Morphological diversity and development of glia in Drosophila

Abstract

Insect glia represents a conspicuous and diverse population of cells and plays a role in controlling neuronal progenitor proliferation, axonal growth, neuronal differentiation and maintenance, and neuronal function. Genetic studies in Drosophila have elucidated many aspects of glial structure, function, and development. Just as in vertebrates, it appears as if different classes of glial cells are specialized for different functions. On the basis of topology and cell shape, glial cells of the central nervous system fall into three classes (Fig. 1A-C): (i) surface glia that extend sheath-like processes to wrap around the entire brain; (ii) cortex glia (also called cell body-associated glia) that encapsulate neuronal somata and neuroblasts which form the outer layer (cortex) of the central nervous system; (iii) neuropile glia that are located at the interface between the cortex and the neuropile, the central domain of the nervous system formed by the highly branched neuronal processes and their synaptic contacts. Surface glia is further subdivided into an outer, perineurial layer, and an inner, subperineurial layer. Likewise, neuropile glia comprises a class of cells that remain at the surface of the neuropile (ensheathing glia), and a second class that forms profuse lamellar processes around nerve fibers within the neuropile (astrocyte-like or reticular glia). Glia also surrounds the peripheral nerves and sensory organs; here, one also recognizes perineurial and subperineurial glia, and a third type called "wrapping glia" that most likely corresponds to the ensheathing glia of the central nervous system. Much more experimental work is needed to determine how fundamental these differences between classes of glial cells are, or how and when during development they are specified. To aid in this work the following review will briefly summarize our knowledge of the classes of glial cells encountered in the Drosophila nervous system, and then survey their development from the embryo to adult.

Fig. 1

Classification and distribution of glia in the Drosophila brain. A. Schematic cross section of brain showing exterior cortex (neuronal and glial cell bodies), central neuropile (neuronal and glial processes), and glial cells. Neurons are subdivided into discrete lineages; each lineage is formed by one neuronal progenitor cell (neuroblast). Cell bodies and processes of neurons that belong to the same lineage form a structural unit, as schematically depicted. The five major classes of glial cells are represented in different colors. B, C. Confocal section of one adult brain hemisphere labeled with synaptic marker Nc82 (B) and glial marker Nervana 2-Gal4>UAS-GFP (Nrv2; C). In B, the interior of compartments shows strong label due to the high density of synapses. Compartment boundaries, formed by glial septa, express a low signal. In C, compartment boundaries which are rich in glial processes are strongly signal-positive. Note also Nrv2-positive cortex glial sheaths surrounding neuronal cell bodies. Bar: 20μm

Fig. 2

Surface glia. A: Confocal section of adult brain hemisphere. GFP is activated by surface glia-specific driver a250-Gal4 (kindly provided by Dr. Julie Simpson, JFRC). Cx cortex; np neuropile. B, B′: Transmission electron microscopic (TEM) photograph of medial part of first instar larval brain. Surface glia comprises an outer layer of perineurial cells (png), covered by a basement membrane (bm), and an inner layer of subperineurial cells (spg). Septate junctions (sj) are formed between subperineurial cells (B′). Note thin nerve (nv) traversing surface glial layer. At the medial position shown, neuronal fiber bundles (np) extending towards the brain commissure are lying directly underneath the surface glial layer. These bundles are surrounded by neuropile glia (npg). C: TEM section showing surface of lateral part of first instar brain. A perineurial layer is lacking; the superineurial glia, recognizable by its characteristic low electron density and the presence of septate junctions (not shown in this picture), forms the outermost layer of glia. Underneath superineurial layer one can distinguish the electron-dense cortex glia (cg) which surrounds neuronal cell bodies (ne). D–K: confocal sections showing clones of surface glia. Upper row (D–G): Perineurial glia. Lower row (H–K): Subperineurial glia. Left two columns (D, E, H, I): Third instar larva. Right two columns (F, G, J, K): Adult. All clones were induced at early first instar larval stage. Perineurial glial cells are elongated, relatively small cells that form the outer layer of surface glia. Arrows in D (inset) and G point at Repo-positive nuclei located within the GFP-labeled perineurial cells; arrowheads indicate Repo-positive, GFP-negative nuclei underneath which represent subperineurial glia. D and G show labeled clones in cross section; E and F are surface views of clones. The series of photographs of clones of subperineurial glia shown in panels H–K is constructed in the same manner as D–G above. Note large size (I, J) and small thickness (H, K) of subperineurial cells. E, I: from Stork et al., 2008. F, G, J, K: from Awasaki et al., 2008. Bar: 20μm (A, D, F, H, J); 10μm (E, I); 5μm (G, K); 0.5μm (B, C)

