Regulated formation and selection of neuronal processes underlie directional guidance of neuronal migration
Introduction
Neuronal migration plays a critical role in the development of the nervous system, and defects in neuronal migration cause a number of human diseases, such as lissencephalies and some types of epilepsy (Rakic, 1971, Hatten and Heinz, 1998, Hatten and Mason, 1990, Kornack and Rakic, 2001, Rice and Curran, 2001, Ross and Walsh, 2001). It is thus imperative to elucidate the cellular and molecular mechanisms that control neuronal migration to further our understanding of neural development and the etiology of neurological diseases.
Secreted guidance molecules such as netrins, ephrins, semaphorins, Slits, and the chemokine SDF have been implicated in controlling neuronal migration (Colamarino and Tessier-Lavigne, 1995, Hu and Rutishauser, 1996, Flanagan and Vanderhaeghen, 1998, Wu et al., 1999, Zhu et al., 1999, Raper, 2000, Klein, 2001, Guan and Rao, 2003, Nguyen-Ba-Charvet et al., 2004). Many of these molecules also play important and well-characterized roles in axon guidance. During development, gradients of these guidance cues are established throughout the nervous system. These gradients are crucial components in long distance pathfinding of migrating neurons and extending axons as they move from their place of origin to their final destination. In an extending axon, the growth cone acts in both a sensory and protrusive manner, while the cell body remains static. Depending on whether the growth cone interprets this cue as an attractant or repellent, the growth cone will gradually turn toward or away from the source of the cue, respectively (Zheng et al., 1996, de la Torre et al., 1997, Ming et al., 1997, Hong et al., 1999, Buck and Zheng, 2002, Ming, 2002). Such turning occurs through asymmetric protrusion of the growth cone during neurite extension, wherein one side of the growth cone extends at a faster rate than the other side, resulting in gradual reorientation of the growth cone and axon shaft. This asymmetric extension of the growth cone in the presence of guidance cues is accomplished through asymmetric formation, stabilization, and invasion of filopodia (Gundersen and Barrett, Zheng et al., 1996). Recent studies have shown that exposure of a growth cone to a guidance cue gradient results in polarization of activated guidance cue receptors and downstream signaling machinery (Hong et al., 2000, Guirland et al., 2004), which is believed to cause changes in the underlying cell cytoskeleton that results in growth cone turning (Gallo and Letourneau, 2003).
Because migrating neurons possess growth cones and respond to the same guidance cues as extending axons, it is possible that migrating neurons sense and respond to gradients of guidance cues in manners similar to extending axons, i.e. with single growth cones turning toward or away from secreted cues. However, in preliminary studies of migrating neurons in brain slices, it has been observed that migrating neurons can turn by sending out new leading processes from either existing leading processes or directly from the cell body (Nadarajah et al., 2001, Murase and Horwitz, 2002, Nadarajah et al., 2002, Polleux et al., 2002, Yacubova and Komuro, 2002, Nadarajah et al., 2003, Tabata and Nakajima, 2003, Tanaka et al., 2003). Similar observations of migrating GABAergic interneurons have been made in vivo (Ang et al., 2003). Although these observations raise the possibility that the turning behaviors of migrating neurons and extending axons differ, their relevance in the context of directed cell migration remains unclear. An alternative possibility is that that the use of neuronal processes in turning is employed by migrating neurons only in specific and/or limited circumstances and that single growth cone reorientation is the predominant mode of turning employed by migrating neurons when turning in response to secreted guidance cues. In addition, prior studies do not address how neuronal processes employed during turning are regulated.
Using time lapse microscopy to study Slit repulsion of primary migrating SVZa neurons, we found that Slit-induced turning did not occur through reorientation of individual growth cones away from the Slit source. Rather, migrating neurons turned through repeated rounds of process extension and retraction that resulted in selection of a dominant process and directional turning away from Slit. These results have broad implications regarding the manner in which migrating neurons sense and interpret guidance cues and may necessitate modification of current models concerning the translation of gradients of extracellular molecules into intracellular polarity during directed neuronal migration.
Section snippets
SVZa cells change migratory direction exclusively through continuous process extension and retraction, not growth cone bending
In postnatal rodents, neuronal precursors migrate tangentially from the anterior subventricular zone (SVZa) toward the olfactory bulb via a prominent pathway known as the Rostral Migratory Stream (RMS) (Doetsch and Alvarez-Buylla, 1996, Kirschenbaum et al., 1999, Liu and Rao, 2003, Nguyen-Ba-Charvet et al., 2004). To visualize the migratory behavior of these SVZa cells in situ, time lapse fluorescent microscopy was used to document the behavior of these neurons as they migrated in RMS slices.
Discussion
These results provide a mechanism for the regulation of SVZa turning in response to Slit, which will provide a framework for analyzing signal transduction during molecular guidance of neuronal migration. Whereas an axon senses extracellular gradients of guidance cues and turns using a single growth cone, our results indicate that a migrating neuron utilizes multiple growth cones, each enriched in guidance cue receptors, to sense and turn in the presence of such gradients. Furthermore, our
Dissection and culture of SVZa explants
Time-delayed co-culture of SVZa cells with Slit aggregates was described previously (Ward and Rao, 2004, Ward et al., 2003). Briefly, SVZa explants were dissected from the RMS of P2–P5 rat brains as previously described (Ward and Rao, 2004). Briefly, coronal sections of the caudal half of the olfactory bulb were made with a tungsten needle, and the RMS was identified by its translucent appearance and dissected out. This tissue was used to make small explants (200–400 μm in diameter), which were
Acknowledgments
Funding for this project was provided by the NIH. We are especially grateful for helpful comments on the manuscript provided by Greg Longmore, Rachel Wong, and Alex Ungewickell and for data analysis provided by Michael DeWulf.
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