. Aspirate residual PBS without drying the tissue.

5. Draw a circle around the skin tissue with a Pap pen to create a hydrophobic border.

6. To block nonspecific antibody binding and endogenous biotin, add 100–200 µL M.O.M blocking buffer with Avidin per section and incubate for 1 h in the humidified staining chamber (see Note 15).

7. Remove the blocking buffer and rinse with PBS.

8. Add 100–200 µL M.O.M diluent buffer per section and incubate for 5 min.

9. Aspirate the diluent.

10. Add 100–200 µL of primary antibody (Mouse α β-catenin, 1:500) in M.O.M diluent buffer with biotin onto the skin section and incubate overnight in the humidified staining chamber at 4 °C.

11. The next day, wash with PBS three times, 5 min each.

3.7.3 Signal Amplification and Development

1. Aspirate excess PBS buffer around the tissue after the last wash.

2. Prepare secondary antibody by diluting α Mouse IgG Biotinylated antibody (1:200, from the M.O.M. kit) in M.O.M. diluent buffer without biotin.

3. Add 100–200 µL of secondary antibody solution per section and incubate for 30 min (see Note 16). At the same time, prepare Vectastain Elite ABC reagent.

4. Wash with PBS three times, 5 min each.

5. Add 100–200 µL ABC reagent mixture onto the skin section and let incubate for 30 min.

6. Wash with PBS two times, 5 min each.

7. For color development, prepare DAB solution and add 100–200 µL DAB substrate to the section. Monitor DAB colorization (brown) under the light microscope until the intensity of the color is satisfactory (between 3 and 5 min, see Note 17) and wash with tap water for 5 min (Fig. 3d).

3.7.4 Hematoxylin Counterstain and Imaging

Hematoxylin counterstain marks the cell’s nucleus to reveal histology and also enhances the contrast from DAB development.

1. Stain the slides in hematoxylin for 30 s.

2. Wash with tap water for 1 min.

3. Incubate in clarifier for 1 min.

4. Wash with tap water for 1 min.

5. Incubate in bluing reagent for 1 min.

6. Wash with tap water for 1 min.

7. Wash four times total with increasing concentrations of Ethanol: 70 % ethanol, 95 % ethanol, 100 % ethanol, 100 % ethanol, then two times with citrus solvent, 1 min each time.

8. Allow evaporation of citrus solvent in the chemical hood for 2–5 min.

9. Mount the slide with a drop (~50 µL) of Permount solution and let solidify overnight.

10. The next day, image slides or store slides at room temperature for future imaging.