Fig. 3

Septate junctions in surface glia: perineurial or subperineurial? A: Part of Fig. 10 of Carlson et al. (2000), showing Drosophila larval brain hemisphere labeled with anti-Neurexin (anti-Nrx) to visualize septate junctions. Cells expressing Nrx are designated as “perineurial glia). B: Part of Fig. 2E of Stork et al. (2008), showing brain hemisphere in which cells forming septate junctions are labeled by Nrx-Gal4>UAS-GFP; this cell type is referred to as “subperineurial glia”.

Fig. 4

Cortex glia. A, A′: Confocal section of adult brain hemisphere. Cx cortex; np neuropile. GFP is activated by cortex glia-specific driver CG4288-Gal4 (kindly provided by Dr. Julie Simpson, JFRC). B: Clone of cortex glia in adult brain. Note location of glial cell body, containing Repo-positive nucleus [arrow; from Awasaki et al., 2008 (with permission) ] C, D: TEM photograph of part of first instar larval brain. Thin, electron-dense process of cortex glial cell (cg) separates three neighboring neuronal somata (ne1-3). High magnification view (D) illustrates small septate junction (sj) between cortex glia and neuron. E: Confocal section of larval brain hemisphere showing clone of cortex glia (from Pereanu et al., 2005). Neuropile is labeled with antibody against Drosophila N-cadherin (DNcad). Note surface lamella (sl) and neuropile lamella (nl). In the deep cortex, primary neurons and early born secondary neurons are individually wrapped by cortex glia; note small diameter of “holes” (each one corresponding to one neuron) surrounded by cortex glia (arrow). In superficial layers, neuroblasts and clusters of ganglion mother cells/late born neurons are all lodged together in larger chambers surrounded by cortex glia (arrowhead). Bar: 20μm (A, E); 0.5μm (C); 0.1μm (D)

Fig. 5

Neuropile glia. A–C: Ensheathing glia labeled in toto in adult brain by the specific driver line NP1243 (A; from Awasaki et al., 2008), as a clone in adult brain (B; from Awasaki et al., 2008), and as a clone in larval brain (C; from Pereanu et al., 2005). Note that ensheathing glial cell bodies (arrow in B) surround the surface of neuropile compartments (cmp) without penetrating into the interior of the compartments. D–F: Reticular (astrocyte-like) glia, labeled in adult brain hemisphere by specific driver line NP6520 (from Awasaki et al., 2008), as a clone in adult (E; from Awasaki et al., 2008), and as a larval clone (F; from Pereanu et al., 2005). In reticular glia, cell bodies lie at the neuropile surface, but numerous processes invade the neuropile (arrow in E and F). G: TEM photograph of neuropile of early larval brain, showing interface of cortex with neuronal cell bodies (ne) and neuropile, formed by the neurites of primary neurons (pn). Note electron-dense glial layer (ng) along cortex-neuropile boundary, and surrounding neurites and trachea (tr) in depth of neuropile. Bars: 20μm (A, C, D, F); 10μm (C, E); 1μm (D)

