1% of the time (n = 42 beads, in 33 retinas), in contrast to only 5.5% of BSA-coated beads (n = 36 beads, in 25 retinas). To assess this interaction in more detail, we performed time-lapse imaging experiments. After Lam1-coated bead implantation, the embryo was allowed to recover for 5–10 hr, and then imaged during the

period of RGC axon extension. Most RGCs that came in contact with the surface of the Lam1 bead consistently showed a very strong interaction (Figure 6C, Movie S10. Lam1 Is Sufficient to Orient RGC Axon Extension In Vivo (Part 1) and Movie S11. Lam1 Is Sufficient Lumacaftor mw to Orient RGC Axon Extension In Vivo (Part 2)). RGCs tightly associated with the beads and extended axons along their surface (70% of experiments,

n = 20 beads/bead clumps, in 14 embryos). The growth cones of these axons subsequently navigated away from the bead, toward the basal surface of the retina, leaving bundles of fasciculated axons hugging the surface of the bead (arrows, Figure 6C). The RGCs generally remained associated with the beads for the length of imaging session, and the RGC layer appeared to organize itself around the Lam1 bead. In contrast to the dramatic effect of the Lam1 beads, BSA-coated beads did not show any substantial interaction with RGCs (n = 6 beads, in five embryos). Instead BSA-coated beads appeared to float aimlessly within the retina, indicating that they do not interact with any retinal cells (compare AZD6738 in vivo Lam1 and BSA-coated beads in Movie S11). In some instances it was possible to track an isolated RGC as it came into contact with a Lam1-coated bead, as is shown in Figure 6D (Movie S12). This young RGC exhibited a typical morphology, with apical and basal processes. The RGC then contacted the Lam1 bead at approximately the midpoint of the basal process (yellow arrowhead). The distal portion of the basal process then Non-specific serine/threonine protein kinase retracted, and short dynamic

neurites were evident at the point of Lam1 contact. The growth cone then sprouted from the contact point, and subsequently navigated away toward the retinal basal surface, demonstrating that Lam1 contact is sufficient to specify the point from which the RGC axon will emerge. The axon shaft remained associated with the bead, and was even observed to split in the example shown (blue arrowhead). This highlights the tight adherence of RGC axon to the Lam1 surface, and the critical importance of Laminin to RGC axons in vivo. A requisite step in axon selection is the differential rearrangement of microtubules in the preaxonal neurite (Witte et al., 2008). This is likely what is visualized using the Kif5c560-YFP microtubule motor construct.