Organization and function of the blood-brain barrier in Drosophila

Abstract

The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. Here, we present a detailed cellular analysis of the glial cell complement constituting the blood-brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, we characterize different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella. To test the functional roles of these sheaths we performed a series of dye penetration experiments in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants allowed to conclude that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). We further compare the requirements of different septate junction components for the integrity of the blood-brain barrier and provide evidence that two of the six Claudin-like proteins found in Drosophila are needed for normal blood-brain barrier function.

Figure 1.

Anatomy of the peripheral nerve. Orthogonal sections through stacks of confocal images of nerves of third instar larvae stained for HRP expression (blue), Repo expression (red), and GFP expression (green). A, The GFP-gene trap insertion in Collagen IV (Viking) labels the neural lamella. B, The Gal4 driver strain c527 activates expression of CD8:GFP predominantly in the perineurial cells, which are just below the neural lamella. C, The SPG-Gal4 driver activates CD8:GFP expression in the subperineurial cells that tightly encircle the axonal fascicles (blue). Note the occurrence of a glial cell nucleus in the fascicle (B, C). D, The GFP-gene trap insertion in neurexinIV (#454) labels a thin stripe along the entire axonal fascicle. E, Repo-Gal4 activates CD8:GFP expression in all glial cells. Note, that some GFP expression is also visible within the nerve. F, A GFP-gene trap insertion in the nervana2 gene labels glial cell membranes within the fascicle. Scale bars: 2 μm.

Figure 2.

Morphology of perineurial and subperineurial glial cells. To label individual cells or cell clones Flp expression was induced in glial cells (repoFlp; repoGal4 UASactin::GFP; Gal80 FRT19A/FRT19A). Larval nervous systems were stained for Repo expression (red) and GFP expression (green), and neurons were labeled with anti HRP (blue). A, MARCM cell clone of perineurial glial cells in the third instar larval brain. Note the extensive cell protrusions generated by these cells (arrowhead). B, A large MARCM clone consisting exclusively of perineurial cells in the abdominal part of the ventral nerve cord of a third instar larval brain. Perineurial glial cells show many fine filopodia-like cell protrusions (arrowhead). C, Small flip-out clone of subperineurial glial cells (repoGal4; UASflp; UAS>CD2 y⁺>mCD8GFP). In this confocal section, two of the nuclei can be identified. In contrast to the perineurial glia, the subperineurial cells never form lateral filopodia-like extensions (arrowhead). D, D', The large and flat subperineurial glial cells are covered by an outer layer of glial cells, the perineurium. D', In an orthogonal section, as indicated by the white line in D, Repo-positive glial nuclei are seen apically to the GFP expressing glial cells (arrowhead). E, Projection of confocal stacks of a third instar brain lobe stained for Repo (red), HRP (blue) and expression of NeurexinIV::GFP fusion protein that is confined to the septate junctions of subperineurial cells. Scale bar, 85 μm. F, F', CNS of a stage 16 embryo stained for Neurexin::GFP and Repo expression. Note that some Repo-positive nuclei are located apically to the GFP expressing subperineurial cells.

Figure 3.

Glial cells in the peripheral nervous system. To label individual cells or cell clones Flp expression was induced in glial cells of animals carrying a UAS>CD2 y⁺>mCD8GFP construct. Larval nervous systems were stained for HRP expression (blue), Repo expression (red), and GFP expression (green). The top panel shows an overview of a ventral nerve cord with attached peripheral nerves. The boxed areas are shown in higher magnification in A–C. A, B, Peripheral nerve with a GFP labeled single wrapping glial cell. This glial cell spreads over at least 200 μm. The position of four orthogonal sections is indicated by small letters (a–d). The wrapping glial cells can form very thin processes that extend over long distances. C, C', The upper nerve shows a wrapping glia that has covered almost the entire fascicle. The lower nerve shows a perineurial glia clone. The arrowhead denotes a fine cell process. C', Different confocal z-section of the same region as shown in C. Note the processes of the perineurial glial cell that cover the nerve. The small letters indicate the position of the orthogonal sections (e, f). Fine perineurial cell processes are indicated by an arrowhead (C', f). g, h, Two additional examples of perineurial glial cells.

Figure 4.

Sensory and motor axons project in distinct fascicles. Preparations of stage 16 nervous systems stained for sensory and motor axons. Anterior is up. A, C, Embryo carrying the 43Gal4 insertion that directs GFP expression to many sensory neurons (green) and some neuropile glia in the CNS (ng) counterstained for the expression of the Fasciclin II protein (red) which is found on all motor axons as well as on some interneurons (in) in the CNS. Note that within the PNS sensory axons and motor axons are running in distinct trajectories (arrowheads). B, D, Embryo carrying a Fasciclin II GFP gene trap insertion (green) counterstained with Mab 22C10 labeling the Futsch protein (red) that is expressed in the peripheral sensory neurons and their axons. A similar separation of sensory axons and motor axons as in A and C can be seen. E, Third instar larvae expressing GFP in motor axons directed by a fasII:Gal4 driver (green). Motor axons are still found in one part of the nerve (sensory axons are labeled with 22C10; Futsch protein, red; all axons are counterstained with anti HRP in blue). F, Third instar larval nerves expressing GFP in sensory axons directed by the cha:Gal4 driver (green) do not intermingle with motor axons (red, anti-Fas II staining). The dotted line indicates the position of the orthogonal section shown on the right.