Fig. 6

Peripheral glia. TEM cross section of peripheral nerve of first instar (A) and late third instar larva (B, B′). Surface glia comprises an outer perineurial glial (png; light green) layer and an inner subperineurial layer (spg; dark green). Axons (ax) are surrounded by wrapping glia (red). Note that perineurial glia has not yet expanded around the entire nerve in early larva, as opposed to late larva where it completely surrounds the nerve (B); similarly, wrapping glia forms processes around individual axons only in late larva. B′ shows magnified view of boxed area in B, illustrating auto-septate junction (sj) of subperineurial glia. Arows in (B) point at protrusions of subperineurial glia contacting axons, a phenomenon not observed in subperineurial glia of CNS. Modified, from Stork et al., 2008. Bar: 1μm

Fig. 7

Embryonic origin of ventral nerve cord glia. A: Overview of glial development. B: Neuroblast map of the ventral nerve cord with glioblasts and neuro-glioblasts indicated (after Goodman and Doe, 1993; Campos-Ortega and Hartenstein, 1997; Ragone et al., 2003). Neuroblasts/neuroglioblasts of one hemineuromere are identified alphanumerically. C–E′: schematic horizontal sections (C–E) and cross sections (C′–E′) of late embryonic ventral nerve cord, showing location of glial cells relative to the boundaries of neuromere, neuropile, and peripheral nerves (modified, from Beckervordersandforth et al., 2008). Boxed areas in C′-E′ indicate dorso-ventral levels of corresponding horizontal sections shown in C–E. Color code used in B (neuroblasts) and C–E′ (differentiated glial cells) allows to discern the type of glial cell produced by a given neuroblast (light green: surface/subperineurial glia; turquoise: cortex glia; dark red: longitudinal neuropile glia; light red: nerve glia; bown: midline neuropile glia; yellow/orange: peripheral glia). F: Schematic rendering of peripheral nerve of one hemisegment (modified, from von Hilchen et al., 2008). Location of peripheral glial cells, in colors allowing to distinguish the neuro-glioblasts (panel B) they are derived from. G, H: Schematic cross sections of ventral nerve cord at embryonic stage 13 (G) and 16 (H), showing location and migration pathways of glial cells.

Fig. 8

Embryonic origin of brain glia. A: Approximate location of the clusters of glial progenitors (outlined in green and red) in relation to the neuropile founder clusters (orange) and the brain neuroblast map (Younossi-Hartenstein et al., 1996; Urbach and Technau, 2003). Neuropile founder (or pioneer) clusters are groups of early differentiating neurons that express the Fasciclin II adhesion molecule, and form an early scaffold of axons prefiguring the brain neuropile (Nassif et al., 1998). The P2l/P2m clusters pioneer the brain commissure; D/T pioneers the longitudinal fiber tracts of the tritocerebrum and deutocerebrum that connect the ventral nerve cord with the brain commissure. P3m/P3l/P4l prefigure the neuropile of the protocerebrum. B, C: lateral view of stage 12 embryo (B) and late stage 14 embryo (C) labeled with an antibody against Repo (brown) and against Fas II protein (neuropile founder clusters; purple; anterior to the left). D–F: Digital models of brain hemispheres of stage 11 (D), late stage 12 (E) and late stage 14 (F) embryos, illustrating the pattern of different populations of glia cell precursors (see color key in D) in lateral view. At stage 11 (D) the BPLG cluster of Repo-positive cells makes its appearance adjacent to the D/T neuropile founder cluster. BPLG comprises the progenitors of the neuropile associated glia cells of the brain. Two clusters located in the ventral (VPSG) and dorsal (DPSG) part of the protocerebrum include precursors of subperineurial glia. During stage 12 (B, E) neuropile glia precursors (BPLG) have increased in number and migrated dorsally, reaching the P2l neuropile founder cluster that pioneers the brain commissure. Ventrally, cells of the BPLG have linked up with longitudinal glia cells of the ventral nerve cord (vnc; LG_lb, LG_mx: longitudinal glial precursors of labial and maxillary neuromere, forming the anterior part of the ventral nerve cord). In addition to VPSG and DPSG, two additional clusters of surface glia precursors are present: ADSG and PDSG. In late embryo (C, F), neuropile glia derived from the BPLG cluster forms a continuous covering of the brain commissure (sec), and the cervical connective (ccn; axon tracts of the deutocerebrum and tritocerebrum in between brain commissure and ventral nerve cord). Surface glia precursors (ADSG, PDSR, VPSG, DPSG) have spread over the entire lateral and dorsal brain hemisphere. Cortex glial cells, probably descendants of the VPSG and DPSG clusters, can be distinguished by their position within the depth of the brain cortex. Other abbreviations: aCC/pCC anterior and posterior corner cells (neuropile founder clusters of anterior neuromeres of ventral nerve cord); ol optic lobe Bar: 20μm

