Figure 3.
DCC-mediated netrin signaling contributes to RGC axon presynaptic differentiation during the development of retinotectal connectivity. A, Sample RGC axons coexpressing Ds-Red (red) and GFP-synaptobrevin (green) imaged by time-lapse confocal microscopy over a 24 h period after microinjection of recombinant netrin-1 (+ netrin) or function-blocking antibodies to DCC (− DCC). Projections on the right show the GFP-synaptobrevin fluorescence only. Scale bar, 50 μm. B, C, Manipulations in netrin signaling alter the number of GFP-synaptobrevin-labeled presynaptic sites and influence RGC axon arbor morphology. B, As RGC axons branch and differentiate, the absolute number of GFP-synaptobrevin-labeled presynaptic sites increases over time (0–4, 0–8, 0–24 h). Microinjection of recombinant netrin-1 induced a significantly higher increase in the number of presynaptic sites relative to controls by 4 h, an effect that persisted for the remainder of the 24 h observation period. Microinjection of DCC function-blocking antibodies led to a smaller increase in presynaptic site number relative to controls from 8 h onward. C, The increase in the number of total branches was significantly higher in RGC axons 8 h after netrin treatment relative to controls, whereas RGC axons had fewer branches 24 h after anti-DCC treatment. *Significance with p ≤ 0.05. #Trend toward significance with 0.05 > p < 0.10. Error bars indicate SEM.