Figure 6. iMDK-treated and mk mutant regenerating limbs display dysregulated wound epidermis gene expression and persistent inflammation.

(A) Beta-III tubulin staining or WE3 staining of DMSO- or iMDK-treated limbs. White dotted line marks boundary of wound epidermis/AEC-blastema. (B) Heatmap of annotated differentially expressed transcripts in DMSO- and iMDK-treated limbs (N = 3 each) reveals two main clusters (colored pink and orange) of transcripts either enriched in DMSO or iMDK treatments. Transcript expression was normalized per row and plotted as a Z-score. Differentially expressed transcripts can be found in Supplementary file 4. (C) Heatmap of normalized TPM expression levels of wound epidermis genes in DMSO- or iMDK-treated regenerating limbs at 11 dpa. (D) Plot of enriched pathways in iMDK-treated limbs. (E) Plot of enriched pathways in DMSO-treated limbs. (F) Quantification of NSE⁺ monocytes at 5 dpa in DMSO/iMDK-treated limbs at 5 dpa (N = 7 DMSO, 6 iMDK). (G) Quantification of the density of NSE⁺ monocytes in mk^WT control and mk^null regenerating limbs at 10 dpa (N = 7 mk^WT, 8 mk^null). (H) NSE staining of DMSO- and iMDK-treated limbs at 5 dpa and 14 dpa. Dotted lines demarcate the amputation plane. (I) Representative NSE stained sections from regenerating limbs of mk^WT control and mk^null mutants at 10 dpa. Higher magnification insets are shown in bottom two panels. Each N represents a limb from a different animal. Data demonstrating rescue of mk^null phenotypes via overexpression of mk during regeneration in mutant limbs as well as electroporation efficiency metrics can be found in Figure 6—figure supplements 1, 2 and 3. A two-tailed unpaired student’s t-test was used for statistical analysis. Graphs are mean ± SD. *p<0.05. Scale bars, A (top): 100 µm, A (bottom), H-I (top): 200 µm, I (bottom): 50 µm. bl, blastema, dpa, days post-amputation.

Figure 6—figure supplement 1. Mk overexpression in mk^null regenerating limbs rescues delayed regeneration.

(A) Experimental schematic of rescue experiment. Either a pCAG-MK and/or pCAG-tdTomato control overexpression construct was injected and electroporated into the regenerating limbs of wildtype or mk^null animals at 3 dpa. (B) Representative image of tdTomato fluorescence in a pCAG-MK/pCAG-tdTomato co-electroporated regenerating limb at 14 dpa (outlined in white dotted line). (C) Quantification of blastema length at 13 dpa. One-way ANOVA analyses were employed on each set of data for statistical analysis (N = 6 mk^WT + tdT, 10 mk^null+tdT, 7 mk^null + MK). (D) Representative time course images of regenerating mk^WT and mk^null limbs electroporated with pCAG-tdTomato and/or pCAG-MK constructs. Two different rescue animals are shown here. Graphs are mean ± SD. **p<0.005, *p<0.05. Scale bars, B, D: 1 mm. n.s., not significant; dpa, days post-amputation.

Figure 6—figure supplement 2. Mk overexpression in mk^null regenerating limbs rescues mutant wound epidermis and monocyte density phenotypes.

(A) Representative images of picro-mallory-stained (top row) and NSE-stained (bottom row) sections from tdTomato and/or mk-overexpressing wildtype and mutant regenerating limbs. Black dotted lines mark the wound epidermis or bone boundary in the top and bottom rows, respectively. (B) Quantification of wound epidermis thickness at 10 dpa. (C) Quantification of NSE⁺ monocyte density at 10 dpa. (D) Representative images of TUNEL-stained sections, focusing on the wound epidermis. Yellow dotted line demarcates the boundary between the wound epidermis and regenerating stump tissues. Yellow asterisk marks the auto-fluorescent bone. White arrows point to green TUNEL⁺ nuclei. (E) Quantification of TUNEL⁺ nuclei in the wound epidermis at 10 dpa. One-way ANOVA analyses were employed on each set of data for statistical analysis (N = 7 mk^WT + tdT, 7 mk^null+tdT, 9 mk^null + MK). Graphs are mean ± SD. **p<0.005, *p<0.05. Scale bars, B (top row), 50 µm, B (bottom row) and D, 100 µm. n.s., not significant; dpa, days post-amputation.

Figure 6—figure supplement 3. Electroporation efficiencies are similar between pCAG-tdTomato and pCAG-MK injected mk^null mutant and mk^WT regenerating limbs.

(A) Validation of pCAG-MK overexpression construct with MK antibody. Western blots of either pCAG-tdTomato or pCAG-MK transfected 293 T cells with the custom axolotl-MK antibody reveals the overexpression and secretion of axolotl MK in pCAG-MK, but not pCAG-tdTomato (tdT), transfected cell lysates and media. (B–C) Quantification of mean fluorescence intensity and the percentage of tdTomato⁺ expressing cells out of total DAPI⁺ cells in the distal-most 500 µm of regenerating tissue at 10 dpa. (D–F) Representative images of anti-tdTomato-stained sections from each condition. One-way ANOVA analyses were employed on each set of data for statistical analysis (N = 7 mk^WT + tdT, 7 mk^null+tdT, 9 mk^null + MK). Scale bars, 500 µm. dpa, days post-amputation, n.s. not significant.