Wound healing and Wnt signaling varies with the hair cycle.

<p>On post-wound day 1, H&E staining of injury sites created in (A) anagen or (B) telogen. Post-wound day 2, (C) H&E staining of anagen (D) versus telogen wounds. Dotted yellow line indicates the healing tongue. Post-wound day 3, (E) H&E staining of anagen (F) versus telogen wounds. Dotted yellow line indicates regenerating epithelium. Post-wound day 4, (G) H&E staining of anagen (H) versus telogen wounds. (I) Percentage of healed wounds expressed as a function of the hair cycle (N = 20 per phase). (J) X-Gal staining of a wound bed in anagen versus (K) telogen. Dotted yellow line indicates auricular cartilage. (L) Hair follicles adjacent to the wound edges, in anagen versus (M) telogen. All mice were male. Abbreviations: we, wound edge; re, regenerating epithelium; es, eschar. Scale bar = 50 µm; asterisk denotes P<0.05.</p

Wnt signaling varies with the hair cycle.

<p>(A) Hemotoxylin staining of 5-week-old ear skin. (B) Gomori trichrome staining of 5-week-old ear skin; anagen-stage hair follicles appear red. (C) X-Gal staining of adjacent tissue from a 5-week-old <i>Axin2^LacZ/+</i> mouse. (D) Hemotoxylin staining, (E) Gomori trichrome staining, and (F) X-Gal staining of ear skin from a 7-week-old <i>Axin2^LacZ/+</i> mouse. (G) Hemotoxylin staining, (H) Gomori trichrome staining, and (I) X-Gal staining of ear skin from a 9-week-old <i>Axin2^LacZ/+</i> mouse. (J) The number of Wnt-responsive dermal cells per ROI was quantified at during anagen, catagen, and telogen (N = 3; 3 sections per mouse, 3 mice per week). All mice were male. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076883#pone-0076883-g001" target="_blank">Fig. 1</a>. Scale bars = 50 µm; asterisk denotes P<0.05.</p

L-Wnt3a stimulates cutaneous repair.

<p>(A) Fluorescent signal in the wound bed on day 3 treated with DiI-labeled liposomes containing PBS or (B) Wnt3a. Dotted lines indicate boundary between the epithelium and dermis. (C) Fluorescent signal in hair follicles and epithelium adjacent to the wound bed, treated with DiI-labeled liposomes containing PBS or (D) Wnt3a. (E–H) X-Gal staining of adjacent tissue sections shown in A–D. (I) BrdU staining in the wound bed treated with L-PBS or (J) L-Wnt3a. Arrows indicate immunopositive cells. (K) TUNEL staining in the wound bed treated with L-PBS or (L) L-Wnt3a. Arrows indicate immunopositive cells. (M) Telogen wounds treated with L-PBS fail to heal. (N) Telogen wounds treated with L-Wnt3a heal. (O) Quantification of the percent of L-PBS versus L-Wnt3a treated wounds that healed (N = 10 per treatment). All mice were male. Scale bars = 50 µm; asterisk denotes P<0.05.</p

Wnt signaling in back skin compared to the ear.

<p>(A) H&E staining of back skin, illustrating hair follicles. (B) Adjacent section from an <i>Axin2^LacZ/+</i> mouse, stained with X-Gal. (C) H&E staining of the epidermis and dermis, and (D) corresponding X-Gal staining. (E) H&E staining of the ear. (F) Adjacent <i>Axin2^LacZ/+</i> section stained with X-Gal. (G) higher magnification of a hair follicle in the ear. (H) X-Gal staining of the epidermis and dermis of the ear. Abbreviations: e, epidermis; df, dermal fibroblasts; HF, hair follicle; dp, dermal papilla; car, cartilage; mc, matrix cells; hs, hair shaft. Scale bars = 50 µm.</p

Elevated Wnt signaling in Axin2^LacZ/LacZ mice correlates with improved cutaneous repair.

