Figure 3. Chemical and genetic perturbations of mk impair limb regeneration.

(A) Experimental design. (B) Brightfield images of DMSO- or iMDK-treated limbs (N = 4/4 iMDK-treated in each condition did not regenerate). (C–D) Alcian blue staining of DMSO- or iMDK-treated limbs at 60 dpa. (E) CRISPR strategy to target the start codon of mk to generate mutants. Control animals were generated using a non-targeted tracrRNA/cas9 complex and were unmodified at the mk locus. (F) Brightfield images of regenerating limbs from control animals or mk F0 mutants. (G) Quantification of blastema length at 14 dpa in control or mk mutant limbs. The severity of the delay in regeneration segregated based on genotype of the animal as either control (mk^WT), mosaic null (mk^null), or partially modified (low or high % WT alleles) (N = 14 control mk^WT, 5 mk^null, 8 low % WT, 4 high % WT). Graph is mean ± SD. (H–I) Representative images of picro-mallory stained sections of regenerating limbs in control animals or mk null mutants. Example genotyping analyses and mk null mutant immunofluorescence validation can be found in Figure 3—figure supplement 1. **p<0.005, two-tailed unpaired Student’s t-test was employed. Each N represents one limb from a different animal. White dotted lines mark amputation plane. Scale bars, B-D, H-I: 500 µm, F: 1 mm. WT, wildtype, dpa, days post-amputation.

Figure 3—figure supplement 1. CRISPR generation of mk mosaic null mutants.

(A) Example genotyping results of fully modified (mosaic null, abbreviated mk^null) or partially modified (low % or high % WT) animals. (B–C) Representative brightfield images of tracrRNA control and mk^null forelimb of four-month old sibling axolotls. (D) Side-by-side brightfield image of tracrRNA control and mk^null axolotls demonstrating no gross observational difference in body size. (E–F) Representative MK immunostaining of a tracrRNA control or mk mosaic null mutant at 7 dpa reveals a loss of staining. Scale bars, B-C: 2 mm, E-F: 500 µm. dpa, days post-amputation.