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Trunk-skin regeneration and wound healing after excision. A: Illustration of a metamorphosed froglet; the red rectangle shows the location for skin excision. B: The skin wound immediately after excision. C: A section of intact trunk skin stained with Elastica van Gieson and Alcian blue. Epi, epidermis; der, dermis; mg, mucous gland; sg, serous gland; mus, muscle (n = 4). D'–G': Magnified views of the rectangles in D–G. Time after incision: D,D', 0 hr (immediately after skin excision) (n = 4); E,E', 24 hr (n = 4); F, F', 4 days (n = 4); G,G' 10 days (n = 4); H,H', 1 month after excision (n = 3). Black arrowheads (B) indicate the wound site. Open arrowheads (D–H) indicate the right-hand border of the wound. Black arrows (E'–G') indicate the basement membrane of the epidermis. Asterisk (H') indicates an exocrine gland duct. B: Bar = 1 mm. C–H,D'–H': Bar = 50 µm.
Forelimb-skin regeneration and wound healing after excision. A: Illustration of a metamorphosed froglet; the red rectangle shows the location for skin excision. B: The skin wound immediately after excision. C: Intact forelimb skin stained with Elastica van Gieson and Alcian blue. Epi, epidermis; der, dermis; mg, mucous gland; sg, serous gland; mus, muscle (n = 1). D'–H': Magnified views of the rectangles in D–H. Time after incision: D,D', 0 hr (immediately after skin excision) (n = 2); E,E', 24 hr (n = 4), F,F', 4 days (n = 4), G,G', 10 days (n = 5); H,H', 1 month after excision (n = 4). Black arrowheads (B) indicate the wound site. Open arrowheads (D–H) indicate the right-hand border of the wound. Asterisk (H') indicates an exocrine gland duct. B: Bar = 1 mm. C–H, D'–H': Bar = 50 µm.
Medial head skin overlies bone, not muscle. A: Brightfield view of the rostral portion of a Xenopus laevis froglet. B: Whole-mount bone staining of a froglet with Alizarin red. C: The head region was stained with Elastica van Gieson and Alcian blue and sectioned through the plane indicated by the dotted line in A. Bra, brain; sku, skull. C',C”: Magnified views of the rectangles in C. The white rectangle in B indicates the medial-dorsal area of the head where the overlying skin was excised in Fig. 1. No subcutaneous muscles were observed in the medial head area C'. Open arrowheads (C”) enclose ocular muscles. A–C: Bar = 1 mm. C',C”: Bar = 100 µm.
Head-skin regeneration and wound healing after excision. A: Illustration of a metamorphosed froglet; the red rectangle shows the excision location. B: The skin wound immediately after excision. C: A section of intact head skin, stained with Elastica van Gieson and Alcian blue. Epi, epidermis; der, dermis; mg, mucous gland; sg, serous gland; sku, skull (n = 4). D'–H': Magnified views of the rectangles in D–H. Time after excision: D, D', 0 hr (immediately after excision) (n = 3); E,E', 24 hr (n = 5); F,F', 4 days (n = 5); G,G', 10 days (n = 5); H,H', 1 month after excision (n = 4). Black arrowheads (B) indicate the wound site. Open arrowheads (D–H) indicate the right-hand border of the wound. Black arrow (G') indicates maturing secretion gland. B: Bar = 1 mm. C–H, D'–H': Bar = 50 µm.
The prx1 enhancer was activated in all trunk, forelimb, and head-skin wound areas 4 days after skin excision in Mprx1-GFP froglets. Sections of regenerating and intact control skin were labeled with an anti-GFP antibody; nuclei were stained with DAPI. Immunostaining of regenerating skin around the original wound area (A,B,E,F,I,J) and intact (control) skin away from the wound area (C,D,G,H,K,L) in the same froglet. Little GFP was detected in the intact control skin (D,H,L), but robust staining for GFP was detected under the wound epidermis (B,F,J). Skin surface is up. Five of five and four of four individuals showed basically the same results in the trunk and forelimb, respectively. Two of four individuals showed robust staining for GFP in wound area of the head (J), but the rest showed relatively weaker staining for GFP. Bar = 100 µm.
Cell-fate tracing of subcutaneous tissues and regenerating skin in a froglet. A: Procedure for cell-fate analysis by skin grafting. The gray froglet represents a J-strain (J/J) individual; the green froglet represents a JG-hybrid (J/GFP) individual. 1: Skin and subcutaneous tissues are ubiquitously labeled with GFP in the J/GFP froglet. 2: A full-thickness skin piece was grafted from the J/J donor to the J/GFP recipient. 3: Once the grafted skin was accepted by the recipient, skin in the center of the graft, including the dermis, was excised. Subcu, subcutaneous tissues. B: Intact skin from a J-strain (n = 1) and (C) JG-hybrid (J/GFP) froglet (n = 1). D: Brightfield view of the skin wound in the graft area immediately after excision. E,E': Brightfield (E) and GFP-fluorescence (E') views of the skin wound 2 weeks after injury. E”: The sample was sectioned through the plane indicated by the dotted line in E' and immunostained with an anti-GFP antibody. The grafted skin was rarely invaded by GFP-positive host-derived cells. Black arrowheads indicate the wound site; white arrowhead indicates the host-graft boundary. B,C,E”: Bar = 200 µm. D,E,E': Bar = 2 mm.
Cells derived from subcutaneous tissues contribute to skin regeneration. A: Illustration of a metamorphosed froglet indicating the transverse planes of sections shown as B-J and as E'-J'. Gray and red rectangles represent the grafted donor skin and excisional skin wound, respectively. B–D: Samples of intact skin regions were sectioned through the plane indicated by the dotted line as “B–D” in A. GFP-positive subcutaneous tissues were observed beneath the GFP-negative grafted skin. E–J: Samples of regenerating skin, including subcutaneous tissues, were sectioned through the plane indicated by the dotted line “E–J” at 2 or 3 weeks after skin excision. E–G: GFP-positive cells had accumulated underneath the GFP-negative wound epidermis at 2 weeks after skin injury (n = 4). Three of four individuals showed basically the same results. H–J: GFP-positive cells contributed to the regenerated skin, especially to the dermis in the center of the wound (n = 4). Four of four individuals showed basically the same results. Asterisks indicate secretion glands. White arrowheads (E–G) indicate the right-hand border of the wound. Pairs of white arrowheads (H–J) indicate the left- and right-hand borders of the wound. Open arrowheads indicate GFP-negative cells contributing to the dermis of the regenerating skin. Bar = 100 µm.
