When they do so, they polarize upside-down, suggesting that the basal lamina is responsible for RGC polarity ( Zolessi et al., 2006). Here we demonstrate that RGC polarization

toward the basal lamina requires the presence of an extracellular cue, Laminin 1 (Lam1). In the absence of Laminin α1 (Lamα1), RGCs exhibit Stage 2 behavior and mispolarization. Contact of newborn RGC processes with Lam1 either in vitro or in vivo is sufficient to cause the specific accumulation of Kif5c560-YFP, a marker of axonal NSC 683864 microtubules, followed by axon extension. Thus, in the normal retina, basally localized Lam1 directs the normal orientation of RGC axon extension in vivo. Live imaging in zebrafish demonstrated that axons extend directly from the most basal portions of RGCs in vivo (Zolessi et al., 2006). This previous study, however, was limited by the unavailability of an intracellular marker of axonogenesis. The

earliest AZD6244 reported marker for definitive axon commitment during hippocampal neuron polarization is the constitutively active motor domain of Kinesin 1 fused to YFP, Kif5c560-YFP. This construct selectively accumulates in axons, directed by biochemical differences in axonal microtubules; perhaps reflecting stabilized microtubules (Hammond et al., 2010, Jacobson et al., 2006 and Konishi and Setou, 2009). During Stage 2, Kif5c560-YFP displays a remarkably dynamic behavior, where YFP signal accumulates in just one (or sometimes a few) neurites, but this accumulation is only transient, passing from one neurite through the cell body to another neurite (Jacobson et al., 2006). As the neuron progresses to Stage 3 and Dipeptidyl peptidase the axon is selected, Kif5c560-YFP accumulates specifically and permanently in the tip of the preaxonal process, and remains there during axon extension (Jacobson et al., 2006). Thus, the oscillatory behavior provides a visual readout of the uncommitted Stage 2 phase, and the cessation of this oscillation and stable Kif5c560 accumulation in one neurite marks axonal commitment. To see whether

this marker of axonogenesis behaves in the same manner in zebrafish RGCs, we performed time-lapse imaging of ath5:GAP-RFP transgenic embryos injected with Kif5c560-YFP RNA at the one-cell stage to label RGCs ( Poggi et al., 2005 and Zolessi et al., 2006). At 30 hours post-fertilization (hpf), the approximate onset of RGC genesis, eyes were dissected and dissociated to obtain isolated cells. After a 12–15 hr incubation at 28.5°C, many RGCs had extended long axons, with bright Kif5c560-YFP signal accumulation within their growth cones ( Figure 1A). This confirms that the rat construct maintains its abilities to recognize axonal microtubules and to accumulate in the axonal growth cones of zebrafish RGCs.