Figure 2. Midkine (mk) is highly expressed in the basal layers of the wound epidermis/AEC and blastemal progenitors.

(A–F’) Timecourse RNAscope in situ hybridization of midkine at 0 (intact), 1, 3, 7, 14, and 21 dpa. Insets in A-F are shown in A’-F’ at higher magnification. Arrowhead in C’ denotes the beginning of the wound epidermis. Dotted line marks amputation plane in E and F or wound epidermis/AEC boundary in B’-F’. (G) In situ hybridization of mk in full skin flap sutured limbs at 7 dpa. Axolotl MK protein expression can be found in Figure 2—figure supplement 1. (H) Breakdown of the percentages of mk⁺ cells that are pax7⁺, prrx-1⁺, and pecam⁺ in regenerating stump tissues during early stages of regeneration. At 3 dpa, a total N of 1579, 444, and 1180 cells were counted for pax7, prrx-1, and pecam double in situs, respectively. At 7 dpa, a total N of 456, 1043, and 274 cells were counted for pax7, prrx-1, and pecam double in situs, respectively (Figure 2—source data 1). (I) Representative images of RNAscope double in situ hybridization of mk with pax7 (left), prrx-1 (middle), or pecam (right) at early (3 dpa) and later blastema (24 dpa) stages. Insets depict where higher magnification images were taken. Black arrowheads mark double positive cells. More detailed analysis of the onset of mk expression in early stages of regeneration, representative images of double in situ hybridization of mk with cell type-specific markers in uninjured tissue, as well as the analysis of mk co-expression with the monocyte marker csf1r can be found in Figure 2—figure supplement 2. Scale bars, A-G: 500 µm, A’-F’: 100 µm, I: 50 µm. dpa, days post-amputation, FSF, full skin flap.

Figure 2—figure supplement 1. MK protein is found throughout the wound epidermis/AEC.

(A) Normalized TPM levels of midkine (mk) in regenerating and full skin flap conditions. (B) Validation of custom polyclonal rabbit anti-MK antibody. Western blotting was performed with increasing volumetric ratios of MK antibody: blocking peptide ratios on 10 dpa protein extracts and revealed lower levels of staining with increased levels of peptide. (C–F’) Immunostaining of axolotl MK protein at 0 (C–C’), 5 (D–D’) and 14 dpa (E–E’). White arrowheads denote the amputation plane. Higher 20x magnification images of the wound epidermis or AEC (14 dpa) are shown in C’-F’. (F–F’) Immunostaining of axolotl MK protein in full skin flap sutured limbs at 7 dpa reveals lower levels of expression. Dotted lines demarcate full skin flap in F and the wound epidermis/stump boundary in C’-F’. Graphs are mean ± SD. **p<0.005, *p<0.05. Scale bars, B, E-H: 500 µm, E’-H’: 100 µm. FSF, full skin flap; dpa, days post-amputation.

Figure 2—figure supplement 2. Mk is expressed in satellite cells, connective tissue, and endothelial cells in intact tissues.

(A) RNAscope in situ hybridization of mk at 24, 56, and 72 hpa. Dotted line denotes wound epidermis boundary. Black arrowheads point to examples of mk⁺ cells. (B) Double RNAscope in situ hybridization of mk in red and pax7 (left) (N = 1073 cells counted), prrx-1 (middle) (N = 1723 cells counted), and pecam (right) (N = 1693 cells counted) in blue in non-regenerating intact limbs. Black arrowheads mark double positive cells. (B) Quantification of mk⁺ only, csf1r⁺ only, and co-positive cells. (C) Double RNAscope in situ hybridization of mk and csf1r at 5 dpa reveals discrete expression patterns (N = 582 cells counted). Black arrowheads denote mk⁺ only cells and black arrows denote csf1r⁺ only cells. Scale bars are 50 µm. hpa, hours post-amputation, dpa, days post-amputation.