500 The Journal of Cell Biology | Volume 159, Number 3, 2002 Figure 1. Elongation of a minor process in a stage 2 neuron induced by experimentally applied tension. (A) Neuron immediately before needle application. Arrowhead marks short process to which needle was attached. (B) Same neuron 0:30 h/min later during early stage of neurite towing. (C) 5:40 after panel B, at the end of towing. Bar, 20 m. hippocampal neurons. Neurons at two stages of develop- ment were used: those in which all neurites were minor processes (stage 2 of Dotti et al., 1988) and neurons that had already specified one process as an axon (stage 3). Minor processes of stage 2 or stage 3 neurons were elon- gated by “towing,” and subsequent identity as an axon was determined using several criteria: rapid rate of spon- taneous growth cone advance, capacity of the towed neu- rite to remain elongated and undergo continued sponta- neous elongation, and the presence of early molecular markers for axons. Figure 2. Elongation of a minor process of a stage 3 neuron induced by experimentally applied tension. (A) Immediately before the beginning of the manipulation, the extant axon was 250 m long, and the minor process (arrowhead) was 25 m. (B) 28 min after needle attachment. (C) Immediately after needle removal, the minor process had been towed to a length of 100 m. (D) After 18 h of incubation following needle detachment, the spontaneous axon had branched and produced a net elongation of 70 m, whereas the experimental neurite elongated an additional 90 m. Bar, 50 m. Results Minor processes of hippocampal neurons elongate in response to applied tension Cultured hippocampal neurons were manipulated at two different stages of polarity development. Stage 2 neurons (which have minor processes but no axon) were used to de- termine whether a particular minor process could be in- duced to differentiate into an axon by experimentally ap- plied tension. A typical example of the elongation that resulted from manipulating a minor process of a stage 2 neu- ron is shown in Fig. 1. Fig. 1 illustrates a routine aspect of these manipulations for stage 2 neurons: the minor process for elongation was explicitly chosen to be the least, or among the least, well developed in terms of neurite length. Because a large growth cone has been reported to be indicative of a developing axon (Bradke and Dotti, 1999), some effort was also made to choose the smallest growth cone, although we found that the size of minor process growth cones varied sig- nificantly over time (Ruthel and Hollenbeck, 2000). Hip- pocampal neurons typically elaborate only a single axon, suggesting that after one process has been chosen as the axon the remaining minor processes are inhibited from becoming axons and become destined to differentiate into dendrites. To determine whether mechanical tension can specify an axon in the presence of an existing axon, minor processes of stage 3 neurons were also experimentally lengthened. Fig. 2 shows an example of the experimental elongation of a minor process from a stage 3 neuron. Elongation of a minor pro- cess of a stage 3 neuron did not result in the loss of the orig- inal axon either during the experimental elongation or dur- ing subsequent unassisted outgrowth (Fig. 2 D). Indeed, both the original axon (as branch growth) and the towed neurite continued to exhibit substantial outgrowth after ten- sion was no longer applied. Like other neuronal types exposed to this experimental growth regime (Zheng et al., 1991; Chada et al., 1997; Lamoureux et al., 1997), tension applied to existing hip-