In contrast to the central nervous system the axons of peripheral nerves are able to regenerate in many vertebrates, including mammals. How this regeneration is achieved and the cellular processes involved have been described in many studies. An important point here is that the cellular environment plays an important role in promoting this axonal regrowth. Among these elements, Schwann cells have been described as required for peripheral axons regeneration. Now, a recent paper from the laboratory of Michael Granato reports on the observation in vivo of axonal regeneration in zebrafish as well as the requirement of Schwann cells to direct those axons towards their original paths (http://www.ncbi.nlm.nih.gov/pubmed/25355219).
Here, the authors use transgenic zebrafish expressing GFP in motor neurons and RFP in Schwann cells to follow in vivo the regeneration of fully transected motor nerves in larvae at 5dpf (days post fertilization). The first thing they describe is how upon transection the amputated distal axons were degraded and fragmented leaving behind axonal debris and denervated Schwann cells. Then, Schwann cells suffered dramatic changes from a tube-like morphology to a shorter and more rounded one. As motor axons regrew into their original paths, Schwann cells membranes also reverted to their normal thinner appearance.
In order to define in vivo the role of Schwann cells the authors used different mutant strains that lack differentiated Schwann cells (mutants for sox10, erbb2, erbb3 and nrg1 type III). In all these mutants, motor axons developed normally through 5 dpf. However, upon transection, axon regeneration was severely affected in all mutants lacking Schwann cells. In them, in 50-80% of the transected nerves the regenerated axons failed to target their original paths and instead ectopically grew into more lateral territories. These results suggest that Schwann cells directed regenerating axons. One possibility could be that proper axonal regeneration would depend on some factor produced by Schwann cells during development. To address this question the authors used a transgenic line in which Schwann cells develop normally but can be eliminated at the requested time point of development. Then, the authors killed the Schwann cells at 3,5 dpf and transected the motor nerves at day 5 dpf. In this context, the regenerated axons also failed to find their original targets as previously described for the sox10 mutants, suggesting that Schwann cells must be present during regeneration to direct re-growing axons. In another set of experiments the authors show how in sox10 mutants the regenerative growth cones initially sprouted and extended from the proximal stump in multiple directions as it happens in wild-type larvae. However, and in contrast to wild-type, axons continued to extend in all directions without going back to their original paths.
Distal denervated Schwann cells could exert this guiding role either by providing a physical substrate for the regenerating axons or by producing some factors that would direct the re-growing axons towards their original paths. To try to distinguish between these two possibilities the authors used a transgenic line in which the degeneration of the amputated distal nerves was delayed by more than 1 week. They crossed this line with the sox10 mutant line so an axonal scaffold distal to the transection gap completely lacking Schwann cells was formed. In mutants for axonal degeneration but with normal Schwann cells, motor axons regenerated normally indicating that those distal amputated axons had no inhibitory action on regeneration. However, in the double mutant with sox10, the regenerating axons failed to find their original paths. In other experiments, the authors transected only half of the axons in the motor nerve to create a continuous axonal scaffold devoid of Schwann cells (in sox10 mutants). There, the presence of this axonal scaffold could only partially compensate for the role of Schwann cells as 50% of the transected nerves showed aberrant abnormal projections compared to the 90% observed in fully transected nerves lacking Schwann cells. Overall, these results suggest that Schwann cells provide more than just a permissive substrate for regeneration.
Finally, the authors analysed the role of dcc (deleted in colorectal cancer) a receptor for netrin, a well-known attractive guidance cue for axonal growth. At 5 dpf dcc was expressed in motor neurons as well as in Schwann cells, whereas netrin1b was expressed in Schwann cells before and after transection. Also, dcc was expressed in motor neurons during the first stages of regeneration. Then the authors used a mutant line for dcc in which, interestingly, motor axons and Schwann cells develop normally. However, upon transection, about 40% of the nerves extended axons not only to their original paths but also ectopically into more lateral territories.
In summary, the authors show here the process of motor axon regeneration in vivo in zebrafish. Schwann cells are required for guiding the regenerated axons to their original paths and do it probably not only by providing some physical substrate but actively producing some factor. Future experiments should determine if netrin could be one of such factors.