CNS-derived glia ensheath peripheral nerves and mediate motor root development

Abstract

Motor function requires that motor axons extend from the spinal cord at regular intervals and that they are myelinated by Schwann cells. Little attention has been given to another cellular structure, the perineurium, which ensheaths the motor nerve, forming a flexible, protective barrier. Consequently, the origin of perineurial cells and their roles in motor nerve formation are poorly understood. Using time-lapse imaging in zebrafish, we show that perineurial cells are born in the CNS, arising as ventral spinal-cord glia before migrating into the periphery. In embryos lacking perineurial glia, motor neurons inappropriately migrated outside of the spinal cord and had aberrant axonal projections, indicating that perineurial glia carry out barrier and guidance functions at motor axon exit points. Additionally, reciprocal signaling between perineurial glia and Schwann cells was necessary for motor nerve ensheathment by both cell types. These insights reveal a new class of CNSborn glia that critically contributes to motor nerve development.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 cord 1 . 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 Retzius 2 . 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 infection 4, 8-10 . Therefore, formation of the perineurium is essential for peripheral nerve function.Correspondence should be addressed to B.A. (b.appel@vanderbilt.edu). AUTHOR CONTRIBUTIONS S.K. produced all of the data except for the electron microscopy images shown in Figure 5c NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptPrevious 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 barrier 11 . 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 cells 12 . Although these studies revealed a critical feature of perineurial cell differentiation, the origin of perineurial cells and how they initially associate ...

nkx2.2apromotes specification and differentiation of a myelinating subset of oligodendrocyte lineage cells in zebrafish

“…We tested this using antisense MOs designed to interfere with nkx2.2a translation. Previous work showed that nkx2.2a MOs reduce or eliminate expression of genes that mark cells born within the p3 domain (Schafer et al, 2007;Kucenas et al, 2008). Accordingly, in nkx2.2a MO-injected embryos, olig2 RNA expression expanded ventrally to include cells adjacent to medial floor plate (see Supplementary Fig.…”

“…Zebrafish strains used in this study included AB,Tg(nkx2.2a:megfp) vu17 (Kirby et al, 2006;Kucenas et al, 2008), Tg(olig2:dsred2) vu19 (Kucenas et al, 2008) and Tg (sox10(7.2):mrfp) vu234 (Kucenas et al, 2008). Embryos were produced by pair-wise matings, raised at 28.5°C in egg water or embryo medium and staged according to hours postfertilization (hpf).…”

“…nkx2.2a MO1 (5′-CCG TCT TTG TGT TGG TCA ACG ACA T-3′) was complementary to a sequence spanning just before and including the translation start codon (Schafer et al, 2007;Kucenas et al, 2008). nkx2.2a MO2 (5′-AAG TTG CTG CAC CAG TTT GAC AAT C-3′) was complementary to a sequence in the 5′ UTR.…”

“…To investigate the relationship between gene expression and OPC formation more directly, we examined transverse sections of Tg(nkx2.2a:megfp);Tg(olig2:dsred2) embryos, which express membrane-tethered EGFP under control of nkx2.2a regulatory DNA (Kirby et al, 2006;Kucenas et al, 2008) and DsRed2 under control of olig2 (Kucenas et al, 2008), respectively. At 24 hpf, EGFP + and DsRed2 + spinal cord cells occupied distinct, but contiguous domains ( Fig.…”

“…We first imaged larvae carrying both a Tg(sox10(7.2):mrfp) transgene, which expresses a membrane tethered Red Fluorescent Protein (mRFP) under the control of sox10 regulatory DNA (Kirby et al, 2006;Kucenas et al, 2008) (Fig. 4A and see Supplementary Movie 3 online).…”