Figure 5.

Ultrastructural morphology of perineurial and subperineurial cells. A–G, The figure shows electron micrographs of stage 16 embryonic nerves (A, B), first larval instar nerves (E–G), and third instar ventral nerve cord (C, D). A, In a peripheral nerve of a stage 16 nerve, perineurial (pg) and subperineurial glial cells (spg) are tightly associated with the axonal fascicles. The wrapping glia (wg) does not ensheath individual axons. The boxed area is shown in magnification in B. B, No glial processes can be detected within the fascicle. The axons are in direct contact with the subperineurial glia (spg) that forms a thin layer around the fascicle (arrowheads). C, In the CNS of a third instar larvae the subperineurial glia (light blue shading) is characterized by its flat appearance. Septate junctions can only be recognized in this glial layer (arrowhead). A thick neural lamella (nl) covers the nervous system. D, In the perineurial glial cell layer (light green shading), numerous cell protrusions can be seen (asterisk). These processes never invade into the subperineurial layer. E, In a first instar larval nerve, septate junctions are formed by the subperineurial cells. The image corresponds to the dotted area in F. F, Only few perineurial glial cells are found along the nerve. They do not fully cover the subperineurial cells as they do in later developmental stages. The wrapping glia has not yet started to individually ensheath every axon. G, The different glial cells are highlighted by shading. nl, Neural lamella; pg, perineurial glia; spg, subperineurial glia; sj, septate junction; wg, wrapping glia; ax, axons. Scale bars: 1 μm.

Figure 6.

Three glial cell layers are present in the larval nerve. A, Electron micrograph of a third larval instar peripheral nerve. The neural lamella (nl) has the same size throughout the nerve circumference. Below are the perineurial glial cells (pg, one cell is highlighted by light blue shading) that show various indentations and profiles of small cell protrusions. The fascicle is encircled by one subperineurial glial cell that forms only short processes toward the axon fascicle (spg, arrows, the cell is labeled in blue). The subperineurial glia forms autocellular septate junctions (boxed area and magnification; white arrowhead points to septate junctions; black arrowhead points to septate junction-free cell–cell contact). Within the fascicle, usually two to three glial cell profiles can be detected (one profile is highlighted in red). B, Third instar larval nerves were stained for HRP (blue), Repo expression (red), and Dlg:GFP expression (green). Scale bar, 1 μm. B, C, Lateral and orthogonal (C) view of a larval nerve expressing a Dlg:GFP fusion protein under the control of the SPG:Gal4 driver. GFP expression accumulates at the septate junctions formed by the subperineurial glial cell.

Figure 7.

Septate junction formation in the PNS. Septate junction formation is followed using the neurexinIV GFP gene trap insertion (green) and confocal analysis. Glial cell nuclei are stained in red (Repo staining) whereas axonal membranes are shown in blue (HRP staining). A, Confocal analysis of the entire nerve, with only the NeurexinIV::GFP expression shown in B. The dotted lines indicate the orthogonal sections shown under (a–e). In level c, septate junctions are formed between two glial cells resulting in a ring-like structure. C, Note the extended and broadened expression of NeurexinIV::GFP, which is in contrast to the discrete NeurexinIV::GFP expression in a corresponding contact point in the tracheal system.

Figure 8.

Different glial layers contribute differently to the blood–brain barrier. Differently sized fluorescent-labeled dextran molecules were injected into stage 17 embryos of the following genotypes: w¹¹¹⁸; gcm^N7–4; nrxIV⁴³⁰⁴; sinu^NWU7, and moody^Δ17. Images shown were taken 2 min after injection. The differently sized dextrans are indicated; the images after injection of 10 and 70 kDa dextran were taken with a Zeiss LSM 5 Live microscope. The scan mode was set at a speed of four frames per second. The images after injection of 500 kDa dextran were taken with a Zeiss LSM 510 Meta. Homozygous embryos were recognized using GFP-labeled balancer chromosomes. A–C, A w¹¹¹⁸ embryo is used as wild-type control. D–F, In gcm mutants, all dyes readily penetrated into the nerve cord. G–I, In nrxIV mutant embryos, larger dextran molecules did not penetrate as easily. J–O, Dye penetration in sinuous mutants is similar to penetration in moody mutants. P, Quantification of 10 kDa dextran uptake over 30 min in the mutants indicated.