<p>(A) Wound on post-injury day 1, stained with Gomori trichrome in <i>Axin2^LacZ/+</i> and (B) <i>Axin2^LacZ/LacZ</i> mice. (C) Wound edge in <i>Axin2^LacZ/+</i> and (D) <i>Axin2^LacZ/LacZ</i> mice. (E) Wound edge stained X-Gal from <i>Axin2^LacZ/+</i> and (F) <i>Axin2^LacZ/LacZ</i> mice. Arrows indicate X-Gal^+ve dermal fibroblasts. (G) Wound on day 3, stained with Gomori in <i>Axin2^LacZ/+</i> and (H) <i>Axin2^LacZ/LacZ</i> mice. (I) Wounds stained with TUNEL in <i>Axin2^LacZ/+</i> and (J) <i>Axin2^LacZ/LacZ</i> mice. Dotted line indicates auricular cartilage. (K) X-Gal in <i>Axin2^LacZ/+</i> and (L) <i>Axin2^LacZ/LacZ</i> mice. (M) Wounds on day 3, stained with X-Gal in <i>Axin2^LacZ/+</i> and (N) <i>Axin2^LacZ/LacZ</i> mice. (O) An eschar covers the wound beds in <i>Axin2^LacZ/+</i> mice. (P) Regenerating epithelium covers the wound beds in <i>Axin2^LacZ/LacZ</i> mice. (Q) X-Gal in <i>Axin2^LacZ/+</i> and (R) <i>Axin2^LacZ/LacZ</i> mice. (S) BrdU in <i>Axin2^LacZ/+</i> and (T) <i>Axin2^LacZ/LacZ</i> mice. (U) Ki67 immunostaining in <i>Axin2^LacZ/+</i> and (V) <i>Axin2^LacZ/LacZ</i> mice. (W) Quantification of X-Gal+ve dermal fibroblasts, and proliferating cells in the wound beds of <i>Axin2^LacZ/+</i> and <i>Axin2^LacZ/LacZ</i> mice. All mice were male. Scale bar = 50 µm; asterisk denotes P<0.05.</p

L-Wnt3a stimulates the survival, proliferation, and engraftment of bone marrow cells.

<p>(A) Aged BMT were treated with L-PBS or (B) L-Wnt3a with an effective concentration = 150 ng/ml, then transplanted into a skeletal defect and after 12 h, analyzed for DNA fragmentation associated with cell apoptosis using TUNEL. (C) Quantification of caspase activity in aged BM treated with L-PBS (grey bar; N = 4) or L-Wnt3a (blue bar; N = 4). (D) Quantification of Ki67 immunostaining for aged BM treated with (E) L-PBS or (F) L-Wnt3a, then transplanted into a skeletal defect and after 12 h analyzed for cell proliferation. (G) FACS analyses of L-PBS treated BMT, harvested from the defect site on post-transplant day 5. (H) FACS analyses of L-Wnt3a treated BMT, harvested from the defect site on post-transplant day 5. (I) GFP immunostaining identifies the L-PBS treated BMT on post-transplant day 7 (J) GFP immunostaining identifies the L-Wnt3a treated BMT on post-transplant day 7. (K) GFP immunostaining shown in higher magnification demonstrating L-PBS treated BMT on post-transplant day 7 (L) GFP immunostaining shown in higher magnification demonstrating L-Wnt3a treated BMT on post-transplant day 7 (M) Histomorphometric quantification of GFP immunopositive cells in the defect site on post-transplant day 7 (N = 5 for each condition). Abbreviations: GFP: green fluorescent protein; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; Scale bars: A,B: 50 µm E,F: 100 µm; I,J: 200 µm; K,L: 50 µm.</p

Wnt3a associates with the liposomal surface.

<p>(A) The rate of Wnt activity partitioning into the liposomal pellet is shown; initially, all Wnt activity (blue lines) is found in the supernatant but within 30 min, the majority of Wnt activity is associated with the liposomal pellet and by 6 h, 90% of Wnt activity is found in the liposomal pellet. (B) Immunoblot analysis using Wnt3a antibody shows that similar to the Wnt activity, initially the majority of the Wnt is found in the supernatant (supnt); within 30 min majority of the majority of Wnt3a segregates into the liposomal pellet and by 6 h 90% of the protein is associated with the liposomal pellet. (C) Following ultra-centrifugation, the majority of the protein is found in the liposomal pellet. (D) Wnt activity (blue bar) is also found in the pellet. Although some Wnt3a is found in the aqueous supernatant, it is inactive (figure C). (E) Sucrose density gradient centrifugation and phospholipid quantification assay (orange line), demonstrate that PBS liposomes migrate to higher density fractions. (F) Sucrose density gradient centrifugation, phosphatidyl choline lipid quantification and Wnt reporter assay, demonstrate that Wnt activity (blue line), the lipids (orange line), co-fractionate on a sucrose density gradient. (G) Immunoblotting analyses using Wnt3a antibody show that Wnt co-migrates with fractions showing maximum Wnt activity and lipid concentration. (H) The stability of L-Wnt3a is measured. At 4°C, L-Wnt3a retains >80% of its activity after extended storage. (I) An anti-Wnt3a immunoblot reveals no evidence of degradation products of L-Wnt3a after extended storage. Data are mean ±SEM from, or are representative of, at least three independent replicates.</p

Wnt3a requires a hydrophobic carrier to maintain its biological activity.

