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Figure 7.
Inhibition of calpain activity promotes RB peripheral axon outgrowth in situ. Open-book skin preparations were used to measure axon outgrowth in situ. a, b, GFP, GFP-capn1-H272A, GFP-FAK-V744G, or GFP-talin-L432G was injected in a single dark blastomere at the eight-cell stage (a) driving expression into one side of the dorsal spinal cord (b). NT, neural tube; NC, notochord. c–g, The skin of 26 hpf embryos was removed, and preparations were labeled with NCAM to observe RB peripheral projections in the skin and flat mounted for confocal imaging. c–g, Representative images of HNK-1 labeling in skin expressing GFP (c), GFP-capn1-H272A (d), GFP-talin-L432G (e), GFP-FAK-V744G (f), or coexpressing GFP-talin-L432G and TagRFP-FAK-V744G (only GFP shown; g). Dashed line represents the dorsal midline. h, Quantification of the maximum axon displacement per 100 μm spinal cord segment on the injected side vs control side for each condition. i, Branching was quantified as the number of axon terminals (growth cones) per 100 μm segment of spinal cord. Scale bar, 50 μm. n > 100 segments and n > 10 embryos for all conditions. *p < 0.05, Mann–Whitney U test. |
