The formation of spinal motor nerves requires coordinated interactions between several types of cells. Motor neurons extend axons into developing muscle fields from the spinal cord through segmentally positioned motor exit points (MEPs). These axons encounter neural crest–derived boundary cap cells clustered at MEPs, which permit the axons, but not the cell bodies, to exit from the spinal cord1
. As motor axons approach their targets, they are sequentially wrapped and then myelinated by Schwann cells, glial cells that also develop from neural crest.
The myelinated motor nerve is surrounded by a flexible cellular sheath called the perineurium, first described in 1841 by Henle and later named by Key and Retzius2
. The perineurium consists of uninterrupted, concentric rings of flattened cells that are connected by tight junctions and encase motor nerves from the MEP to the neuromuscular junction (NMJ)2–7
. The perineurial sheath serves as a barrier, protecting axons from ionic flux, toxins and infection4,8–10
. Therefore, formation of the perineurium is essential for peripheral nerve function.
Previous studies have suggested that, during development, the perineurium appears to form by a series of steps in which nearby mesenchymal cells first assemble as a loosely organized tube around the nerve and then mature to create a multilayered barrier11
. The maturation step requires the signaling molecule Desert hedgehog (Dhh), which is expressed by Schwann cells. In the absence of Dhh, the perineurium is disorganized and is permeable to macromolecules and inflammatory cells12
. Although these studies revealed a critical feature of perineurial cell differentiation, the origin of perineurial cells and how they initially associate with motor nerves remain unknown.
, motor axon–ensheathing glia are born in the lateral edges of the CNS and then migrate out in a chain-like fashion along the motor nerve13–17
. The peripheral ensheathing glia of flies and peri-neurial cells of vertebrates have similar functional properties, raising the possibility that they have similar developmental origins. We tested this hypothesis by investigating the origin and development of perineurial cells in zebrafish. Using transgenic reporter genes and time-lapse imaging, we directly determined that, as in Drosophila
, glial cells born in the CNS migrate into the periphery to ensheath motor nerves. Specifically, we observed that ventral spinal-cord glia emerged from MEPs and migrated along the entire length of motor axons, ensheathing both axons and Schwann cells and forming the perineurium. In the absence of these perineurial cells, we saw that motor axons exited the spinal cord ectopically and Schwann cells failed to wrap motor nerves, indicating that these cells help direct development of spinal ventral nerve roots. Finally, we observed that perineurial glia failed to ensheath motor nerves in mutant embryos with defective Schwann cell development, suggesting that the wrapping behavior of perineurial glia is instructed by signals derived from Schwann cells. These studies reveal intimate, orchestrated interactions between motor neurons, Schwann cells and perineurial glia in the formation of peripheral motor nerves.