Glial ensheathment of peripheral axons in Drosophila

Abstract

The ensheathment of neurons and their axons creates an ion-sensitive microenvironment that allows rapid conduction of nerve impulses. One of the fundamental questions about axonal ensheathment is how insulating glial cells wrap around axons. The mechanisms that underlie insulation of axons in invertebrates and vertebrates are not fully understood. In the present article we address cellular aspects of axonal ensheathment in Drosophila by taking advantage of glial mutants that illustrate a range of phenotypic defects including ensheathment of axons. From the findings of these mutant studies, we summarize that loss of glial cells, defects in glial membrane wrapping, failure of glial migration, and loss of specialized ladderlike septate junctions between ensheathing glial membranes result in axon-glial functional defects. These studies provide a broad perspective on glial ensheathment of axons in Drosophila and key insights into the anatomical and cellular aspects of axonal insulation. Given the powerful genetic approaches available in Drosophila, the axonal ensheathment process can be dissected in great detail to reveal the fundamental principles of ensheathment. These observations will be relevant to understanding the very similar processes in vertebrates, where defects in glial cell functions lead to devastating neurological diseases.

Fig. 1

Distribution, migration and process extension of glial cells around axons in the Drosophila embryonic PNS. A–D: Embryonic segments from wild-type stage 12 (A), stage 14 (B), stage 15 (C), and stage 16 (D) embryos immunostained for sensory neuronal marker 22C10 (22C; green, white arrowheads) and glial nuclear marker Repo (REP; red) showing profile of sensory axon trajectories in the PNS. The sensory neurons were born in the periphery (A) and start sending their axons toward the CNS (B–D). The peripheral glial cells ensheath the sensory axon tracts as they grow toward the CNS. E–H: Portion of embryonic segments from wild-type stage 12 (E), stage 14 (F), stage 15 (G), and stage 16 (H) embryos immunostained for motor neuronal markers Fas II (FAS; green) and REP (red) shows motor axonal trajectories. As soon as the neurons and glia were specified in the CNS (E), they expressed these markers. The glial cells are in close proximity to the motor neurons in the neurectoderm, which produces both neuroblasts and glioblasts. F: Embryonic stage 14 shows migration of the glia toward periphery along the motor axons that exit the CNS (white arrowhead). G: Glial cells are lined along the axons as they are still in the process of migrating to their final destination. H: Stage 16 embryo marks the completion of glial migration to the periphery along with the motor axons. I–K: Whole-mount embryos of the genotype repo-GAL4::UAS-mCD8GFP at stage 12 (I), stage 14 (J), and stage 16 (K) show glial membrane ensheathment, highlighted by anti-GFP (green) around the peripheral motor axons stained for FAS (red). L–N: Higher magnification of embryos at stage 12 (L, arrowhead points to cone-shaped glia exiting CNS), stage 14 (M), and stage 16 (N) showing extension of glial processes around the motor axons as they migrated out of the CNS and made their way to the periphery. Not all of the axon tracts at stage 16 (N) were ensheathed as glial ensheathment is completed during the larval stages. White lines in B–H and L–N refer to the CNS/PNS transition zones. O: Schematic showing distribution of 7 peripheral glial cells in a single segment at stage 17 of embryonic development in the PNS. The glial cells nearer to the CNS region are exit glia (EG). One of the exit glia is closet to the segmental nerve (SN), and the rest of the glia line the intersegmental nerve (ISN). The neuronal clusters ventral (v), ventral’ (v’), lateral (l), and dorsal (d) are shown as a reference. The most prominent neuronal cluster is the lateral cluster with 5 chordotonal neurons (lch). Both the ISN and SN contain motor and sensory axons.

Fig. 2

Peripheral nerve insulation in Drosophila. A: Transverse section through a Drosophila larval peripheral nerve showing individual axons (a) being wrapped by the membrane (m) of the inner glial cell. This in turn is wrapped by another glial cell, the perineurial glial cell. Extensive SJs were established between perineurial and inner glial membranes (arrowhead; Banerjee et al., 2006a). There is an outer-most layer of neural lamella (L) surrounding the nerve fibers. Scale bar = 1 μm. B: Schematic of a Drosophila peripheral nerve showing the axons and fascicles (a) surrounded by a sheath formed by the inner ensheathing glial cell (i), which in turn is surrounded by the outer perineurial glial cells (o). Arrowhead points to the areas where septate junctions formed between glial membranes.