In support of this, proximal centrosome localization was sometimes unstable, and the centrosome could reorient during Stage 3. This was especially obvious in cases where the bead became dislodged from its original position after neurite contact and was pulled onto the surface of the cell body. The neurite that had originally contacted the bead remained committed to form the axon, while the centrosome tracked

the bead as it moved around the cell body. This indicates that Laminin-dependent axon commitment selleckchem is an early event that only transiently depends on localized Laminin contact, and is separable from the persistent effect of Laminin on centrosome localization. Although we have established that Lam1 is sufficient to direct axon commitment in vitro, we wanted to know if this was also the case in vivo. To answer this question we developed a system to implant polystyrene beads into the retina of 24 hpf zebrafish embryos using a sharp glass needle. This system allowed us to reintroduce Lam1 into a Lamα1-deficient embryo, to unambiguously identify where the ectopic Lam1 was located, and to assess its influence on polarizing RGCs. The bead implantation procedure did not have a dramatic effect on the structure of Ibrutinib nmr the retina, which had no noticeable structural defects, and appeared normal with a bead, or a clump of beads, suspended within it (data

not shown). Lam1-coated beads were implanted into 24–28 hpf lamα1 morphant embryos ( Figure 6A). Embryos were grown until 3 dpf, and we imaged

them by confocal microscopy to look for an interaction between the beads and RGC axons. In many cases an interaction between the beads and RGC axons was obvious, where large axon bundles were observed in contact with the beads/bead clumps. Axons hugged the surface of the beads, often causing them to lie within the axon fascicle ( Figure 6B). Beads were generally positioned at the base of the RGC axon bundles, close to RGC cell bodies, consistent Oxalosuccinic acid with the hypothesis that Lam1 is acting to direct polarization and RGC axon sprouting. Axon growth can be directed by the physical nature of a substrate. Therefore, it is possible that the physical presence of a polystyrene bead, rather than the Lam1 coating, is able to influence RGC polarization and axon extension. To control for this possibility, we implanted BSA-coated beads into Lamα1 morphant embryos. BSA-coated beads very rarely showed an association with RGC axons. To quantify this observation, confocal stacks from retinas implanted with either Lam1 or BSA-coated beads were blinded and classified as either showing a clear and dramatic interaction with RGC axons, where many RGC axons were seen in contact with the surface of the bead (similar to those shown in Figure 6B), or not.