Figure 6. ARF suppresses fin regeneration by inducing apoptosis and cell-cycle arrest.
(A) Quantification of EdU staining at 2, 4, and 6 dpa in wild type (WT) and hs:ARF fins exposed to heat shock (left). At 2 dpa, 6.0% ± 1.1% of cells in WT regenerates were EdU + compared with approximately 2.2% ± 0.8% in Heat shock (hs):ARF regenerates. At 4 dpa, approximately 7.4% ± 0.6% of cells in WT regenerates were EdU + compared with 4.2% ± 0.6% in hs:ARF regenerates. At 6 dpa, approximately 6.4% ± 0.9% of cells in WT regenerates were EdU + compared with 2.7% ± 0.3% in hs:ARF regenerates. Significantly fewer cycling cells are detected with ARF expression (N = 10 fins, p<0.001). Representative (left – sagittal confocal, right – longitudinal) images of EdU staining at 2 dpa in WT and hs:ARF fins exposed to heat shock (right). Scale bars: left – 50 μm, right – 25 μm. Dashed lines represent amputation planes. (B) Quantification of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 2, 4, and 6 dpa in WT and hs:ARF fins exposed to heat shock (left). At 2 dpa, 2.2% ± 1.2% of cells in WT regenerates were TUNEL + , while 6.7% ± 3.7% of cells in hs:ARF regenerates were TUNEL + . At 4 dpa, only 2.7% ± 1.2% of cells in WT regenerates were TUNEL + compared with 4.8% ± 0.8% in hs:ARF regenerates. At 6 dpa, 2.4% ± 0.6% of cells in WT regenerates were TUNEL +, while 3.1% ± 0.5% of cell in hs:ARF regenerates were TUNEL +. Significantly more apoptosis is detected with ARF expression (N = 10 fins, p<0.001). Representative images (left – sagittal, right – longitudinal) of TUNEL staining at 2 dpa in WT and hs:ARF fins exposed to heat shock (right). Image quantification was performed on regenerates only. Dashed lines represent amputation planes. Scale bars: left – 50 μm, right – 25 μm. (C) Quantification of EdU staining in WT and hs:ARF fins 3 hr after a single heat shock or 24 hr of heat shock delivered at 4 dpa. After a single heat shock, 3.3% ± 1.5% of cells in WT regenerates were EdU + compared with 1.9% ± 0.6% in hs:ARF regenerates. After 24 hr of heat shock, 3.0% ± 0.7% of cells in WT regenerates were EdU + compared with 1.2% ± 0.4% in hs:ARF regenerates. Significantly fewer cycling cells are detected with ARF expression after blastema formation (N = 10 fins, p<0.001). Results are shown as mean ± standard deviation.