506 The Journal of Cell Biology | Volume 159, Number 3, 2002 Figure 11. De novo initiation of short experimental neurite and subsequent spontaneous elongation and dephospho-tau compartmentation. (A) A neuron with several fairly long minor process is stimulated by experimental application of tension to the cell margin (arrow). (B) After 3 h of towing, a neurite of 50 m has been initiated, and the growth cone has moved beyond the needle (arrowhead) as in Fig. 5. This was taken as an opportunity to remove the needle (C). (D) 5 h after needle removal, the experimental neurite has elongated an additional 100 m. (E) The next day, the experimental neurite elongated 100 m further. (F) Shortly thereafter, the neuron was fixed and processed for dephospho-tau localization. This particular experimental neurite shows clear compartmentation of the antigen but not a gradient (see discussion of dephospho-tau localization in Results). This is also another instance (Fig. 5) in which one other minor processes elongated, but interestingly none of the spontaneous neurites show dephospho-tau localization. Bar, 20 m. unmanipulated sibling minor process ever showed rapid out- growth of this kind during the course of our observations. Growth cones of towed neurites were clearly behaving quite differently than those of the untowed siblings and in a way that is characteristic of early axonal specification. In addition to rapid spontaneous growth cone activity, the difference between an axon and a minor process is also partly defined by the persistence of growth by an axon, leading to its substantial increase in length over time. In our experi- ments, when the towing needle was removed successfully and without damaging the neurite the extent and persistence of outgrowth by experimentally induced neurites were at least as good as that of the axons of early stage 3 axons that were spontaneously specified. Among experimental neurites, 80% remained extended or grew, whereas among spontane- ous axons 67% of initial axons remained extended or grew. Among experimental neurites, 30% doubled their length after the experimental intervention (Figs. 2, 6, 7, and 11), whereas 15% of spontaneous axons doubled their length over the next day or two and, surprisingly, another 15% grew substantially by elongating a second axon. In this latter regard, Ruthel and Hollenbeck (2000) found that 4% of in- dividually identified stage 2 (not early stage 3 as in our ob- servations) spontaneously grew two axons in only 14 h of observation. In addition to elongation characteristics, the frequency of “failure” in axonal specification was also similar among experimental and spontaneous axons at early times of specification. Some 20% of experimentally induced neurites failed to persist, whereas 33% of spontaneous stage 3 neu- rons showed regression of an initially specified axon. Thus, at early times of apparent axonal specification both experi- mentally induced neurites and spontaneous axons showed very similar extents and persistence of growth. Dephospho-tau and L1 are among the earliest molecular markers for axonal specification (Van den Pol and Kim, 1993; Mandell and Banker, 1996; Ruthel and Hollenbeck, 2000). Both showed compartmentation to experimental neurites at the end of the towing period with uniform distri- bution for dephospho-tau or uniformly punctate distribu- tion for L1 (Fig. 9). Because dephospho-tau compartmenta- tion and gradient have been reported to take additional days of outgrowth after axonal specification, we analyzed dephos- pho-tau compartmentation in several experimental cells al- lowed to incubate after towing. Most experimental neurites that were incubated after towing showed the distal to proxi- mal gradient of dephospho-tau characteristic of hippocampal axons. This gradient of dephospho-tau in experimental neu- rites was similar in appearance to that of spontaneously spec- ified axons, and both showed good reproducibility of the phenomena. Thus, the identity of the experimental neurites as axons is further supported by immunostain for the molec- ular marker dephospho-tau. Attainment of a critical length by one minor process, and therefore the breaking of symmetry, has been hypothesized to be the point at which an axon is specified (Goslin and Banker, 1989). As our studies have shown, tension and ax- onal elongation are very closely related. However, because growth cone advance and neurite lengthening can only oc- cur as a result of a force, from a physical point of view force necessarily precedes lengthening. We were able to experi- mentally separate these two closely related parameters by showing that tension could induce axonal fate even when we ceased towing while the experimental neurite was shorter than its siblings. Our best success was achieved by initiating neurites de novo where, once again, alternation of tension and relaxation stimulated growth cone activity. These exper-