<p>To generate “Wnt3a+CHAPS” (red), 30 ng/µL of Wnt3a protein was diluted in a buffer containing 1× PBS, 0.5 M NaCl, and 1% CHAPS yielding a final CHAPS concentration of 1% and final Wnt3a concentration of 1.95 ng/µL. To generate “Wnt3a−CHAPS” (green), 30 ng/µL Wnt3a protein was diluted in a buffer containing 1× PBS and 0.5 M NaCl yielding a final CHAPS concentration of 0.004% and final Wnt3a concentration of 1.95 ng/µL. Aliquots of each solution were incubated at 23°C for the indicated times, then applied to LSL cells (see Methods). The output of the LSL assay is luciferase activity/beta galactosidase activity (i.e., luc/lac); percent activity is defined as % activity = , where 100% activity = 0.05 ng/µL Wnt3a. (A) After 24 h at room temperature, Wnt3a+CHAPS retains of its 98.0% activity (red line) whereas Wnt3a−CHAPS (green line) retains 11.4% of its activity. (B) Anti-Wnt3a immunoblotting confirms that equivalent amounts of Wnt3a protein were present in both +CHAPS and −CHAPS conditions at the beginning (0 h) and conclusion (24 h) of the incubation period. (C) Immunoblot analysis confirms that in the presence of CHAPS Wnt co-fractionates with lower density sucrose fractions (#1–3). In the absence of CHAPS the protein precipitates as the majority of the protein is found in a high density fraction (lower panel). (D) Sucrose density gradient centrifugation and Wnt reporter assay, demonstrate that in the presence of CHAPS, Wnt activity (red line) migrate to lower density fractions. Without CHAPS, Wnt activity is not detected (green line) (E) To generate “Wnt3a+lipids” (blue), 30 ng/µL of Wnt3a protein was diluted in DMPC∶cholesterol 90∶10 lipid mixture containing 1× PBS yielding a final lipid concentration of 14 µmoles and final Wnt3a concentration of 1.95 ng/µL. To generate “Wnt3a−lipids” (green), 30 ng/µL Wnt3a protein was diluted in a buffer containing 1× PBS and 0.5 M NaCl yielding a final CHAPS concentration of 0.004% and final Wnt3a concentration of 1.95 ng/µL. Both the samples were incubated at room temperature for 24 h. (F) Anti-Wnt3a immunoblotting confirms that equivalent amounts of Wnt3a protein were present in both +lipids and −lipids conditions at the beginning (0 h) and conclusion (24 h) of the incubation period. Data are mean ±SEM from, or are representative of, at least three independent replicates. (G) Wnt3a incubated in the presence of CHAPS (red bar) and lipids (blue bar) for 6 h at room temperature. Wnt3a samples were prepared as described in (A) and (E). (H) Wnt3a incubated in the presence of CHAPS (red bar) and lipids (blue bar) for 6 h at 37°C.</p

L-Wnt3a activates Wnt/beta-catenin pathway responses in a variety of cell types.

<p>Quantitative RT-PCR analyses demonstrate that in (A) primary mouse embryonic fibroblasts and (B) BM-derived mesenchymal stem cells, <i>Axin2</i> expression shows a dose-dependent increase in response to increasing L-Wnt3a concentrations. (C) MEFs were incubated with L-PBS (grey line) or L-Wnt3a for varying amounts of time. After 2 h, L-Wnt3a induces <i>Axin2</i> expression, just above baseline. After 4 h, L-Wnt3a induced <i>Axin2</i> expression has increased 3-fold (blue line). Following a 2 h-incubation with L-Wnt3a the media is removed and incubation continues for varying amounts of time (dashed blue line). 2 h after wash off, <i>Axin2</i> expression starts steadily declining and returns to baseline at 12 h. (D) MEFs were incubated with vehicle (grey line) or L-Wnt3a (blue line) for up to 24 h. After 3 h, L-Wnt3a represses <i>Sox9</i> expression and keeps it repressed through 24 h of incubation.</p