Fig. 2
Ensheathing glial cells cover the neuropil and contact neurons at its margin. a Ventral nerve cord of a third-instar larva with the genotype (UAS-CD8GFP; R83E12-Gal4) stained for expression of GFP (green), Repo (red), and neuronal membranes (blue, anti-HRP staining). R83E12-Gal4 expressing ensheathing glial cells cover the entire neuropil. The white dashed line indicates the position of the orthogonal section below. b, c Single cell clones show individual ensheathing glial cells. An ensheathing glial cell covering the nerve root is labeled in green, an ensheathing glial cell covering the neuropil only is shown in magenta. d Schematic of the GFP reconstitution across synaptic partners technique (GRASP). Expression of GFP1–10 in glial cells is controlled by Gal4/UAS, whereas expression of GFP11 is controlled by LexA/LexOP. Only when GFP1–10 and GFP11 expressing cells contact each other, GFP is reconstituted and fluorescence can be observed. e Pan-neuronal expression of GFP as detected by antibody staining. The white dashed line indicates the position of the orthogonal section below. Note that GFP expression is also found in neuronal cell bodies (arrowhead). f–h GFP reconstitution in the third-instar larval CNS. Animals expressing the two GFP fragments in combinations as indicated. Upper panels show maximum intensity projection of the reconstituted GFP signal (no antibody staining). Middle: A single confocal section also stained for horseradish peroxidase (HRP; neuronal membranes, blue). The white dashed lines indicate the positions of the orthogonal sections shown below. f GFP reconstitution indicating neuronal membranes and astrocytes contacting each other is found throughout the neuropil (red asterisks). g Ensheathing glial cells and neurons contact each other only in the nerve roots (arrows) and at the margin of the neuropil (arrowheads). h Ensheathing glial cells and vGlut expressing motor neurons contact each other just at the dorsal margin of the neuropil. Scale bars are 20 µm
