Injection and in vivo electroporation of msxb morpholinos. A: Fin electroporated with 1.2 mM msxb morpholino. B: Fin electroporated with 1.2 mM msxb 5-mismatch morpholino. C: Fin electroporated with 1.2 mM (0.6 mM of each) msxb splice morpholinos. D: Whole mount in situ hybridization using msxc probe of fin electroporated with 1.2 mM msxb morpholino. Note the equal distribution of msxc expression across the fin. Black line, distal edge of fin re-growth at the time of fin injection/electroporation. For A-D, the dorsal (experimental) side is shown on the left and denoted by an asterisk. In each case, the ventral side of the fin was also electroporated, but it was not injected with a morpholino. E: Graph comparing areas of outgrowth of dorsal (experimental) and ventral (control) sides at 72 hpa. On the left, bars depict the percent inhibition as calculated over the entire 72 hpa. On the right is the second 1.2-mM experiment for which percent inhibition only over the final 24 hr of outgrowth was calculated. *Significant difference in outgrowth compared to fins injected with the corresponding concentration of the appropriate 5-mismatch control morpholino (P less than or equal to 0.001). At least five fins were examined for each concentration of morpholino. F: RT-PCR analysis of altered msxb transcript splicing by the pair of msxb splice morpholinos. Top: RT-PCR analysis on embryos injected with both msxb splice morpholinos. Bottom: RT-PCR analysis on fins injected/electroporated with 1.2 mM of the msxb splice morpholinos. For both panels, the PCR conditions were the same. The primers used are listed above the lanes at top, with their approximate binding location to msxb depicted in a cartoon below the bottom panel. G: PCNA-immunolocalization in fins injected/electroporated with 1.2-mM msxb morpholino. On the left, representative images of PCNA-immunostaining in longitudinal sections from dorsal (experimental) and ventral (control) halves of the fin. White bars that transverse the low-magnification images depict the cut site. Scale bar = 50 μm. On the right is a graph depicting the average number of PCNA-positive cells in the distal 150 μm of the dorsal and ventral blastemas. *Significant difference (P < 0.0001) between the dorsal (experimental) and ventral (control) halves of the fin.
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