Fig. 9

Glial proliferation during the larval period. A, B: Confocal sections of third larval instar brains. In A, mitotic cells are labeled by anti-phospho-histone H3 (red), and glial cells by Nrv2-Gal4 > UAS-GFP (green). Arrow indicates mitotic glial cell. In B, larva had been fed BrdU for 12h prior to dissection. BrdU label appears in secondary neural lineages (sn), and in all three classes of glial cells (arrowheads: surface glia; open arrows: cortex glia; solid arrows: neuropile glia). C: GFP labeled clone of type II DPMm1 lineage (green) containing Gcm-positive (blue) glial precursors (from Izergina et al., 2009). D: 3D digital model of lineages of larval brain hemisphere; posterior view; medial to the right. Arrowheads indicate midline. Type II lineages are rendered in color and are identified alphanumerically (after Pereanu and Hartenstein, 2006; Fung et al., 2009). E, F: GFP labeled clones of secondary lineages with adjacent glial cells in third larval instar brains (from Pereanu et al., 2006). E: Cortex glia (open arrow), located directly adjacent to neuroblast (open arrowhead). F: Surface glia (solid arrowhead) forming part of secondary lineage (open arrowhead). Scale bars: 20μm

Fig. 10

Glia of the optic lobe. A, B: Schematic sections of the larval (A) and adult (B) optic lobe (la lamina; lp lamina primordium; me medulla; mp medulla primordium; lo lobula; lp lobula plate; lop lobula/lobula plate primordium). Glia and glial progenitors are depicted, following color code of previous figures. C–E: Frontal confocal sections of late larval brain hemisphere, labeled with glial marker anti-Repo (red). In C and D, neurons are labeled by Elav-Gal4>UAS-GFP (green). In E, glial membranes are labeled by Nrv2-Gal4>UAS-GFP (green). In C, red channel (Repo-positive glial cells) is shown as Z-projection. Here, rows of glial progenitors associated with the lamina primordium and medulla primordium appear as continuous bands, similar as in schematic (A) above. In the sections shown in D and E, rows of glial progenitors appear twice, at a dorsal level and a ventral level. Note in E lack of differentiation of optic lobe glia (Nrv2-Gal4 driven GFP is faint or absent), with the exception of several cells associated with the lobula neuropile glia (lng). White arrows point at scattered, small Repo-positive cells which appear among the columns of freshly produced medulla neurons (mp). Abbreviations: cb central brain; cbcg cortex glia of central brain; cbng neuropile glia of central brain; eg epithelial glia (neuropile glia of lamina); gpc glial progenitor center; la lamina; lng neuropile glia of lobula/lobula plate; lo lobula; lop lobula/lobula plate primordium; lp lamina primordium; lpl lobula plate; mg marginal glia (neuropile glia of lamina); mng medulla neuropile glia; mp medulla primordium; os optic stalk; png perineurial glia; sg sattelite glia (cortex glia of lamina); spg subperineurial glia; xg glia of outer chiasma.