Figure 7. Overexpression of mk in regenerating limbs leads to wound epidermis expansion, decreased blastemal cell proliferation, and improper resolution of inflammation.

(A) Schematic of mk overexpression experiment. (B–E) Representative brightfield images of tdTomato (tdT)- (B–C) or mk-overexpressing (D–E) regenerating limbs at 7 and 16 dpa. White dotted line denotes amputation planes and white arrowhead in D denotes small aberrant skin growth. (F) Representative picro-mallory stained sections of tdT- or mk-overexpressing regenerating limbs. Asterisk marks protruding bone. (G) Quantification of wound epidermis thickness in tdTomato- or mk-overexpressing limbs at 8 dpa (N = 5 each) and 16 dpa (N = 5 tdT, N = 4 MK). (H) Quantification of the percentage of EdU⁺ nuclei in the wound epidermis of tdTomato- or mk-overexpressing limbs at 8 dpa (N = 5 each). (I) Representative images of EdU-stained sections of tdTomato- or mk-overexpressing limbs. Wound epidermis/AEC are outlined with yellow dotted lines. (J) Quantification of the percentage of EdU⁺ nuclei in regenerating stump tissues of tdT- or mk-overexpressing limbs. (K) Quantification of the density of NSE⁺ monocytes in tdT- or mk-overexpressing limbs. (L–M) Representative images of NSE-stained sections in tdT- or mk-overexpressing limbs. Black dotted line denotes wound epidermis boundary. Asterisk denotes protruding bone. Quantification of EdU⁺ cells in the wound epidermis and blastemal cells of DMSO/iMDK-treated and WT/mk^null regenerating limbs is shown in Figure 7—figure supplement 1. Electroporation efficiency data can be found in Figure 7—figure supplement 2. Data showing that mk-overexpressing regenerating limbs display delayed regeneration is shown in Figure 7—figure supplement 3. Data demonstrating that overexpression of mk in non-regenerating intact limbs does not affect cellular proliferation or monocyte density can be found in Figure 7—figure supplement 4. Graphs are mean ± SD. Two-tailed unpaired student’s t-tests were used for statistical analysis. *p<0.05, **p<0.01, ***p<0.005. Scale bars, B-E: 500 µm, F, I, L-M: 200 µm. bl, blastema, dpa, days post-amputation.

Figure 7—figure supplement 1. Mk is not required for proliferation in the wound epidermis or blastemal cells.

(A–B) Quantification of the percentage of EdU⁺ cells in the wound epidermis (WE) of DMSO/iMDK-treated limbs (5dpa: N = 6 DMSO, 6 iMDK; 14 dpa: N = 4 DMSO, 5 iMDK) (A) or mk^WT/mk^null limbs (7 dpa: N = 12 mk^WT, 8 mk^null; 10 dpa: N = 12 mk^WT, 13 mk^null) (B). (C–D) Quantification of the percentage of EdU⁺ blastemal cells in DMSO/iMDK-treated limbs (C) or mk^WT/mk^null limbs (D). Two-tailed unpaired t-tests were performed at each time point between the two conditions for statistical analysis. Graphs are mean ± SD. **p<0.005, *p<0.05. n.s., not significant; dpa, days post-amputation.

Figure 7—figure supplement 2. Electroporation efficiencies of pCAG-tdT and pCAG-MK injected regenerating limbs are similar.

(A–B) Quantification of mean fluorescence intensity (MFI) (A) and the percentage of tdTomato⁺ cells out of the total DAPI⁺ cells (B) in the distal most 500 µm of regenerating pCAG-tdT (N = 8) and/or pCAG-MK (N = 9) electroporated limbs at 6 dpa. (C–D) Representative immunostaining against tdTomato of tdTomato- or MK-electroporated limbs. Two-tailed unpaired t-tests were performed for statistical analysis. Scale bar, 250 µm. dpa, days post-amputation, n.s., not significant.

Figure 7—figure supplement 3. Mk-overexpressing limbs resolve epithelial overgrowth and undergo delayed regeneration.

Representative time course images of control tdTomato (top) or mk-overexpressing (bottom) regenerating limbs at 20, 27, 36, and 42 dpa (N = 9/12 animals exhibited a substantial delay in regeneration). Alcian-stained regenerated limbs at 51 dpa are shown on the far right. Scale bars, 1 mm. dpa, days post-amputation.

Figure 7—figure supplement 4. Overexpression of mk in non-regenerating intact limbs does not affect cellular proliferation or monocyte density.

(A) Experimental schematic of overexpression experiment in non-regenerating limbs. (B) Mean fluorescence intensity (MFI) quantification of electroporated limbs. (C) Quantification of the percentage of EdU⁺ cells in tdT- or mk-overexpressing intact limbs. (D) Representative images of tdT and EdU-stained cross-sections from electroporated limbs. (E) Quantification of monocyte density in overexpression limbs. (F–G) Representative images from NSE⁺ stained sections from non-regenerating overexpression limbs. Two-tailed unpaired t-tests were performed for statistical analysis. Graphs are mean ± SD. Scale bars, D, F-G: 200 um. n.s., not significant.