Fig 1.
DNA methylation and methyltransferase expression in ectopic limb blastemas.
(A) Total DNA methylation levels as determined through ELISA format assay. Ectopic blastema tissue samples were collected at 10 days post nerve deviation. n = 6 for each sample, totaling 24 samples. (B, C) qPCR analysis of DNMT1 and DNMT3a expression in uninjured tissues (skin and muscle), regenerating tissues (wound epithelium, ectopic blastema mesenchyme), and developing limb buds. Ectopic blastema tissue samples were collected at 10 days post nerve deviation. n = 10 for uninjured skin, muscle, wound epithelium, and mesenchyme; n = 4 for limb bud, totaling 44 samples. (* = p < 0.05; ** = p < 0.005).
Fig 2.
DNMT3a expression is modulated by signaling from nerves.
DNMT3a expression (qPCR) in the epithelium of wounds created on the arm of axolotls that either healed without forming a blastema (lateral wound; n = 8 for day 0, 24hr, 72hr, 5 days. n = 10 for 10 days) or received a surgically deviated nerve to form an ectopic blastema (nerve deviated; n = 8 for day 0, 24hr, 72hr, 5 days. n = 10 for 10 days). In addition the nerve supply to the limb was severed proximally (denervated) prior to making wounds either with (NdevWE; n = 4) or without (LatWE; n = 4) surgically deviating a nerve distally. (* = p < 0.05; *** = p < 0.0005).
Fig 3.
Inhibition of DNMT activity induces expression of the WE/AEC marker gene, Sp9.
Sp9 expression (qPCR) in the epithelium of wounds created on the arm of axolotls that healed without forming a blastema (lateral wound epithelium, LatWE; n = 5); received a surgically deviated nerve to induce formation of an ectopic blastema (nerve deviated wound epithelium, NdevWE; n = 7); received an implanted bead containing 2’deoxycytidine without a deviated nerve (LatWE + 2’dC; n = 5); or received an implanted bead containing decitabine without a deviated nerve (LatWE + decitabine; n = 7). (* = p < 0.05).
Fig 4.
Inhibition of DNMT activity inhibits reformation of the basal lamina.
Trichrome staining of wounds six days post-surgery. Wounds were either untreated (A, B; mock; n = 4); received an implanted bead containing 2’deoxycytidine without a deviated nerve (C, D; 2’dC; n = 8); received a surgically deviated nerve to induce formation of an ectopic blastema (E, F; NDev; n = 4); or received an implanted bead containing decitabine without a deviated nerve (G, H; Dec; n = 8). Images in (B, D, F and H are higher magnifications of the boxed areas in (A, D, E, and G correspondingly). In order to increase the visibility of the basal lamina (stained blute) in the original images, the high magnification images (panels B,D,F,H) have been color adjusted by placing the mid-tone colors on the blue end of the spectrum and highlights on the yellow end of the spectrum. All four panels were adjusted as one image, and thus have been treated equally. The lower magnification images were not adjusted. Dotted lines indicate the transition between the uninjured skin (right) and the wound (left). Arrows indicate the region beneath the epidermis/WE where the basal lamina structure can be detected. Scale bars = 200 microns.
Fig 5.
Inhibition of Collagen IV expression in decitabine-treated wound epithelia.
Immunohistochemistry staining for Collagen IV, a marker for the basal lamina, in wounds six days post-surgery. Wounds were either untreated (A, mock); received an implanted bead containing 2’deoxycytidine without a deviated nerve (B, 2’dC); received a surgically deviated nerve to induce formation of an ectopic blastema (C, NDev); or received an implanted bead containing decitabine without a deviated nerve (D, Dec). White arrowheads indicate areas within the wound that are negative for ColIV staining; yellow arrowheads indicate areas that are positive for Col IV staining. Dotted line in (D) indicates the transition between the uninjured skin (right) and the wound (left). Scale bars = 200 microns.
Fig 6.
Experimental design to test whether decitabine-treated wound epithelia can participate in blastema formation.
Cartoon illustrating the sequence of surgical procedures for making the wound, implanting beads, and grafting the treated and control wound epithelia to a new wound with a deviated nerve.
Fig 7.
Decitabine treated wound epithelia can participate in blastema formation.
Participation of a decitabine-treated WE in blastema formation as observed at day 3 (A), day 7 (B), day 11 (C) and day 16 (D) after grafting; n = 6. Lack of formation of an ectopic blastema when a 2’deoxycytidine-treated WE was grafted as observed at day 3 (E), day 7 (F), day 11 (G) and day 16 (H) after grafting; n = 8. Arrows indicate the position of the deviated nerve (A, E) and the formation of an ectopic blastema (C, D). Scale bars = 1mm.
Table 1.
Ability of decitabine-treated wound epithelia to participate in blastema formation.