Proliferation is arrested at the amputated caudal fin fold margin of Shp2-deficient embryos. At 2 dpf, the caudal fin folds of embryos from a ptpn11a+/− ptpn11b−/− incross were amputated and allowed to regenerate. The embryos were fixed at 2 dpa (4 dpf, 2 dpa) and subjected to whole-mount immunohistochemistry using an antibody specific for the cell proliferation marker PCNA (red). The embryos were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (blue). Maximum-intensity projection images of the caudal fin folds were taken, and all the embryos were genotyped. (A) Representative images of amputated embryo caudal fin folds, with the edges of the fin folds indicated with dashed lines. The number of embryos showing similar patterns/total number of embryos analyzed are indicated in the bottom right corners of the images in the right-hand column. Scale bars, 100 μm. (B) PCNA immunofluorescence between the tip of the notochord and the edge of the caudal fin fold was quantified by mean particle count, with thresholding and size restriction to remove background signal. Equivalent uncut controls were also quantified, and the mean values of all the caudal fin folds are shown. The statistical significance of the means was determined relative to ptpn11a+/+ ptpn11b−/− zebrafish embryos within the amputated group, and likewise within the uncut group. *, P < 0.05; the error bars represent standard deviations.
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