Fig. 6. The mps1 regeneration defect is caused by severe blastemal proliferative abnormalities. (A) Indices of proliferation at 2 days postamputation. (Left) BrdU incorporation data were obtained from counting 500-3,000 mesenchymal nuclei from 6-10 sections of each of five whole-mount immunostained regenerates. (Middle) A total of 14 regenerating rays from six wild-type fish and 21 rays from eight mps1 animals were used to count H3P-positive nuclei. (Right) A total of 1,355 H3P-positive nuclei from eight wild-type regenerates and 704 H3P-positive nuclei from 10 mps1 regenerates were scored for mitotic phases at 500x magnification. Results are shown as mean ± s.e.m. (t-test; ^*P<0.05, ^**P<<0.001). (B) (Left) Confocal projections of 2-day postamputation fin regenerates stained with anti-H3P to indicate mesenchymal mitoses. The bright points are individual mitotic nuclei, severely reduced in mps1 regenerates. Both fins show non-specific epidermal fluorescence at the distal edge (see Materials and Methods). (Middle) High magnification confocal images of H3P-positive mesenchymal nuclei. An mps1 fin ray with an unusually high number of mitoses is shown. Arrowheads point to late phase mitoses, deficient in mps1 fin regenerates. (Right) Projections of single 4-day postamputation fin rays from animals that have incorporated BrdU for the final 5 hours of regeneration. Note the reduced incorporation, and unusually large nuclei in cycling mps1 cells that are suggestive of aneuploidy (arrowheads in right image). Original magnification is 150x (left panels) and 945x (middle and right panels).