Rapid Changes in the Distribution of GAP-43 Correlate With the Expression of Neuronal Polarity during Normal Development and under Experimental Conditions

Hippocampal neurons growing in culture initially extend several, short minor processes that have the potential to become either axons or dendrites. The first expression of polarity occurs when one of these minor processes begins to elongate rapidly, becoming the axon. Before axonal outgrowth, the growth-associated protein GAP-43 is distributed equally among the growth cones of the minor processes; it is preferentially concentrated in the axonal growth cone once polarity has been established (Goslin, K., D. Schreyer, J. Skene, and G. Banker. 1990. J. Neurosci. 10:588-602). To determine when the selective segregation of GAP-43 begins, we followed individual cells by video microscopy, fixed them as soon as the axon could be distinguished, and localized GAP-43 by immunofluorescence microscopy. Individual minor processes acquired axonal growth characteristics within a period of 30-60 min, and GAP-43 became selectively concentrated to the growth cones of these processes with an equally rapid time course. We also examined changes in the distribution of GAP-43 after transection of the axon. After an axonal transection that is distant from the soma, neuronal polarity is maintained, and the original axon begins to regrow almost immediately. In such cases, GAP-43 became selectively concentrated in the new axonal growth cone within 12-30 min. In contrast, when the axon is transected close to the soma, polarity is lost; the original axon rarely regrows, and there is a significant delay before a new axon emerges. Under these circumstances, GAP-43 accumulated in the new growth cone much more slowly, suggesting that its ongoing selective routing to the axon had been disrupted by the transection. These results demonstrate that the selective segregation of GAP-43 to the growth cone of a single process is closely correlated with the acquisition of axonal growth characteristics and, hence, with the expression of polarity. T HE development of distinct axonal and dendritic domains is essential for the functional polarization of neurons. The differential outgrowth of these two types of processes can be readily observed in dissociated cell cultures derived from the hippocampal region of the cerebral cortex. Like their counterparts in situ, the axons and dendrites that develop in hippocampal cultures differ from one another in light and electron microscopic morphology, in the macromolecular composition of their cytoskeletons and membranes, and in synaptic polarity (Banker and Waxman, 1988; Goslin et al., 1988). Early in development, hippocampal neurons extend several short "minor processes 7 which are approximately equal in length (stage 2 of development). These stage 2 neurons show no indication of polarity as judged by their growth characteristics, light and electron microscopic appearance, and immunostaining for axonal and dendritic markers (Dotti et al., 1988; Goslin et al., 1990; Deitch, J., and G. Banker, unpublished observations). Moreover, experimental evidence indicates that at this stage the minor processes are not irreversibly specified as axons or dendrites (Dotti and Banker, 1987; Goslin and Banker, 1989). Polarity is first expressed when, rather abruptly, one minor process begins to grow rapidly and acquires axonal characteristics (stage 3 of development). The transition between stage 2 and stage 3 is thus a critical point in the expression of neuronal polarity. Somewhat later, during stage 4 of development, the remaining minor processes begin to elongate and take on the properties of dendrites. To investigate possible mechanisms underlying the establishment of polarity, it is essential to identify molecular differences between axons and dendrites that precede or accompany the transition between stage 2 and stage 3, when hippocampal neurons first express a polarity. One protein of potential interest in this regard is the nervous system-specific phosphoprotein GAP-43 (also termed B-50, F1, P57, or pp46), a protein with possible growth regulatory functions (for reviews see Benowitz and Routtenberg, 1987; Skene, 1989; Gordon-Weeks, 1989). GAP-43 is an important constituent of axonal growth cones (Skene et al., 1986; Meiri et ai . , 1986, 1988) but is not detectable in dendrites or dendritic growth cones (Goslin et al., 1988). We have recently shown that GAP-43 becomes selectively distributed to the © The Rockefeller University Press, 0021-9525/90/04/1319/13 $2.00 The Journal of Cell Biology, Volume 110, April 199