Figure 2. Osteoclasts are identified during skeletal resorption.

(A) Apotome image of immunofluorescence for anti-cathepsin K (CTSK; red) in zeugopod section at 8 dpa. Calcein was used for calcified cartilage labeling (green) and Hoechst for nuclear staining (white). Scale bar: 50 µm (n=2). (B) Tartrate-resistant acid phosphatase (TRAP) enzymatic staining in zeugopod section at 9 dpa. Scale bar: 50 µm (n=2). (C) RT-qPCR for Trap, Ctsk, and Dcstamp at different dpa upon zeugopodial amputation. Solid line represents mean, each dot is an animal (n=6. ** p<0.01, * p<0.05, one-way ANOVA, Bonferroni’s multiple comparisons test, each timepoint versus 0 dpa). (D) In vivo confocal imaging of Ctsk:mRuby3 (red) upon digit amputation. Calcein was used for calcified cartilage labeling (green). Image represents a maximum intensity projection of 10 images (3 µm interval). White arrowhead: mRuby3⁺ cells (osteoclasts). Black arrowhead: break in the skeletal tissue. Dashed line: amputation plane. Asterisk: multinucleated osteoclast. Scale bar: 100 µm (n=3). (E) In vivo confocal imaging of Sox9 × Ctsk at 7 dpa. Images were taking at time 0, 1, and 2hr. Image represents a maximum intensity projection of four images (3µm interval). White arrowhead: eGFP⁺ osteoclast engulfing mCherry⁺ chondrocyte. Scale bar: 50 µm (n=6). (F) Orthogonal view of in vivo confocal imaging from the Sox9 × Ctsk line at 13 dpa. Image is composed of 15 planes with a voxel depth of 2 µm. Center of cross shows mCherry⁺ cell phagocytosed by eGFP⁺ cell. Scale bar: 50 µm (n=6).

Figure 2—figure supplement 1. Characterization of Ctsk:eGFP transgenic line and assessment of apoptosis in skeleton during regeneration.

(A) Confocal image of immunofluorescence (IF) for anti-F4/80 (magenta) in Ctsk:eGFP zeugopod section at 7 dpa. eGFP endogenous fluorescence was acquired (green), and Hoechst was used for nuclear staining (white). Inset: multinucleated eGFP⁺/F4/80⁺ cell. Scale bar: 100µm (n=4). (B) Confocal image of IF for anti-F4/80 (magenta) in Ctsk:eGFP zeugopod section at 7 dpa. eGFP endogenous fluorescence was acquired (green), and Hoechst was used for nuclear staining (white). Arrowheads: eGFP⁺/F4/80⁻ perichondral cell. C: cartilage. Dashed line: cartilage boundaries. Scale bar: 100µm (n=4). (C) Confocal image of TUNEL assay (magenta) in Ctsk:eGFP (green) zeugopod section at 11 dpa. Hoechst was used for nuclear staining (white). Arrowheads: TUNEL⁺/eGFP⁺ osteoclasts. Scale bar: 100µm (n=3). (D) Confocal image of TUNEL assay in limbs at 5 and 7 dpa. C: cartilage. Yellow dashed line: cartilage boundaries. Asterisk: Staining noise. Scale bar: 250µm (n=3).