Figure 3.
Time-lapse video microscopy images of neurites regenerating from dissociated wild-type (WT) and map1b-/- (KO) DRG neurons. The four 90 min sequences represent the evolution of individual neurites after an initial culture period of 15-18 hr (set as To), in 10 min intervals between frames. An overview of each neuron at the end of the acquisition time is presented at the bottom (Tf). Scale bar, 10 μm. a, Terminal branching on a map1b+/+ neurite. After neurite extension for the first 20 min, two particularly long filopodia are distinguished on the growth cone that will give rise to two new, separately extending branches (arrows). After 60 min, new filopodia emerge from the upper growth cone, which after two separate branching events give rise to three new processes. Two of them are stabilized (arrows), and the third one retracts (arrowhead). Frequent retraction of unstabilized new processes leads to a straight aspect of wild-type neurites (arrowheads; see To +90′ and panel Tf). b, Terminal branching and filopodia dynamics on map1b-/- neurites. The neurite shown presents numerous thin, filopodia-like processes and a particularly large growth cone (asterisk) with several actively extending and retracting filopodia. In a sequence of terminal branching events between To +30′ and To +90′, numerous new processes are elaborated (arrows) that, in contrast to map1b+/+ neurons, do not retract, eventually giving rise to the highly branched aspect of the map1b-/- neuron. c, Three smooth, straight neurites extending from the same map1b+/+ neuron. Their growth cones are small, typical for rapidly extending neurites. Thin, short new processes forming by growth cone bifurcation are always retracted (arrowheads), so that the final morphology of all three neurites remains rectilinear and smooth (Tf). d, Collateral branching events on a map1b-/- neurite. At To, a proximal segment on the neurite shaft (arrow) undergoes a swelling, followed by the appearance of filopodia (To +10′) that finally give rise to a new growth cone (To +30′). During extension of this new growth cone, it comes into contact with another process (arrowhead), originating from a previous terminal branching event (To +60′). This latter process then retracts, whereas the new collateral branch is obviously stabilized and continues to progress (To +90′; see also Tf 2.5 hr later).