Following injury, peripheral nerves reestablish neuromuscular connections with their developmental targets. While the molecular pathways that govern peripheral nerve development and neuromuscular synapse formation are well understood, it is unclear whether the same pathways are reemployed to promote regeneration. For example, neuromuscular synapses require the evolutionarily conserved Agrin-Lrp4-MuSK signaling pathway to cluster acetylcholine receptors (AChRs) beneath motor axon terminals, yet their in vivo role in peripheral nerve regeneration has not been examined. To determine whether this pathway is re-utilized during regeneration, we used a previously established assay that enables us to transect spinal motor nerves and continuously monitor regeneration in live, intact zebrafish (Rosenberg et al., 2012).
We find that animals harboring a TALEN-induced lrp4 null allele display defects in neuromuscular synapse formation, while motor axon growth and guidance is unaffected. In contrast, after nerve transection, these axons largely fail to regrow to their muscle targets. Using the same assay, we find that the obligate Lrp4 co-receptor MuSK, which is essential for neuromuscular synapse development, is dispensable for motor axon regeneration, demonstrating that Lrp4 promotes axon regeneration through a MuSK-independent pathway. Moreover, using a second lrp4 mutant allele we find that the Lrp4 transmembrane domain, while critical for neuromuscular synapse development, is dispensable for axon regeneration, consistent with the idea that Lrp4 promotes regeneration through a novel, tether-independent pathway. Restoring lrp4 expression only to muscle cells in animals otherwise lacking lrp4 rescues neuromuscular synapse development but does not rescue motor axon regeneration, suggesting that Lrp4 acts in another cell type to promote axon regeneration. We will present ongoing efforts to further characterize this novel MuSK-independent pathway through which Lrp4 promotes peripheral nerve regeneration.