504 The Journal of Cell Biology | Volume 159, Number 3, 2002 Figure 8. Immunofluorescent localization of dephospho-tau in experimental neurite of stage 3 neuron after 1 d of incubation. (A) Immediately after needle attachment to experimental neurite. (B) At the end of towing, 9 h 43 min later, experimental neurite is 400 m long. (C and D) Matched phase and fluorescent images of experimental cell fixed after 20 h of incubation after needle removal. Note that the spontaneous axon elongated from 125 m to 190 m during these 20 h. The experimental neurite shortened immediately after and incident to needle removal, but then remained essentially stable at the length shown in C and D. nipulated neurons undergo, including ones we had pre- sumed were artifacts of towing. One example of the latter is that we saw two instances among the 45 in which rapid elongation of the axon was accompanied by the transloca- tion of the somatic cytoplasm and nucleus into the axonal shaft, leaving a still motile “ghost” of the cell body. We had reported this phenomenon previously in brain neurons sub- jected to hard towing (see Fig. 6 in Ingber et al., 2000). Thus, we were surprised by the similarity of spontaneous and experimental neurites at early times of axonal specifica- tion in life course and phenomenology. Experimentally elongated neurites show molecular markers for axons An early marker for axons in hippocampal neurons is the en- richment of dephospho-tau in a distal to proximal gradient in the axon (Mandell and Banker, 1996; Ruthel and Hollen- beck, 2000). Among 30 neurons examined, 16 stage 2 and 14 stage 3 neurons, all experimentally elongated neurites ex- amined showed compartmentation of dephospho-tau anti- gen. All six cells fixed immediately after towing showed compartmentation as a general enrichment of dephospho- tau to the axon, with fairly uniform distribution (data not shown). However, after a day in incubation 16 out of 24 cells showed a clear gradient of dephospho-tau immunofluo- rescence, whereas the remaining 8 neurons continued to show uniform compartmentation (see Fig. 11). Fig. 7 shows the distal to proximal dephospho-tau gradient seen in the experimental neurite of a stage 2 neuron permitted to grow for an additional 24 h after towing. Fig. 8 shows a similar re- sult for dephospho-tau compartmentation in both the towed neurite and spontaneous axon of a stage 3 neuron after 24 h. Another molecular marker that is enriched in axons at early times after specification is the cell adhesion molecule, L1 (Van den Pol and Kim, 1993). Fig. 9 shows an example in which a minor process of a stage 2 neurite is elongated for 3 h, then fixed immediately and stained for L1. As shown here, L1 is rapidly compartmentalized to the axon that has been experimentally specified. As reported previously for spontaneous axons of hippocampal neurons (Van den Pol and Kim, 1993), we found that L1 has a punctate distribu- tion along the experimental neurite of our cells. Even short tension-induced neurites grow and develop as axons One hypothesis for the mechanism of axonal specification of minor processes is the achievement of a critical length. In this scenario, relative length is a “symmetry breaker” and ax- onal specification occurs when a minor process becomes 15 m longer than any of its sibling neurites (Goslin and Banker, 1989). As the foregoing data indicate, the very close relationship between tension and length that characterizes neurite outgrowth makes separating the two difficult. How- Figure 9. Immunofluorescent localization of L1 in experimental neurite of stage 3 neuron immediately after towing. (A) A minor process (arrow) was attached to a needle and elongated 50 m over 3 h and 40 min (B). (C) After needle removal, the cell was fixed and processed for L1 localization. As shown here, the experimental neurite shows the punctate distribution of L1. Bar, 30 m.