Prx-1 Expression in Xenopus laevis Scarless Skin-Wound Healing and Its Resemblance to Epimorphic Regeneration  Hitoshi Yokoyama, Tamae Maruoka, Akio Aruga, Takanori Amano, Shiro Ohgo, Toshihiko Shiroishi, Koji Tamura  Journal of Investigative Dermatology  Volume 131, Issue 12, Pages 2477-2485 (December 2011) DOI: 10.1038/jid.2011.223 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Skin regeneration and wound healing in Xenopus froglets. Skin wound in limb (a) and trunk (b) immediately after surgery (0hours). Limb (c) and trunk (d) skin-wound sections stained with H&E and Alcian blue. (e, f) Limbs of froglets with similar wounds after 2 months. (g, h) H&E- and Alcian blue-stained sections. (g′, h′) High-power views of boxed areas in g and h. Black arrowheads (a, b, e, f) indicate the wound site. Open arrowheads (c, d, g, h) indicate the right-hand border of the wound. Asterisk (g′, h′) indicates an exocrine gland duct. (a, b, e, f) Bar=5mm; (c, d, g, g′, h, h′) bar=100μm. D, dermis; E, epidermis; H&E, hematoxylin and eosin; M, muscle. Journal of Investigative Dermatology 2011 131, 2477-2485DOI: (10.1038/jid.2011.223) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Limb skin regeneration and wound healing after excision. Sections were stained with hematoxylin and eosin and Alcian blue. Boxed areas in a–d are shown at high power in a′–d′. (a, a′) Immediately after skin excision (0hours). (b, b′) Twenty-four hours after excision. (c, c′) Four days after excision. Note the long, slender cells beneath the epidermis. (d, d′) Ten days after excision. Organized collagen fibrils and maturating exocrine glands are visible in the regenerated dermal layer. Open arrowheads indicate the right-hand border of the original wound. Bar=100μm. Journal of Investigative Dermatology 2011 131, 2477-2485DOI: (10.1038/jid.2011.223) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of prx1 4 days after skin wounding or limb amputation. (a, c, e) In situ hybridization for prx1. Boxed regions are shown at high power in a′, c′, e′, and e″. (b, d, f) Corresponding serial sections stained with hematoxylin and eosin and Alcian blue. (a, c) Expression of prx1 in accumulated mononuclear cells. (e) Expression of prx1 in limb blastema. Cells beneath the wound epidermis (e′) and perichondrial cells (e″) expressed prx1. Open arrowheads indicate the right-hand border of the original wound in a and b; arrowheads flank the wound in c and d. Black arrowheads indicate the amputation plane. Bar=200μm. (g–i) Results from quantitative PCR for prx1. *P<0.005 by Student's t-test. Journal of Investigative Dermatology 2011 131, 2477-2485DOI: (10.1038/jid.2011.223) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Colocalization of GFP and BrdU in Mprx1-GFP froglet trunk skin 4 days after excision. Sections of healing and control skin double labeled with anti-GFP and anti-BrdU antibodies. DAPI staining shows nuclei. (a–c) Intact-skin control. (b) Little GFP was detected. (c) Only a few BrdU-positive cells were found in the skin. (d–f) Sections of wounded trunk skin. (e′, f′) High-power views of the boxed regions in e and f. (e, e′) Robust staining for GFP and (f, f′) BrdU. (g) Merged picture of e′ and f′. Multiple cells were positive for BrdU and GFP (arrowheads). Bar=100μm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein. Journal of Investigative Dermatology 2011 131, 2477-2485DOI: (10.1038/jid.2011.223) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Mprx1 enhancer activation was not detected in healing adult mouse skin. (a) Mouse Mprx1-LacZ construct. (b) Limb-specific reporter activity in a mouse embryo. At 24hours (c, d) and 4 days (e, f) after skin excision, X-gal-positive cells were not detected at the wound site but were seen outside it (outside). The X-gal-positive cells seemed to be arrector pili muscles and dermal papilla cells. Black arrowheads (e) indicate the boundary between the wound and overlying scab (S). Hematoxylin and eosin (H&E) staining of healing skin at 24hours (g) and 4 days (h) after skin excision. Open arrowheads indicate the right border of the original excision wound (wounded area is to the left). Bar=200μm. Journal of Investigative Dermatology 2011 131, 2477-2485DOI: (10.1038/jid.2011.223) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Model for the involvement of the prx1 limb enhancer in limb regeneration and skin-wound healing.Amphibian, top, limb regeneration: Wound epidermis covers the stump very quickly, and the prx1 limb enhancer is activated in blastema cells. If the blastema is fully capable of limb patterning, a complete limb is regenerated, as in urodeles. If the blastema has a limb-patterning deficiency, a hypomorphic limb will result, as in Xenopus froglets. Amphibian, bottom, skin-wound healing: Wound epidermis covers the injury very quickly. The prx1 limb enhancer is activated in the accumulated mononuclear cells under the wound epidermis. Mammal: The wound is covered first by a clot, which becomes a scab, and several days later by the epidermis (re-epithelization). The prx1 limb enhancer is not activated. Journal of Investigative Dermatology 2011 131, 2477-2485DOI: (10.1038/jid.2011.223) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions