Figure 7
Sema3A-induced axon retraction in wt , ferDR/DR, fps-/- or fpsKR/KRembryonic DRG neurons. (A), Sema3A-induced ferDR/DRaxon retraction in response to NGF. Sema3A was added to the NGF-containing media at time 0, and axon retraction quantified. X-axis indicates time of observation with the "zero" point corresponding to axon ends at time = 0. The Y-axis indicates the change in length of individual axons with decreasing or increasing distance values indicating axon retraction or growth, respectively. The black line indicates the mean value of wt axon behavior and the red line indicates the mean value of ferDR/DRaxon response to Sema3A stimulation. Gray areas indicate standard deviations. (wt, n = 47 axons; ferDR/DR, n = 51 axons; p < 0.0005). Data was from 8 independent experiments using 12–18 embryos of each genotype. (B), Sema3A-induced ferDR/DRaxon retraction in the absence of NGF. The analysis was performed essentially as in (A), except that before Sema3A stimulation, the NGF-containing media was removed and replaced with NGF-free media (wt, n = 35 axons; ferDR/DR, n = 39 axons; p < 0.001). Data was from 6 independent experiments using 8–12 embryos of each genotype. (C), ferDR/DRaxon growth in the presence of NGF, retraction in response to NGF-depletion, and further retraction in response to Sema3A addition. The analysis was performed essentially as in A, except that axon outgrowth in the presence of NGF was monitored between 1–150 minutes (p < 0.3). The media was then replaced with NGF-free media and monitoring was continued between 150–300 minutes (p < 0.01). Finally, Sema3A was added and monitoring was continued between 300–450 minutes (p < 0.01) (wt, n = 29 axons; ferDR/DR, n = 27 axons). Data was from 5 independent experiments using 8–12 embryos for each genotype. (D), Sema3A-induced axon retraction of fps-/- axons in the presence of NGF. This analysis was performed essentially as described in A, except using DRGs from wt and fps-/- E14 embryos. (wt, n = 31 axons; fps-/-, n = 35 axons; p < 0.07). Data was from 4 independent experiments using 8–12 embryos per genotype. (E), Growth of fpsKR/KRaxons in the presence of NGF, followed by retraction in response to Sema3A. This experiment was done essentially as described above except axon outgrowth from wt and fpsKR/KRDRGs in the presence of NGF was monitored from 0–60 minutes. Sema3A was then added to the NGF-containing media and axon retraction was monitored from 60–150 minutes (wt, n = 22 axons; fpsKR/KR, n = 19 axons; p < 0.2). Data was from 2 independent experiments using 4–6 embryos for each genotype. (F), fps-/- axon growth in the presence of NGF, followed by retraction in response to NGF-depletion, and further retraction in response to Sema3A addition. This experiment was done as described in C, except DRGs were from wt and fps-/- E14 embryos. Axon outgrowth in the presence of NGF was monitored between 1–75 minutes (p < 0.4). The media was then replaced with NGF-free media and monitoring was continued between 75–225 minutes (p < 0.3). Finally, Sema3A was added and monitoring was continued between 225–375 minutes (p < 0.01) (wt, n = 36 axons; fps-/-, n = 32 axons). Data was from 4 independent experiments using 8–12 embryos for each genotype.