Fig. 3.

Failed blastema maintenance in nbl.(A) Whole-mount in situ hybridization of msxb in WT and nbl fin regenerates. Fish were incubated for 5 days at 25°C before an 8-h incubation at 33°C. msxb expresson is absent in the nbl distal blastema (red arrowhead). (B) Sections of WT and nbl regenerates immunostained for BrdUrd (green) and DAPI (blue). Fish were incubated with BrdUrd during the final 2 h at 33°C. Note that an 8-h incubation at 33°C does not affect mesenchymal and epidermal cell proliferation levels during regenerative outgrowth but distal blastemal cells disappear in nbl (red arrowhead). (C) Apoptosis of blastema cells in nbl. WT and nbl fish regenerated for 5 days at 25°C followed by 4 h at 33°C. Fins were immunostained with active Caspase-3 (red) and DAPI (blue). Note that distal blastema cells undergo apoptosis in nbl. (D) Electron micrographs of distal blastema in WT and nbl fish. Fins regenerated for 5 days at 25°C followed by 8-h incubations at 33°C. Note the larger mitochondria with empty matrix (red arrowhead) in nbl mutants. (Bars, 2 μm.)