Figure 8.

NGF Downregulation Restores R3KO Axon Trajectory in the Skin

(A) Example of Wthindpaw epidermis transfected with control shRNA plasmid. (Left) Superimposition of EGFP (green) and TrkA (magenta). (Middle) EGFP-positive areas representing successful transfection and long-term integration of the shRNA sequence into the genome of skin epidermal cells (mostly keratinocytes). (Right) Immunofluorescence of TrkA axons in the epidermis. Scale bar, 20 μm.

(B and C) Two examples of R3KO hindpaw epidermis (taken from the same animal at different locations) with EGFP fluorescence representing areas of shRNA-mediated NGF downregulation. TrkA axons contacting EGFP-positive areas are straighter (arrowheads) compared with axons in remote areas. Color images show EGFP (green) and TrkA (magenta).

(D) Curviness indexes of WT (top) and R3KO (bottom) axons within (“within EGFP area”) or outside (“outside EGFP area”) EGFP-positive areas. EGFP delineates areas transfected with either control or NGF shRNA. Left four panels: each line representing a difference in the average curviness index between the two areas of one animal. ∗∗p < 0.01; ns, not significant; paired t test. Right four panels: scatterplots in which each dot represents the curviness index of a single axon segment. The numbers in parentheses indicate the numbers of axon segments analyzed.

See also Figure S4.